ASMSPoster collection
Clinical, Forensic and Pharmaceutical Applications
• Page 4Rapid development of analytical method for anti-epileptic drugs in plasma using UHPLC method scouting system coupled to LC/MS/MS
• Page 11Determination of ∆9-tetrahydrocannabinol and two of its metabolites in whole blood, plasma and urine by UHPLC-MS/MS using QuEChERS sample preparation
• Page 17Determination of opiates, amphetamines and cocaine in whole blood, plasma and urine by UHPLC-MS/MS using a QuEChERS sample prepa-ration
• Page 23Simultaneous analysis for forensic drugs in human blood and urine using ultra-high speed LC-MS/MS
• Page 29Simultaneous screening and quantitation of amphetamines in urine by on-line SPE-LC/MS method
• Page 36Single step separation of plasma from whole blood without the need for centrifugation ap-plied to the quantitative analysis of warfarin
• Page 42Development and validation of direct analysis method for screening and quantitation of amphetamines in urine by LC/MS/MS
• Page 48Next generation plasma collection technology for the clinical analysis of temozolomide by HILIC/MS/MS
• Page 54Application of a sensitive liquid chromatography-tandem mass spectrometric method to pharma-cokinetic study of telbivudine in humans
• Page 60Accelerated and robust monitoring for immu- nosuppressants using triple quadrupole mass spectrometry
• Page 66Highly sensitive quantitative analysis of felodip-ine and hydrochlorothiazide from plasma using LC/MS/MS
• Page 73Highly sensitive quantitative estimation of geno-toxic impurities from API and drug formulation using LC/MS/MS
• Page 80Development of 2D-LC/MS/MS method for quan-titative analysis of 1�,25-Dihydroxylvitamin D3 in human serum
• Page 86Analysis of polysorbates in biotherapeutic prod-ucts using two-dimensional HPLC coupled with mass spectrometer
• Page 93A rapid and reproducible Immuno-MS platform from sample collection to quantitation of IgG
• Page 99Simultaneous determinations of 20 kinds of common drugs and pesticides in human blood by GPC-GC-MS/MS
• Page 103Low level quantitation of loratadine from plasma using LC/MS/MS
PO-CON1452E
Rapid development of analyticalmethod for antiepileptic drugs inplasma using UHPLC method scoutingsystem coupled to LC/MS/MS
ASMS 2014 ThP 672
Miho Kawashima1, Satohiro Masuda2, Ikuko Yano2,
Kazuo Matsubara2, Kiyomi Arakawa3, Qiang Li3,
Yoshihiro Hayakawa3
1 Shimadzu Corporation, Tokyo, JAPAN,
2 Kyoto University Hospital, Kyoto, JAPAN,
3 Shimadzu Corporation, Kyoto, JAPAN
2
Rapid development of analytical method for antiepileptic drugs in plasma using UHPLC method scouting system coupled to LC/MS/MS
IntroductionMethod development for therapeutic drug monitoring (TDM) is indispensable for managing drug dosage based on the drug concentration in blood in order to conduct a rational and ef�cient drug therapy. Liquid chromatography coupled with tandem quadrupole mass spectrometry is increasingly used in TDM because it can perform selective and sensitive analysis by simple sample pretreatment. The UHPLC method scouting system coupled to tandem
quadrupole mass spectrometer used in this study can dramatically shorten the total time for optimization of analytical conditions because this system can make enormous combinatorial analysis methods and run batch program automatically. In this study, we developed a high-speed and sensitive method for measurement of seventeen antiepileptics in plasma by UHPLC coupled with tandem quadrupole mass spectrometer.
Figure 1 Antiepileptic drugs used in this assay
Experimental
UHPLC based method scouting system (Nexera X2 Method Scouting System, Shimadzu Corporation, Japan) is configured by Nexera X2 UHPLC modules. For the detection, tandem quadrupole mass spectrometer (LCMS-8050, Shimadzu Corporation, Japan) was used. The system can be operated at a maximum pressure of 130 MPa, and it enables to automatically select up to 96 unique combinations of eight different mobile phases and six different columns. A
dedicated software was newly developed to control the system (Method Scouting Solution, Shimadzu Corporation, Japan), which provides a graphical aid to configure the different type of columns and mobile phases. The software is integrated into the LC/MS/MS workstation (LabSolutions, Shimadzu Corporation, Japan) so that selected conditions are seamlessly translated into method files and registered to a batch queue, ready for analysis instantly.
Instruments
N
O NH2
Carbamazepine Carbamazepine- 10,11-epoxide
N
O NH2
O
Diazepam
N
N
O
CH3
Cl
Ethomuximide
NHCH3
CH3
O
O
Felbamate
O ONH2
O
NH2
O
Gabapentin
NH2
OH
O
N
N
NCl
Cl
NH2
NH2
Lamotrigine Levetiracetam
N O
CH3
NH2
O
Phenobarbial
NH
NH
O
O
O
CH3
Primidone
NH
NH
O
CH3 O
Phenytoin
NH NH
O
O
Tiagabine
SCH3
NS OH
O
CH3
Zonisamide
ON
S
O
O
CH3O
O
OO
O
CH3
CH3
CH3
CH3
OS
O
ONH2
Topiramate Vigabatrin
CH2
NH2
OH
O
Clonazepam
NH
N
N+
O
O O
Cl
-
NH
N
N+
O
O O
Nitrazepam
-
3
Rapid development of analytical method for antiepileptic drugs in plasma using UHPLC method scouting system coupled to LC/MS/MS
Figure 2 Nexera Method Scoutuing System and LCMS-8050 triple quadrupole mass spectrometer
Result
The MS condition optimization was performed by flow injection analysis (FIA) of ESI positive and negative ionization mode, and the compound dependent parameters such as CID and pre-bias voltage were adjusted using automatic
MRM optimization function. The transition that gave highest intensity was used for quantification. The MRM transitions used in this assay are listed in Table 1.
The main standard mixture was prepared in methanol from individual stock solutions. The calibration standards were prepared by diluting the standard mixture with methanol. QC sample was prepared by adding 4 volume of acetonitrile to 1 volume of control plasma, thereby precipitating proteins, and subsequently adding the standard mixture to the supernatant to contain plasma concentration equivalents stated in Table 4. The QC samples were further diluted 100 times (10 μL sample
added to 990μL methanol) before injection. Next step of preparation procedure was divided into three groups by the intensity of each compound. For ethomuximide, phenobarbial and phenytoin, the supernatant was used for the LC/MS/MS analysis without further dilution. For zonisamide, 10 μL supernatant was further diluted with 990 μL methanol. For others, 100 μL supernatant was further diluted with 900 μL methanol. The diluted solutions were used for the LC/MS/MS analysis.
MRM condition optimization
Calibration standards and QC samples
4
Rapid development of analytical method for antiepileptic drugs in plasma using UHPLC method scouting system coupled to LC/MS/MS
Figure. 3 Schematic representation and features of the Nexera Method Scouting System.
Table 1 Compounds, Ionization polarity and MRM transition
Retaintion (min)Compound Polarity Precursor m/z Product m/z
3.84
3.24
3.93
4.79
2.50
2.86
2.27
2.96
2.32
3.90
3.06
3.64
2.83
4.28
3.14
0.82
2.58
+
+
+
+
+
+
+
+
+
+
+
+
+
-
-
-
-
237.1
253.1
316.1
284.9
239.3
172.2
256.2
171.2
281.9
219.2
376.2
130.2
213.1
140.0
231.0
337.9
143.1
194.2
180.15
269.55
154.15
117.20
154.25
211.05
126.15
236.20
162.15
111.15
71.15
132.10
42.00
42.05
78.00
143.10
Carbamazepine
Carbamazepine-10,11-epoxide
Clonazepam
Diazepam
Ethomuximide
Felbamate
Gabapentin
Lamotrigine
Levetiracetam
Nitrazepam
Phenobarbial
Phenytoin
Primidone
Tiagabine
Topiramate
Vigabatrin
Zonisamide
36 analytical conditions, comprising combinations of 9 mobile phase and 4 columns, were automatically investigated using Method Scouting System. Schematic representation of scouting system was shown in Figure 3. From the result of scouting, the combination of 10 mM
ammonium acetate water and methanol for mobile phase and Inertsil-ODS4 for separation column were selected. Using this combination of mobile phase and column, the gradient condition was further optimized. The final analytical condition was shown in Table 2.
UHPLC condition optimization
Auto SamplerLPGE Unit
Column Oven
LCMS-8050
Pump A
Pump B
1 2 3 4
1 2 3 4
(A) 1 – 10mM Ammonium Acetate 2 – 10mM Ammonium Formate 3 – 0.1%FA - 10mM Ammonium Acetate(B) 1 – Methanol 2 – Acetonitrile 3 – Methanol/Acetonitrile=1/1
Kinetex XB-C18 (Phenomenex)
Kinetex PFP (Phenomenex)
InertsilODS-4 (GL Science)
Discovery HS F5-5 (SPELCO)
2.1 x 50 mm
2.1 x 50 mm
2.1 x 50 mm
2.1 x 50 mm
5
Rapid development of analytical method for antiepileptic drugs in plasma using UHPLC method scouting system coupled to LC/MS/MS
Table.2 UHPLC analytical conditions
Figure. 4 Chromatogram of 17 AEDs calibration standards
Column : Inertsil ODS-4 (50 mmL. x 2.1mmi.d., 2um)
Mobile phase : A) 10mM Ammonium Acetate
B) Methanol
Binary gradient : B conc. 3% (0.65 min) → 40% (1.00 min) → 85% (5.00 min)
→ 100% (5.01-8.00 min) → 3% (8.01-10.00 min)
Flow Rate : 0.4 mL/min
Injection vol. : 1 μL
Column Temp. : 40 deg. C
Figure 4 shows MRM chromatograms of the 17 AEDs. It took only 10 minutes per one UHPLC/MS/MS analysis, including column rinsing.
Precision, accuracy and linearity of AEDs
0.0 1.0 2.0 3.0 4.0 5.0 min
Vigabatrin130.20>71.15(+)
0.0 1.0 2.0 3.0 4.0 5.0 min
Gabapentin172.20>154.25(+)
0.0 1.0 2.0 3.0 4.0 5.0 min
Levetiracetam171.20>126.15(+)
0.0 1.0 2.0 3.0 4.0 5.0 min
Ethomuximide140.00>42.00(-)
0.0 1.0 2.0 3.0 4.0 5.0 min
Zonisamide213.10>132.10(+)
0.0 1.0 2.0 3.0 4.0 5.0 min
Primidone 219.20>162.15(+)
0.0 1.0 2.0 3.0 4.0 5.0 min
Felbamate239.30>117.20(+)
0.0 1.0 2.0 3.0 4.0 5.0 min
Lamotrigine256.20>211.05(+)
0.0 1.0 2.0 3.0 4.0 5.0 min
Phenobarbial231.00>42.05(-)
0.0 1.0 2.0 3.0 4.0 5.0 min
Topiramate337.85>78.00(-)
0.0 1.0 2.0 3.0 4.0 5.0 min
Carbamazepine-10,11-epoxide253.10>180.15(+)
0.0 1.0 2.0 3.0 4.0 5.0 min
Phenytoin251.00>208.20(-)
0.0 1.0 2.0 3.0 4.0 5.0 min
Carbamazepine237.10>194.20(+)
0.0 1.0 2.0 3.0 4.0 5.0 min
Nitrazepam 281.90>236.20(+)
0.0 1.0 2.0 3.0 4.0 5.0 min
Clonazepam 316.10>269.55(+)
0.0 1.0 2.0 3.0 4.0 5.0 min
Tiagabine376.20>111.15(+)
0.0 1.0 2.0 3.0 4.0 5.0 min
Diazepam284.90>154.15
Rapid development of analytical method for antiepileptic drugs in plasma using UHPLC method scouting system coupled to LC/MS/MS
6
Table.3 Linearity of 17 AEDs QC sample
Compound Linarity (ng/mL) r2
0.25
0.25
0.005
0.01
25
0.5
2
0.25
0.5
0.005
5
5
0.25
0.25
0.5
0.5
0.5
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
50
50
2.5
5
2500
100
50
50
100
1
500
500
10
50
100
50
20
0.999
0.998
0.998
0.999
0.998
0.998
0.999
0.999
0.999
0.999
0.996
0.998
0.996
0.998
0.998
0.998
0.996
Carbamazepine
Carbamazepine-10,11-epoxide
Clonazepam
Diazepam
Ethomuximide
Felbamate
Gabapentin
Lamotrigine
Levetiracetam
Nitrazepam
Phenobarbial
Phenytoin
Primidone
Tiagabine
Topiramate
Vigabatrin
Zonisamide
Table 3 illustrates linearity of 17 AEDs and Table 4 illustrates accuracy and precision of the QC samples at three concentration levels. Determination coefficient (r2) of all calibration curves was larger than 0.995, and the precision
and accuracy were within +/- 15%. Excellent linearity, accuracy and precision for all 17 AEDs were obtained at only 1 μL injection volume.
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Rapid development of analytical method for antiepileptic drugs in plasma using UHPLC method scouting system coupled to LC/MS/MS
Table.4 Accuracy and precision of 17 AEDs QC sample
Compound
Plasma concentrationequivalents (µg/mL)
Precision (%) Accuracy (%)
HighMiddleLow
1.8
1.8
0.04
0.1
18
3.6
18
1.8
3.6
0.04
3.6
3.6
1.8
1.8
3.6
8.9
36
71
71
1.8
2.9
714
179
143
71
179
1.4
143
143
45
71
143
89
179
2.2
2.4
3.3
3.2
7.8
1.7
1.3
10.5
2.1
3.3
3.5
7.8
3.2
1.8
12.5
1.4
3.3
0.9
1.9
0.7
1.7
1.5
0.4
0.7
1.2
0.5
1.4
6.2
1.9
0.7
1.8
1.5
1.1
1.3
18
18
0.9
0.7
446
89
36
45
89
0.4
71
89
18
18
36
18
89
0.9
1.3
0.5
1.4
1.4
0.8
0.7
1.7
1.1
1.5
1.6
1.2
0.7
1.0
1.2
2.1
1.6
106.1
104.2
106.7
105.8
104.3
97.1
85.8
107.7
99.5
105.0
100.9
103.2
99.5
107.6
105.4
105.9
111.7
103.9
105.0
102.1
106.6
99.9
106.3
98.8
98.4
104.9
105.2
108.4
100.1
112.6
105.7
101.6
101.6
100.4
95.8
98.2
90.1
100.6
97.0
91.7
89.5
99.2
90.4
97.9
95.8
96.2
97.1
97.5
96.1
88.8
95.2
Carbamazepine
Carbamazepine-10,11-epoxide
Clonazepam
Diazepam
Ethomuximide
Felbamate
Gabapentin
Lamotrigine
Levetiracetam
Nitrazepam
Phenobarbial
Phenytoin
Primidone
Tiagabine
Topiramate
Vigabatrin
Zonisamide
HighMiddleLowHighMiddleLow
Conclusions• We could select the most suitable combination of mobile phase and column from 36 analytical condition without
time-consuming investigation.• We have measured plasma sample as it is after 100-10,000 times dilution by methanol without making tedious sample
pretreatment. Excellent linearity, precision and accuracy for all 17 AEDs were obtained at only 1 uL injection volume.
PO-CON1446E
Determination of Δ9-tetrahydrocannabinoland two of its metabolites in whole blood,plasma and urine by UHPLC-MS/MS usingQuEChERS sample preparation
ASMS 2014 ThP600
Sylvain DULAURENT1, Mikaël LEVI2, Jean-michel GAULIER1,
Pierre MARQUET1,3 and Stéphane MOREAU2
1 CHU Limoges, Department of Pharmacology and Toxicology,
Unit of clinical and forensic toxicology, Limoges, France ; 2 Shimadzu France SAS, Le Luzard 2, Boulevard Salvador
Allende, 77448 Marne la Vallée Cedex 23 Univ Limoges, Limoges, France
2
Determination of Δ9-tetrahydrocannabinol and two of its metabolites in whole blood, plasma and urine by UHPLC-MS/MS using QuEChERS sample preparation
IntroductionIn France, as in other countries, cannabis is the most widely used illicit drug. In forensic as well as in clinical contexts, ∆9-tetrahydrocannabinol (THC), the main active compound of cannabis, and two of its metabolites [11-hydroxy-∆9-tetrahydrocannabinol (11-OH-THC) and 11-nor-∆9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH)] are regularly investigated in biological �uids for example in Driving Under the In�uence of Drug context (DUID) (�gure 1). Historically, the concentrations of these compounds were determined using a time-consuming extraction procedure
and GC-MS. The use of LC-MS/MS for this application is relatively recent, due to the low response of these compounds in LC-MS/MS while low limits of quanti�cation need to be reached. Recently, on-line Solid-Phase-Extraction coupled with UHPLC-MS/MS was described, but in our hands it gave rise to signi�cant carry-over after highly concentrated samples. We propose here a highly sensitive UHPLC-MS/MS method with straightforward QuEChERS sample preparation (acronym for Quick, Easy, Cheap, Effective, Rugged and Safe).
Methods and MaterialsIsotopically labeled internal standards (one for each target compound in order to improve method precision and accuracy) at 10 ng/mL in acetonitrile, were added to 100 µL of sample (urine, whole blood or plasma) together with 50 mg of QuEChERS salts (MgSO4/NaCl/Sodium
citrate dehydrate/Sodium citrate sesquihydrate) and 200 µL of acetonitrile. Then the mixture was shaken and centrifuged for 10 min at 12,300 g. Finally, 15 µL of the upper layer were injected in the UHPLC-MS-MS system. The whole acquisition method lasted 3.4 min.
Figure 1: Structures of THC and two of its metabolites
OH
O
H
HCH3
CH3
OHO
THC-COOH
OH
O
H
H
CH2
CH3CH3
OH
11-OH-THC
OH
O
H
H
CH3
CH3CH3
THC
3
Determination of Δ9-tetrahydrocannabinol and two of its metabolites in whole blood, plasma and urine by UHPLC-MS/MS using QuEChERS sample preparation
UHPLC conditions (Nexera MP system)
Column : Kinetex C18 50x2.1 mm 2.6 µm (Phenomenex)
Mobile phase A : 5mM ammonium acetate in water
B : CH3CN
Flow rate : 0.6 mL/min
Time program : B conc. 20% (0-0.25 min) - 90% (1.75-2.40 min) - 20% (2.40-3.40 min)
Column temperature : 50 °C
MS conditions (LCMS-8040)
Ionization : ESI, negative MRM mode
Ion source temperatures : Desolvation line: 300°C
Heater Block: 500°C
Gases : Nebulization: 2.5 L/min
Drying: 10 L/min
MRM Transitions:
Compound MRM Dwell time (msec)
THC 313.10>245.25 (Quan) 60
313.10>191.20 (Qual) 60
313.10>203.20 (Qual) 60
THC-D3 316.10>248.30 (Quan) 5
316.10>194.20 (Qual) 5
11-OH-THC 329.20>311.30 (Quan) 45
329.20>268.25 (Qual) 45
329.20>173.20 (Qual) 45
11-OH-THC-D3 332.30>314.40 (Quan) 5
332.30>271.25 (Qual) 5
THC-COOH 343.20>245.30 (Quan) 50
343.20>325.15 (Qual) 50
343.20>191.15 (Qual) 50
343.20>299.20 (Qual) 50
THC-COOH-D3 346.20>302.25 (Quan) 5
346.20>248.30 (Qual) 5
Pause time : 3 msec
Loop time : 0.4 sec (minimum 20 points per peak for each MRM transition)
4
Determination of Δ9-tetrahydrocannabinol and two of its metabolites in whole blood, plasma and urine by UHPLC-MS/MS using QuEChERS sample preparation
Figure 1: Chromatogram obtained after an injection of a 15 µL whole blood extract spiked at 50 µg/L
Results
A typical chromatogram of the 6 compounds is presented in figure 1.
Chromatographic conditions
Figure 2: in�uence of QuEChERS salts on urine extraction/partitioning: A: acetonitrile with urine sample lead to one phase / B: acetonitrile, QuEChERS salts and urine lead to 2 phases.
As described by Anastassiades et al. J. AOAC Int 86 (2003) 412-31, the combination of acetonitrile and QuEChERS salts allowed the extraction/partitioning of compounds of interest from matrix. This extraction/partitioning process is not only
obtained with whole blood and plasma-serum where deproteinization occurred and allowed phase separation, but also with urine as presented in figure 2.
Extraction conditions
A B
5
Determination of Δ9-tetrahydrocannabinol and two of its metabolites in whole blood, plasma and urine by UHPLC-MS/MS using QuEChERS sample preparation
Figure 3: Chromatogram obtained after an injection of a 15 µL whole blood extract spiked at 0.5 µg/L (lower limit of quanti�cation).
One challenge for the determination of cannabinoids in blood using LC-MS/MS is the low quantification limits that need to be reached. The French Society of Analytical Toxicology proposed 0.5 µg/L for THC et 11-OH-THC and 2.0 µg/L for THC-COOH. With the current application, the
lower limit of quantification was fixed at 0.5 µg/L for the three compounds (3.75 pg on column). The corresponding extract ion chromatograms at this concentration are presented in figure 3.
Validation data
The upper limit of quantification was set at 100 µg/L. Calibration graphs of the cannabinoids-to-internal standard peak-area ratios of the quantification transition versus
expected cannabinoids concentration were constructed using a quadratic with 1/x weighting regression analysis (figure 4).
Contrary to what was already observed with on-line Solid-Phase-Extraction no carry-over effect was noted using the present method, even when blank samples were
injected after patient urine samples with concentrations exceeding 2000 µg/L for THC-COOH.
THC11-OH-THCTHC-COOH
Figure 4: Calibration curves of the three cannabinoids
THC11-OH-THCTHC-COOH
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Determination of Δ9-tetrahydrocannabinol and two of its metabolites in whole blood, plasma and urine by UHPLC-MS/MS using QuEChERS sample preparation
Conclusions• Quick sample preparation based on QuEChERS salts extraction/partitioning, almost as short as on-line Solid Phase
Extraction.• Low limit of quanti�cation compatible with determination of DUID.• No carry over effect noticed.
PO-CON1445E
Determination of opiates, amphetaminesand cocaine in whole blood, plasmaand urine by UHPLC-MS/MS usinga QuEChERS sample preparation
ASMS 2014 ThP599
Sylvain DULAURENT1, Mikaël LEVI2, Jean-michel GAULIER1,
Pierre MARQUET1,3 and Stéphane MOREAU2
1 CHU Limoges, Department of Pharmacology and Toxicology,
Unit of clinical and forensic toxicology, Limoges, France ; 2 Shimadzu France SAS, Le Luzard 2, Boulevard Salvador
Allende, 77448 Marne la Vallée Cedex 23 Univ Limoges, Limoges, France
2
Determination of opiates, amphetamines and cocaine in whole blood, plasma and urine by UHPLC-MS/MS using a QuEChERS sample preparation
IntroductionThe determination of drugs of abuse (opiates, amphetamines, cocaine) in biological �uids is still an important issue in toxicology, in cases of driving under the in�uence of drugs (DUID) as well as in forensic toxicology. At the end of the 20th century, the analytical methods able to determine these three groups of narcotics were mainly based on a liquid-liquid-extraction with derivatization followed by GC-MS. Then LC-MS/MS was proposed,
coupled with off-line sample preparation. Recently, on-line Solid-Phase-Extraction coupled with UHPLC-MS/MS was described, but in our hands it gave rise to signi�cant carry-over after highly concentrated samples. We propose here another approach based on the QuEChERS (acronym for Quick, Easy, Cheap, Effective, Rugged and Safe) sample preparation principle, followed by UHPLC-MS/MS.
Methods and MaterialsThis method involves 40 compounds of interest (13 opiates, 22 amphetamines, as well as cocaine and 4 of its
metabolites) and 18 isotopically labeled internal standards (designed with *) (Table1).
Table 1: list of analyzed compounds with their associate internal standard (*)
Cocaine and metabolitesAmphetamines or related
compounds Opiates
• Anhydroecgonine methylester• Benzoylecgonine*• Cocaethylene*• Cocaine*• Ecgonine methylester*
• 2-CB• 2-CI• 4-MTA• Ritalinic acid• Amphetamine*• BDB• Ephedrine*• MBDB• m-CPP• MDA*• MDEA*• MDMA*• MDPV• Mephedrone• Metamphetamine*• Methcathinone• Methiopropamine• Methylphenidate• Norephedrine• Norfen�uramine• Norpseudoephedrine• Pseudoephedrine
• 6-monoacetylmorphine*• Dextromethorphan• Dihydrocodeine*• Ethylmorphine• Hydrocodone• Hydromorphone• Methylmorphine*• Morphine*• Naloxone*• Naltrexone*• Noroxycodone*• Oxycodone*• Pholcodine
3
Determination of opiates, amphetamines and cocaine in whole blood, plasma and urine by UHPLC-MS/MS using a QuEChERS sample preparation
UHPLC conditions (Nexera MP system, �gure 1)
Column : Restek Pinnacle DB PFPP 50x2.1 mm 1.9 µm
Mobile phase A : 5mM Formate ammonium with 0.1% formic acid in water
B : 90% CH3OH/ 10% CH3CN (v/v) with 0.1 % formic acid
Flow rate : 0.474 mL/min
Time program : B conc. 15% (0-0.16 min) - 20% (1.77 min) - 90% (2.20 min) –
100% (4.00 min) – 15% (4.10-5.30 min)
Column temperature : 50 °C
MS conditions (LCMS-8040, �gure 1)
Ionization : ESI, Positive MRM mode
Ion source temperatures : Desolvation line: 300°C
Heater Block: 500°C
Gases : Nebulization: 2.5 L/min
Drying: 10 L/min
MRM Transitions : 2 Transitions per compounds were dynamically scanned for 1 min except
pholcodine (2 min)
Pause time : 3 msec
Loop time : 0.694 sec (minimum 17 points per peak for each MRM transition)
To 100 µL of sample (urine, whole blood or plasma) were added isotopically labeled internal standards (in order to improve method precision and accuracy) at 20 µg/L in acetonitrile (20 µL), and 200 µL of acetonitrile. After a 15 s shaking, the mixture was placed at -20°C for 10 min. Then approximately 50 mg of QuEChERS salts (MgSO4/NaCl/Sodium citrate dehydrate/Sodium citrate
sesquihydrate) were added and the mixture was shaken again for 15 s and centrifuged for 10 min at 12300 g. The upper layer was diluted (1/3; v/v) with a 5 mM ammonium formate buffer (pH 3). Finally, 5 µL were injected in the UHPLC-MS/MS system. The whole acquisition method lasted 5.5 min.
Figure 1: Shimadzu UHPLC-MS/MS Nexera-8040 system
4
Determination of opiates, amphetamines and cocaine in whole blood, plasma and urine by UHPLC-MS/MS using a QuEChERS sample preparation
Figure 2: Chromatograms obtained after an injection of a 5 µL whole blood extract spiked at 200 µg/L. Order of retention - A: norephedrine and norpseudoephedrine / B: ephedrine and pseudoephedrine
Figure 3: Chromatogram obtained after an injection of a 5 µL whole blood extract spiked at 200 µg/L
Results
The analytical conditions allowed the chromatographic separation of two couples of isomers: norephedrine and norpseudoephedrine; ephedrine and pseudoephedrine
(figure 2). A typical chromatogram of the 58 compounds is presented in figure 3.
Chromatographic conditions
A B
5
Determination of opiates, amphetamines and cocaine in whole blood, plasma and urine by UHPLC-MS/MS using a QuEChERS sample preparation
Figure 4: in�uence of QuEChERS salts on urine extraction/partitioning: A: acetonitrile with urine sample lead to one phase / B: acetonitrile, QuEChERS salts and urine lead to 2 phases.
As described by Anastassiades et al. J. AOAC Int 86 (2003) 412-31, the combination of acetonitrile and QuEChERS salts allowed the extraction/partitioning of compounds of interest from matrix. This extraction/partitioning process is not only
obtained with whole blood and plasma-serum where deproteinization occurred and allowed phase separation, but also with urine as presented in figure 4.
Extraction conditions
Among the 40 analyzed compounds, 38 filled the validation conditions in term of intra- and inter-assay precision and accuracy were less than 20% at the lower limit of quantification and less than 15% at the other concentrations.Despite the quick and simple sample preparation, no significant matrix effect was observed and the lower limit of quantification was 5 µg/L for all compounds, while the upper limit of quantification was set at 500 µg/L. The
concentrations obtained with a reference (GC-MS) method in positive patient samples were compared with those obtained with this new UHPLC-MS/MS method and showed satisfactory results.Contrary to what was already observed with on-line Solid-Phase-Extraction, no carry-over effect was noted using the present method, even when blank samples were injected after patient urine samples with analytes concentrations over 2000 µg/L.
Validation data
A B
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Determination of opiates, amphetamines and cocaine in whole blood, plasma and urine by UHPLC-MS/MS using a QuEChERS sample preparation
Conclusions• Separation of two couples of isomers with a run duration less than 6 minutes and using a 5 cm column.• Quick sample preparation based on QuEChERS salts extraction/partitioning, almost as short as on-line Solid Phase
Extraction.• Lower limit of quanti�cation compatible with determination of DUID.• No carry over effect noticed.
PO-CON1442E
Simultaneous analysis for forensic drugs in human blood and urine using ultra-high speed LC-MS/MS
ASMS 2014 ThP-592
Toshikazu Minohata1, Keiko Kudo2, Kiyotaka Usui3, Noriaki Shima4, Munehiro Katagi4, Hitoshi Tsuchihashi5, Koichi Suzuki5, Noriaki Ikeda2
1Shimadzu Corporation, Kyoto, Japan 2Kyushu University, Fukuoka, Japan 3Tohoku University Graduate School of Medicine, Sendai, Japan 4Osaka Prefectural Police, Osaka, Japan 5Osaka Medical Collage, Takatsuki, Japan
2
Simultaneous analysis for forensic drugs in human blood and urine using ultra-high speed LC-MS/MS
IntroductionIn Forensic Toxicology, LC/MS/MS has become a preferred method for the routine quantitative and qualitative analysis of drugs of abuse. LC/MS/MS allows for the simultaneous analysis of multiple compounds in a single run, thus enabling a fast and high throughput analysis. In this study, we report a developed analytical system using ultra-high
speed triple quadrupole mass spectrometry with a new extraction method for pretreatment in forensic analysis. The system has a sample preparation utilizing modi�ed QuEChERS extraction combined with a short chromatography column that results in a rapid run time making it suitable for routine use.
Figure 1 Scheme of the modi�ed QuEChERS procedure
[ ref.] (1) Usui K et al, Legal Medicine 14 (2012), 286-296
Methods and Materials
Whole blood sample preparation was carried out by the modified QuEChERS extraction method (1) using Q-sep™ QuEChERS Sample Prep Packets purchased from RESTEK (Bellefonte, PA).
1) Add 0.5 mL of blood and 1 mL of distilled water into the 15 mL centrifugal tube and agitate the mixture using a vortex mixer.
2) Add two 4 mm stainless steel beads, 1.5 mL of acetonitrile and 100 µL of acetonitrile solution containing 1 ng/µL of Diazepam-d5. Then agitate using the vortex mixer.
3) Add 0.5 g of the filler of the Q-sep™ QuEChERS Extraction Salts Packet.
4) Vigorously shake the tube by hand several times, agitate well using the vortex mixer for approximately 20 seconds. Then centrifuge the tube for 10 minutes at 3000 rpm.
5) Move the supernatant to a different 15 mL centrifugal tube and add 100 µL of 0.1 % TFA acetonitrile solution. Then, dry using a nitrogen-gas-spray concentration and drying unit or a similar unit.
6) Reconstitute with 200 µL of methanol using the vortex mixer. Then move it to a microtube, and centrifuge for 5 minutes at 10,000 rpm.
7) Transfer 150 µL of the supernatant to a 1.5 mL vial for HPLC provided with a small-volume insert.
Sample Preparation
Sample0.5 mL
Water 1 mL ACN 1.5 mL Diazepam-d5 (IS) 100ng Stainless-Steel Beads (4mm x 2)
[Shake] [Centrifuge]
Transfer supernatant Add 100uL of 0.1% TFA
Dry
Reconstitution with 200 uL MeOH
LC/MS/MS analysis
Q-sep QuEChERSExtraction Salts(MgSO4,NaOAc)
3
Simultaneous analysis for forensic drugs in human blood and urine using ultra-high speed LC-MS/MS
Analytical Conditions
LC-MS/MS Analysis
HPLC (Nexera UHPLC system)
Column : YMC Triart C18 (100x2mm, 1.9μm)
Mobile Phase A : 10 mM Ammonium formate - water
Mobile Phase B : Methanol
Gradient Program : 5%B (0 min) - 95%B (10 min - 13min) - 5%B (13.1 min - 20 min)
Flow Rate : 0.3 mL / min
Column Temperature : 40 ºC
Injection Volume : 5 uL
Mass (LCMS-8050 triple quadrupole mass spectrometry)
Ionization : heated ESI
Polarity : Positive & Negative
Probe Voltage : +4.5 kV (ESI-Positive mode); -3.5 kV (ESI-Negative mode)
Nebulizing Gas Flow : 3 L / min
Drying Gas Pressure : 10 L / min
Heating gas �ow : 10 L / min
DL Temperature : 250 ºC
BH Temperature : 400 ºC
MRM parameter :
Treated samples were analyzed using a Nexera UHPLC system coupled to a LCMS-8050 triple quadrupole mass spectrometer (Shimadzu Corporation, Japan) with LC/MS/MS Rapid Tox. Screening Database. The Database contains product ion scan spectra for 106 forensic and toxicology-related compounds of Abused drugs, Psychotropic drugs and Hypnotic drugs etc (Table 1) and
provides Synchronized Survey Scan® parameters (product ion spectral data acquisition parameters based on the MRM intensity as threshold) optimized for screening analysis.Samples were separated on a YMC Triart C18 column. A �ow rate of 0.3 mL/min was used together with a gradient elution.
Analytes Ret. Time Q1 m/z Q3 m/zCollisionEnergy
-27
-34
-24
-41
-23
-30
-24
-37
-30
-19
-24
-36
-24
-39
9.338
8.646
5.378
8.408
9.350
8.786
8.253
Diazepam-d5
Alprazolam
Atropine
Estazolam
Ethyl lo�azepate
Etizolam
Haloperidol
154.05
198.20
281.10
205.10
124.15
93.20
267.15
205.25
259.10
287.15
314.10
138.15
165.15
123.10
290.15
290.15
309.10
309.10
290.15
290.15
295.05
295.05
361.15
361.15
343.05
343.05
376.15
376.15
Analytes Ret. Time Q1 m/z Q3 m/zCollisionEnergy
-28
-55
-27
-25
25
14
21
15
19
14
23
16
7.993
8.573
8.093
5.243
6.762
8.883
Risperidone
Triazolam
Amobarbital(neg)
Barbital(neg)
Phenobarbital(neg)
Thiamylal(neg)
191.05
69.05
315.00
308.20
42.00
182.00
42.10
140.10
42.20
85.10
58.10
101.00
411.20
411.20
343.05
343.05
225.15
225.15
183.10
183.10
231.10
231.10
253.00
253.00
4
Simultaneous analysis for forensic drugs in human blood and urine using ultra-high speed LC-MS/MS
Results and DiscussionEtizolam Risperidone TriazolamAlprazolam
0.1ng/mL
Conc. Area Accuracy %RSD9,0048,2889,51975,23675,98374,023829,519831,098849,597
112.1105.1119.389.689.680.699.999.6
104.2
0.01
0.1
1
6.57
6.04
2.53
Conc. Area Accuracy %RSD4,8655,1094,321
48,03849,15254,497
604,640581,207579,390
114.4119.9105.784.085.187.0103.799.2101.2
0.01
0.1
1
8.71
1.82
2.22
Conc. Area Accuracy %RSD29,83232,43630,461335,202309,273343,172
3,826,3733,718,8543,705,165
108.4116.7110.891.383.785.6102.899.4101.4
0.01
0.1
1
5.14
4.74
1.66
Conc. Area Accuracy %RSD3,0473,0643,35627,99125,54226,317288,776297,332294,788
107.0109.2118.594.885.781.599.0101.5102.9
0.01
0.1
1
5.63
7.83
1.96
negative
positive
Figure 2 LCMS-8050 triple quadrupole mass spectrometer
0.01ng/mL
S/N 39.5
309.10>281.10(+)
309.10>281.10(+)
(x103)
(x104)
2.0
1.0
0.5
0.0
1.0
0.5
0.0
8.0
0.00 0.25 0.75 Conc. Ratio0.50
8.5 9.0 9.5
1.0
0.0
Area Ratio
r2=0.998
0.00 0.25 0.75 Conc. Ratio0.50
7.5
5.0
2.5
0.0
Area Ratio (x0.1)
r2=0.998
0.00 0.25 0.75 Conc. Ratio0.50
Area Ratio
r2=0.9985.0
2.5
0.0
4.0
2.0
3.0
1.0
0.00.00 0.25 0.75 Conc. Ratio0.50
Area Ratio (x0.1)
r2=0.998
8.0 8.5 9.0 9.5 8.0 8.5 9.0 9.57.0 7.5 8.0 8.5
0.0
(x104)0.0
0.5
(x103)
1.0
343.05>314.10(+)
343.05>314.10(+)
S/N 145.5
0.0
(x104)0.0
(x103)
2.5
2.5
411.20>191.05(+)
411.20>191.05(+)
S/N 107.6
0.0
(x103)0.0
(x102)
2.5
2.5
S/N 18.8
343.05>315.00(+)
343.05>315.00(+)
5
Simultaneous analysis for forensic drugs in human blood and urine using ultra-high speed LC-MS/MS
In this experiment, two different matrices consisting of human whole blood and urine were prepared and 18 drugs were spiked into extract solution. Calibration curves constructed in the range from 0.01 to 1 ng/mL for 12 drugs (Alprazolam, Aripiprazole, Atropine, Brotizolam, Estazolam, Ethyl lo�azepate, Etizolam, Flunitrazepam,
Haloperidol, Nimetazepam, Risperidone and Triazolam) and from 1 to 100 ng/mL for 6 drugs (Bromovalerylurea, Amobarbital, Barbital, Loxoprofen, Phenobarbital and Thiamylal). All calibration curves displayed linearity with an R2 > 0.997 and excellent reproducibility was observed for all compounds (CV < 12%) at low concentration level.
Conc. Area Accuracy %RSD1,8371,8622,04121,68522,16920,654227,698223,480225,079
100.299.1
105.899.6
102.492.5
101.398.3
100.9
1
10
100
4.53
5.30
1.62
Conc. Area Accuracy %RSD521464509
5,0785,0335,424
55,42055,65853,484
108.796.6103.495.695.499.4101.4100.898.7
1
10
100
7.10
2.38
1.42
Conc. Area Accuracy %RSD725693617
7,9098,5647,93981,98783,27482,656
106100.2
9198.8107.596.799.299.7100.8
1
10
100
9.82
5.82
0.85
Conc. Area Accuracy %RSD2,5202,1922,28830,80829,62331,379318,233317,214313,399
10795.397.5101.498.3100.6100.799.3100
1
10
100
8.99
1.68
0.71
Phenobarbital (neg) Thiamylal (neg)Amobarbital (neg) Barbital (neg)
Figure 3 Results of 8 drugs spiked in human whole blood using LCMS-8050
7.5 8.0 8.5 9.0
10ng/mL
1ng/mL
2.5
(x102)
0.0(x103)
2.5
0.0
225.15>42.00(-)
225.15>42.00(-)
Area Ratio (x0.1)
r2=0.999
0.0 25.0 Conc. Ratio50.0
2.0
1.0
0.0
Area Ratio (x0.01)
0.0 25.0 Conc. Ratio50.0
5.0
2.5
0.0
r2=0.999Area Ratio (x0.1)
r2=0.999
0.0 25.0 Conc. Ratio50.00.00
0.25
0.50
0.75
1.00
0.0 25.0 Conc. Ratio50.00.0
1.0
2.0
3.0
4.0Area Ratio (x0.1)
r2=0.999
S/N 40.2 S/N 38.2 S/N 167.95.0
(x10)
0.0(x102)
5.0
2.5
0.0
183.10>42.10(-)
183.10>42.10(-)
S/N 15.3
231.10>42.20(-)
231.10>42.20(-)
1.0
(x102)
0.0
0.5
(x103)
1.0
0.5
0.0
5.0
(x102)
0.0
2.5
(x103)
5.0
2.5
0.0
253.00>58.10(-)
253.00>58.10(-)
4.5 5.0 5.5 6.0 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Simultaneous analysis for forensic drugs in human blood and urine using ultra-high speed LC-MS/MS
Conc. Area Accuracy %RSD1,4681,2331,24517,24120,54618,689211,917251,963234,789
102.286.687.6104.4114.7106.996.810397.9
1
10
100
12.73
5.10
3.34
Conc. Area Accuracy %RSD651695654
4,9895,6135,443
55,39269,48166,327
93.696.189
105.2109.6108.692.6104
101.3
1
10
100
2.77
2.07
5.98
Conc. Area Accuracy %RSD612545609
5,6566,6326,38471,96588,68582,091
103.689.499.397.9106.1104.495.210599.1
1
10
100
8.16
4.24
4.95
Conc. Area Accuracy %RSD3,1423,4703,15327,25734,37732,933365,563431,826390,719
95.1100.591.494.9110.8108.598.5104.196.1
1
10
100
4.54
8.15
4.15
Figure 4 Results of 4 drugs spiked in human urine using LCMS-8050
Conclusions• The validated sample preparation protocol can get adequate recoveries in quantitative works for all compounds ranging
from acidic to basic. • The combination of the modi�ed QuEChERS extraction method and high-speed triple quadrupole LC/MS/MS with a
simple quantitative method enable to acquire reliable data easily.
7.5 8.0 8.5 9.0
Phenobarbital (neg) Thiamylal (neg)Amobarbital (neg) Barbital (neg)
Area Ratio (x0.1)
r2=0.999Area Ratio (x0.1)
r2=0.999Area Ratio (x0.1)
r2=0.999Area Ratio (x0.1)
r2=0.999
2.0
3.0
1.0
0.00.0 25.0 Conc. Ratio50.0 0.0 25.0 Conc. Ratio50.0 0.0 25.0 Conc. Ratio50.0 0.0 25.0 Conc. Ratio50.0
0.50
0.75
0.25
0.00
1.0
0.5
0.0
5.0
2.5
0.0
10ng/mL
1ng/mL
2.5
(x102)
0.0(x103)
2.5
0.0
225.15>42.00(-)
225.15>42.00(-)
S/N 14.7 S/N 9.4 S/N 18.3 S/N 97.41.0
(x102)
(x102)
5.0
2.5
0.0
183.10>42.10(-)
183.10>42.10(-)
231.10>42.20(-)
231.10>42.20(-)
253.00>58.10(-)
253.00>58.10(-)
1.0
0.0
(x102)
(x103)
1.0
0.5
0.0
2.5
5.0
0.0
(x102)
(x103)
5.0
2.5
0.0
4.5 5.0 5.5 6.0 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
PO-CON1460E
Simultaneous Screening and Quantitationof Amphetamines in Urine by On-line SPE-LC/MS Method
ASMS 2014 ThP587
Helmy Rabaha1, Lim Swee Chin1, Sun Zhe2,
Jie Xing2 & Zhaoqi Zhan2
1Department of Scienti�c Services, Ministry of Health,
Brunei Darussalam;2Shimadzu (Asia Paci�c) Pte Ltd, Singapore, SINGAPORE
2
Simultaneous Screening and Quantitation of Amphetamines in Urine by On-line SPE-LC/MS Method
IntroductionAmphetamines belong to stimulant drugs and are also controlled as illicit drugs worldwide. The conventional analytical procedure of amphetamines in human urine includes initial immunological screening followed by GCMS con�rmation and quantitation [1]. With new SAMHSA guidelines effective in Oct 2010 [2], screening, con�rmation and quantitation of illicit drugs including amphetamines were allowed to employ LC/MS and LC/MS/MS, which usually does not require a derivatization step as used in the GCMS method [1]. The objective of this study was to develop an on-line SPE-LC/MS method for
analysis of �ve amphetamines in urine without sample pre-treatment except dilution with water. The compounds studied include amphetamine (AMPH), methamphetamine (MAMP) and three newly added MDMA, MDA and MDEA by the new SAMHSA guideline (group A in Table 1). Four potential interferences (group B in) and PMPA (R) as a control reference were also included to enhance the method reliability in identi�cation of the �ve targeted amphetamines from those structurally similar analogues which potentially present in forensic samples.
ExperimentalThe test stock solutions of the ten compounds (Table 1) were prepared in the toxicology laboratory in the Department of Scienti�c Services (MOH, Brunei). Five urine specimens were collected from healthy adult volunteers. The urine samples used as blank and matrix to prepare spiked amphetamine samples were not pre-treated off-line by any means except dilution of 10 times with pure water. An on-line SPE-LC/MS was set up on the LCMS-2020, a single quadrupole system, with a switching valve and a trapping column kit (Shimadzu Co-Sense con�guration) installed in the column oven and controlled by the LabSolutions workstation. The analytical column used was Shim-pack VP-ODS 150 x 2mm (5um) and the trapping column was Synergi Polar-RP 50 x 2mm (2.5um), instead of
a normal SPE cartridge. The injected sample �rst passed through the trapping column where the amphetamines were trapped, concentrated and washed by pure water for 3 minutes followed by switching to the analytical �ow line. The trapped compounds were then eluted out with a gradient program: 0.01min, valve at position 0 & B=5%; 3 min, valve at position 1; 3.01-10 min, B=5% → 15%; 10.5-12 min, B=65%; 12.1 min, B=5%; 14 min stop, valve to position 0. The mobile phases A and B were water and MeOH both with 0.1% formic acid and mobile C was pure water. The total �ow rates of the trapping line and analytical line are 0.6 and 0.3 mL/min, respectively. The injection volume was 20uL in all experiments.
3
Simultaneous Screening and Quantitation of Amphetamines in Urine by On-line SPE-LC/MS Method
Figure 1: Schematic diagram of on-line SPE-LC/MS system
Table 1: Amphetamines & relevant compounds
Name Abbr. Name Formula Structure
Amphetamine
Methampheta-mine
3,4-methylene-dioxyamphetamine
3,4-methylene-dioxymetham phetamine
3,4-methylene dioxy-N-ethyl amphetamine
Nor pseudo-ephedrine
Ephedrine
Pseudo-Ephedrine
Phentermine
Propyl-amphetamine
AMPH
MAMP
MDA
MDMA
MDEA
Nor pseudo-E
Ephe
Pseudo-E
Phent
PAMP
No
A1
A2
A3
A4
A5
B1
B2
B3
B4
R
C9H13N
C10H15N
C10H13NO2
C11H15NO2
C12H17NO2
C9H13NO
C10H15NO
C10H15NO
C10H15N
C12H19N
Manual injectorPump A
SPE Trapping Column
5
13
Mixer
Switching Valve
LCMS-2020
Waste
Pump B Auto sampler
Analytical column
Pump C
4
Simultaneous Screening and Quantitation of Amphetamines in Urine by On-line SPE-LC/MS Method
Results and Discussion
With ESI positive SIM and scan mode, all of the 10 compounds formed protonated ions [M+H]+ which were used as quantifier ions. The scan spectra were used for confirmation to reduce false positive results. Mixed standards of the ten compounds in Table 1 spiked in urine was used for method development. An initial difficulty encountered was that the normal reusable SPE cartridges
(10-30 mmL) for on-line SPE could not trap all of the ten compounds. With using a 50mmL C18-column to replace the SPE cartridge, the ten compounds studied were trapped efficiently. Furthermore, the trapped compounds were well-separated and eluted out in 8~13 minutes as sharp peaks (Figure 2) by the fully automated on-line SPE-LC/MS method established.
Calibration curves of the on-line SPE-LC/MS method were established using mixed standard samples with concentrations from 2.5 ppb to 500 ppb. Linear calibration
curves with R2> 0.999 were obtained for every compound (Figure 3 & Table 2).
Development of on-line SPE-LC/MS method
Figure 2: SIM chromatograms of urine blank (a) and �ve amphetamines and related compounds (125 ppb each) spiked in urine (b) by on-line SPE-LC/MS.
0.0 2.5 5.0 7.5 10.0 12.5 min0.0
0.5
1.0
1.5
2.0(x1,000,000)
2:152.10(+)2:166.10(+)2:208.20(+)2:194.10(+)2:180.10(+)2:178.10(+)2:150.10(+)2:136.10(+)
(a) Urine blank (b) spiked samples
0.0 2.5 5.0 7.5 10.0 12.5 min
0.0
0.5
1.0
1.5
2.0(x1,000,000)
2:152.10(+)2:166.10(+)2:208.20(+)2:194.10(+)2:180.10(+)2:178.10(+)2:150.10(+)2:136.10(+)
Nor
pseu
doEp
hedr
ine
Pseu
do
MD
EA
MD
MA
MD
A
PAM
P
MA
MP
Phen
t
AM
PH
Figure 3: Calibration curves of �ve amphetamines and �ve related compounds with concentrations from 2.5 ppb to 500 ppb by on-line SPE-LC/MS method
0 250 Conc.0.0
2.5
5.0
7.5
Area (x1,000,000)
0 250 Conc.0.0
0.5
1.0
1.5
Area (x10,000,000)
0 250 Conc.0.0
0.5
1.0Area (x10,000,000)
0 250 Conc.0.0
1.0
2.0
Area (x10,000,000)
0 250 Conc.0.0
0.5
1.0
Area (x10,000,000)
0 250 Conc.0.0
1.0
2.0
Area (x10,000,000)
0 250 Conc.0.0
1.0
2.0
3.0Area (x10,000,000)
0 250 Conc.0.0
0.5
1.0
1.5
Area (x10,000,000)
0 250 Conc.0.0
0.5
1.0
1.5
Area (x10,000,000)
0 250 Conc.0.0
2.5
5.0
Area (x1,000,000)
AMPH MAMP
Phent PAMP
MDA MDMA MDEA
Ephedrine Pseudo-ENor pseudo-E
Simultaneous Screening and Quantitation of Amphetamines in Urine by On-line SPE-LC/MS Method
5
Table 2: Peak detection, retention, calibration curves and method performance evaluation
NameRec. %
(62.5ppb)RSD%(n=6)(62.5ppb)
LOD/LOQ(ppb)
Norpseudo-E
Ephe
Pseudo-E
AMPH
MAMP
MDA
MDMA
MDEA
Phent
PAMP (Ref)
97.3
84.4
78.9
85.6
76.5
71.8
72.2
74.8
74.5
69.5
M.E %(62.5ppb)
69.3
111.0
109.2
71.1
96.8
70.3
116.3
107.1
69.9
96.8
Linearity(r2)
0.9982
0.9960
0.9976
0.9983
0.9968
0.9989
0.9973
0.9908
0.9960
0.9912
1.67
0.54
0.41
0.98
0.94
1.94
1.08
2.18
1.82
5.30
S/N(2.5ppb)
11.3
33.7
28.5
17.5
30.3
18.2
36.6
41.9
12.7
37.7
0.71/2.17
0.25/0.76
0.29/0.88
0.48/1.46
0.26/0.80
0.45/1.36
0.23/0.70
0.19/0.57
0.66/2.01
0.22/0.66
SIM ion(+)
152.1
166.1
166.1
136.1
150.1
180.1
194.1
208.1
150.1
178.1
RT(min)
8.0
8.4
9.0
9.6
10.2
10.4
10.8
12.2
12.4
12.7
Conc. range(ppb)
2.5 - 500
2.5 - 500
2.5 - 500
2.5 - 500
2.5 - 500
2.5 - 500
2.5 - 500
2.5 - 500
2.5 - 500
2.5 - 500
The trapping efficiency of the on-line SPE is critical and must be evaluated first, because it determines the recovery of the method. In this study, the recovery of the on-line SPE was determined by injecting a same mixed standard sample from a manual injector installed before the analytical column (by-pass on-line SPE) and also from the Autosampler (See Figure 1). The peaks areas obtained by the two injections were used to calculate recovery value of the on-line SPE method. As shown in Table 2, the recovery obtained with 62.5 ppb mixed standards are at 69.5% ~ 97.3%. The recovery with 250 ppb and 500 ppb mixed samples were also determined and similar results were obtained. Matrix effect was determined with 62.5 ppb and 250 ppb levels of mixed samples in clear solution and in urine. The results (Table 2) show a variation between 69.3% and 116% with compounds. The matrix effect with different
urine specimens did not show significant differences. Repeatability was evaluated with spiked mixed samples of 62.5 ppb and 250 ppb. The results of 62.5 ppb is shown in Table 2, RSD between 0.41% and 5.3%. The sensitivity of the on-line SPE-LC/MS method was evaluated with spiked sample of 2.5 ppb level. The SIM chromatograms are shown in Figure 4. The S/N ratios obtained ranged 11.3~42, which were suitable to determine LOQ (S/N = 10) and LOD (S/N = 3). Since the urine samples were diluted for 10 times with water before injection, the LOD and LOQ of the method for source urine samples were at 1.9~7.1 and 5.7~21.7 ng/mL, respectively. The confirmation cutoff values of the five targeted amphetamines (Group A) in urine enforced by the new SMAHSA guidelines are 250 ng/mL [2]. The on-line SPE-LC/MS method established has sufficient allowance in terms of sensitivity and confirmation reliability for analysis of actual urine samples.
Performance evaluation of on-line SPE-LCMS method
Figure 4: SIM chromatograms of 10 compounds with 2.5 ppb each by on-line SPE-LC/MS method.
7.5 10.0 12.5 min
1.0
2.0
3.0
4.0
5.0
6.0(x10,000)
2:152.10(+)2:166.10(+)2:208.20(+)2:194.10(+)2:180.10(+)2:178.10(+)2:150.10(+)2:136.10(+)
Nor
pseu
do
Ephe
drin
e
Pseu
do
MD
EA
MD
MA
MD
A
PAM
P
MA
MP
Phen
t
AM
PH
Simultaneous Screening and Quantitation of Amphetamines in Urine by On-line SPE-LC/MS Method
6
Figure 5: Durability test of on-line SPE-LC/MS method, comparison of 1st and 200th injections.
The durability of the trapping column was tested purposely by continuous injections of spiked urine samples (125 ppb) for 200 times in a few days. Figure 5 shows the chromatograms of the first and 200th injections of a same
spiked sample. The results show that the variations of peak area and retention time of the 200th injection compared to the 1st injection were at 89.5%~117.8% and 89.5%~99.8% respectively.
Durability of on-line SPE trapping column
Confirmation reliability of LC/MS and LC/MS/MS methods must be proven to be equivalent to the GCMS method according to the SMAHSA guidelines [2]. Validation of confirmation reliability of the on-line SPE-LC/MS method has not be carried out systematically. The high sensitivity of MS detection in SIM mode is a key factor to ensure no false-negative and the scan spectra acquired
simultaneously is used for excluding false-positive. In this work, the confirmation reliability was evaluated using five different urine specimens as matrix to prepare spiked samples of 2.5 ppb (correspond 25 ng/mL in source urine) and above. The results show that false-positive and false negative results were not found.
Con�rmation Reliability
ConclusionsA novel high sensitivity on-line SPE-LC/MS method was developed for screening, conformation and quanti�cation of �ve amphetamines: AMPH, MAMP, MDMA, MDA and MDEA in urines. The recovery of the on-line SPE by employing a 50mmL Synergi Polar-RP column was at 72%~86% for the �ve amphetamines, which are considerably high if comparing with conventional on-line
SPE cartridges. The method performance was evaluated thoroughly with urine spiked samples. The results demonstrate that the on-line SPE-LC/MS method is suitable for direct analysis of the amphetamines and relevant compounds in urine samples without off-line sample pre-treatment.
0.0 2.5 5.0 7.5 10.0 12.5 min
0.0
0.5
1.0
1.5
2.0
(x1,000,000)
2:152.10(+)2:166.10(+)2:208.20(+)2:194.10(+)2:180.10(+)2:178.10(+)2:150.10(+)2:136.10(+)
Nor
pseu
do Ephe
drin
ePs
eudo
MD
EA
MD
MA
MD
A
PAM
P
MA
MP
Phen
t
AM
PH
0.0 2.5 5.0 7.5 10.0 12.5 min
0.0
0.5
1.0
1.5
2.0(x1,000,000)
2:152.10(+)2:166.10(+)2:208.20(+)2:194.10(+)2:180.10(+)2:178.10(+)2:150.10(+)2:136.10(+)
Nor
pseu
do Ephe
drin
ePs
eudo
MD
EA
MD
MA
MD
A
PAM
P
MA
MP
Phen
t
AM
PH
1st injection spiked mixed std 125ppb in urineinj vol: 20 µL
200th injection spiked mixed std 125ppb in urineinj vol: 20 µL
Simultaneous Screening and Quantitation of Amphetamines in Urine by On-line SPE-LC/MS Method
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
References1. Kudo K, Ishida T, Hara K, Kashimura S, Tsuji A, Ikeda N, J Chromatogr B, 2007, 855, 115-120. 2. SAMHSA “Manual for urine laboratories, National laboratory certi�cation program”, Oct 2010, US Department of
Health and Human Services.
PO-CON1481E
Single step separation of plasma from whole blood without the need for centrifugation applied to the quantitative analysis of warfarin
ASMS 2014 MP762
Alan J. Barnes1, Carrie-Anne Mellor2,
Adam McMahon2, Neil J. Loftus1
1Shimadzu, Manchester, UK 2WMIC, University of Manchester, UK
2
Single step separation of plasma from whole blood without the need for centrifugation applied to the quantitative analysis of warfarin
IntroductionDried plasma sample collection and storage from whole blood without the need for centrifugation separation and refrigeration opens new opportunities in blood sampling strategies for quantitative LC/MS/MS bioanalysis. Plasma samples were generated by gravity �ltration of a whole blood sample through a laminated membrane stack allowing plasma to be collected, dried, transported and analysed by LC/MS/MS. This novel plasma separation card (PSC) technology was applied to the quantitative LC/MS/MS analysis of warfarin, in blood samples. Warfarin is a coumarin anticoagulant vitamin-K antagonist used for the treatment of thrombosis and thromboembolism. As a
result of vitamin-K recycling being inhibited, hepatic synthesis is in-turn inhibited for blood clotting factors as well as anticoagulant proteins. Whilst the measurement of warfarin activity in patients is normally measured by prothrombin time by international normalized ratio (INR) in some cases the quantitation of plasma warfarin concentration is needed to con�rm patient compliance, resistance to the anticoagulant drug, or diet related issues. In this preliminary evaluation, warfarin concentration was measured by LC/MS/MS to evaluate if PSC technology could complement INR when sampling patient blood.
Materials and Methods
Warfarin standard was dissolved in water containing 50% ethanol + 0.1% formic acid, spiked (60uL) to whole human blood (1mL) and mixed gently. 50uL of spiked blood was deposited onto the PSC. After 3 minutes, the primary filtration overlay was removed followed by 15 minutes air drying at room temperature. The plasma sample disc was prepared directly for analysis after drying. LC/MS/MS sample preparation involved vortexing the sample disk in
40uL methanol, followed by centrifugation 16,000g 5 min. 20uL supernatant was added directly to the LCMS/MS sample vial already containing 80uL water (2uL analysed). Control plasma comparison was prepared by centrifuging remaining blood at 1000g for 10min. 2.5uL supernatant plasma was taken, 40uL methanol added, and prepared as PSC samples. LCMS/MS sample injection volume, 2uL.
Sample preparation
Warfarin was measured by MRM, positive negative switching mode (15msec).
LC-MS/MS analysis
LC/MS/MS System : Nexera UHPLC system + LCMS-8040 Shimadzu Corporation
Flow rate : 0.4mL/min (0-7.75min), 0.5mL/min (7.5-14min), 0.4mL/min (15min)
Mobile phase : A= Water + 0.1% formic acid
B= Methanol + 0.1% formic acid
Gradient : 20% B (0-0.5 min), 100% B (8-12 min), 20% B (12.01-15 min)
Analytical column : Phenomenex Kinetex XB C18 100 x 2.1mm 1.7um 100A
Column temperature : 50ºC
Ionisation : Electrospray, positive, negative switching mode
Desolvation line : 250ºC
Drying/Nebulising gas : 10L/min, 2L/min
Heating block : 400ºC
3
Single step separation of plasma from whole blood without the need for centrifugation applied to the quantitative analysis of warfarin
Design of plasma separator technology
Plasma separation work�ow
Control Spot:[Determines whether enough blood was placed on the card].
Filtration Layer[Filtration layer captures blood cells by a combination of �ltration and adsorption. The average linear vertical migration rate is approximately 1um/sec].
Collection Layer[Loads with a speci�c aliquot of plasma onto a 6.35mm disc]. Although �ow through the �ltration membrane is unlikely to be constant throughout the plasma extraction process, the average loading rate of the Collection Disc was 13 nL/sec. This corresponds to a volumetric �ow rate into the Collection Disc of 400 pL/mm2/sec.
Isolation Screen[Precludes lateral wicking along the card surface].
Spreading Layer[Lateral spreading layer rapidly spreads blood so it will enter the �ltration layer as a front while adding buffers and anticoagulants. The lateral spreading rate is 150um/sec].
1 3 42
A NoviPlex card is removed from foil packaging.
Approximately 50uL of whole blood is added to the test area.
After 3 minutes, the top layer is completely removed (peeled back).
The collection disc contains 2.5uL of plasma. Card is air dried for 15 minutes.
The collection disc is removed from the card and is ready for extraction for LC-MS/MS analysis.
Figure 1. Noviplex work�ow.
4
Single step separation of plasma from whole blood without the need for centrifugation applied to the quantitative analysis of warfarin
Figure 2. Applying a blood sample, either as a �nger prick or by accurately measuring the blood volume, to the laminated membrane stack retains red cells and allows a plasma sample to be collected. The red cells are retained by a combination of adsorption and �ltration whilst plasma advances through the membrane stack
by capillary action. After approximately three minutes the plasma Collection Disc was saturated with an aliquot of plasma and was ready for LC/MS/MS analysis.
Figure 3. Comparison between the warfarin response in both positive and negative ion modes for warfarin calibration standards at 2.5ug/mL and 0.4ug/mL extracted from the plasma separation cards and a conventional plasma sample. There is a broad agreement in ion signal intensity between
the 2 sample preparation techniques.
ResultsComparison between plasma separation cards (PSC) and plasma
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
(x100,000)
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min
0.000.250.500.751.001.251.501.752.002.252.502.753.00
(x100,000)
1.0 2.0 3.0 4.0 5.0 6.0 7.0 min
0.00.10.20.30.40.50.60.70.80.91.01.11.2(x100,000)
1.0 2.0 3.0 4.0 5.0 6.0 7.0 min
0.00
0.25
0.50
0.75
1.00
1.25
1.50
(x100,000)
Plasma separation cardPositive ionWarfarin m/z 309.20 > 163.05
Q1 (V) -22Collision energy -15Q3 (V) -15
Plasma separation cardNegative ionWarfarin m/z 307.20 > 161.25
Q1 (V) 14Collision energy 19 Q3 (V) 30
PlasmaNegative ionWarfarin m/z 307.20 > 161.05
Q1 (V) 14Collision energy 19 Q3 (V) 30
Plasma Positive ionWarfarin m/z 309.20 > 163.05
Q1 (V) -22Collision energy -15Q3 (V) -15
2.5ug/mL Calibration standard
0.4ug/mLCalibration standard
2.5ug/mL Calibration standard
0.4ug/mLCalibration standard
2.5ug/mL Calibration standard
0.4ug/mLCalibration standard
2.5ug/mL Calibration standard
0.4ug/mLCalibration standard
5
Single step separation of plasma from whole blood without the need for centrifugation applied to the quantitative analysis of warfarin
The drive to work with smaller sample volumes offers significant ethical and economical advantages in pharmaceutical and clinical workflows and dried blood spot sampling techniques have enabled a step change approach for many toxicokinetic and pharmacokinetic studies. However, the impressive growth of this technique in the quantitative analysis of small molecules has also discovered several limitations in the case of sample
instability (some enzyme labile compounds, particularly prodrugs, analyte stability can be problematic), hematocrit effect and background interferences of DBS. DBS also shows noticeable effects on many lipids dependent on the sample collection process. To compare PSC to plasma lipid profiles the same blood sample extraction procedure applied for warfarin analysis was measured by a high mass accuracy system optimized for lipid profiling.
Plasma separation card comparison
Figure 4. In both ion modes, the calibration curve was linear over the therapeutic range studied for warfarin extracted from PSC’s (calibration range 0-3ug/mL, single point calibration standards at each level with the exception of replicate calibration points at 2.5ug/mL and 0.4ug/mL (n=3); r2>0.99 for
PSC analysis [r2>0.99 for a conventional plasma extraction]).
Figure 5. Matrix blank comparison. In both ion modes, the MRM chromatograms for PSC and plasma are comparable. Warfarin ion signals were not detected in the any PSC or plasma matrix blank.
Plasma separation cardNegative ionWarfarin m/z 309.20 > 163.05Replicate calibration points at 2.5ug/mL and 0.4ug/mL (n=3)
Plasma separation cardPositive ionWarfarin m/z 309.20 > 163.05Replicate calibration points at 2.5ug/mL and 0.4ug/mL (n=3)
Linear regresson analysisy = 246527x + 14796
R² = 0.9986
0
100,000
200,000
300,000
400,000
500,000
600,000
700,000
800,000
0 0.5 1 1.5 2 2.5 3 3.5
Linear regression analysisy = 133197x + 15795
R² = 0.9954
0
50000
100000
150000
200000
250000
300000
350000
400000
450000
0 0.5 1 1.5 2 2.5 3 3.5
Blood concentration ( ug/mL) Blood concentration ( ug/mL)
0.0 2.5 5.0 min
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75(x10,000)
2.5 5.0 min
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75(x10,000)
Matrix blank comparisonPositive ionPlasma separation card matrix blankPlasma matrix blank
Matrix blank comparisonNegative ionPlasma separation card matrix blankPlasma matrix blank
Single step separation of plasma from whole blood without the need for centrifugation applied to the quantitative analysis of warfarin
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Conclusions• In this limited study, plasma separation card (PSC) sampling delivered a quantitative analysis of warfarin spiked into
human blood.• PSC generated a linear calibration curve in both positive and negative ion modes (r2>0.99; n=5); • The warfarin plasma results achieved by using the PSC technique were in broad agreement with conventional plasma
sampling data.• The plasma generated by the �ltration process appears broadly similar to plasma derived from conventional
centrifugation.• Further work is required to consider the robustness and validation in a routine analysis.
References• Jensen, B.P., Chin, P.K.L., Begg, E.J. (2011) Quanti�cation of total and free concentrations of R- and S-warfarin in
human plasma by ultra�ltration and LC-MS/MS. Anal Bioanal Chem., 401, 2187-2193• Radwan, M.A., Bawazeer, G.A., Aloudah, N.M., Aluadeib, B.T., Aboul-Enein, H.Y. (2012) Determination of free and total
warfarin concentrations in plasma using UPLC MS/MS and its application to patient samples. Biochemical Chromatography, 26, 6-11
Figure 6. Lipid pro�les from the same human blood sample extracted using a plasma separation card (left hand pro�le) compared to a conventional plasma samples (centrifugation). Both lipid pro�les are comparable in terms of distribution and the number of lipids detected (the scaling has been
normalized to the most intense lipid signal).
Conventional plasma samplePositive ionLCMS-IT-TOFLipid pro�ling
Diacylglycero-phosphocholines
Ceramidephosphocholines
7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 min
MonoacylglycerophosphoethanolaminesMonoacylglycerophosphocholines
7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 min
Plasma separation card samplePositive ionLCMS-IT-TOFLipid pro�ling
PO-CON1462E
Development and Validation of Direct Analysis Method for Screeningand Quantitation of Amphetamines in Urine by LC/MS/MS
ASMS 2014 MP535
Zhaoqi Zhan1, Zhe Sun1, Jie Xing1, Helmy Rabaha2
and Lim Swee Chin2 1Shimadzu (Asia Paci�c) Pte Ltd, Singapore, SINGAPORE;2Department of Scienti�c Services, Ministry of Health,
Brunei Darussalam
2
Development and Validation of Direct Analysis Method for Screening and Quantitation of Amphetamines in Urine by LC/MS/MS
IntroductionAmphetamines are among the most commonly abused drugs type worldwide. The conventional analytical procedure of amphetamines in human urine in forensic laboratory involves initial immunological screening followed by GCMS con�rmation and quantitation [1]. The new guidelines of SAMHSA under U.S. Department of Health and Human Services effective in Oct 2010 [2] allowed use of LC/MS/MS for screening, con�rmation and quantitation of illicit drugs including amphetamines. One of the advantages by using LC/MS/MS is that derivatization of amphetamines before analysis is not needed, which was a standard procedure of GCMS method. Since analysis speed and throughput could be enhanced signi�cantly, development and use of LC/MS/MS methods are in
demand and many such efforts have been reported recently [3]. The objective of this study is to develop a fast LC/MS/MS method for direct analysis of amphetamines in urine without sample pre-treatment (except dilution with water) on LCMS-8040, a triple quadrupole system featured as ultra fast mass spectrometry (UFMS). The compounds studied include amphetamines (AMPH), methamphetamine (MAMP) and three newly added MDMA, MDA and MDEA by the new SAMHSA guidelines, four potential interferences as well as PMPA as a control reference (Table 1). Very small injection volumes of 0.1uL to 1uL was adopted in this study, which enabled the method suitable for direct injection of untreated urine samples without causing signi�cant contamination to the ESI interface.
ExperimentalThe stock standard solutions of amphetamines and related compounds as listed in Table 1 were prepared in the Toxicology Laboratory in the Department of Scienti�c Services (MOH, Brunei). Five urine specimens were collected from healthy adult volunteers. The urine samples used as blank and spiked samples were not pre-treated by any means except dilution of 10 times with Milli-Q water.An LCMS-8040 triple quadrupole coupled with a Nexera UHPLC system (Shimadzu Corporation) was used. The analytical column used was a Shim-pack XR-ODS III UHPLC column (1.6 µm) 50mm x 2mm. The mobile phases used
were water (A) and MeOH (B), both with 0.1% formic acid. A fast gradient elution program was developed for analysis of the ten compounds: 0-1.6min, B=2%->14%; 1.8-2.3min, B=70%; 2.4min, B=2%; end at 4min. The total �ow rate was 0.6 mL/min. Positive ESI ionization mode was applied with drying gas �ow of 15 L/min, nebulizing gas �ow of 3 L/min, heating block temperature of 400 ºC and DL temperature of 250 ºC. Various injection volumes from 0.1 uL to 5 uL were tested to develop a method with a lower injection volume to reduce contamination of untreated urine samples to the interface.
Results and Discussion
MRM optimization of the ten compounds (Table 1) was performed using an automated MRM optimization program with LabSolutions workstation. Two MRM transitions were selected for each compound, one for quantitation and second one for confirmation (Table 1). The ten compounds were separated and eluted in 0.75~2.2 minutes as sharp peaks as shown in Figure 1. In addition to analysis speed and detection sensitivity, this method development was also focused on evaluation of small to ultra-small injection volumes to develop a method suitable for direct injection of urine samples without any
pre-treatment while it should not cause significant contamination to the interface. The Nexera SIL-30A auto-sampler enables to inject as low as 0.10 uL of sample with excellent precision.Figure 1 shows a few selected results of direct injection of urine blank (a) and mixed standards spiked in urine with 1 uL (c and d) and 0.1 uL (b) injection. It can be seen that all compounds (12.5 ppb each in urine) could be detected with 0.1uL injection except MDA and Norpseudo-E. With 1uL injection, all of them were detected.
Method development of direct injection of amphetamines in urine
3
Development and Validation of Direct Analysis Method for Screening and Quantitation of Amphetamines in Urine by LC/MS/MS
Figure 1: MRM chromatograms of urine blank (a) and spiked samples of amphetamines and related compounds in urine by LC/MS/MS method with 1uL and 0.1uL injection volumes.
Table 1: MRMs of amphetamines and related compounds
Compound Abbr. RT (min) MRM
Nor pseudo ephedrine
Ephedrine
Pseudo ephedrine
Amphetamine
Methampheta-mine
3,4-methylenedi oxyamphetamine
3,4-methylene dioxymeth amphetamine
3,4-methylene dioxy-N-ethyl amphetamine
Phentermine
Propyl amphetamine
Nor pseudo-E
Ephe
Pseudo-E
AMPH
MAMP
MDA
MDMA
MDEA
Phent
PAMP
Cat.
B1
B2
B3
A1
A2
A3
A4
A5
B4
R
0.75
0.94
1.01
1.20
1.42
1.49
1.59
1.94
1.93
2.20
152>134
152>115
166>148
166>91
166>148
166>91
136>91
136>119
150>91
150>119
180>163
180>163
194>163
194>105
208>163
208>105
150>91
150>119
178>91
178>65
CE (V)
-13
-23
-14
-31
-14
-30
-20
-14
-20
-14
-12
-38
-13
-22
-12
-24
-20
-40
-22
-47
0.0 0.5 1.0 1.5 2.0 2.5 min0.0
1.0
2.0
3.0
(x10,000)
Phen
t
Nor
pseu
do
Pseu
doEp
hedr
ine
MD
EA
MD
MA
MD
A
PAM
P
MA
MP
AM
PH
0.0 0.5 1.0 1.5 2.0 2.5 min0.0
1.0
2.0
3.0
(x100,000)
Phen
t
Nor
pseu
do
Pseu
doEp
hedr
ine
MD
EA
MD
MA
MD
A
PAM
P
MA
MP
AM
PH
0.0 0.5 1.0 1.5 2.0 2.5 min0.0
0.5
1.0
1.5
(x1,000,000)
Phen
t
Nor
pseu
do
Pseu
doEp
hedr
ine
MD
EA
MD
MA
MD
A
PAM
P
MA
MP
AM
PH
(a) Urine blank, 1 uL inj (b) 12.5ppb in urine, 0.1uL inj
(c) 12.5ppb, 1uL inj (d) 62.5ppb in urine, 1uL inj
0.0 0.5 1.0 1.5 2.0 2.5 min0.0
1.0
2.0
3.0
(x10,000)
4
Development and Validation of Direct Analysis Method for Screening and Quantitation of Amphetamines in Urine by LC/MS/MS
Figure 2: Calibration Curves of amphetamines spiked in urine with 0.1uL injection
Linear calibration curves were established for the ten compounds spiked in urine with different injection volumes: 0.1, 0.2, 0.5, 1, 2 and 5 uL. Good linearity of calibration curves (R2>0.999) were obtained for all injection volumes including 0.1uL, an ultra-small injection
volume. The calibration curves with 0.1 uL injection volume are shown in Figure 2. The linearity (r2) of all compounds with 0.1 uL and 1 uL injection volumes are equivalently good as shown in Table 2.
Calibration curves with small and ultra-small injection volumes
Repeatability of peak area was evaluated with a same loading amount (6.25 pg) but with different injection volumes. The RSD shown in Table 2 were 1.6% ~ 7.9% and 1.6 ~ 7.8% for 0.1uL and 1uL injection, respectively. It is worth to note that the repeatability of every compounds with of 0.1uL injection is closed to that of 1uL injection as well as 5uL injection (data not shown).Matrix effect of the method was determined by comparison of peak areas of mixed standards in pure water and in urine matrix. The results of 62.5ppb with 1uL injection were at 102-115% except norpseudoephedrine (79%) as shown in Table 2.Accuracy and sensitivity of the method were evaluated with spiked samples of low concentrations. The results of
LOD and LOQ of the ten compounds in urine are shown in Table 3. Since the working samples (blank and spiked) were diluted for 10 times with water before injection, the concentrations and LOD/LOQ of the method described above for source urine samples have to multiply a factor of 10. Therefore, the LOQs of the method for urine specimens are at 2.1-17.1 ng/mL for AMPH, PAMP, MDMA and MDEA and 53 ng/mL for MDA. The LOQs for the potential interferences (Phentermine, Ephedrine, Pseudo-Ephedrine and Norpseudo-Ephedrine) are at 17-91 ng/mL, 2.4 ng/mL for the internal reference MAMP. The sensitivity of the direct injection LC/MS/MS method are significantly higher than the confirmation cutoff (250 ng/mL) required by the SAMHSA guidelines.
Performance validation
0 250 Conc.0.0
1.0
2.0
3.0
Area (x100,000)
0 250 Conc.0.0
2.5
5.0
Area (x100,000)
0 250 Conc.0.0
2.5
5.0
Area (x100,000)
0 250 Conc.0.0
2.5
5.0
7.5
Area (x100,000)
0 250 Conc.0.0
2.5
5.0
Area (x100,000)
0 250 Conc.0.0
2.5
5.0Area (x100,000)
0 250 Conc.0.0
0.5
1.0
1.5
Area (x1,000,000)
0 250 Conc.0.0
2.5
5.0
7.5
Area (x100,000)
0 250 Conc.0.00
0.25
0.50
0.75
1.00
1.25Area (x1,000,000)
0 250 Conc.0.0
2.5
5.0
7.5
Area (x100,000)
AMPH MAMP
Phent PAMP
MDA MDMA MDEA
Ephedrine Pseudo-ENor pseudo-E
Development and Validation of Direct Analysis Method for Screening and Quantitation of Amphetamines in Urine by LC/MS/MS
5
Table 2: Method Performance with different inj. volumes
NameCalibration curve, R2
(0.1uL)
RSD% area (n=6)
(0.1uL)
M.E. %1
(1uL)
Norpseudo-E
Ephe
Pseudo-E
AMPH
MAMP
MDA
MDMA
MDEA
Phent
PAMP
0.9992
0.9995
0.9994
0.9997
0.9998
0.9978
0.9993
0.9996
0.9998
0.9998
(1uL)
0.9996
0.9998
0.9986
0.9998
0.9999
0.9995
0.9998
0.9998
0.9998
0.9932
(ppb)2
1-500
2.5-500
1-500
1-500
1-500
2.5-500
1-500
1-500
2.5-500
1-500
4.5
3.2
3.7
3.5
1.6
7.9
1.8
3.5
4.1
2.9
(1uL)
5.7
2.9
3.3
2.4
2.3
7.8
4.5
2.9
1.6
2.0
79
115
113
102
110
103
115
115
106
102
The method operational stability with 1uL injection was tested with spiked samples of 25 ppb in five urine specimens, corresponding to 250 ng/mL in the source urine samples. Continuous injections of accumulated 120 times was carried out in about 10 hours. The purpose of the experiment was to evaluate the operational stability against the ESI source contamination by urine samples without pre-treatment. Figure 3 shows the first injection and the
120th injection of the same spiked sample (S1) as well as other spiked samples (S2, S3, S4 and S5) in between. Decrease in peak areas of the compounds occurred, but the degree of the decrease in average was about 17% from the first injection to the last injection. This result indicates that it is possible to carry out direct analysis of urine samples (10 times dilution with water) by the high sensitivity LC/MS/MS method with a very small injection volume.
Method operational stability
1: Measured with mixed stds of 62.5 ppb in clear solution and spiked in urine2: For 0.1uL injection, the lowest conc. is 2.5 or 12.5 ppb
Table 3: Method performance: sensitivity & accuracy (1uL)
NameMeas. S/N LOQ
Norpseudo-E
Ephe
Pseudo-E
AMPH
MAMP
MDA
MDMA
MDEA
Phent
PAMP
1.2
2.2
1.0
1.1
1.0
2.4
1.1
1.1
2.6
1.0
Accuracy
(%)
118.7
88.2
99.5
114.1
103.6
96.3
106.4
111.8
105.3
101.7
Conc. (ppb)
Prep.
1.0
2.5
1.0
1.0
1.0
2.5
1.0
1.0
2.5
1.0
2.3
2.7
5.9
6.7
21.8
4.5
51.9
28.5
2.9
42.2
Sensitivity (ppb)
LOD
1.53
2.41
0.50
0.51
0.14
1.60
0.06
0.12
2.73
0.07
5.09
8.04
1.67
1.71
0.47
5.34
0.21
0.39
9.10
0.24
Development and Validation of Direct Analysis Method for Screening and Quantitation of Amphetamines in Urine by LC/MS/MS
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Figure 3: Selected chromatograms of continuous injections of spiked samples (25 ppb) with 1 µL injection. Five urine specimens S1, S2, S3, S4 and S5 were used to prepare these spiked samples.
References1. Kudo K, Ishida T, Hara K, Kashimura S, Tsuji A, Ikeda N, J Chromatogr B, 2007, 855, 115-120. 2. Mandatory guidelines for Federal Workplace Drug Testing Program, 73 FR 71858-71907, Nov. 25, 2008. 3. Huei-Ru Lina, Ka-Ian Choia, Tzu-Chieh Linc, Anren Hu,, Journal of Chromatogr B, 2013, 929, 133–141.
ConclusionsIn this study, we developed a fast LC/MS/MS method for direct analysis of �ve amphetamines and related compounds in human urine for screening and quantitative con�rmation. Very small injection volumes of 0.1~1.0 uL were adopted to minimize ESI contamination and enhance
operational stability. The good performance results observed reveals that screening and con�rmation of amphetamines in human urine by direct injection to LC/MS/MS is possible and the method could be an alternative choice in forensic and toxicology analysis.
0.0 1.0 2.0 min
0.0
2.5
5.0
7.5
(x100,000)
Phen
t
Nor
pseu
do
Pseu
doEp
hedr
ine
MD
EA
MD
MA
MD
A
PAM
P
MA
MP
AM
PH
0.0 1.0 2.0 min
0.0
2.5
5.0
7.5
(x100,000)
Phen
t
Nor
pseu
do Pseu
doEp
hedr
ine
MD
EA
MD
MA
MD
A
PAM
P
MA
MP
AM
PH
0.0 1.0 2.0 min
0.0
2.5
5.0
7.5
(x100,000)
Phen
t
Nor
pseu
doPs
eudo
Ephe
drin
e
MD
EA
MD
MA
MD
A
PAM
P
MA
MP
AM
PH
0.0 1.0 2.0 min
0.0
2.5
5.0
(x100,000)
Phen
t
Nor
pseu
do Pseu
do
Ephe
drin
e
MD
EA
MD
MA
MD
A
PAM
P
MA
MP
AM
PH
0.0 1.0 2.0 min
0.0
2.5
5.0
(x100,000)
Phen
t
Nor
pseu
do Pseu
doEp
hedr
ine
MD
EA
MD
MA
MD
A
PAM
P
MA
MP
AM
PH
0.0 1.0 2.0 min
0.0
2.5
5.0
(x100,000)
Phen
t
Nor
pseu
doPs
eudo
Ephe
drin
e
MD
EA
MD
MA
MD
A
PAM
P
MA
MP
AM
PH
S1 (1st inj)
S1 (110th inj)
S2 (11th inj) S3 (21st inj)
S4 (31st inj) S5 (41st inj)
PO-CON1482E
Next generation plasma collectiontechnology for the clinical analysis oftemozolomide by HILIC/MS/MS
ASMS 2014 WP641
Alan J. Barnes1, Carrie-Anne Mellor2,
Adam McMahon2, Neil Loftus1
1Shimadzu, Manchester, UK 22WMIC, University of Manchester, UK
2
Next generation plasma collection technology for the clinical analysis of temozolomide by HILIC/MS/MS
IntroductionPlasma extraction technology is a novel technique achieved by applying a blood sample to a laminated membrane stack which allows plasma to �ow through the asymmetric �lter whilst retaining the cellular components of the blood sample.Plasma separation card technology was applied to the quantitative analysis of temozolomide (TMZ); an oral imidazotetrazine alkylating agent used for the treatment of Grade IV astrocytoma, an aggressive form of brain tumour.
Under physiological conditions TMZ is rapidly converted to 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide (MTIC) which in-turn degrades by hydrolysis to 5-aminoimidazole-4-carboxamide (AIC). Storage of plasma has previously shown that both at -70C and 4C degradation still occurs. In these experiments, whole blood containing TMZ standard was applied to NoviPlex plasma separation cards (PSC). The aim was to develop a robust LC/MS/MS quantitative method for TMZ.
Materials and Methods
TMZ spiked human blood calibration standards (50uL) were applied to the PSC as described below in figure 1.
Plasma separation
1 3 42
A NoviPlex card is removed from foil packaging.
Approximately 50uL of whole blood is added to the test area.
After 3 minutes, the top layer is completely removed (peeled back).
The collection disc contains 2.5uL of plasma. Card is air dried for 15 minutes.
The collection disc is removed from the card and is ready for extraction for LC-MS/MS analysis.
Figure 1. Noviplex plasma separation card work�ow
3
Next generation plasma collection technology for the clinical analysis of temozolomide by HILIC/MS/MS
Control Spot:[Determines whether enough blood was placed on the card].
Filtration Layer[Filtration layer captures blood cells by a combination of �ltration and adsorption. The average linear vertical migration rate is approximately 1um/sec].
Collection Layer[Loads with a speci�c aliquot of plasma onto a 6.35mm disc]. Although �ow through the �ltration membrane is unlikely to be constant throughout the plasma extraction process, the average loading rate of the Collection Disc was 13 nL/sec. This corresponds to a volumetric �ow rate into the Collection Disc of 400 pL/mm2/sec.
Isolation Screen[Precludes lateral wicking along the card surface].
Spreading Layer[Lateral spreading layer rapidly spreads blood so it will enter the �ltration layer as a front while adding buffers and anticoagulants. The lateral spreading rate is 150um/sec].
Figure 1. Noviplex plasma separation card work�ow (Cont'd)
Figure 2. Applying a blood sample, either as a �nger prick or by accurately measuring the blood volume, to the laminated membrane stack retains red cells and allows a plasma sample to be collected. The red cells are retained by a combination of adsorption and �ltration whilst plasma advances through the membrane stack
by capillary action. After approximately three minutes the plasma Collection Disc was saturated with an aliquot of plasma and was ready for LC/MS/MS analysis.
TMZ was extracted from the dried plasma collection discs by adding 40uL acetonitrile + 0.1% formic acid, followed by centrifugation 16,000g for 5 min. 30uL supernatant was added directly to the LC/MS/MS sample vial for analysis.
As a control, conventional plasma samples were prepared by centrifuging the human blood calibration standards at 1000g for 10min. TMZ was extracted from 2.5uL of plasma using the same extraction protocol as applied for PSC.
Sample preparation
4
Next generation plasma collection technology for the clinical analysis of temozolomide by HILIC/MS/MS
Figure 3. HILIC LC/MS/MS chromatograms for PSC TMZ analysis at 0.5 and 8ug/mL. The PSC calibration curve was linear between 0.2-10ug/mL (r2>0.99).HILIC was considered in response to previous published data and to minimize potential stability issues. However, to reduce sample cycle times a reverse
phase method was also developed.
Results
Temozolomide is known to be unstable under physiological conditions and is converted to 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide (MTIC) by
a nonenzymatic, chemical degradation process. Previous studies have used HILIC to analyze the polar compound and to avoid degradation in aqueous solutions.
HILIC LC/MS/MS
LC/MS/MS analysis
Ionisation : Electrospray, positive mode
MRM 195.05 >138.05 CE -10
HPLC : HILIC
Nexera UHPLC system
Flow rate : 0.5mL/min (0-7min), 1.8mL/min (7.5min-17.5min)
Mobile phase : A= Water + 0.1% formic acid
B= Acetonitrile + 0.1% formic acid
Gradient : 95% B – 30%% B (6.5 min),
30% B (7.5 min), 95% B (18 min)
Analytical column : ZIC HILIC 150 x 4.6mm 5um 200ª
Column temperature : 40ºC
Injection volume : 10uL
Reverse Phase
Nexera UHPLC system
0.4mL/min
A= Water + 0.1% formic acid
B= methanol + 0.1% formic acid
5% B – 100%% B (3 min),
100% B (7 min), 5% B (10 min)
Phenomenex Kinetex XB C18 100 x 2.1mm 1.7um 100A
50ºC
2µL
Desolvation line : 300ºC
Drying/Nebulising gas : 10L/min, 2L/min
Heating block : 400ºC
Linear regression analysisy = 64578x + 18473
R² = 0.9988
0
100000
200000
300000
400000
500000
600000
700000
0 2 4 6 8 10 12
Peak Area
Blood Concentration (ug/mL)
Plasma separation cardHILIC analysisTMZ Single point calibration standardsCalibration curve 0.2-10ug/mL
0.0 2.5 5.0 min
0.0
1.0
2.0
3.0
4.0
5.0(x10,000)
Plasma separation cardHILIC analysisTMZ m/z 195.05 > 138.05
Q1 (V) -20 Collision energy -10 Q3 (V) -12
8.0ug/mL calibn std
0.5ug/mL calibn std
5
Next generation plasma collection technology for the clinical analysis of temozolomide by HILIC/MS/MS
Figure 4. Reverse phase LC/MS/MS chromatograms for PSC TMZ analysis at 0.5 and 8ug/mL. The PSC calibration curve was linear between 0.2-10ug/mL (r2>0.99; replicate samples were included in the liner regression analysis at 0.5 and 8ug/mL; n=3).
Due to the relatively long cycle time (18 min), a faster reversed phase method was developed (10 min). Sample preparation was modified with PSC sample disk placed in 40uL methanol + 0.1% formic acid, followed by centrifugation 16,000g 5 min. 20uL supernatant was
added directly to the LC/MS sample vial plus 80uL water + 0.1% formic acid. In addition to reversed phase being faster, the sample injection volume was reduced to just 2uL as a result of higher sensitivity from narrower peak width (reversed phase,13 sec; HILIC, 42 sec).
Reversed Phase LC/MS/MS
Figure 5. Human blood TMZ calibration standards were prepared using PSC and conventional plasma. Using the con�rmatory ion transition 195.05>67.05 both the PSC and plasma sample are in broad agreement with regard to matrix ion signal distribution.
Comparison between PSC and plasma
Linear regression analysisy = 72219x - 355.54
R² = 0.9997
0
100,000
200,000
300,000
400,000
500,000
600,000
700,000
800,000
0 2 4 6 8 10 12
Peak Area
Blood Concentration (ug/mL)
Plasma separation cardRP analysisTMZ m/z 195.05 > 138.05
Q1 (V) -20Collision energy -10Q3 (V) -12
8.0ug/mL Calibration standard
0.5ug/mLCalibration standard
Plasma separation cardRP analysisTMZ calibration curveReplicate calibration points at 0.5ug/mL and 8ug/mL (n=3)
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 min
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
(x10,000)
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min
0.00
0.25
0.50
0.75
1.00
1.25
1.50
(x1,000)
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
(x100) Matrix blank comparisonMRM 195.05>67.05Plasma separation card matrix blank
Plasma matrix blank
500ng/mL comparisonMRM 195.05>67.05Plasma separation card 500ng/mL calibration standard
Plasma500ng/mL calibration standard
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Next generation plasma collection technology for the clinical analysis of temozolomide by HILIC/MS/MS
ConclusionsThis technology has the potential for a simplified clinical sample collection by the finger prick approach, with future work aimed to evaluate long term sample stability of PSC samples, stored at room temperature. Quantitation of drug metabolites MTIC and AIC also could help provide a measure of sample stability.
References• Andrasia, M., Bustosb, R., Gaspara,A., Gomezb, F.A. & Kleknerc, A. (2010) Analysis and stability study of
temozolomide using capillary electrophoresis. Journal of Chromatography B. Vol. 878, p1801-1808• Denny, B.J., Wheelhouse, R.T., Stevens, M.F.G., Tsang, L.L.H., Slack, J.A., (1994) NMR and molecular modeliing
investigation of the mechanism of activation of the antitumour drug temozolomide and its Interaction with DNA. Biochemistry, Vol. 33, p9045-9051
Figure 6. Human blood TMZ calibration standards were prepared using PSC and conventional plasma. Using the quantitation ion transition 195.05>138.05 both the PSC and plasma sample are in broad agreement in signal distribution and intensity including the presence of
a matrix peak at 2.4mins.
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min
0.00
0.25
0.50
0.75
1.00
1.25
1.50
(x10,000)
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min
0.00
0.25
0.50
0.75
1.00
1.25
1.50
(x10,000) Matrix blank comparisonMRM 195.05>138.05Plasma separation card matrix blank
Plasma matrix blank
500ng/mL comparisonMRM 195.05>138.05Plasma separation card 500ng/mL calibration standard
Plasma500ng/mL calibration standard
TMZ
TMZRt
1.7mins
Matrix peak Matrix peak
PO-CON1475E
Application of a Sensitive Liquid Chromatography-Tandem Mass SpectrometricMethod to Pharmacokinetic Study of Telbivudine in Humans
ASMS 2014 WP 629
Bicui Chen1, Bin Wang1, Xiaojin Shi1, Yuling Song2,
Jinting Yao2, Taohong Huang2, Shin-ichi Kawano2,
Yuki Hashi2
1 Pharmacy Department, Huashan Hospital,
Fudan University,
2 Shimadzu Global COE, Shimadzu (China) Co., Ltd.
2
Application of a Sensitive Liquid Chromatography-Tandem Mass Spectrometric Method to Pharmacokinetic Study of Telbivudine in Humans
IntroductionTelbivudine is a synthetic L-nucleoside analogue, which is phosphorylated to its active metabolite, 5’-triphosphate, by cellular kinases. The telbivudine 5’-triphosphate inhibits HBV DNA polymerase (a reverse transcriptase) by competing with the natural substrate, dTTP. Incorporation
of 5’-triphosphorylated-telbivudine into viral DNA obligates DNA chain termination, resulting in inhibition of HBV replication. The objectives of the current studies were to develop a selective and sensitive LC-MS/MS method to determine of telbivudine in human plasma.
Method
(1) Add 100 μL of plasma into the polypropylene tube, add 40 μL of internal standard working solution (33 µg/mL, with thymidine phosphorylase) to all other tubes.
(2) Incubate the tubes for 1 h at 37 ºC in dark.(3) Add 200 μL of acetonitrile to all tubes, seal and vortex for 1 minutes.(4) Centrifuge the tubes for 5 minutes at 13000 rpm.(5) Transfer 200 μL supernatant to a clean glass bottle and inject into the HPLC-MS/MS system.
Sample Preparation
The analysis was performed on a Shimadzu Nexera UHPLC instrument (Kyoto, Japan) equipped with LC-30AD pumps, CTO-30A column oven, DGU-30A5 on-line egasser, and SIL-30AC autosampler. The separation was carried out on GL Sciences InertSustain C18 column (3.0 mmI.D. x 100
mmL.) with the column temperature at 40 ºC. A triple quadruple mass spectrometer (Shimadzu LCMS-8050, Kyoto, Japan) was connected to the UHPLC instrument via an ESI interface.
LC-MS/MS Analysis
Analytical Conditions
HPLC (Nexera UHPLC system)
Column : InertSustain (3.0 mmI.D. x 100 mmL., 2 μm, GL Sciences)
Mobile Phase A : water with 0.1% formic acid
Mobile Phase B : acetonitrile
Gradient Program : as shown in Table 1
Flow Rate : 0.4 mL/min
Column Temperature : 40 ºC
Injection Volume : 2 µL
Table 1 Time Program
Time (min) Module Command Value
0.00
4.00
4.10
6.00
Pumps
Pumps
Pumps
Controller
Pump B Conc.
Pump B Conc.
Pump B Conc.
Stop
5
80
5
3
Application of a Sensitive Liquid Chromatography-Tandem Mass Spectrometric Method to Pharmacokinetic Study of Telbivudine in Humans
Results and DiscussionHuman plasma samples containing telbivudine ranging from 1.0 to 10000 ng/mL were prepared and extracted by protein precipitation and the �nal extracts were analyzed by LC-MS/MS. MRM chromatograms of telbivudine (1 ng/mL) and deuterated internal standard are presented in Fig. 1 (blank) and Fig. 2 (spiked). The linear regression for telbivudine was found to be >0.9999. The calibration curve with human plasma as the matrix were shown in Fig. 3. Excellent precision and accuracy were maintained for four orders of magnitude, demonstrating a linear dynamic range suitable for real-world applications. LLOQ for telbivudine was 1.0 ng/mL, which met the criteria for bias (%) and precision within ±15% both within run and between run. The
intra-day and inter-day precision and accuracy of the assay were investigated by analyzing QC samples. Intra-day precision (%RSD) at three concentration levels (3, 30, and 8000 ng/mL) were below 2.5% and inter-day precision (%RSD) was below 2.5%. The recoveries of telbivudine were 100.6±2.5 %, 104.5±1.5% and 104.3±1.6% at three concentration levels, respectively. The stability data showed that the processed samples were stable at the room temperature for 8 h, and there was no signi�cant degradation during the three freeze/thaw cycles at -20 ºC. The reinjection reproducibility results indicated that the extracted samples could be stable for 72 h at 10 ºC.
MS (LCMS-8050 triple quadrupole mass spectrometer)
Ionization : ESI
Polarity : Positive
Ionization Voltage : +0.5 kV (ESI-Positive mode)
Nebulizing Gas Flow : 3.0 L/min
Heating Gas Flow : 8.0 L/min
Drying Gas Flow : 12.0 L/min
Interface Temperature : 250 ºC
Heat Block Temperature : 300 ºC
DL Temperature : 350 ºC
Mode : MRM
Table 2 MRM Parameters
CompoundPrecursor
m/z
243.10
246.10
Productm/z
127.10
130.10
Dwell Time(ms)
100
100
Q1 Pre Bias(V)
-26
-16
Q3 Pre Bias(V)
-13
-25
CE (V)
-10
-9
Telbivudine
Telbivudine-D3
4
Application of a Sensitive Liquid Chromatography-Tandem Mass Spectrometric Method to Pharmacokinetic Study of Telbivudine in Humans
Figure 1 Representative MRM chromatograms of blank human plasma(left: transition for telbivudine, right: transition for internal standard)
Figure 2 Representative MRM chromatograms of telbivudine (left, 1 ng/mL) and internal standard (right) in human plasma
Figure 3 Calibration curve of telbivudine in human plasma
0.0 1.0 2.0 3.0 4.0 5.0 min
0.0
1.0
2.0
3.0
4.0(x100)
1:Telbivudine 243.10>127.10(+) CE: -10.0
1.0 2.0 3.0 4.0 5.0 min
0.0
1.0
2.0
3.0
4.0
(x1,000)2:Telbivudine-D3 246.10>130.10(+) CE: -9.0
0.0 1.0 2.0 3.0 4.0 5.0 min
0.0
2.5
5.0
7.5
(x100)1:Telbivudine 243.10>127.10(+) CE: -10.0
Telb
ivud
ine
1.0 2.0 3.0 4.0 5.0 min
0.00
0.25
0.50
0.75
1.00
1.25
1.50(x1,000,000)
2:Telbivudine-D3 246.10>130.10(+) CE: -9.0
Telb
ivud
ine-
D3
0 2500 5000 7500 Conc. Ratio0.0
0.5
1.0
1.5
2.0
2.5
Area Ratio
Application of a Sensitive Liquid Chromatography-Tandem Mass Spectrometric Method to Pharmacokinetic Study of Telbivudine in Humans
5
Figure 4 Representative MRM chromatograms of real-world sample
CompoundCalibration
Curve
Y = (2.77×10-4)X + (3.39×10-5)
Linear Range(ng/mL)
1~10000
Accuracy(%)
93.1~116.6%
r
0.9998Telbivudine
Table 3 Accuracy and precision for the analysis of amlodipine in human plasma(in pre-study validation, n=3 days, six replicates per day)
Added Conc.(ng/mL)
3
400
8000
Intra-day Precision(%RSD)
2.18
1.52
1.76
Inter-day Precision(%RSD)
2.11
1.58
1.68
Accuracy(%)
107.7~114.4
91.6~95.9
95.4~101.3
Table 5 Matrix effect for QC samples (n=6)
QC Level
LQC
MQC
HQC
Added Conc.(ng/mL)
3
400
8000
Matrix Factor
82.3%
81.7%
90.8%
IS-NormalizedMatrix Factor
99.0%
101.0%
101.5%
Table 4 Recovery for QC samples (n=6)
QC Level
LQC
MQC
HQC
Concentartion(ng/mL)
3
400
8000
Recovery(%)
100.6
104.5
104.3
0.0 1.0 2.0 3.0 4.0 5.0 min
0.0
1.0
2.0
3.0
(x10,000)1:Telbivudine 243.10>127.10(+) CE: -10.0
1.0 2.0 3.0 4.0 5.0 min
0.00
0.25
0.50
0.75
1.00
(x1,000,000)2:Telbivudine-D3 246.10>130.10(+) CE: -9.0
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Application of a Sensitive Liquid Chromatography-Tandem Mass Spectrometric Method to Pharmacokinetic Study of Telbivudine in Humans
ConclusionResults of parameters for method validation such as dynamic range, linearity, LLOQ, intra-day precision, inter-day precision, recoveries, and matrix effect factors were excellent. The sensitive LC-MS/MS technique provides a powerful tool for the high-throughput and highly selective analysis of telbivudine in clinical trial study.
PO-CON1449E
Accelerated and robust monitoringfor immunosuppressants using triplequadrupole mass spectrometry
ASMS 2014 WP628
Natsuyo Asano1, Tairo Ogura1, Kiyomi Arakawa1
1 Shimadzu Corporation. 1, Nishinokyo Kuwabara-cho,
Nakagyo-ku, Kyoto 604–8511, Japan
2
Accelerated and robust monitoring for immunosuppressants using triple quadrupole mass spectrometry
IntroductionImmunosuppressants are drugs which lower or suppress activity of the immune system. They are used to prevent the rejection after transplantation or treat autoimmune disease. To avoid immunode�ciency as adverse effect, it is recommended to monitor blood level of therapeutic drug with high throughput and high reliability. There are several analytical technique to monitor drugs, LC/MS is superior in terms of cross-reactivity at low level and throughput of
analysis. Therefore, it is important to analyze these drugs in blood by using ultra-fast mass spectrometer to accelerate monitoring with high quantitativity. We have developed analytical method for four immunosuppressants (Tacrolimus, Rapamycin, Everolimus and Cyclosporin A) with two internal standards (Ascomycin and Cyclosporin D) using ultra-fast mass spectrometer.
Figure 1 Structure of immunosuppressants and internal standards (IS)
O
HO
O
O OH
ON
OO
OHO
O
H O
HOO
O
O O OH
OOO
N
OO
O
HO
O
HO
O
O O OH
OOO
N
OO
O
HO
O
TacrolimusMW: 804.02
EverolimusMW: 958.22
RapamycinMW: 914.17
N
O
N O
NH
OHN
O
N
OHN
O
N
N
O
O
N
HO
HN
O
O
N
O
H
O
O
HO HO
N
O
OO
O
O
H
OH
O
HO
N N
OO
HN
O
N
O
N O
N
ON
OH
O
NH
OHN
O
NO
HNO
Cyclosporin AMW: 1202.61
Ascomycin (IS)MW: 792.01
Cyclosporin D (IS)MW: 1216.64
3
Accelerated and robust monitoring for immunosuppressants using triple quadrupole mass spectrometry
Methods and MaterialsStandard samples of each compound were analyzed to optimize conditions of liquid chromatograph and mass spectrometer. Whole blood extract was prepared based on liquid-liquid extraction described bellow.
2.7 mL of Whole blood and 20.8 mL of Water ↓Vortex for 15 seconds ↓Add 36 mL of MTBE/Cyclohexane (1:3) ↓Vortex for 15 seconds and Centrifuge with 3000 rpm at 20 ºC for 10 minutes ↓Extract an Organic phase ↓Evaporate and Dry under a Nitrogen gas stream ↓Redissolve in 1.8 mL of 80 % Methanol solution with 1 mmol/L Ammonium acetate ↓Vortex for 1 minute and Centrifuge with 3000 rpm at 4 ºC for 5 minutes ↓Filtrate and Transfer into 1 mL glass vial
Table 1 Analytical conditions
UHPLC
Liquid Chromatograph : Nexera (Shimadzu, Japan)
Analysis Column : YMC-Triart C18 (30 mmL. × 2 mmI.D.,1.9 μm)
Mobile Phase A : 1 mmol/L Ammonium acetate - Water
Mobile Phase B : 1 mmol/L Ammonium acetate - Methanol
Gradient Program : 60 % B. (0 min) – 75 % B. (0.10 min) – 95 % B. (0.70 – 0.90 min) –
60 % B. (0.91 – 1.80 min)
Flow Rate : 0.45 mL/min
Column Temperature : 65 ºC
Injection Volume : 1.5 µL
MS
MS Spectrometer : LCMS-8050 (Shimadzu, Japan)
Ionization : ESI (negative)
Probe Voltage : -4.5 ~ -3 kV
Nebulizing Gas Flow : 3.0 L/min
Drying Gas Flow : 5.0 L/min
Heating Gas Flow : 15.0 L/min
Interface Temperature : 400 ºC
DL Temperature : 150 ºC
HB Temperature : 390 ºC
4
Accelerated and robust monitoring for immunosuppressants using triple quadrupole mass spectrometry
ResultImmunosuppressants, which we have developed a method for monitoring of, has been often observed as ammonium or sodium adduct ion by using positive ionization. In general, protonated molecule (for positive) or deprotonated molecule (for negative) is more preferable for reliable quantitation than adduct ions such as ammonium, sodium, and potassium adduct. In this study,
each compound was detected as deprotonated molecule in negative mode by using heated ESI source of LCMS-8050 (Table 2).The separation of all compounds was achieved within 1.8 min, with a YMC-Triart C18 column (30 mmL. × 2 mmI.D.,1.9 μm) and at 65 ºC of column oven temperature.
Figure 2 MRM chromatograms of immnosuppresants in human whole blood (50 ng/mL)
Peak No.
1
2
3
4
5
6
Compound
Ascomysin (IS)
Tacrolimus
Rapamycin
Everolimus
Cyclosporin A
Cyclosporin D (IS)
Porality
neg
neg
neg
neg
neg
neg
Precursor ion (m/z)
790.40
802.70
912.70
956.80
1200.90
1215.10
Product ion (m/z)
548.20
560.50
321.20
365.35
1088.70
1102.60
Table 2 MRM transitions
0.75 1.00 1.25 min
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
(x100,000)
2
4
3
1
5
6
Accelerated and robust monitoring for immunosuppressants using triple quadrupole mass spectrometry
5
Figure 3 MRM chromatograms at LLOQ and ISTD (left), and calibration curves (right) for four immnosuppresants in human whole blood
a) Tacrolimus
0.5 – 1000 ng/mL
0.5 ng/mL
Ascomycin40 ng/mL
c) Everolimus
0.5 ng/mL
Ascomycin40 ng/mL 0.5 – 100 ng/mL
b) Rapamycin
0.5 ng/mL
Ascomycin40 ng/mL 0.5 – 500 ng/mL
d) Cyclosporin A
Cyclosporin D100 ng/mL
0.5 ng/mL
0.5 – 1000 ng/mL
Accelerated and robust monitoring for immunosuppressants using triple quadrupole mass spectrometry
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Figure 3 illustrates both a calibration curve and chromatogram at the lowest calibration level for all immunosuppressants analyzed. Table 3 lists both the reproducibility and accuracy for each immunosuppressant that has been simultaneously measured in 1.8 minutes.
In high speed measurement condition, we have achieved high sensitivity and wide dynamic range for all analytes. Additionally, the accuracy of each analyte ranged from 88 to 110 % and area reproducibility at the lowest calibration level of each analyte was less than 20%.
Conclusions• Monitoring with negative mode ionization permitted more sensitive, robust and reliable quantitation for four
immunosuppressants.• A total of six compounds were measured in 1.8 minutes. The combination of Nexera and LCMS-8050 provided a faster
run time without sacri�cing the quality of results.• Even with a low injection volume of 1.5 μL, the lower limit of quantitation (LLOQ) for all compounds was 0.5 ng/mL. • In this study, it is demonstrated that LCMS-8050 is useful for the rugged and rapid quantitation for immunosuppressants
in whole blood.
AcknowledgementWe appreciate suggestions from Prof. Kazuo Matsubara and Assoc. Prof. Ikuko Yano from the department of pharmacy, Kyoto University Hospital, and Prof. Satohiro Masuda from the department of pharmacy, Kyusyu University Hospital.
Table 3 Reproducibility and Accuracy
Compound
Tacrolimus
Concentration
Low (0.5 ng/mL) Low-Mid (2 ng/mL)High (1000 ng/mL)
CV % (n = 6)
18.013.02.87
Accuracy %
99.499.588.7
RapamycinLow (0.5 ng/mL)
Low-Mid (5 ng/mL)High (500 ng/mL)
6.872.883.41
95.6109.390.0
EverolimusLow (0.5 ng/mL)
Low-Mid (5 ng/mL)High (100 ng/mL)
10.45.112.26
95.3104.493.3
Cyclosporin ALow (0.5 ng/mL)
Low-Mid (10 ng/mL)High (1000 ng/mL)
7.312.362.67
95.199.994.9
PO-CON1468E
Highly sensitive quantitative analysis of Felodipine and Hydrochlorothiazidefrom plasma using LC/MS/MS
ASMS 2014 TP497
Shailendra Rane, Rashi Kochhar, Deepti Bhandarkar,
Shruti Raju, Shailesh Damale, Ajit Datar,
Pratap Rasam, Jitendra Kelkar
Shimadzu Analytical (India) Pvt. Ltd., 1 A/B Rushabh
Chambers, Makwana Road, Marol, Andheri (E),
Mumbai-400059, Maharashtra, India.
2
Highly sensitive quantitative analysis of Felodipine and Hydrochlorothiazide from plasma using LC/MS/MS
IntroductionFelodipine is a calcium antagonist (calcium channel blocker), used as a drug to control hypertension[1]. Hydrochlorothiazide is a diuretic drug of the thiazide class that acts by inhibiting the kidney’s ability to retain water. It is, therefore, frequently used for the treatment of hypertension, congestive heart failure, symptomatic edema, diabetes insipidus, renal tubular acidosis and the prevention of kidney stones[2].Efforts have been made here to develop high sensitive
methods of quantitation for these two drugs using LCMS-8050 system from Shimadzu Corporation, Japan.Presence of heated Electro Spray Ionization (ESI) probe in LCMS-8050 ensured good quantitation and repeatability even in the presence of a complex matrix like plasma. Ultra high sensitivity of LCMS-8050 enabled development quantitation method at low ppt level for both Felodipine and Hydrochlorthiazide.
Method of Analysis
To 100 µL of plasma, 500 µL of cold acetonitrile was added for protein precipitation then put in rotary shaker at 20 rpm for 15 minutes for uniform mixing. It was centrifuged
at 12000 rpm for 15 minutes. Supernatant was collected and evaporated to dryness at 70 ºC and finally reconstituted in 200 µL Methanol.
Preparation of matrix matched plasma by protein precipitation method using cold acetonitrile
Figure 2. Structure of Hydrochlorothiazide
HydrochlorothiazideHydrochlorothiazide, abbreviated HCTZ (or HCT, HZT), is a diuretic drug of the thiazide class that acts by inhibiting the kidney‘s ability to retain water. Hydrochlorothiazide is 6-chloro-1,1-dioxo-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide.Its empirical formula is C7H8ClN3O4S2 and its structure is shown in Figure 2.
Figure 1. Structure of Felodipine
FelodipineFelodipine is a calcium antagonist (calcium channel blocker). Felodipine is a dihydropyridine derivative that is chemically described as ± ethyl methyl 4-(2,3-dichlorophenyl)1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate. Its empirical formula is C18H19Cl2NO4 and its structure is shown in Figure 1.
3
Highly sensitive quantitative analysis of Felodipine and Hydrochlorothiazide from plasma using LC/MS/MS
LC/MS/MS analysisCompounds were analyzed using Ultra High Performance Liquid Chromatography (UHPLC) Nexera coupled with LCMS-8050 triple quadrupole system (Shimadzu
Corporation, Japan), The details of analytical conditions are given in Table 1 and Table 2.
• Felodipine Calibration Std : 5 ppt, 10 ppt, 50 ppt, 100 ppt, 500 ppt, 1 ppb and 10 ppb• HCTZ Calibration Std : 2 ppt, 5 ppt, 10 ppt, 50 ppt, 100 ppt, and 500 ppt
To 500 µL plasma, 100 µL sodium carbonate (1.00 mol/L) and 5 mL of diethyl ether : hexane (1:1 v/v) was added. It was placed in rotary shaker at 20 rpm for 15 minutes for uniform mixing and centrifuged at 12000 rpm for 15
minutes. Supernatant was collected and evaporated to dryness at 60 ºC. It was finally reconstitute in 1000 µL Methanol.
Preparation of matrix matched plasma by liquid-liquid extraction method using diethyl ether and hexane mixture (1:1 v/v)
Response of Felodipine and Hydrochlorothiazide were checked in both above mentioned matrices. It was found that cold acetonitrile treated plasma and diethyl ether: hexane (1:1 v/v) treated plasma were suitable for
Felodipine and Hydrochlorothiazide molecules respectively. Calibration standards were thus prepared in respective matrix matched plasma.
Preparation of calibration standards in matrix matched plasma
Figure 3. LCMS-8050 triple quadrupole mass spectrometer by Shimadzu Figure 4. Heated ESI probe
LCMS-8050 triple quadrupole mass spectrometer by Shimadzu (shown in Figure 3), sets a new benchmark in triple quadrupole technology with an unsurpassed sensitivity (UFsensitivity), Ultra fast scanning speed of 30,000 u/sec (UFscanning) and polarity switching speed of 5 msecs (UFswitching). This system ensures highest quality of data, with very high degree of reliability.
In order to improve ionization efficiency, the newly developed heated ESI probe (shown in Figure 4) combines high-temperature gas with the nebulizer spray, assisting in the desolvation of large droplets and enhancing ionization. This development allows high-sensitivity analysis of a wide range of target compounds with considerable reduction in background.
4
Highly sensitive quantitative analysis of Felodipine and Hydrochlorothiazide from plasma using LC/MS/MS
Results
LC/MS/MS method for Felodipine was developed on ESI positive ionization mode and 383.90>338.25 MRM transition was optimized for it. Checked matrix matched plasma standards for highest (10 ppb) as well as lowest concentrations (5 ppt) as seen in Figure 5 and Figure 6
respectively. Calibration curves as mentioned with R2 = 0.998 were plotted (shown in Figure 7). Also as seen in Table 3, % Accuracy was studied to confirm the reliability of method. Also, LOD as 2 ppt and LOQ as 5 ppt was obtained.
LC/MS/MS analysis results of Felodipine
• Column : Shim-pack XR-ODS (75 mm L x 3 mm I.D.; 2.2 µm)
• Flow rate : 0.3 mL/min
• Oven temperature : 40 ºC
• Mobile phase : A: 10 mM ammonium acetate in water
B: methanol
• Gradient program (%B) : 0.0 – 3.0 min → 90 (%); 3.0 – 3.1 min → 90 – 100 (%);
3.1 – 4.0 min → 100 (%); 4.0– 4.1 min → 100 – 90 (%)
4.1 – 6.5 min → 90 (%)
• Injection volume : 10 µL
• MS interface : ESI
• Nitrogen gas �ow : Nebulizing gas 1.5 L/min; Drying gas 10 L/min;
• Zero air �ow : Heating gas 10 L/min
• MS temperature : Desolvation line 200 ºC; Heating block 400 ºC
Interface 200 ºC
Table 1. LC/MS/MS conditions for Felodipine
• Column : Shim-pack XR-ODS (100 mm L x 3 mm I.D.; 2.2 µm)
• Flow rate : 0.2 mL/min
• Oven temperature : 40 ºC
• Mobile phase : A: 0.1% formic acid in water
B: acetonitrile
• Gradient program (%B) : 0.0 – 1.0 min → 80 (%); 1.0 – 3.5 min → 40 – 100 (%);
3.5 – 4.5 min → 100 (%); 4.5– 4.51min → 100 – 80 (%)
4.51 – 8.0 min → 90 (%)
• Injection volume : 25 µL
• MS interface : ESI
• Nitrogen gas �ow : Nebulizing gas 2.0 L/min; Drying gas 10 L/min;
• Zero air �ow : Heating gas 15 L/min
• MS temperature : Desolvation line 250 ºC; Heating block 500 ºC
Interface 300 ºC
Table 2. LC/MS/MS conditions for Hydrochlorothiazide
5
Highly sensitive quantitative analysis of Felodipine and Hydrochlorothiazide from plasma using LC/MS/MS
Figure 5. Felodipine at 10 ppb in matrix matched plasma Figure 6. Felodipine at 5 ppt in matrix matched plasma
Figure 7. Calibration curve of Felodipine
LC/MS/MS method for Hydrochlorothiazide was developed on ESI negative ionization mode and 296.10>204.90 MRM transition was optimized for it. Checked matrix matched plasma standards for highest (500 ppt) as well as lowest (2 ppt) concentrations as seen in Figures 8 and 9 respectively.
Calibration curves as mentioned with R2=0.998 were plotted (shown in Figure 10). Also as seen in Table 4, % Accuracy was studied to confirm the reliability of method. Also, LOD as 1 ppt and LOQ as 2 ppt were obtained.
LC/MS/MS analysis results of Hydrochlorothiazide
Table 3: Results of Felodipine calibration curve
Nominal Concentration (ppb)
Measured Concentration (ppb)
% Accuracy(n=3)
% RSD for area counts (n=3)
0.005
0.01
0.05
0.1
0.5
1
10
Standard
STD-FEL-01
STD-FEL-02
STD-FEL-03
STD-FEL-04
STD-FEL-05
STD-FEL-06
STD-FEL-07
Sr. No.
1
2
3
4
5
6
7
0.005
0.010
0.053
0.103
0.469
0.977
10.023
97.43
103.80
104.47
103.13
94.88
97.33
100.90
9.87
8.76
2.24
1.23
1.33
0.95
0.60
0.0 2.5 5.0
0.0
2.5
5.0(x100,000)383.90>338.25(+)
FELO
DIP
INE
0.0 2.5 5.0
0.0
0.5
1.0
1.5
2.0
(x1,000)383.90>338.25(+)
FELO
DIP
INE
0.0 2.5 5.0 7.5 Conc.0.0
0.5
1.0
1.5
2.0Area (x1,000,000)
1 2 3 4 5
6
7
0.05 0.10 Conc.0.0
0.5
1.0
1.5
2.0
2.5
3.0Area (x10,000)
1 2
3
4
Highly sensitive quantitative analysis of Felodipine and Hydrochlorothiazide from plasma using LC/MS/MS
6
Figure 8. Hydrochlorothiazide at 500 ppt in matrix matched plasma Figure 9. Hydrochlorothiazide at 2 ppt in matrix matched plasma
Figure 10. Calibration curve of Hydrochlorothiazide
Conclusion• Highly sensitive LC/MS/MS method for Felodipine and Hydrochlorothiazide was developed on LCMS-8050 system.• LOD of 2 ppt and LOQ of 5 ppt was achieved for Felodipine and LOD of 1 ppt and LOQ of 2 ppt was achieved for
Hydrochlorothiazide by matrix matched methods.• Heated ESI probe of LCMS-8050 system enables drastic augment in sensitivity with considerable reduction in
background. Hence, LCMS-8050 system from Shimadzu is an all rounder solution for bioanalysis.
Table 4. Results of Hydrochlorothiazide calibration curve
Nominal Concentration (ppb)
Measured Concentration (ppb)
% Accuracy(n=3)
% RSD for area counts (n=3)
0.002
0.005
0.01
0.05
0.1
0.5
Standard
STD-HCTZ-01
STD-HCTZ-02
STD-HCTZ-03
STD-HCTZ-04
STD-HCTZ-05
STD-HCTZ-06
Sr. No.
1
2
3
4
5
6
0.0020
0.0048
0.0099
0.0512
0.1019
0.4944
102.03
95.50
100.07
102.67
102.11
102.13
6.65
3.53
3.80
1.60
3.58
1.68
0.0 2.5 5.0 7.5
0.0
0.5
1.0
1.5
(x10,000)296.10>204.90(-)
HC
TZ
0.0 2.5 5.0 7.5
0.0
0.5
1.0
1.5
2.0
2.5(x100)
296.10>204.90(-)
HC
TZ
0.0 0.1 0.2 0.3 0.4 Conc.0.00
0.25
0.50
0.75
1.00Area (x100,000)
1 2 3
4
5
6
0.000 0.025 0.050 Conc.0.0
0.5
1.0
1.5
Area (x10,000)
1 2 3
4
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Highly sensitive quantitative analysis of Felodipine and Hydrochlorothiazide from plasma using LC/MS/MS
References[1] YU Peng; CHENG Hang, Chinese Journal of Pharmaceutical Analysis, Volume 32, Number 1, (2012), 35-39(5).[2] Hiten Janardan Shah, Naresh B. Kataria, Chromatographia, Volume 69, Issue 9-10, (2009), 1055-1060.
PO-CON1467E
Highly sensitive quantitative estimationof genotoxic impurities from API and drug formulation using LC/MS/MS
ASMS 2014 TP496
Shruti Raju, Deepti Bhandarkar, Rashi Kochhar,
Shailesh Damale, Shailendra Rane, Ajit Datar,
Pratap Rasam, Jitendra Kelkar
Shimadzu Analytical (India) Pvt. Ltd.,
1 A/B Rushabh Chambers, Makwana Road, Marol,
Andheri (E), Mumbai-400059, Maharashtra, India.
2
Highly sensitive quantitative estimation of genotoxic impurities from API and drug formulation using LC/MS/MS
IntroductionThe toxicological assessment of Genotoxic Impurities (GTI) and the determination of acceptable limits for such impurities in Active Pharmaceutical Ingredients (API) is a dif�cult issue. As per European Medicines Agency (EMEA) guidance, a Threshold of Toxicological Concern (TTC) value of 1.5 µg/day intake of a genotoxic impurity is considered to be acceptable for most pharmaceuticals[1]. Dronedarone is a drug mainly used for indications of cardiac arrhythmias. GTI of this drug has been quantitated here. Method has been optimized for simultaneous analysis of DRN-IA {2-n-butyl-3-[4-(3-di-n-butylamino-propoxy)benzoyl]-5-nitro
benzofuran}, DRN-IB {5-amino-3-[4-(3-di-n-butylamino-propoxy)benzoyl}-2-n-butyl benzofuran} and BHBNB {2-n-butyl-3-(4-hydroxy benzoyl)-5-nitro benzofuran}. Structures of Dronedarone and its GTI are shown in Figure 1.As literature references available on GTI analysis are minimal, the feature of automatic MRM optimisation in LCMS-8040 makes method development process less tedious. In addition, the lowest dwell time and pause time and ultrafast polarity switching of LCMS-8040 ensures uncompromised and high sensitive quantitation.
Figure 1. Structures of Dronedarone and its GTI
O
OOH
NO2
C4H9
O
O O
C4H9
NH2
N
C4H9
C4H9
O
O O
C4H9
NO2
N
C4H9
C4H9
DRN-IA
O
O O
C4H9
NHSO2Me
N
C4H9
C4H9
Dronedarone
DRN-IB BHBNB
3
Highly sensitive quantitative estimation of genotoxic impurities from API and drug formulation using LC/MS/MS
LC/MS/MS Analytical ConditionsAnalysis was performed using Ultra High Performance Liquid Chromatography (UHPLC) Nexera coupled with LCMS-8040 triple quadrupole system (Shimadzu Corporation, Japan), shown in Figure 2. Limit of GTI for Dronedarone is 2 mg/kg. However, general dosage of Dronedarone is 400 mg, hence, limit for GTI is 0.8 µg/400 mg. This when reconstituted in 20 mL system, gives an
effective concentration of 40 ppb. For analytical method development it is desirable to have LOQ at least 30 % of limit value, which in this case corresponds to 12 ppb. The developed method gives provision for measuring GTI at much lower level. However, recovery studies have been done at 12 ppb level.
Figure 2. Nexera with LCMS-8040 triple quadrupole system by Shimadzu
Method of Analysis
• Preparation of DRN-IA and DRN-IB and BHBNB stock solutions 20 mg of each impurity standard was weighed separately and dissolved in 20 mL of methanol to prepare stock solutions
of each standard.
• Preparation of calibration levels GTI mix standards (DRN-IA, DRN-IB and BHBNB) at concentration levels of 0.5 ppb, 1 ppb, 5 ppb, 10 ppb, 40 ppb, 50
ppb and 100 ppb were prepared in methanol using stock solutions of all the three standards.
• Preparation of blank sample 400 mg of Dronedarone powder sample was weighed and mixed with 20 mL of methanol. Mixture was sonicated to
dissolve sample completely.
• Preparation of spiked (at 12 ppb level) sample 400 mg of sample was weighed and spiked with 60 µL of 1 ppm stock solution. Solution was then mixed with 20 mL of
methanol. Mixture was sonicated to dissolve sample completely.
Sample Preparation
4
Highly sensitive quantitative estimation of genotoxic impurities from API and drug formulation using LC/MS/MS
Table 1. LC/MS/MS analytical conditions
Results
LC/MS/MS method was developed for simultaneous quantitation of GTI mix standards. MRM transitions used for all GTI are given in Table 2. No peak was seen in diluent (methanol) at the retention times of GTI for selected MRM transitions which confirms the absence of any interference from diluent (shown in Figure 3). MRM chromatogram of GTI mix standard at 5 ppb level is shown in Figure 4. Linearity studies were carried out using external standard
calibration method. Calibration graphs of each GTI are shown in Figure 5. LOQ was determined for each GTI based on the following criteria – (1) % RSD for area < 15 %, (2) % Accuracy between 80-120 % and (3) Signal to noise ratio (S/N) > 10. LOQ of 0.5 ppb was achieved for DRN-IB and BHBNB whereas 1 ppb was achieved for DRN-IA. Results of accuracy and repeatability for all GTI are given in Table 3.
LC/MS/MS analysis
• Column : Shim-pack XR-ODS II (75 mm L x 3 mm I.D.; 2.2 µm)
• Mobile phase : A: 0.1% formic acid in water
B: acetonitrile
• Flow rate : 0.3 mL/min
• Oven temperature : 40 ºC
• Gradient program (B%) : 0.0–2.0 min → 35 (%); 2.0–2.1 min → 35-40 (%);
2.1–7.0 min → 40-60 (%); 7.0–8.0 min → 60-100 (%);
8.0–10.0 min → 100 (%); 10.0–10.01 min → 100-35 (%);
10.01–13.0 min → 35 (%)
• Injection volume : 1 µL
• MS interface : Electro Spray Ionization (ESI)
• MS analysis mode : MRM
• Polarity : Positive and negative
• MS gas �ow : Nebulizing gas 2 L/min; Drying gas 15 L/min
• MS temperature : Desolvation line 250 ºC; Heat block 400 ºC
Note: Flow Control Valve (FCV) was used for the analysis to divert HPLC �ow towards waste during elution of Dronedarone so as to prevent contamination of Mass Spectrometer.
Table 2: MRM transitions selected for all GTI
Name of GTI MRM transition Retention time (min) Mode of ionization
DRN-IB
DRN-IA
BHBNB
479.15>170.15
509.10>114.10
338.20>244.05
1.83
5.85
8.77
Positive ESI
Positive ESI
Negative ESI
Below mentioned table shows the analytical conditions used for analysis of GTI.
5
Highly sensitive quantitative estimation of genotoxic impurities from API and drug formulation using LC/MS/MS
Figure 4. MRM chromatogram of GTI mix standard at 5 ppb level
Figure 5. Calibration graphs for GTI
Figure 3. MRM chromatogram of diluent (methanol)
0.0 2.5 5.0 7.5 10.0 min
0
5000
10000
15000
20000
25000
30000
35000
40000 3:BHBNB 338.20>244.05(-) CE: 20.02:DRA-IA 509.10>114.10(+) CE: -41.01:DRA-IB 479.15>170.15(+) CE: -29.0
BHBN
B 33
8.20
>24
4.05
DRN
-IA 5
09.1
0>11
4.10
DRN
-IB 4
79.1
5>17
0.15
0.0 2.5 5.0 7.5 10.0 min
0
250
500
750
1000
3:BHBNB 338.20>244.05(-) CE: 20.02:DRA-IA 509.10>114.10(+) CE: -41.01:DRA-IB 479.15>170.15(+) CE: -29.0
0.0 25.0 50.0 75.0 Conc.0
250000
500000
750000Area
DRN-IB R2-0.9989
0.0 25.0 50.0 75.0 Conc.0
250000
500000
750000
1000000
1250000
Area
DRN-IA R2-0.9943
0.0 25.0 50.0 75.0 Conc.0
50000
100000
150000
Area
BHBNB R2-0.9922
Highly sensitive quantitative estimation of genotoxic impurities from API and drug formulation using LC/MS/MS
6
Figure 6. MRM chromatogram of blank sample
Table 3: Results of accuracy and repeatability for all GTI
Standard concentration (ppb)
Calculated concentration from calibration graph
(ppb) (n=6)
% Accuracy (n=6)
% RSD for area counts (n=6)
0.5
1
5
12
40
50
100
1
5
12
40
50
100
0.5
1
5
12
40
50
100
Name of GTI
DRN-IB
DRN-IA
BHBNB
Sr. No.
1
2
3
0.492
1.044
4.961
12.014
38.360
49.913
103.071
0.994
4.916
11.596
37.631
48.605
100.138
0.486
1.062
4.912
11.907
37.378
48.518
96.747
98.40
104.40
99.22
100.12
95.90
99.83
103.07
99.40
98.32
96.63
94.08
97.21
100.14
97.20
106.20
98.24
99.23
93.45
97.04
96.75
9.50
6.62
3.10
2.97
1.17
1.08
0.86
5.02
2.82
2.43
1.27
1.40
0.99
4.88
6.97
2.16
1.31
0.37
0.43
0.91
Recovery studiesFor recovery studies, samples were prepared as described previously. MRM chromatogram of blank and spiked samples are shown in Figures 6 and 7 respectively. Results
of recovery studies have been shown in Table 4. Recovery could not be calculated for DRN-IB as blank sample showed higher concentration than spiked concentration.
0.0 2.5 5.0 7.5 10.0 min
0
50000
100000
150000
200000
250000
300000
350000
4000003:BHBNB 338.20>244.05(-) CE: 20.02:DRA-IA 509.10>114.10(+) CE: -41.01:DRA-IB 479.15>170.15(+) CE: -29.0
BHBN
B 33
8.20
>24
4.05
DRN
-IA 5
09.1
0>11
4.10
DRN
-IB 4
79.1
5>17
0.15
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Highly sensitive quantitative estimation of genotoxic impurities from API and drug formulation using LC/MS/MS
Figure 7. MRM chromatogram of spiked sample
Conclusion• A highly sensitive method was developed for analysis of GTI of Dronedarone.• Ultra high sensitivity, ultra fast polarity switching (UFswitching) enabled sensitive, selective, accurate and reproducible
analysis of GTI from Dronedarone powder sample.
References[1] Guideline on The Limits of Genotoxic Impurities, (2006), European Medicines Agency (EMEA).
Table 4. Results of the recovery studies
Concentration of GTI mix standard spiked
in blank sample (ppb)
Average concentration obtained from calibration graph for blank sample (ppb) (A) (n=3)
Average concentration obtained from calibration graph
for spiked sample (ppb) (B) (n=3)
% Recovery = (B-A)/ 12 * 100
12
12
12
Name of Impurity
DRN-IB
DRN-IA
BHBNB
94.210
3.279
1.241
NA
12.840
12.723
NA
79.678
95.689
0.0 2.5 5.0 7.5 10.0 min
0
25000
50000
75000
100000
125000 3:BHBNB 338.20>244.05(-) CE: 20.02:DRA-IA 509.10>114.10(+) CE: -41.01:DRA-IB 479.15>170.15(+) CE: -29.0
BHBN
B 33
8.20
>24
4.05
DRN
-IA 5
09.1
0>11
4.10
DRN
-IB 4
79.1
5>17
0.15
PO-CON1470E
Development of 2D-LC/MS/MS Method for Quantitative Analysis of1α,25-Dihydroxylvitamin D3 in Human Serum
ASMS 2014 WP449
Daryl Kim Hor Hee1, Lawrence Soon-U Lee1,
Zhi Wei Edwin Ting2, Jie Xing2, Sandhya Nargund2,
Miho Kawashima3 & Zhaoqi Zhan2
1 Department of Medicine Research Laboratories,
National University of Singapore, 6 Science Drive 2,
Singapore 1175462 Customer Support Centre, Shimadzu (Asia Paci�c) Pte
Ltd, 79 Science Park Drive, #02-01/08, Singapore 1182643 Global Application Development Centre, Shimadzu
Corporation, 1-3 Kanda Nishihiki-cho, Chiyoda-ku,
Tokyo 101-8448, Japan
2
Development of 2D-LC/MS/MS Method for Quantitative Analysis of 1α,25-Dihydroxylvitamin D3 in Human Serum
IntroductionDevelopments of LC/MS/MS methods for accurate quantitation of low pg/mL levels of 1α,25-dihydroxy vitamin D2/D3 in serum were reported in recent years, because their levels in serum were found to be important indications of several diseases associated with vitamin D metabolic disorder in clinical research and diagnosis [1]. However, it has been a challenge to achieve the required sensitivity directly, due to the intrinsic dif�culty of ionization of the compounds and interference from matrix [2,3]. Sample extraction and clean-up with SPE and immunoaf�nity methods were applied to remove the interferences [4] prior to LC/MS/MS analysis. However, the
amount of serum required was normally rather high from 0.5mL to 2mL, which is not favourite in the clinical applications. Direct analysis methods with using smaller amount of serum are in demand. Research efforts have been reported in literatures to enhance ionization ef�ciency by using different interfaces such as ESI, APCI or APPI and ionization reagents to form purposely NH3 adduct or lithium adduct [4,5]. Here, we present a novel 2D-LC/MS/MS method with APCI interface for direct analysis of 1α,25-diOH-VD3 in serum. The method achieved a detection limit of 3.1 pg/mL in spiked serum samples with 100 uL injection.
ExperimentalHigh purity 1α,25-dihydroxyl Vitamin D3 and deuterated 1α,25-dihydroxyl-d6 Vitamin D3 (as internal standard) were obtained from Toronto Research Chemicals. Charcoal-stripped pooled human serum obtained from Bioworld was used as blank and matrix to prepare spiked samples in this study. A 2D-LC/MS/MS system was set up on LCMS-8050 (Shimadzu Corporation) with a column switching valve installed in the column oven and controlled by LabSolutions workstation. The details of columns, mobile phases and gradient programs of 1st-D and 2nd-D LC
separations and MS conditions are compiled into Table 1. The procedure of sample preparation of spiked serum samples is shown in Figure 1. It includes protein precipitation by adding ACN-MeOH solvent into the serum in 3 to 1 ratio followed by vortex and centrifuge at high speed. The supernatant collected was �ltered before standards with IS were added (post-addition). The clear samples obtained were then injected into the 2-D LC/MS/MS system.
Table 1: 2D-LC/MS/MS analytical conditions
LC condition
1st D: FC-ODS (2.0mml.D. x 75mm L, 3μm)2nd D: VP-ODS (2.0mmI.D. x 150mm L, 4.6μm)
A: Water with 0.1% formic acidB: Acetontrile
C: Water with 0.1% formic acidD: MeOH with 0.1% formic acid
B: 40% (0 to 0.1min) → 90% (5 to 7.5min) → 15% (11 to 12min) → 40% (14 to 25min); Total �ow rate: 0.5mL/min
D: 15% (0min) → 80% (20 to 22.5min) → 15% (23 to 25min); Peak cutting: 3.15 to 3.40; Total �ow rate: 0.5 mL/min
45ºC
100 uL
Column
Mobile Phase of 1st D
Mobile Phase of 2nd D
1st D gradient pro-gram & �ow rate
2nd D gradient pro-gram & �ow rate
Oven Temp.
Injection Vol.
MS Interface condition
APCI, 400ºC
Positive, MRM
300ºC & 200ºC
Ar (270kPa)
N2, 2.5 L/min
N2, 7.0 L/min
Interface
MS mode
Heat Block & DL Temp.
CID Gas
Nebulizing Gas Flow
Drying Gas Flow
3
Development of 2D-LC/MS/MS Method for Quantitative Analysis of 1α,25-Dihydroxylvitamin D3 in Human Serum
Figure 1: Flow chart of serum sample pre-treatment method
150µL of serum 450µL of ACN/MeOH (1:1)
Shake and Vortex 10mins
Centrifuge for 10 minutes at 13000rpm
480µL of Supernatant
0.2µm nylon �lter
400µL of �ltered protein precipitated Serum
500µL of calibrate50µL of of Std stock
50µL of IS stock
Results and Discussion
An APCI interference was employed for effective ionization of 1α,25-diOH-VitD3 (C27H44O3, MW 416.7). A MRM quantitation method for 1α,25-diOH-VitD3 with its deuterated form as internal standard (IS) was developed. MRM optimization was performed using an automated MRM optimization program with LabSolutions workstation. Two MRM transitions for each compound were selected
(Table 2), the first one for quantitation and the second one for confirmation. The parent ion of 1α,25-diOH-VitD3 was the dehydrated ion, as it underwent neutral lost easily in ionization with ESI and APCI [2,3]. The MRM used for quantitation (399.3>381.3) was dehydration of the second OH group in the molecule.
Development of 2D-LC/MS/MS method
Table 2: MRM transitions and CID parameters of 1α,25-diOH-VitD3 and deuterated IS
Q1 Pre Bias Q3 Pre BiasName
1α,25-dihydroxyl Vitamin D3
1α,25-dihydroxyl-d6 Vitamin D3 (IS)
RT1 (min)
22.74
22.71
Transition (m/z)
399.3 > 381.3
399.3 > 157.0
402.3 > 366.3
402.3 > 383.3
-20
-20
-20
-20
CID Voltage (V)
CE
-13
-29
-12
-15
-14
-17
-18
-27
1, Retention time by 2D-LC/MS/MS method
4
Development of 2D-LC/MS/MS Method for Quantitative Analysis of 1α,25-Dihydroxylvitamin D3 in Human Serum
Figure 2: 1D-LC/MS/MS chromatograms of 22.7 pg/mL 1α,25-diOH-VitD3 (top) and 182 pg/mL internal
standard (bottom) in serum (injection volume: 50uL)
0.0 2.5 5.0 7.5 10.0 min0
1000
2000
3000
4000
5000 1:OH2D3 399.30>105.00(+) CE: -44.01:OH2D3 399.30>157.00(+) CE: -29.01:OH2D3 399.30>381.30(+) CE: -13.0
OH
2-V
D3
2.5 5.0 7.5 10.0 min0
100
200
300
400
500
600
700 2:OH2D3-D6 402.30>366.30(+) CE: -12.02:OH2D3-D6 402.30>383.30(+) CE: -15.0
OH
2-V
D3-
D3
Peak cutting (125 uL) in 1st D separationand transferred to 2nd D LC
The reason to develop a 2-D LC separation for a LC/MS/MS method was the high background and interferences occurred with 1D LC/MS/MS observed in this study and also reported in literatures. Figure 2 shows the MRM chromatograms of 1D-LC/MS/MS of spiked serum sample. It can be seen that the baseline of the quantitation MRM (399.3>381.3) rose to a rather high level and interference peaks also appeared at the same retention time. The 2-D LC/MS/MS method developed in this study involves “cutting the targeted peak” in the 1st-D separation precisely (3.1~3.4 min) and the portion retained in a stainless steel sample loop (200 uL) was transferred into the 2nd-D column for further separation. The operation was accomplished by switching the 6-way valve in and out by a time program. Both 1st-D and 2nd-D separations were carried out in gradient elution mode. The organic mobile phase of 2nd-D (MeOH with 0.1% formic acid) was different from that of 1st-D (pure ACN). The interference peaks co-eluted with the analyte in 1st-D were separated from the analyte peak (22.6 min) as shown in Figure 3.
Two sets of standard samples were prepared in serum and in clear solution (diluent). Each set included seven levels of 1α,25-diOH-VitD3 from 3.13 pg/mL to 200 pg/mL, each added with 200 pg/mL of IS (See Table 3). The chromatograms of the seven spiked standard samples in serum are shown in Figure 3. A linear IS calibration curve (R2 > 0.996) was established from these 2D-LC/MS/MS analysis results, which is shown in Figure 4. It is worth to
note that the calibration curve has a non-zero Y-intercept, indicating that the blank (serum) contains either residual 1α,25-diOH-VitD3 or other interference which must be deducted in the quantitation method. The peak area ratios shown in Table 3 are the results after deduction of the background peaks. The accuracy of the method after this correction is between 92% and 117%.
Calibration curve (IS), linearity and accuracy
Figure 3: Overlay of 2nd-D chromatograms of 7 levels from 3.13 pg/mL to 200 pg/mL spiked in serum.
Figure 4: Calibration curves of1α,25-diOH VD3 in serum by IS method.
0 10 20 min
0
1000
2000
3000
4000
22.0 23.0 min
1000
2000
3000
4000
1α,25-diOH-VitD3
0.00 0.25 0.50 0.75 Conc. Ratio0.0
1.0
2.0
3.0
4.0
5.0
Area Ratio
R2 = 0.9967
Non-zero intercept
Development of 2D-LC/MS/MS Method for Quantitative Analysis of 1α,25-Dihydroxylvitamin D3 in Human Serum
5
Table 3: Seven levels of standard samples for calibration curve and performance evaluation
Figure 5: MRM peaks of 1α,25-diOH-VitD3 spiked in pure diluent (top) and in serum (bottom) of L1, L3, L5 and L7 (spiked conc. refer to Table 3)
Matrix effect of the 2D-LC/MS/MS method was determined by comparison of peak area ratios of standard samples in diluent and in serum at the seven levels. The results are compiled into Table 3. The matrix effect of the method are between 58% and 95%. It seems that the matrix effect is stronger at lower concentrations than at higher concentrations. Repeatability of peak area of the method was evaluated with L2 and L3 spiked serum samples for both target and IS. The Results of RSD (n=6) are displayed in Table 4. The MRM peaks of 1α,25-diOH VD3 in clear solution and in serum are displayed in pairs (top and bottom) in Figure 5. It can be seen from the first pair (diluent and serum blank) that a peak appeared at the same retention of 1α,25-diOH VD3 in the blank serum. As pointed out above, this peak is
from either the residue of 1α,25-diOH VD3 or other interference present in the serum. Due to this background peak, the actual S/N ratio could not be calculated. Therefore, it is difficult to determine the LOD and LOQ based on the S/N method. Tentatively, we propose a reference LOD and LOQ of the method for 1α,25-diOH VD3 to be 3.1 pg/mL and 10 pg/mL, respectively. The specificity of the method relies on several criteria: two MRMs (399>381 and 399>157), their ratio and RT in 2nd-D chromatogram. The MRM chromatograms shown in Figure 5 demonstrate the specificity of the method from L1 (3.1 pg/mL) to L7 (200 pg/mL). It can be seen that the results of spiked serum samples (bottom) meet the criteria if compared with the results of samples in the diluent (top).
Matrix effect, repeatability, LOD/LOQ and speci�city
Conc. Level of Std.
L1
L2
L3
L4
L5
L6
L7
1α,25-diOH VD3 (pg/mL)
3.13
6.25
12.5
25.0
50.0
100.0
200.0
Conc. Ratio1 (Target/IS)
0.0156
0.0313
0.0625
0.1250
0.2500
0.5000
1.0000
Area Ratio2
(in serum)
0.243
0.321
0.456
0.757
1.188
2.168
4.531
Area Ratio2
(in clear solu)
0.414
0.481
0.603
0.914
1.354
2.580
4.740
Accuracy3
(%)
103.8
100.0
117.3
115.9
95.5
92.15
102.0
Matrix Effect (%)
58.7
66.8
75.6
82.9
87.7
84.0
95.6
1, Target = 1α,25-diOH VD3; 2, Area ratio = area of target / area of IS; 3, Based on the data of spiked serum samples
22.5 24.7
0
250
500
7501:399.30>157.00(+)1:399.30>381.30(+)
OH
2VD
3/22
.565
22.5 24.7
0
250
500
7501:399.30>157.00(+)1:399.30>381.30(+)
OH
2VD
3/22
.565
22.5 24.7
0
500
1000
1:399.30>157.00(+)1:399.30>381.30(+)
OH
2VD
3/22
.573
22.5 24.7
0
1000
2000
3000
4000 1:399.30>157.00(+)1:399.30>381.30(+)
OH
2VD
3/22
.598
22.5 24.7
0
250
500
7501:399.30>157.00(+)1:399.30>381.30(+)
OH
2VD
3/22
.595
22.5 24.7
0
250
500
7501:399.30>157.00(+)1:399.30>381.30(+)
22.5 24.7
0
250
500
750
10001:399.30>157.00(+)1:399.30>381.30(+)
OH
2VD
3/22
.619
22.5 24.7
0
1000
2000
3000
4000 1:399.30>157.00(+)1:399.30>381.30(+)
OH
2VD
3/22
.630
22.5 24.7
0
250
500
7501:399.30>157.00(+)1:399.30>381.30(+)
OH
2VD
3/22
.622
22.5 24.7
0
250
500
7501:399.30>157.00(+)1:399.30>381.30(+)
OH
2VD
3/22
.602L1 L3 L5 L7 Diluent
L1 L3 L5 L7 Serum blank
Development of 2D-LC/MS/MS Method for Quantitative Analysis of 1α,25-Dihydroxylvitamin D3 in Human Serum
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
ConclusionsA 2D-LC/MS/MS method with APCI interface has been developed for quantitative analysis of 1α,25-dihydroxylvitamin D3 in human serum without of�ine extraction and cleanup. The detection and quantitation range of the method is from 3.1 pg/mL to 200 pg/mL, which meets the diagnosis requirements in clinical applications. The performance of the method was evaluated thoroughly, including linearity, accuracy,
repeatability, matrix effect, LOD/LOQ and speci�city. The results indicate that the 2D-LC/MS/MS method is sensitive and reliable in detection and quantitation of trace 1α,25-dihydroxylvitamin D3 in serum. Further studies to enable the method for simultaneous analysis of both 1α,25-dihydroxylvitamin D3 and 1α,25-dihydroxylvitamin D2 are needed.
References1. S. Wang. Nutr. Res. Rev. 22, 188 (2009).2. T. Higashi, K. Shimada, T. Toyo’oka. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. (2010) 878, 1654.3. J. M. El‐Khoury, E. Z. Reineks, S. Wang. Clin. Biochem. 2010. DOI: 10.1002/jssc.20200911.4. Chao Yuan, Justin Kosewick, Xiang He, Marta Kozak and Sihe Wang, Rapid Commun. Mass Spectrom. 2011, 25,
1241–12495. Casetta, I. Jans, J. Billen, D. Vanderschueren, R. Bouillon. Eur. J. Mass Spectrom. 2010, 16, 81.
For Research Use Only. Not for use in diagnostic procedures.
Table 4: Repeatability Test Results (n=6)
Sample
L2
L3
Compound
1α,25-diOH VD3
IS
1α,25-diOH VD3
IS
Spiked Conc. (pg/mL)
6.25
200
12.5
200
%RSD
10.10
7.66
9.33
6.28
PO-CON1450E
Analysis of polysorbates in biotherapeuticproducts using two-dimensional HPLC coupled with mass spectrometer
ASMS 2014 WP 182
William Hedgepeth, Kenichiro Tanaka Shimadzu Scienti�c Instruments, Inc., Columbia MD
2
Analysis of polysorbates in biotherapeutic products using two-dimensional HPLC coupled with mass spectrometer
IntroductionPolysorbate 80 is commonly used for biotherapeutic products to prevent aggregation and surface adsorption, as well as to increase the solubility of biotherapeutic compounds. A reliable method to quantitate and characterize polysorbates is required to evaluate the quality and stability of biotherapeutic products. Several methods for polysorbate analysis have been reported, but most of
them require time-consuming sample pretreatment such as derivatization and alkaline hydrolysis because polysorbates do not have suf�cient chromophores. Those methods also require an additional step to remove biotherapeutic compounds. Here we report a simple and reliable method for quantitation and characterization of polysorbate 80 in biotherapeutic products using two-dimensional HPLC.
Fig.1 Typical structure of polysorbate 80
Materials
Reagents: Tween® 80 (Polysorbate 80), IgG from human serum, potassium phosphate monobasic, potassium phosphate dibasic, and ammnonium formate were purchased from Sigma-Aldrich. Water was made in house using a Millipore Milli-Q Advantage A10 Ultrapure Water Purification System. Isopropanol was purchased from Honeywell. Standard solutions: 10 mmol/L phosphate buffer (pH 6.8) was prepared by dissolving 680 mg of potassium
phosphate monobasic and 871 mg of potassium phosphate dibasic in 1 L of water. Polysorbate 80 was diluted with 10 mmol/L phosphate buffer (pH 6.8) to 200, 100, 50, 20, 10 mg/L and transferred to 1.5 mL vials for analysis.Sample solutions: A model sample was prepared by dissolving 2 mg of IgG in 0.1 mL of a 100 mg/L polysorbate 80 standard solution. The sample was centrifuged and transferred to a 1.5 mL vial for analysis.
Reagents and standards
w+x+y+z=approx. 20
OO
OH
OOH
O
OOH
O
O
CH3
yz
x
w
3
Analysis of polysorbates in biotherapeutic products using two-dimensional HPLC coupled with mass spectrometer
Fig.2 Flow diagram of Co-Sense for BA
The standard and sample solutions were injected into a Shimadzu Co-Sense for BA system consisting of two LC-20AD pumps and a LC-20AD pump equipped with a solvent switching valve, DGU-20A5R degassing unit, SIL-20AC autosampler, CTO-20AC column oven equipped with a 6-port 2-position valve, and a CBM-20A system controller. Polysorbate 80 was detected by a LCMS-2020 single quadrupole mass spectrometer or a LCMS-8050 triple quadrupole mass spectrometer because polysorbates do not have any chromophores and are present at low concentrations in antibody drugs. A SPD-20AV UV-VIS
detector was used to check protein removal.Fig. 2 shows the flow diagram of the Co-Sense for BA system. In step 1, a sample pretreatment column “Shim-pack MAYI-ODS” traps polysorbate 80 in the sample. Proteins (antibody) cannot enter the pore interior that is blocked by a hydrophilic polymer bound on the outer surface. Other additives and excipients such as sugars, salts, and amino acids cannot be retained by the ODS phase of the inner surface due to their polarity. In step 2, the trapped polysorbate 80 is introduced to the analytical column by valve switching.
System
Step 1 : Protein removal
Step 2 : Analyzing the trapped polysorbate
Autosampler
Valve(Position 0)
Pump 1
Pump 2
Sample pretreatment column
Analytical column
Mass spectrometer
UV-VIS detector
Mobile phase C
Mobile phase D
Mobile phase A(solution for sample injection)
Mobile phase B(solution for rinse)
Protein,Salts,
Amino acids,Sugars
Polysorbate80
Autosampler
Valve(Position 1)
Pump 1
Pump 2
Sample pretreatment column
Analytical column
Mass spectrometer
UV-VIS detector
Mobile phase C
Mobile phase D
Mobile phase B(solution for rinse)
Mobile phase A(solution for sample injection)
Polysorbate80
4
Analysis of polysorbates in biotherapeutic products using two-dimensional HPLC coupled with mass spectrometer
Results
A fast analysis for quantitation will be shown here. Table 1 shows the analytical conditions and Fig. 3 shows the TIC chromatogram of a 100 mg/L polysorbate 80 standard solution and the mass spectrum of the peak at 4.4 min. Polysorbates contain many by-products, so several peaks appeared on the TIC chromatogram. The peak at 4.4 min was identified as polyoxyethylene sorbitan monooleate (typical structure of polysorbate 80) based on E. Hvattum et al 2011. The ion at 783 was used as a marker for detection in selected ion mode (SIM). This ion is attributable to the 2NH4
+ adduct of polyoxyethylene sorbitan monooleate containing 25 polyoxyethylene groups. Fig. 4 shows the SIM chromatogram of the model sample (20 g/L of IgG, 100 mg/L of polysorbate 80 in 10
mmol/L phosphate buffer pH6.8). Polysorbate 80 in the model sample was successfully analyzed. The peak at 4.4 min was used for quantitation.Six replicate injections for the model sample were made to evaluate the reproducibility. The relative standard deviations of retention time and peak area were 0.034 % and 1.11 %, respectively. The recovery ratio was obtained by comparing the peak area of the model sample and a 100 mg/L polysorbate 80 standard solution and was 99 %. Five different levels of polysorbate 80 standard solutions ranging from 10 to 200 mg/L were used for the linearity evaluation. The correlation coefficient (R2) of determination was higher than 0.999.
Quantitation method
Table 1 Analytical Conditions
System : Co-Sense for BA equipped with LCMS-2020
[Sample Injection]
Column : Shim-pack MAYI-ODS (5 mm L. x 2.0 mm I.D., 50 μm)
Mobile Phase : A: 10 mmol/L ammonium formate in water
B: Isopropanol
Solvent Switching : A (0-1.5 min), B (1.5-3.5 min), A (3.5-9 min)
Flow Rate : 0.6 mL/min
Valve Position : 0 (0-1 min, 7-9 min), 1 (1-7 min)
Injection Volume : 1 µL
[Separation]
Column : Kinetex 5u C18 100A (50 mm L. x 2.1 mm I.D., 5 μm)
Mobile Phase : A: 10 mmol/L ammonium formate in water
B: Isopropanol
Time Program : B. Conc 5 % (0-1 min) - 100 % (6-7 min) -5 % (7.01-9 min)
Flow Rate : 0.3 mL/min
Column Temperature : 40 ºC
[UV Detection]
Detection : 280 nm
Flow Cell : Semi-micro cell
[MS Detection]
Ionization Mode : ESI Positive
Applied Voltage : 4.5 kV
Nebulizer Gas Flow : 1.5 mL/min
DL Temperature : 250 ºC
Block Heater Temp. : 400 ºC
Scan : m/z 300-2000
SIM : m/z 783
5
Analysis of polysorbates in biotherapeutic products using two-dimensional HPLC coupled with mass spectrometer
Fig.4 SIM chromatogram of the model sample
An analysis for characterization will be shown here. Table 2 shows the analytical conditions and Fig. 5 shows the TIC chromatogram of the model sample and mass spectra of the peaks from 10 to 30 min. A longer column and gradient were applied to obtain better resolution. Polysorbate 80 consists of not only monooleate (typical structure of polysorbate 80), but also many by-products such as polyoxyethylene, polyoxyethylene sorbitan, polyoxyethylene isosorbide, dioleate, trioleate, tetraoleate
and others. The peaks on the TIC chromatogram are assumed to correspond to those by-products. For example, the peaks from 10 to 22 min correspond to polyoxyethylene and polyoxyethylene isosorbide and the peaks from 22 to 30 min correspond to polyoxyethylene sorbitan. This method is helpful for characterization as well as checking degradation such as auto-oxidation and hydrolysis.
Characterization method
Fig.3 TIC Chromatogram of 100 mg/L polysorbate 80 standard solution and mass spectrum of the peak at 4.4 min
500 550 600 650 700 750 800 850 900 950 m/z0.0
0.5
1.0
1.5
Inten.(x100,000)
601587 616631
572645
660557
783675 805 827543849761689 739528 871 893704 915717
Doubly charged ions
Triply charged ions
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 min
1000000
2000000
3000000
4000000
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 min0
25000
50000
75000
100000
Analysis of polysorbates in biotherapeutic products using two-dimensional HPLC coupled with mass spectrometer
6
Fig.5 TIC chromatogram of the model sample
Table 2 Analytical Conditions
System : Co-Sense for BA equipped with LCMS-8050
[Sample Injection]
Column : Shim-pack MAYI-ODS (5 mm L. x 2.0 mm I.D., 50 μm)
Mobile Phase : A: 10 mmol/L ammonium formate in water
B: Isopropanol
Solvent Switching : A (0-1.5 min), B (1.5-3.5 min), A (3.5-9 min)
Flow Rate : 0.6 mL/min (0-10 min, 95.01-110 min), 0.1 mL/min (10.01-95 min)
Valve Position : 0 (0-3 min, 100-110 min), 1 (3-100 min)
Injection Volume : 5 µL
[Separation]
Column : Kinetex 5u C18 100A (100 mm L. x 2.1 mm I.D., 5 μm)
Mobile Phase : A: 10 mmol/L ammonium formate in water
B: Isopropanol
Time Program : B. Conc 5 % % (0-3min) – 35% (15min) – 100% (100min) – 5% (100.01-110min)
Flow Rate : 0.2 mL/min
Column Temperature : 40 ºC
[UV Detection]
Detection : 280 nm
Flow Cell : Semi-micro cell
[MS Detection]
Ionization Mode : ESI Positive
Applied Voltage : 4.5 kV
Nebulizer Gas Flow : 2 mL/min
Drying Gas Flow : 10 mL/min
Heating Gas Flow : 10 mL/min
Interface Temperature : 300 ºC
DL Temperature : 250 ºC
Block Heater Temp. : 400 ºC
Q1 Scan : m/z 300-2000
0 10 20 30 40 50 60 70 80 90 100 min
0.0
1.0
2.0
3.0
4.0
(x100,000,000)1:TIC(+)
10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 min
0.0
2.5
5.0
7.5
(x10,000,000)1:TIC(+)
Polyoxyethylene sorbitan
Polyoxyethylene isosorbide
Polyoxyethylene
400 500 600 700 800 m/z0.0
1.0
2.0
3.0
4.0
5.0
6.0Inten.(x100,000)
513.6528.3498.9 543.0
484.2557.6
469.5651.0673.0628.9 695.0572.3
717.1454.8 606.9 739.0587.0761.1
440.2 783.1805.1425.4 827.1
300 400 500 600 700 800 900 m/z0.0
1.0
2.0
3.0
Inten.(x100,000)
692.8648.8
736.8604.7
560.7 780.9421.7443.8399.7 465.8 564.7 608.8 652.8520.7377.6 824.9516.6
696.9445.4 740.9355.6 423.5401.6 869.0379.5
784.9913.0
O
O OOH
OOH
y
z
OHO
H
x
OO
OH
OOH
O
OOH
OH
yz
x
w
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Analysis of polysorbates in biotherapeutic products using two-dimensional HPLC coupled with mass spectrometer
Fig.6 Chromatogram of elution from the sample pretreatment column
Fig. 6 shows the chromatogram of elution from the sample pretreatment column. Protein (IgG) was successfully removed from the sample by using the MAYI-ODS column.
Con�rmation of protein removal
E. Hvattum, W.L. Yip, D. Grace, K. Dyrstad, Characterization of polysorbate 80 with liquid chromatography mass spectrometry and nuclear magnetic resonance spectroscopy: Specific determination of oxidation products of thermally oxidized polysorbate 80, J Pharm Biomed Anal 62, (2012) 7-16
Reference
Conclusions1. Co-Sense for BA system automatically removed protein from the sample and enabled quantitation and characterization
of polysorbate 80 in a protein formulation.2. The quantitation method was successfully applied to the model sample with excellent reproducibility and recovery.3. The high-resolution chromatogram was obtained by the characterization method. This method is helpful for
characterization as well as checking degradation such as auto-oxidation and hydrolysis.
5uL injection of model sample
1uL injection of model sample
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 min
0
250000
500000
750000
1000000
1250000
uV
PO-CON1457E
A Rapid and Reproducible Immuno-MSPlatform from Sample Collection to Quantitation of IgG
ASMS 2014 WP161
Rachel Lieberman1, David Colquhoun1, Jeremy Post1,
Brian Feild1, Scott Kuzdzal1, Fred Regnier2, 1Shimadzu Scienti�c Instruments, Columbia, MD, USA 2Novilytic L.L.C, North Webster, IN, USA
2
A Rapid and Reproducible Immuno-MS Platform from Sample Collection to Quantitation of IgG
Sample Work�ow
Using rapid, automated processing, coupled to the speed and sensitivity of the LCMS-8050 allows for improved analysis of Immunoglobulin G.
Introduction
Novel Aspect
Dried blood spot analysis (DBS) has provided clinical laboratories a simple method to collect, store and transport samples for a wide variety of analyses. However, sample stability, hematocrit effects and inconsistent spotting techniques have limited the ability for wide spread adoption in clinical applications. Dried plasma spots (DPS) offer new opportunities by providing stable samples that
avoid variability caused by the hematocrit. This presentation focuses on an ultra-fast-immuno-MS platform that combines next generation plasma separator cards (Novilytic L.L.C., North Webster, IN) with fully automated immuno-af�nity enrichment and rapid digestion as an upfront sample preparation strategy for mass spectrometric analysis of immunoglobulins.
LC/MS/MSAffinitySelection
EnzymeDigestion Desalting
Automates and integrates key proteomic workflow steps: - Affinity Selection (15 min) - Trypsin digestion (1-8 min) - Online Desalting - Reversed phase LCExceptional reproducibility (CV less than 10%)
Rapid plasma extraction technology from whole blood (~ 18 minutes) - 2.5 uL of plasma collected (3 min) - Air dry for 15 minutes - Extract plamsa disc for analysis
- Ultrafast MRM methods - Up to 555 MRM transitions per run - Heated electrospray source - Scan speeds up to 30,000 u/sec - Polarity switching 5 msec
Perfinity WorkstationNoviplexTM Card LCMS-8050 Triple Quadrupole MS
BufferExchange
PlasmaGeneration
3
A Rapid and Reproducible Immuno-MS Platform from Sample Collection to Quantitation of IgG
MethodsIgG was weighed out and then diluted in 500 μL of 0.5% BSA solution. Approximately15 uL of IgG standard was spiked into mouse whole blood and processed using the Noviplex card. The resulting plasma collection disc was extracted with 30 uL of buffer and each sample was
reduced and alkylated to yield a total sample volume of 100 uL. IgG standards and QC samples were directly injected onto the Per�nity-LCMS-8050 platform for af�nity pulldown with a Protein G column followed by trypsin digestion and LC/MS/MS analysis.
Noviplex Cards
Approximately 50 uL of the spiked whole blood was pipetted onto the Noviplex card test area (1). The spot was allowed to dry for 3 minutes (2). The top layer of the card was then peeled back (3) to reveal the plamsa collection
disc. The plasma collection disc was allowed to dry for an additional 15 minutes. Once the disc was dry (4), it was placed into an eppendorf tube for solvent extraction.
IgG concentrations for calibration levels. LCMS gradient conditions.
0
20
40
60
80
100
0 2 4 6 8 10 12 14 16
%B
Time (minutes)
MRM transitions on LCMS-8050 for two IgG peptides monitored.
Compound Name
TTPPVLDSDGSFFLYSK
VVSVLTVLHQDWLNGK
Transitions
937.70>836.25
937.70>723.95
603.70>805.7
+/-
+
+
+
Q1 Rod Bias(V)
-27
-27
-22
CE (V)
-28
-30
-16
Q3 Rod Bias(V)
-26
-22
-13
Level
1
2
3
4
5
6
7
Conc.(μg/mL)
465
315
142.5
127.5
102
60
22.5
Amount oncolumn (μg)
34.88
23.63
10.69
9.56
7.65
4.50
1.69
Time (min)
0
0.2
8
9.5
10
12.5
12.51
16
%B
2
2
50
50
90
90
2
2
Amount oncolumn (pmol)
581.25
393.75
178.13
159.37
127.50
75.00
28.12
(1)
(2) (3) (4)
4
A Rapid and Reproducible Immuno-MS Platform from Sample Collection to Quantitation of IgG
Results - Chromatograms
Total Ion Chromatogram for IgG
Optimization of Collision Energies for peptides of interest
MRM Chromatogram for Level 4 standard of spiked IgG in whole blood.
VVSVLTVLHQDWLNGKTTPPVLDSDGSFFLYSK
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 min
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 min
0
25000000
50000000
75000000
100000000
125000000
150000000
175000000
200000000
225000000
250000000
275000000
300000000
6.200 6.225 6.250 6.275 6.300 6.325 6.350 6.375 6.400 6.425 6.450 6.475 6.500 6.525 6.550 6.575 6.600 6.625 6.650 6.675 min
0
250000
500000
750000
1000000
1250000
300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00Inten.
938
836915510
397
938
937
836
836
724283
891379
397
836 1046640591283
809443
352295 524
723407 851
407337 724466 756658
837
1163561397369
449
Range CE: -50 to -10 VTTPPVLDSDGFFLYSK
[M+2H]+2
[P1+2H]+2
[P2+2H]+2
Carryover Assessment
Blank InjectionControl - Mouse blood
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 min
0
100
200
300
400
500
600
700
800
900
1000
1100
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 min
0
10
20
30
40
50
60
70
80
90
5
A Rapid and Reproducible Immuno-MS Platform from Sample Collection to Quantitation of IgG
Results - Calibration Curves
VVSVLTVLHQDWLNGK
Sample
QC 1
QC 2
QC 3
QC 4
Ret. Time
6.49
6.516
6.514
6.492
Area
32,492
11,726
8,507
2,727
Calc. Conc.
502.804
167.189
115.155
21.745
Std. Conc.
465
142.5
102
22.5
% Accuracy
108.1
117.3
112.9
96.6
TTPPVLDSDGSFFLYSK
Sample
QC 1
QC 2
QC 3
QC 4
Ret. Time
6.029
6.052
6.047
6.029
Area
61,525
25,355
16,900
6,502
Calc. Conc.
416.447
155.568
94.58
19.587
Std. Conc.
465
142.5
102
22.5
% Accuracy
89.6
109.2
92.7
87.1
0 100 200 300 400 Conc.0
25000
50000
Area
r2 = 0.979
TTPPVLDSDGSFFLYSK VVSVLTVLHQDWLNGK
r2 = 0.989
0 100 200 300 400 Conc.0
5000
10000
15000
20000
25000
30000
Area
Level 7
5.50 5.75 6.00 6.25 6.50
0
500
1000
1500
2000 937.70>723.95(+)937.70>836.25(+)Level 1
5.50 5.75 6.00 6.25 6.50
0
5000
10000
15000
20000
25000937.70>723.95(+)937.70>836.25(+) Level 7
6.00 6.25 6.50 6.75
0
100
200
300
400
500
600603.70>805.70(+)Level 1
6.00 6.25 6.50 6.75
0
2500
5000
7500
10000603.70>805.70(+)
Calibration Curve and MS Chromatograms
Results - Tables and Replicates
QC data and Calculations for IgG Peptides
A Rapid and Reproducible Immuno-MS Platform from Sample Collection to Quantitation of IgG
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
VVSVLTVLHQDWLNGK TTPPVLDSDGSFFLYSK
Skyline Data - Retention Time Replicates
839
AM
_226
2014
...L1
...00
5
839
AM
_226
2014
...L2
...00
4
839
AM
_226
2014
...L3
...00
3
839
AM
_226
2014
...L4
...00
2
1433
PM
_225
2014
...L5
...00
8
1433
PM
_225
2014
...L6
...00
6
1433
PM
_225
2014
...L7
...00
4
Replicate
5.90
5.95
6.00
6.05
6.10
6.15
6.20
Ret
enti
on
Tim
e
y15 - 836.4169++
839
AM
_226
2014
...L1
...00
5
839
AM
_226
2014
...L2
...00
4
839
AM
_226
2014
...L3
...00
3
839
AM
_226
2014
...L4
...00
2
1433
PM
_225
2014
...L5
...00
8
1433
PM
_225
2014
...L6
...00
6
1433
PM
_225
2014
...L7
...00
46.35
6.40
6.45
6.50
6.55
6.60
6.65y14 - 805.4385++
839
AM
_226
2014
...L1
...00
5
839
AM
_226
2014
...L2
...00
4
839
AM
_226
2014
...L3
...00
3
839
AM
_226
2014
...L4
...00
2
1433
PM
_225
2014
...L5
...00
8
1433
PM
_225
2014
...L6
...00
6
1433
PM
_225
2014
...L7
...00
4
Replicate
6.35
6.40
6.45
6.50
6.55
6.60
6.65
Ret
enti
on
Tim
e
y14 - 805.4385++
Integration of Skyline Software into LabSolutions allows for further interrogation of data. Here are representative �gures showing the retention time reproducibility for each peptide monitored during the analysis.
ConclusionsCombining the sample collection technique of next generation plasma separator Noviplex cards for quick plamsa collection from whole blood, with the automated af�nity selection and trypsin digestion of the Per�nity workstation coupled to LCMS-8050, provides a very rapid and reproducible Immuno-MS platform for quantitation of IgG peptides. Furthermore, this rapid immuno-MS platform can be applied to many other peptide/protein applications.
PO-CON1473E
Simultaneous Determinations of 20 kindsof common drugs and pesticides in human blood by GPC-GC-MS/MS
ASMS 2014 TP 757
Qian Sun, Jun Fan, Taohong Huang,
Shin-ichi Kawano, Yuki Hashi,
Shimadzu Global COE, Shanghai, China
2
Simultaneous Determinations of 20 kinds of common drugs and pesticides in human blood by GPC-GC-MS/MS
IntroductionOn-line gel permeation chromatography-gas chromatography/mass spectrometry (GPC-GC-MS) is a unique technique to cleanup sample that reduce the time of sample preparation. GPC can ef�ciently separates fats, protein and pigments from samples, due to this advantage, on-line GPC is widely used for pesticide analysis. Meanwhile, compared to widely used GC-MS, GC-MS/MS
techniques provide much better selectivity thus signi�cantly lower detection limits. In this work, a new method was developed for rapid determination of 20 common drugs and pesticides in human blood by GPC-GC-MS/MS. The modi�ed QuEChERS method was used for sample preparation.
ExperimentalThe human blood samples were extracted with acetonitrile, then was puri�ed by PSA, C18 and MgSO4 to remove most of the fats, protein and pigments in samples, then after on-line GPC-GC-MS/MS analysis which further removed
macromolecular interference material, such as protein and cholesterol, the background interference brought about by the complex matrix in samples was effectively reduced.
Figure 1 Schematic �ow diagram of the sample preparation
Sample pretreament
PSA/C18/MgSO4
vortex
centrifuge
CH3CN
vortex
human blood 2 mL
evaporate
GPC-GC-MS/MS
supernatant
set volume using moblie phase
3
Simultaneous Determinations of 20 kinds of common drugs and pesticides in human blood by GPC-GC-MS/MS
ResultsFor all of analytes, recoveries in the acceptable range of 70~120% and repeatability (relative standard deviations, RSD)≤5% (n=3) were achieved for matrices at spiking levels of 0.01 µg/mL. The limitis of detection were 0.03~4.4 µg/L.
The method is simple, rapid and characterized with acceptable sensitivity and accuracy to meet the requirements for the analysis of common drugs and pesticides in the human blood.
Figure 2 MRM chromatograms of standard mixture
Instrument
GPC
Mobile phase : acetone/cyclohexane (3/7, v/v)
Flow rate : 0.1mL/min
Column : Shodex CLNpak EV-200 (2 mmI.D. x 150 mmL.)
Oven temperature : 40 ºC
Injection volume : 10 μL
GCMS-TQ8030
Column : deactivated silica tubing [0.53 mm(ID) x 5 m(L)]
+pre-column Rtx-5ms [0.25 mm(ID) x 5 m(L)]
Rtx-5ms [0.25mm(ID) x 30 m(L), Thickness: 0.25 μm]
Injector : PTV
Injector time program : 120 ºC(4.5min)-(80 ºC/min)-280 ºC(33.7 min)
Oven temperature program : 82 ºC(5min)-(8 ºC/min)-300 ºC(7.75 min)
Linear velocity : 48.8 cm/sec
Ion Source temperature : 210 ºC
Interface temperature : 300 ºC
15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5
0.00
0.25
0.50
0.75
1.00
(x10,000,000)
Simultaneous Determinations of 20 kinds of common drugs and pesticides in human blood by GPC-GC-MS/MS
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Table 1 Results of method validation for drugs and pesticides(Concentration range: 5-100 μg/L, LODs: S/N≥3, LOQs: S/N≥10, RSDs: n=3)
ConclusionA very quick, easy, effective, reliable method in human blood based on modi�ed QuEChERS method was developed using GPC-GCMS-TQ8030. The performance of the method was very satisfactory with results meeting
validation criteria. The method has been successfully applied for determination of human blood samples and ostensibly has further application opportunities, e.g. biological samples.
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Compound Name
Dichlorvos
Methamidophos
Barbital
Sulfotep
Dimethoate
Malathion
Chlorpyrifos
Phenobarbital
Parathion
Triazophos
Zopiclone deg.
Diazepam
Midazolam
Zolpidem
Clonazepam
Estazolam
Clozapine
Alprazolam
Zolpidem
Triazolam
10.795
11.800
15.210
17.580
18.310
21.555
21.715
22.000
22.180
25.675
26.025
27.635
29.250
31.225
31.795
32.335
32.400
32.730
33.095
33.700
tR
(min)
0.9993
0.9994
0.9994
0.9995
0.9993
0.9997
0.9996
0.9995
0.9993
0.9994
0.9993
0.9992
0.9994
0.9993
0.9995
0.9994
0.9991
0.9993
0.9995
0.9992
CorrelationCoef�cient*
0.103
0.023
0.018
0.011
0.400
0.005
0.010
0.353
0.003
0.046
0.189
0.007
0.048
1.298
0.432
0.092
0.050
0.028
1.027
0.027
LOD(µg/L)
0.345
0.076
0.058
0.037
1.333
0.016
0.033
1.177
0.009
0.155
0.631
0.022
0.160
4.325
1.440
0.305
0.167
0.095
3.425
0.091
LOQ(µg/L) Recovery (%)
72.9
85.3
72.4
110.7
103.7
82.7
85.7
79.6
92.3
87.7
83.5
98.3
87.1
99.3
110.0
103.7
100.6
103.3
87.3
81.3
RSD (%)
2.99
3.58
1.72
2.27
3.10
2.52
3.57
3.25
3.17
1.32
1.28
1.55
2.01
1.01
1.57
1.37
3.12
1.48
1.75
2.56
0.01 µg/mL
PO-CON1466E
Low level quantitation of Loratadinefrom plasma using LC/MS/MS
ASMS 2014 TP498
Shailesh Damale, Deepti Bhandarkar, Shruti Raju,
Rashi Kochhar, Shailendra Rane, Ajit Datar,
Pratap Rasam, Jitendra Kelkar
Shimadzu Analytical (India) Pvt. Ltd., 1 A/B Rushabh
Chambers, Makwana Road, Marol, Andheri (E),
Mumbai-400059, Maharashtra, India.
2
Low level quantitation of Loratadine from plasma using LC/MS/MS
IntroductionLoratadine is a histamine antagonist drug used for the treatment of itching, runny nose, hay fever and such other allergies. Here, an LC/MS/MS method has been developed for high sensitive quantitation of this molecule from plasma using LCMS-8050, a triple quadrupole mass spectrometer from Shimadzu Corporation, Japan. Presence
of heated Electro Spray Ionization (ESI) interface in LCMS-8050 ensured good quantitation and repeatability even in the presence of a complex matrix like plasma. Ultra high sensitivity of LCMS-8050 enabled development of a low ppt level quantitation method for Loratadine.
Method of AnalysisThis bioanalytical method was developed for measuring Loratadine in therapeutic concentration range for the analysis of routine samples. It was important to develop a
simple and accurate method for estimation of Loratadine in human plasma.
To 100 µL of plasma 500 µL cold acetonitrile was added for protein precipitation. It was placed in rotary shaker at 20 rpm for 15 minutes for uniform mixing. This solution
was centrifuged at 12000 rpm for 15 minutes. Supernatant was taken and evaporated to dryness at 70 ºC . The residue was reconstituted in 200 µL Methanol.
Preparation of matrix matched plasma by protein precipitation method using cold acetonitrile
1 ppt, 5 ppt, 50 ppt, 100ppt, 500 ppt, 1 ppb, 5 ppb and 10 ppb of Loratadine calibration standards were prepared
in cold acetonitrile treated matrix matched plasma.
Preparation of calibration standards in matrix matched plasma
Figure 1. Structure of Loratadine
LoratadineLoratadine, a piperidine derivative, is a potent long-acting, non-sedating tricyclic antihistamine with selective peripheral H1-receptor antagonist activity. It is used for relief of nasal and non-nasal symptoms of seasonal allergies and skin rashes[1,2,3]. Due to partial distribution in central nervous system, it has less sedating power compared to traditional H1 blockers. Loratadine is given orally, is well absorbed from the gastrointestinal tract, and has rapid �rst-pass hepatic metabolism; it is metabolized by isoenzymes of the cytochrome P450 system, including CYP3A4, CYP2D6, and, to a lesser extent, several others. Loratadine is almost totally (97–99 %) bound to plasma proteins and reaches peak plasma concentration (Tmax) in ~ 1–2 h[4,5].
Ethyl 4- (8-chloro-5, 6-dihydro-11H-benzo [5, 6] cyclohepta [1, 2-b] pyridin-11-ylidene) -1-piperidinecarboxylate
3
Low level quantitation of Loratadine from plasma using LC/MS/MS
LC/MS/MS analysisLCMS-8050 triple quadrupole mass spectrometer by Shimadzu Corporation, Japan (shown in Figure 2A), sets a new benchmark in triple quadrupole technology with an unsurpassed sensitivity (UFsensitivity) with Scanning speed of 30,000 u/sec (UFscanning) and polarity switching speed of 5 msecs (UFswitching). This system ensures highest quality of data, with very high degree of reliability.In order to improve ionization ef�ciency, the newly developed heated ESI probe combines high-temperature gas with the nebulizer spray, assisting in the desolvation of large droplets and enhancing ionization. This development allows high-sensitivity analysis of a wide
range of target compounds with considerable reduction in background.Presence of heated Electro spray interface in LCMS-8050 (shown in Figure 2B) ensured good quantitative sensitivity even in presence of a complex matrix like plasma.The parent m/z of 382.90 giving the daughter m/z of 337.10 in the positive mode was the MRM transition used for quantitation of Loratadine. MS voltages and collision energy were optimized to achieve maximum transmission of mentioned precursor and product ion. Gas �ow rates, source temperature conditions and collision gas were optimized, and linearity graph was plotted for 4 orders of magnitude.
Figure 2A. LCMS-8050 triple quadrupole mass spectrometer by Shimadzu Figure 2B. Heated ESI probe
Table 2. LCMS conditions
ESI
Positive
2.0 L / min (nitrogen)
10.0 L / min (nitrogen)
15.0 L / min (zero air)
300 ºC
250 ºC
400 ºC
382.90 > 337.10
MS Interface
Polarity
Nebulizing Gas Flow
Drying Gas Flow
Heating Gas Flow
Interface Temp.
Desolvation Line Temp.
Heater Block Temp.
MRM Transition
B conc. (%)Time (min)
60
100
100
60
0.01
1.50
4.00
4.10
13.00
A conc. (%)
40
0
0
40
Stop
Table 1. LC conditions
Shim-pack XR-ODS (100 mm L x 2.0 mm ID ; 2.2 µm)
A : 0.1% formic acid in water
B : acetonitrile
0.15 mL/min
40 ºC
20 µL
Column
Mobile Phase
Gradient Program
Flow Rate
Oven Temperature
Injection Volume
Low level quantitation of Loratadine from plasma using LC/MS/MS
4
Figure 4A. Mass chromatogram 10 ppb Figure 4B. Mass chromatogram 0.001 ppb
Figure 5. Overlay chromatogram
Results
LC/MS/MS method for Loratadine was developed on ESI +ve ionization mode and 382.90>337.10 MRM transition was optimized for Loratadine. Checked matrix matched plasma standards for highest (10 ppb) as well as lowest (0.001 ppb) concentrations as seen in Figures 4A and 4B respectively. Optimized MS method to ensure no plasma interference at the retention time of Loratadine (Figure 5).
Calibration curve was plotted for Loratadine concentration range. Also as seen in Table 3, % Accuracy was studied to confirm the reliability of method. Linear calibration curves were obtained with regression coefficients R2 > 0.998. % RSD of area was within 15 % and accuracy was within 80-120 % for all calibration levels.
LC/MS/MS Analysis
Speci�city and interference
0.0 2.5 5.0 7.5
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5(x1,000,000)382.90>337.10(+)
LORA
TAD
INE/
3.39
1
0.0 2.5 5.0 7.5-1.0
0.0
1.0
2.0
3.0
4.0
5.0
(x10,000)382.90>337.10(+)
LORA
TAD
INE/
3.37
7
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 min
-0.3
-0.2
-0.1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2(x10,000)
1:LORATIDINE 382.90>337.10(+) CE: -23.0 LORA_PLASMA_002.lcd1:LORATIDINE 382.90>337.10(+) CE: -23.0 LORA_PLASMA_003.lcd
------ LOQ Level
------ Blank
5
Low level quantitation of Loratadine from plasma using LC/MS/MS
Figure 6. Loratadine calibration curve
Conclusion• Highly sensitive LC/MS/MS method for Loaratadine was developed on LCMS-8050 system.• Calibration was plotted from 10 ppb to 0.001 ppb, and LOQ was computed as 0.001 ppb.
Table 3. Results of Loratadine calibration curve
Nominal Concentration (ppb)
Measured Concentration (ppb)
% Accuracy(n=3)
% RSD for area counts (n=3)
0.001
0.005
0.05
0.1
0.5
1.0
5.0
10.0
Standard
STD-01
STD-02
STD-03
STD-04
STD-05
STD-06
STD-07
STD-08
Sr. No.
1
2
3
4
5
6
7
8
0.00096
0.0050
0.057
0.095
0.048
0.986
5.077
9.983
0.62
5.24
0.98
1.81
1.40
0.11
1.07
1.96
95.83
100.73
114.83
95.40
95.70
98.53
101.53
99.37
Result Table
0.0 2.5 5.0 7.5 Conc.0.0
1.0
2.0Area (x10,000,000)
1 2 3 4 5
6
7
8
0.05 0.10 Conc.0.0
1.0
2.0
Area (x100,000)
1 2
3
4
Low level quantitation of Loratadine from plasma using LC/MS/MS
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
References[1] Bhavin N. Patel, Naveen Sharma, Mallika Sanyal, and Pranav S. Shrivastav, Journal of chromatographic Sciences,
Volume 48, (2010), 35-44.[2] J. Chen, YZ. Zha, KP. Gao, ZQ. Shi, XG. Jiang, WM. Jiang, XL. Gao, Pharmazie, Volume 59, (2004), 600-603.[3] M. Haria, A. Fitton, and D.H. Peters, Drugs, Volume 48, (1994), 617-637.[4] J. Hibert, E. Radwanski, R. Weglein, V. Luc, G. Perentesis, S. Symchowicz, and N. Zampaglione, J.clin. Pharmacol,
Volume 27, (1987), 694-698.[5] S.P.Clissold, E.M. Sorkin, and K.L. Goa, Drugs, Volume 37,(1989), 42-57.
Food
• Page 111An LCMS method for the detection of cocoa butter substitutes, replacers, and equivalents in commercial chocolate-like products
• Page 116Highly sensitive and robust LC/MS/MS method for quantitative analysis of articial sweeteners in beverages
• Page 122Highly sensitive and rapid simultaneous method for 45 mycotoxins in baby food samples by HPLC-MS/MS using fast polarity switching
• Page 129High sensitivity analysis of acrylamide in potato chips by LC/MS/MS with modified QuEChERS sample pre-treatment procedure
• Page 135Determination of benzimidazole residues in animal tissue by ultra high performance liquid chromatography tandem mass spectrometry
• Page 141High sensitivity quantitation method of dicyandi-amide and melamine in milk powders by liquid chromatography tandem mass spectrometry
• Page 147Multiresidue pesticide analysis from dried chili powder using LC/MS/MS
• Page 154Multi pesticide residue analysis in tobacco by GCMS/MS using QuEChERS as an extraction method
• Page 161Simultaneous quantitative analysis of 20 amino acids in food samples without derivatization using LC-MS/MS
PO-CON1458E
An LCMS Method for the Detectionof Cocoa Butter Substitutes,Replacers, and Equivalents inCommercial Chocolate-like Products
ASMS 2014 ThP632
Jared Russell, Liling Fang and Willard Bankert
Shimadzu Scienti�c Instruments., Columbia, MD
2
An LCMS Method for the Detection of Cocoa Butter Substitutes,Replacers, and Equivalents in Commercial Chocolate-like Products
IntroductionThere is increasing demand for genuine cocoa butter (CB) in chocolate products in developed nations, however, this demand has created a shortage of CB and raised its costs. To overcome this, chocolate manufactures sometimes add vegetable-derived fats to some chocolate products to reduce costs while still maintaining desirable physical characteristics. It is of current interest to have a reliable method to detect, identify, and quantify the triacylglycerol (TAG) components of cocoa butter substitutes, replacers, and equivalents (CBEs) in chocolate products. Traditionally GC was used for this task, but due to the low volatility of triacylglycerides and their susceptibility to thermal decomposition, retention time is the only identifying factor
for the TAGs and typical GC analyses of this type can take 40 minutes. LCMS is able to not only provide faster throughput, but also has the additional advantage of allowing characterization of the TAG, including qualitative regiospeci�c analysis. We have developed a single, UHPLC column-based LCMS method to analyze the TAG components in commercial chocolate and chocolate-like products. This analysis has a runtime of 17minutes, making it suitable for relatively high throughput. Additionally, the method was very repeatable, with an interday variability of <7% for the absolute area counts of the three major TAGs in CB (POP,POS,SOS).
Materials and MethodA Shimadzu Nexera UHPLC coupled to a Shimadzu LCMS-8040 triple quadrupole mass spectrometer was utilized for this analysis. A pure CB standard was used as a
reference. Chocolate and chocolatey products were purchased in retail stores over a range of cocoa content.
For analysis, we slightly modified a sample preparation method originally used for algal oils. For analysis, 5mg of sample was weighed and then dissolved in a 3:1 Toluene-Isopropyl Alcohol solution. We then sonicated the
mixture for 5 minutes. The solution was filtered through a Thomson filter vial (P/N 35538-100) to remove sugars and other insoluble materials and diluted 5-fold using 3:1 Toluene-IPA and injected into the UHPLC-MS system.
Sample Preparation
Chromatography
Instrument : Shimadzu Nexera UHPLC system
Column : Shimadzu Shim-Pack XR-ODSIII (200x2.1mm,)
Mobile Phase A : LC/MS Acetonitrile
Mobile Phase B : 1:1 Dichloromethane-Isopropyl Alcohol
Gradient Program : 48% B (initially) – gradient to 51% B (0-8.0 min) – gradient to 54% B
(8.0 – 11.0 min) – gradient to 74% B (11.0-14.0 min) – hold at 74% B
(14.0-15.0 min) – reequilibrate at 48% B (15.1-17 min)
Flow Rate : 0.33 mL/min
Column Temperature : 30°C
Injection Volume : 1 μL
Mass Spectrometry
Instrument : Shimadzu LCMS-8040 Triple Quadrupole Mass Spectrometer
Ionization : APCI
Polarity : Positive
Scan Mode : Q3 Scan
3
An LCMS Method for the Detection of Cocoa Butter Substitutes,Replacers, and Equivalents in Commercial Chocolate-like Products
ResultsRetail Chocolates from Hershey’s, Lindt and Tcho, as well as a chocolatey candy - Charleston Chew - were compared against pure cocoa butter. The chocolates used were selected to cover a range of Cocoa content and purity. We speci�cally chose to use Hershey’s Mr. Goodbar and Charleston Chews because they listed the use of vegetable oils in their ingredients list. As you can see in the chromatograms, the products that market themselves as pure chocolate have similar chromatograms in comparison to the pure CB.We used an MS library that was provided to us by Dr. John Carney and Mona Koutchekinia to identify the types of TAGs contained in the chocolates using the spectral information captured in the Q3 scans. A minimum similarity of 70 was required for a result to be considered a
match. In order to identify usage of CBEs, we applied the equation: %POP<44.025-0.733*%SOS, which was determined by the European Commission Joint Research Centre, which can detect around 2% CBE usage in CB content, or approximately 0.4% CBE content in chocolate.The chocolate products we tested all agreed with the expected results: All of the dark chocolate products we tested passed this speci�cation, as well as Hershey’s Milk Chocolate. The two products which had a higher %POP than is allowable, Mr. Goodbar and Charleston Chew, were selected speci�cally for the inclusion of vegetable oils. It may be informative to further test the accuracy of this testing method by adulterating cocoa butter with known quantities of CBEs. The data has been summarized in Table 1.
Table 1: Percentage of the major TAGs in CB in various chocolate products
Product
Cocoa Butter
Lindt 85% Cocoa
TCHO 70% from Ghana
TCHO 65% from Ecuador
Hershey's Special Dark
Hershey's Milk Chocolate
Hershey's Mr Goodbar
Charleston Chew
%POP
23.7%
16.9%
17.8%
20.9%
20.0%
18.6%
44.8%
100.0%
%POS
46.9%
46.4%
46.1%
46.2%
47.1%
46.6%
21.1%
0.0%
%SOS
29.5%
36.6%
36.1%
32.9%
32.9%
34.8%
34.1%
0.0%
%POP needs tobe less than
43.8
43.8
43.8
43.8
43.8
43.8
43.8
44.0
4
An LCMS Method for the Detection of Cocoa Butter Substitutes,Replacers, and Equivalents in Commercial Chocolate-like Products
Figure 1. Chromatograms of the various chocolate products analyzed versus pure cocoa butter
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 min
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2
1.3
1.4
1.5
(x100,000,000)
1:TIC(+) Hershey's Milk Chocolate.lcd1:TIC(+) Hershey's Special Dark 45% Cacao.lcd1:TIC(+) TCHO 65% from Ecuador.lcd1:TIC(+) TCHO 70% from Ghana.lcd1:TIC(+) Lindt 85% Cocoa.lcd1:TIC(+) Cocoa Butter.lcd
PLP
OO
P
POP
OO
S
POS
SOS*
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 min
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0(x10,000,000)
1:TIC(+) Charleston Chew.lcd1:TIC(+) Hershey's Mr. Goodbar.lcd1:TIC(+) Cocoa Butter.lcd
PLP
OO
P
POP
OO
S
POS
SOS*
An LCMS Method for the Detection of Cocoa Butter Substitutes,Replacers, and Equivalents in Commercial Chocolate-like Products
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
ConclusionsWe have developed a 17 minute method for the rapid determination of CBE usage in chocolate products by using a UHPLC column and Q3 ion scans to analyze samples and then matching spectral information with an MS library of ion ratios for identifying TAGs.Further studies could add a calibration curve to enable quanti�cation of TAGs. This method should also provide a base method which can be modi�ed to support TAG analysis in other product types.
ReferencesCo ED, Koutchekinia M, Carney J et al. Matching the Functionality of Single-Cell Algal Oils with Different Molecular Compositions. 2014. Buchgraber M and Anklam E. Validation of a Method for the Detection of Cocoa Butter Equivalents in Cocoa Butter and Plain Chocolate. 2003.
AcknowledgementsDr. John Carney and Mona Koutchekinia for the invaluable information they provided.
PO-CON1471E
Highly Sensitive and Robust LC/MS/MSMethod for Quantitative Analysis of Arti�cial Sweeteners in Beverages
ASMS 2014 MP351
Jie Xing1, Wantung Liw1, Zhi Wei Edwin Ting1,
Yin Ling Chew*2 & Zhaoqi Zhan1
1 Customer Support Centre, Shimadzu (Asia Paci�c)
Pte Ltd, 79 Science Park Drive, #02-01/08, SINTECH IV,
Singapore Science Park 1, Singapore 1182642 Department of Chemistry, Faculty of Science,
National University of Singapore, 21 Lower Kent
Ridge Road, Singapore 119077, *Student
2
Highly Sensitive and Robust LC/MS/MS Method for Quantitative Analysis of Arti�cial Sweeteners in Beverages
IntroductionArti�cial sweeteners described as intense, low-calorie and non-nutritive are widely used as sugar substitutes in beverages and foods to satisfy consumers’ desire to sweet taste while concerning about obesity and diabetes. As synthetic additives in food, the use of arti�cial sweeteners must be approved by authority for health and safety concerns. For example, Aspartame, Acesulfame-K, Saccharin, Sucralose and Neotame are the FDA approved arti�cial sweeteners on the US market. However, there are also many other arti�cial sweeteners allowed to use in EU and many other countries (Table 2), but not in the US. In this regard, analysis of arti�cial sweeteners in beverages and foods has become essential due to the relevant regulations in protection of consumers’ bene�ts and safety concerns in many countries [1, 2]. Recently, arti�cial
sweeteners are found as emerging environmental contaminants in surface water and waste water [3]. Initially, HPLC analysis method with ELSD detection was adopted, because many arti�cial sweeteners are non-UV absorption compounds [2]. Recently, LC/MS/MS methods have been developed and used for identi�cation and quantitation of arti�cial sweeteners in food and beverages as well as water for its high sensitivity and selectivity [3, 4]. Here we report a high sensitivity LC/MS/MS method for identi�cation and quantitation of ten arti�cial sweeteners (Table 2) in beverage samples. An ultra-small injection volume was adopted in this study to develop a very robust LC/MS/MS method suitable for direct injection of beverage samples without any sample pre-treatment except dilution with solvent.
ExperimentalTen arti�cial sweeteners of high purity as listed in Table 2 were obtained from chemicals suppliers. Stock standard solutions and a set of calibrants were prepared from the chemicals with methanol/water (50/50) solvent as the diluent. Three brand soft-drinks and a mouthwash bought from local supermarket were used as testing samples in this study. The samples were not pretreated by any means
except dilution with the diluent prior to injection into LCMS-8040 (Shimadzu Corporation, Japan), a triple quadrupole LC/MS/MS system. The front-end LC system connected to the LCMS-8040 is a high pressure binary gradient Nexera UHPLC. The details of analytical conditions of LC/MS/MS method are shown in Table 1.
Table 1: LC/MC/MS analytical conditions of arti�cial sweeteners on LCMS-8040
Synergi, Polar-RP C18 (100 x 2 mm, 2.5µm )
0.25 mL/min
A: water with 0.1% Formic acid - 0.03% TA
B: MeOH with 0.1% FA - 0.03% Trimethylamine
B: 10% (0.01 to 0.5 min) → 95% (8 to 9 min) → 10% (9.01 to 11min)
ESI, MRM, positive-negative switching
Nebulizing gas: 3L/min, Drying gas: 15L/min, Heating block: 400ºC, DL: 250ºC
0.1uL, 0.5uL, 1uL, 5uL and 10uL
Column
Flow Rate
Mobile Phase
Gradient program
MS mode
ESI condition
Inj. Vol.
3
Highly Sensitive and Robust LC/MS/MS Method for Quantitative Analysis of Arti�cial Sweeteners in Beverages
Table 2: Arti�cial Sweeteners, MRM transitions and calibration curves on LCMS-8040
Results and Discussion
First, precursor selection and MRM optimization of the ten sweeteners studied was carried out using an automated MRM optimization program of the LabSolutions. Six compounds were ionized in negative mode and four in positive mode as shown in Table2. For each compound, two optimized MRM transitions were selected and used, with the first one for quantitation and the second one for confirmation.The ten compounds were well-separated as sharp peaks between 2 min and 8.2 min as shown in Figure 1. Linear calibration curves of wide concentration ranges were established with mixed standards in diluent as summarized
in Table 2. We also investigated the performance of the LC/MS/MS method established by employing very small injection volumes (0.1, 0.5, 1 and 5 uL). This is because actual beverages usually contain very high contents of sweeteners (>>1ppm) to MS detection. Analysts normally dilute the samples before injection into LC/MS/MS. An alternative way is to inject a very small volume of samples even without dilution. Figs 2 & 3 show a chromatogram and calibration curves established with 0.1uL injection, which demonstrates the feasibility of an ultra-small injection volume combined with high sensitivity LC/MS/MS.
Method development
Compd. & Abbr. Name
Acesulfame K (Ace-K)
Cyclamate (CYC)3
Saccharin (SAC)
Sucralose2 (SUC)
Aspartame (ASP)
Neotame (NEO)
Alitame (ALI)
Dulcin (DUL)
NeohespiridinDihydrochalcone (NHDC)
Glycyrrhi-Zinate (GLY)
Pola. (+/-)
-
-
-
-
-
-
-
-
+
+
+
+
+
+
+
+
-
-
-
-
Q1 (V)
11
11
19
12
13
13
20
20
-19
-19
-18
-18
-23
-23
-22
-21
30
30
22
22
Trans. (m/z)
161.9 >82.1
161.9 >78.0
178.3 >80.1
178.3 >79.0
181.9 >106.1
181.9 >42.1
441.0 >395.1
441.0 >359.1
295.1 >120.1
295.1 >180.1
379.3 >172.2
379.3 >319.3
332.2 >129.1
332.2 >187.1
181.1 >108.1
181.1 >136.1
611.3 >303.1
611.3 >125.3
821.5 >351.2
821.5 >193.2
Cat1
A2
A5
A3
A4
A1
A6
B1
B3
B2
C1
CE (V)
14
32
24
27
20
36
11
15
-25
-14
-23
-18
-19
-16
-25
-18
38
47
46
52
Q3 (V)
29
28
30
10
15
13
25
23
-25
-20
-20
-24
-26
-21
-21
-26
30
20
20
19
RT (min)
1.99
2.87
3.28
4.61
5.15
7.51
5.44
5.58
6.71
8.19
Conc. R. (ug/L)
1 - 20000
5 - 20000
1 - 20000
5 - 20000
0.1 - 2000
0.05 - 1000
0.1 - 2000
5 - 10000
0.5 - 2000
5 - 1000
R2
0.9999
0.9996
0.9984
0.9983
0.9999
0.9998
0.9995
0.999
0.9988
0.9996
MRM parameter RT & Calibration Curve4
1. A1~A6: US FDA, EU and others approval; B1~B3: only EU and other countries approval. C1: natural sweetener, info not available.2. Sucralose precursor ion m/z 441.0 is formic acid adduct ion. 3. Sodium cyclamate known as “magic sugar” was initially banned in the US in 2000. FDA lifted the ban in 2013.4. Injection volume: 10 uL
4
Highly Sensitive and Robust LC/MS/MS Method for Quantitative Analysis of Arti�cial Sweeteners in Beverages
Figure 3: Calibration curves of arti�cial sweeteners on LCMS-8040 with an ultra-small injection volume (0.1 uL) of same set of calibrants as shown in Table 2.
Figure 1: MRM Chromatogram of ten sweeteners by LC/MS/MS with 10uL injection: Asp & Ali 1ppb, Neo 0.5ppb, Dul, Gly, Ace-K, Sac, Suc and Cyc 10ppb, NHDC 1ppb.
Figure 2: MRM Chromatogram of ten sweeteners by LC/MS/MS with 0.1uL injection: Asp & Ali 0.1ppm, Neo 0.05ppm, Dul, Gly, Ace-K, Sac, Suc and Cyc 1ppm, NHDC 0.1ppm.
0.0 2.5 5.0 7.5 10.0 min
0.0
1.0
2.0
3.0
4.0
5.0
(x10,000)
Cyc
lam
ate
NH
DC
Sucr
alos
e
Sacc
harin
Ace
sulfa
me
K
Gly
cyrr
hizi
c
Dul
cin
Neo
tam
e
Alit
ame
Asp
arta
me
0 10000 Conc.0.0
1.0
2.0
Area (x100,000)
Ace-K r2=0.9977
0 10000 Conc.0.0
1.0
2.0
3.0
4.0Area (x10,000)
0 10000 Conc.0.0
2.5
5.0
Area (x10,000)
0 10000 Conc.0.0
0.5
1.0
1.5
Area (x100,000)
0 1000 Conc. 0.0
0.5
1.0
1.5 Area (x100,000)
0 500 Conc.0.0
2.5
5.0Area (x100,000)
0 1000 Conc.0.0
0.5
1.0
1.5
Area (x100,000)
0 10000 Conc.0.0
1.0
2.0
3.0
Area (x100,000)
0 1000 Conc.0.0
1.0
2.0
3.0
4.0
Area (x10,000)
0 10000 Conc.0.0
2.5
5.0
7.5Area (x10,000)
CYC r2=0.9948
SAC r2=0.9977
SUC r2=0.9991
ASP r2=0.9983
NEO r2=0.9982
ALI r2=0.9990
DUL r2=0.9987
NHDC r2=0.9991
GLY r2=0.9997
0 500 Conc.0.0
1.0
Area(x10,000)
0 500 Conc.0.0
0.5
1.0
Area(x1,000)
24 Conc.0.0
2.5
5.0Area(x1,000)
0 Conc.0.0
5.0
Area(x1,000)
0 Conc.0.0
2.5
Area(x1,000)
0.0 25.0 Conc.0.0
1.0
Area(x10,000)
0 Conc.0.0
0.5
1.0Area(x10,000)
0 500 Conc.0.0
1.0
Area(x10,000)
0.0 25.0 Conc.0.0
0.5
1.0Area(x1,000)
0 500 Conc.0.0
2.5
Area(x1,000)
0.0 2.5 5.0 7.5 10.0 min
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5(x1,000)
Cyc
lam
ate
NH
DC
Sucr
alos
e
Sacc
harin
Ace
sulfa
me
K
Gly
cyrr
hizi
c
Dul
cin
Neo
tam
e
Alit
ame
Asp
arta
me
Highly Sensitive and Robust LC/MS/MS Method for Quantitative Analysis of Arti�cial Sweeteners in Beverages
5
Table 3 summarizes the results of repeatability and sensitivity of the method with mixed standards. The method was not evaluated with beverage spiked samples. However, because beverage samples are normally diluted many times,
matrix effect and interferences can be ignored for high sensitivity LC/MS/MS analysis. The results indicate that the method with ultra-small injection volume exhibits good linearity, repeatability and sensitivity.
Method performance
The LC/MS/MS method established was applied for screening and quantitation of the targeted sweeteners in three brand beverages: S1, S2 and S3, and a mouthwash
S4. The results are shown in Figure 4 and Table 4. It is interested to note that glycyrrizinate was found in the mouthwash.
Analysis of beverage samples
Table 3: Repeatability and Sensitivity of LC/MS/MS method of arti�cial sweeteners
RSD%
5.2
8.1
5.8
2.7
3.0
1.0
1.7
3.1
4.6
5.4
LOQ/LOD (0.1 µL inj)
200
800
250
200
80
5
40
160
100
400
Conc. (ug/L)
100
100
100
100
10
5
10
100
10
100
RSD%
5.1
11.7
8.0
7.5
7.8
5.3
8.6
7.5
9.2
8.2
Conc. (ug/L)
20
20
20
20
2
1
2
20
2
20
Name
Ace-K
CYC
SAC
SUC
ASP
NEO
ALI
DUL
NHDC
GLY
50
500
100
100
20
3
25
50
25
150
LOQ/LOD (0.5 µL inj)
40
200
50
50
20
2
10
30
40
15
10
90
20
15
4
1
5
10
6
5
LOQ/LOD 10 (µL inj)
4.0
14
4.5
2.4
0.5
0.03
0.2
1.4
0.5
5.0
1.33
4.5
1.5
0.8
0.17
N.A.
N.A.
0.5
0.18
1.8
Repeatability (peak area), 10uL Sensitivity (ug/L)
Table 4: Screening and quantitation results for ten arti�cial sweeteners in beverages and mouthwash (mg/L)
S4
ND
ND
208.7
ND
449.3
ND
S3
ND
97.2
ND
183.4
ND
ND
S2
127.9
165.9
ND
ND
ND
ND
S1
116.9
143.9
ND
55.1
ND
ND
Arti�cial Sweetener
ASP
Ace-K
Saccharin
SUC
GLY
Others
1. S2 was diluted 100 times, the rests were diluted 10 times. 1 uL injection.2. ND = not detected.
Highly Sensitive and Robust LC/MS/MS Method for Quantitative Analysis of Arti�cial Sweeteners in Beverages
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
References1. http://en.wikipedia.org/wiki/Sugar_substitute and EU directive 93/35/EC, 96/83/EC, 2003/115/EC, 2006/52/EC and
2009/163/EU.2. Buchgraber and A. Wasik, Report EUR 22726 EN (2007).3. F.T. Large, M. Scheurer and H.-J Brauch, Anal Bioanal Chem, 403: 2503-2518 (2012) 4. Ho-Soo Lim, Sung-Kwan Park, In-Shim Kwak, Hyung-Ll Kim, Jun-Hyun Sung, Mi-Youn Byun and So-Hee Kim, Food Sci,
Biotechnol, 22(S):233-240 (2013)
ConclusionsA MRM-based LC/MS/MS method was developed and evaluated for screening and quantitation of ten arti�cial sweeteners in beverages. This high sensitivity LC/MS/MS method combined with small or ultra-small injection volume (0.1~1.0 uL) was proven to be feasible and reliable in actual samples analysis of the targeted sweeteners in beverages, achieving high throughput and free of sample
pre-treatment (except dilution). The method is expected to be applicable to surface water and drinking water samples. For wastewater and various foods, sample pre-treatment is usually required. However, the advantages of the method in high sensitivity and ultra-small injection volume are expected to enable it tolerates relatively simple sample pre-treatment procedures.
Figure 4: Screening and quantitation for 10 targeted arti�cial sweeteners in beverage and mouthwash samples by LC/MS/MS with 1uL injection.
0.0 2.5 5.0 7.5 10.0 min
0.0
1.0
2.0
3.0
4.0
5.0(x1,000,000)
Sucr
alos
e (x
10)
Ace
sulfa
me
K (x
10)
Asp
arta
me
S1
0.0 2.5 5.0 7.5 10.0 min
0.0
1.0
2.0
3.0
4.0
5.0
(x100,000)
Ace
sulfa
me
K (x
10)
Asp
arta
me
S2
0.0 2.5 5.0 7.5 10.0 min
0.0
1.0
2.0
3.0
(x100,000)
Sucr
alos
e
Ace
sulfa
me
K
S3
0.0 2.5 5.0 7.5 10.0 min
0.0
0.5
1.0
1.5
(x100,000)
Sacc
harin
Gly
cyrr
hizi
c
S4
PO-CON1480E
Highly sensitive and rapid simultaneous method for 45 mycotoxinsin baby food samples by HPLC-MS/MS using fast polarity switching
ASMS 2014 MP345
Stéphane MOREAU1 and Mikaël LEVI2
1 Shimadzu Europe, Albert-Hahn Strasse 6-10,
Duisburg, Germany 2 Shimadzu France SAS, Le Luzard 2, Boulevard Salvador
Allende, 77448 Marne la Vallée Cedex 2, France
2
Highly sensitive and rapid simultaneous method for 45 mycotoxins in baby food samples by HPLC-MS/MS using fast polarity switching
IntroductionMycotoxins are toxic metabolites produced by fungal molds on food crops. For consumer food safety, quality control of food and beverages has to assay such contaminants. Depending on the potency of the mycotoxin and the use of the food, the maximum allowed level is de�ned by legislation. Baby food is particularly critical. For example, European Commission has �xed the maximum level of A�atoxin B1 and M1 to 0.1 and 0.025 µg/kg, respectively, in baby food or milk.
Therefore, a sensitive method to assay mycotoxins in complex matrices is mandatory. In order to ensure productivity of laboratory performing such assays, a unique rapid method able to measure as much mycotoxins as possible independently of the sample origin is also needed.In this study, we tested three kind of samples: baby milk powder, milk thickening cereals (�our, rice and tapioca) and a vegetable puree mixed with cereals.
Materials and Methods
Sample preparation was performed by homogenization followed by solid phase extraction using specific cartridges (Isolute® Myco, Biotage, Sweden) covering a large spectrum of mycotoxins.Sample (5g) was mixed with 20 mL of water/acetonitrile (1/1 v/v), sonicated for 5 min and agitated for 30 min at room temperature. After centrifugation at 3000 g for 10 min, the supernatant was diluted with water (1/4 v/v). Columns (60mg/3 mL) were conditioned with 2 mL of acetonitrile then 2 mL of water. 3 mL of the diluted supernatant were loaded at the lowest possible flow rate.
Then column was washed with 3 mL of water followed by 3 mL of water/acetonitrile (9/1 v/v). After drying, compounds were successively eluted with 2 mL of acetonitrile with 0.1% of formic acid and 2 mL of methanol.The eluate was evaporated under nitrogen flow at 35 ºC until complete drying (Turbovap, Biotage, Sweden).The sample was reconstituted in 150 µL of a mixture of water/methanol/acetonitrile 80/10/10 v/v with 0.1% of formic acid.
Sample preparation
Extracts were analysed on a Nexera X2 (Shimadzu, Japan) UHPLC system and coupled to a triple quadrupole mass spectrometer (LCMS-8050, Shimadzu, Japan). Analysis was
carried out using selected reaction monitoring acquiring 2 transitions for each compound.
LC-MS/MS analysis
3
Highly sensitive and rapid simultaneous method for 45 mycotoxins in baby food samples by HPLC-MS/MS using fast polarity switching
Table 1 – LC conditions
Table 2 – MS/MS conditions
Analytical column : Shimadzu GLC Mastro™ C18 150x2.1 mm 3µm
Mobile phase : A = Water 2mM ammonium acetate and 0.5% acetic acid
B = Methanol/Isopropanol 1/1 + 2mM ammonium acetate
and 0.5% acetic acid
Gradient : 2%B (0.0min), 10%B (0.01min), 55%B (3.0min), 80%B (7.0 -8.0min),
2%B (8.01min), Stop (11.0min)
Column temperature : 50ºC
Injection volume : 10 µL
Flow rate : 0.4 mL/min
Ionization mode : Heated ESI (+/-)
Temperatures : HESI: 400ºC
Desolvation line: 250ºC
Heat block: 300ºC
Gas �ows : Nebulizing gas (N2): 2 L/min
Heating gas (Air): 15 L/min
Drying gas (N2): 5 L/min
CID gas pressure : 270 kPa (Ar)
Polarity switching time : 5 ms
Pause time : 1 ms
Dwell time : 6 to 62 ms depending on the number of concomitant transitions
to ensure a minimum of 30 points per peak in a maximum loop time
of 200 ms (including pause time and polarity switching)
4
Highly sensitive and rapid simultaneous method for 45 mycotoxins in baby food samples by HPLC-MS/MS using fast polarity switching
Name
15-acetyldeoxynivalenol (15ADON) [M+H]+
3-acetyldeoxynivalenol (3ADON) [M+H]+
A�atoxine B1 (AFB1) [M+H]+
A�atoxine B2 (AFB2) [M+H]+
A�atoxine G1 (AFG1) [M+H]+
A�atoxine G2 (AFG2) [M+H]+
A�atoxine M1 (AFM1) [M+H]+
Alternariol [M-H]-
Alternariol monomethyl ether [M-H]-
Beauvericin (BEA) [M+H]+
Citrinin (CIT) [M+H]+
D5-OTA (ISTD)
Deepoxy-Deoxynivalenol (DOM-1) [M-H]-
Deoxynivalenol (DON) [M-CH3COO]-
Deoxynivalenol 3-Glucoside (D3G) [M+CH3COO]-
Deoxynivalenol 3-Glucoside (D3G) [M+CH3COO]-
Diacetoxyscirpenol (DAS) [M+NH4]+
Enniatin A (ENN A) [M+H]+
Enniatin A1 (ENN A1) [M+H]+
Enniatin B (ENN B) [M+H]+
Enniatin B1 (ENN B1) [M+H]+
Fumagillin (FUM) [M+H]+
Fumonisine B1 (FB1) [M+H]+
Fumonisine B2 (FB2) [M+H]+
Fumonisine B3
Fusarenone-X (FUS-X) [M+H]+
HT2 Toxin [M+Na]+
Moniliformin (MON) [M-H]-
Neosolaniol (NEO) [M+NH4]+
Nivalenol (NIV) [M+CH3COO]-
Ochratoxin A (OTA) [M+H]+
Ochratoxin B (OTB) [M+H]+
Patulin (PAT) [M-H]-
Sterigmatocystin (M+H]+
T2 Tetraol [M+CH3COO]-
T2 Toxin [M+NH4]+
Tentoxin [M-H]-
Tenuazonic acid (TEN) [M-H]-
Wortmannin (M-H)
Zearalanol (alpha) (ZANOL) [M-H]-
Zearalanol (beta) (ZANOL) [M-H]-
Zearalanone (ZOAN) [M-H]-
Zearalenol (alpha) (ZENOL) [M-H]-
Zearalenol (beta) (ZENOL) [M-H]-
Zearalenone (ZON) [M-H]-
Ret. Time (min)
3.37
3.37
3.78
3.57
3.46
3.26
3.30
4.78
5.81
8.03
4.16
5.22
3.02
2.61
2.45
2.45
1.20
8.51
8.22
7.57
7.92
6.16
4.10
4.71
4.38
2.84
4.58
1.16
2.90
2.41
5.53
4.83
2.35
5.60
1.64
4.94
4.77
4.51
3.95
5.17
4.85
5.43
5.25
4.94
5.52
MRM Quan
339 > 297.1
339 > 231.1
312.6 > 284.9
315.1 > 259
329.1 > 242.9
330.9 > 244.9
329.1 > 273
257 > 214.9
271.1 > 255.9
784 > 244.1
251.3 > 233.1
409.2 > 239.1
279.2 > 249.3
355.3 > 295.2
517.5 > 457.1
517.5 > 457.1
384 > 283.3
699.2 > 682.2
685.3 > 668.3
657 > 640.4
671.2 > 654.2
459.2 > 131.1
722.1 > 334.2
706.2 > 336.3
706.2 > 336.2
355.1 > 247
446.9 > 344.9
97.2 > 40.9
400.2 > 215
371.2 > 280.9
404.2 > 239
370.2 > 205.1
153 > 81.2
325.3 > 310
356.8 > 297.1
484.2 > 215
413.1 > 140.9
196.1 > 138.8
426.9 > 384
321.3 > 277.2
321.3 > 277.2
319 > 275.1
319.2 > 275.2
319.2 > 275.2
316.8 > 174.9
MRM Qual
339 > 261
339 > 231.1
312.6 > 240.9
315.1 > 286.9
329.1 > 199.9
330.9 > 313.1
329.1 > 229
257 > 213.1
271.1 > 228
784 > 262
251.3 > 205.1
N/A
279.2 > 178.4
355.3 > 265.1
517.5 > 427.1
517.5 > 427.1
384 > 343
699.3 > 210
685.3 > 210.1
657 > 195.9
671.2 > 196
459.2 > 338.7
722.1 > 352.2
706.2 > 318.1
706.2 > 688.1
355.1 > 175
446.9 > 285
N/A
400.2 > 185
371.2 > 311.1
404.2 > 358.1
370.2 > 187
153 > 53
325.3 > 281.1
356.8 > 59.1
484.2 > 305
413.1 > 271.1
196.1 > 112
426.9 > 282.1
321.3 > 303.2
321.3 > 303.1
319 > 301.1
319.2 > 160.1
319.2 > 160.1
316.8 > 131.1
Table 3 – MRM transitions
5
Highly sensitive and rapid simultaneous method for 45 mycotoxins in baby food samples by HPLC-MS/MS using fast polarity switching
Figure 1 – Structure of the Mastro™ column
Figure 2 – Parameters selection view in the Interface Setting Support Software
Results and discussion
LC conditions were transferred from a previously described method (Tamura et al., Poster TP-739, 61st ASMS). In particularly, the column was chosen to provide very good peak shape for chelating compounds like fumonisins thanks to its inner PEEK lining.
Small adjustments in the mobile phase and in the gradient program were made to handle more mycotoxins, especially the isobaric ones. These modifications are reported in the Table 1.
Method development
Also, autosampler rinsing conditions were kept to ensure carry-over minimisation of some difficult compounds.Electrospray parameters (gas flows and temperatures) were cautiously optimized to find the optimal combination for the most critical mycotoxins (aflatoxins). Since these parameters act in a synergistic way, a factorial design experiment is needed to find it. Manually testing all combinations in the chromatographic conditions is very
time consuming. Therefore, new assistant software (Interface Setting Support) was used to generate all possible combinations and generate a rational batch analysis. Optimal combination was found in chromatographic conditions. The difference observed between optimum and default or worst parameters was of 200 and 350%, respectively.
Stationary phase
Stainless steel Body Polymer lining
Polymer frit
Highly sensitive and rapid simultaneous method for 45 mycotoxins in baby food samples by HPLC-MS/MS using fast polarity switching
6
Extraction and ionisation recovery for aflatoxins was measured in the three matrices by comparing peak areas of the raw sample extract to extract spiked at 50 ppb after or before extraction and to standard solution. Results in table
4 showed that the total recovery was quite acceptable to ensure accurate quantification. Results from other matrices were not significatively different.
Results
Repeatability was evaluated at low level for aflatoxins. Figure 3 shows an overlaid chromatogram (n=4) for aflatoxins.
Table 4 – Extraction and ionisation recoveries in puree
Figure 3 – Chromatogram of a�atoxins at 0.1 ppb in milk thickening cereals
Figure 4 – Chromatogram of the 45 mycotoxins in standard at 50 ppb (2 ppb for a�atoxins and ochratoxines)
Extraction recovery
Ionisation recovery
Total recovery
AFB1
101%
49%
49%
AFB2
109%
90%
98%
AFG1
104%
96%
100%
AFG2
114%
106%
121%
AFM1
118%
91%
108%
3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.0 min
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
2.50
(x10,000)
1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 min-500000
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
6000000
6500000
7000000
7500000
8000000
8500000
9000000
9500000
10000000
10500000
11000000
11500000
12000000
Highly sensitive and rapid simultaneous method for 45 mycotoxins in baby food samples by HPLC-MS/MS using fast polarity switching
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Conclusion• A very sensitive method for multiple mycotoxines was set up to ensure low LOQ in baby food sample,• Thanks to high speed polarity switching, a high number of mycotoxines can be assayed using the same method in a
short time, • The extraction method demonstrate good recoveries to ensure accurate quanti�cation.
PO-CON1461E
High Sensitivity Analysis of Acrylamidein Potato Chips by LC/MS/MS with Modi�ed QuEChERS Sample Pre-treatment Procedure
ASMS 2014 MP342
Zhi Wei Edwin Ting1; Yin Ling Chew*2;
Jing Cheng Ng*2; Jie Xing1; Zhaoqi Zhan1
1 Shimadzu (Asia Paci�c) Pte Ltd, Singapore, SINGAPORE; 2 Department of Chemistry, Faculty of Science,
National University of Singapore, 21 Lower Kent
Ridge Road, Singapore119077, *Student
2
High Sensitivity Analysis of Acrylamide in Potato Chips by LC/MS/MS with Modi�ed QuEChERS Sample Pre-treatment Procedure
IntroductionAcrylamide was found to form in fried foods like potato-chips via the so-called Maillard reaction of asparagine and glucose (reducing sugar) at higher temperature (120ºC) in 2002 [1,2]. The health risk of acrylamide present in many processing foods became a concern immediately, because it is known that the compound is a neurotoxin and a potential carcinogen to humans [3]. Various analytical methods, mainly LC/MS/MS and GC/MS based methods, were established and used in analysis of acrylamide in foods in recent years [4]. We
present a novel LC/MS/MS method for quantitative determination of acrylamide in potato chips with using a modi�ed QuEChERS procedure for sample extraction and clean-up, achieving high sensitivity and high recovery. A small sample injection volume (1uL) was adopted purposely to reduce the potential contamination of samples to the interface of MS system, so as to enhance the operation stability in a laboratory handling food samples with high matrix contents.
ExperimentalAcrylamide and isotope labelled acrylamide-d3 (as internal standard) were obtained from Sigma-Aldrich. The QuEChERS kits were obtained from RESTEK. A modi�ed procedure of the QuEChERS was optimized and used in the sample extraction of acrylamide (Q-sep Q100 packet, original unbuffered) in potato chips and clean-up of matrix with d-SPE tube (Q-sep Q250, AOAC 2007.01). Acrylamide and acrylamide-d3 (IS) stock solutions and diluted calibrants were prepared using water as the solvent.
Method development and performance evaluation were carried out using spiked acrylamide samples in the extracted potato chip matrix. A LCMS-8040 triple quadrupole LC/MS/MS (Shimadzu Corporation, Japan) was used in this work. A polar-C18 column of 2.5µm particle size was used for fast UHPLC separation with a gradient elution method. Table 1 shows the details of analytical conditions on LCMS-8040 system,.
Table 1: LC/MS/MS analytical conditions of LCMS-8040 for acrylamide
LC condition
Phenomenex Synergi 2.5u Polar-Rp 100A (100 x 2.00mm)
0.2 mL/min
A: waterB: 0.1% formic acid in Methanol
Gradient elution, B%: 1% (0 to 1 min) → 80% (3 to 4.5 min) → 1% (5.5 to 10min)
40ºC
1.0 µL
Column
Flow Rate
Mobile Phase
Elution Mode
Oven Temp.
Injection Vol.
MS Interface condition
ESI
Positive, MRM, 2 transitions each compound
400ºC
200ºC
Ar (230kPa)
N2, 1.5L/min
N2, 10.0L/min
Interface
MS mode
Block Temp.
DL Temp.
CID Gas
Nebulizing Gas Flow
Drying Gas Flow
3
High Sensitivity Analysis of Acrylamide in Potato Chips by LC/MS/MS with Modi�ed QuEChERS Sample Pre-treatment Procedure
Results and Discussion
The details of a modified QuEChERS procedure for potato chips are shown in Figure 1. Hexane was used to defat potato chips, removing oils and non-polar components. In the extraction step with Q-sep Q100Packet extraction salt (contain 4g MgSO4 & 0.5g NaCl), additional 4g of MgSO4 was added to absorb the water completely (aqueous phase disappeared). Acrylamide is soluble in both aqueous and organic phases. With this modification, high recovery of acrylamide was obtained. It is believed that this is because complete removal of water in the mixed extract solution could promote acrylamide transferring into the organic phase. Dispersive SPE tube was used as PSA to remove organic acids which may decompose acrylamide in the process.
QuEChERS Sample Pre-treatment
As acrylamide is a more polar compound, a Polar-RP type column was selected. Isotope labeled internal standard (acrylamide-d3) was used to compensate the variation of acrylamide peak area caused by system fluctuation and inconsistency in sample preparation of different batches.The precursor ions of acrylamide and acrylamide-d3 (IS) were their protonated ions (m/z72.1 and m/z75.1). The MRM optimization was carried out using an automated program of the LabSolutions workstation, which could generate a list of all MRM transitions with optimized CID voltages accurate to (+/-) 1 volt in minutes. Two MRM transitions of acrylamide and acryl-amide-d3 were selected as quantifier and confirmation ion as shown in Table 2.The obtained extract solution of potato chips was used as “blank” and also matrix for preparation of post-spiked calibrants for establishment of calibration curve with IS (acrylamide-d3). To obtain reliable results, the blank and each post-spiked calibrant as shown in Table 3 were injected three times and the average peak area ratios were calculated and used.
Method Development
Figure 1: Flow chart of sample pre-treatment with modi�ed QuEChERS.
Table 3: Acrylamide spiked samples and peak area ratios of measured by IS method
Acrylamide post-spiked
IS post-spiked
Conc. RatioCalculated
Area Ratio measured*
L0, Blank
L1, 1ppb
L2, 5ppb
L3, 10ppb
L4, 50ppb
L5 100ppb
L6, 500ppb
50ppb
0
0.02
0.10
0.20
1.00
2.00
10.00
0.6033
0.6120
0.6786
0.8239
1.7686
2.8196
11.8330
*= Area (acrylamide) / Area (IS)
Table 2: MRM transitions and CID voltages
Name MRM (m/z)Q1 Q3
Acrylamide-d3
Acrylamide
75.1 > 58.0*
75.1 > 30.1
72.1 > 55.0*
72.1 > 27.1
-29
-29
-17
-17
CID Voltage (V)
CE
-15
-24
-16
-22
-22
-30
-24
-30
*MRM transition as quanti�er
[1] Weigh 2.0g of sample in a 50mL centrifuge tube Add 5mL hexane, 10mL water
and 10mL acetonitrile[2] Vortex and shake vigorously for 1min Add Q-sep Q100Packet salt Additional 4g MgSO4 (anhydrous)[3] Vortex and shake vigorously for 5min
[4] Discard the hexane (top layer)
[5] Transfer the solution into a 20mL volumetric �ask wash extraction salt with ACN
in the centrifuge tube[6] Combine the washing solution into the
volumetric �ask (above)
[7] Transfer 1mL of solution into the 2mL Q-sep Q250 QuEChERS dSPE tube
[8] Vortex and centrifuge for 10min at 13000rpm
[9] Transfer 500uL extract to a 1.5mL vial Evaporate to dryness by N2 blow[10] Reconstitute with 250uL of Milli Q water
[11] Analyze by Shimadzu LCMS-8040
4
High Sensitivity Analysis of Acrylamide in Potato Chips by LC/MS/MS with Modi�ed QuEChERS Sample Pre-treatment Procedure
It was found that the potato chips used as “blank” in this study was not free of acrylamide. Instead, it contained 27.1 ng/mL of acrylamide in the extract solution. A linear calibration curve was established with an intercept of
0.594 at zero spiked concentration (L0) as shown in Figure 2. Good linearity with correlation coefficient (R2) greater than 0.9999 across the range of 1.0 ng/mL– 500.0 ng/mL was obtained.
Figure 2: Calibration curve (left) and MRM peaks (right) of acrylamide spiked into potato chips matrix, 1-500 ppb with 50 ppb IS added.
It was hard to estimate the LOD and LOQ of the analytical method due to the presence of acrylamide (27.1 ng/mL) in the “blank” (extract of potato chips). However, as reported also by other researchers, it is difficult to obtain potato chips free of acrylamide actually. To obtain actual concentration, it is normally subtracting the background content of acrylamide of a “blank” sample used as reference from a measurement of testing sample. The same way was used to estimate actual S/N value in this work. As a result, the LOD and LOD of acrylamide of this method with 1ul injection volume were estimated to be lower than 1ng/mL and 3ng/mL, respectively. This is consistence with the results estimated with the IS.The repeatability of the method was evaluated with L2 and L4 spiked samples. The results are shown in Table 4 and
Figure 3. The peak area %RSD of acrylamide and IS were below 4%. The matrix effect (M.E.), recovery efficiency (R.E.) and process efficiency (P.E.) of the method were determined with a duplicate set of spiked samples of 50 ng/mL level except for the non-spiked sample. The chromatograms of “set 2”, i.e., non-spiked extract, pre-spiked, post-spiked and the standard in neat solution are shown in Figure 4. Noted that, the existing acrylamide in the extract of the potato chips used as reference was accounted for 27.1 ng/mL, corresponding to 135.5 ng per gram of potato chips. The average R.E, M.E and P.E of the method for extraction and analysis of acrylamide obtained are shown in Table 6.
Method Performance Evaluation
spiked Sample Compound Conc. (ng/mL) %RSD
L2
L4
Acrylamide
Acrylamide-d3
Acrylamide
Acrylamide-d3
5
50
50
50
3.5
3.8
3.9
3.6
Table 4: Repeatability Test Results (n=6)
2.5 5.0 7.5 min
0
100000
200000
3000002:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 500ppb 01a.lcd2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 100ppb 01a.lcd2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 50ppb 01a.lcd2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 10ppb 01a.lcd2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 5ppb 01a.lcd2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 1ppb 01a.lcd
1.5 2.0 2.5 min
0
50000
100000
150000
0.0 2.5 5.0 7.5 Conc. Ratio0.00
0.25
0.50
0.75
1.00
1.25Area Ratio (x10)
Y= 1.1239X + 0.594168 R2 = 0.9999
0.00 Conc. Ratio0.0
0.5
1.0
Area Ratio
High Sensitivity Analysis of Acrylamide in Potato Chips by LC/MS/MS with Modi�ed QuEChERS Sample Pre-treatment Procedure
5
Figure 3: Overlay MRM chromatograms of 5 ng/mL acrylamide spiked in potato chips extract (total: 27.1+5 = 32.1 ng/mL)
2.5 5.0 7.5 min
0
10000
20000
30000
2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 5ppb R06.lcd2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 5ppb R05.lcd2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 5ppb R04.lcd2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 5ppb R03.lcd2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 5ppb R02.lcd2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 5ppb R01.lcd
1.0 1.5 2.0 2.5
Figure 4: The MRM peaks of acrylamide detected in “blank” extract of potato chips (a), neat standard of 50ppb (b) post-spiked sample of 50ppb (c) and pre-spiked sample of 50ppb.
ConclusionsAcrylamide is formed unavoidably in starch-rich food in cooking and processing at high temperature like potato chips, French fries, cereals and roasted coffee etc. The analysis method established in this work can be used to monitor the levels of acrylamide in processing food accurately and reliably. The QuEChERS method is proven to be fast and effective in extraction of acrylamide from potato chips. The excellent performance of the method in terms of sensitivity, linearity, repeatability and recovery are
related to the outstanding performance of the LC/MS/MS used which features ultra fast mass spectrometry (UFMS) technology. The high sensitivity of the method allows the analysis to be performed with a very small injection volume (1µL or below), which would be a great advantage in running heavily food samples with high matrix contents and strong matrix effects. Maintenance of the interface of a mass spectrometer could also be reduced signi�cantly.
Table 6: Method evaluation of at 50.0ng/mL concentration in potato chips matrix
Parameter Set 1 Set 2 Average
R.E.
M.E.
P.E.
104.7%
96.5%
100.8%
112.0%
84.6%
94.5%
108.4%
90.5%
97.6%
(d) pre-spiked
1.5 2.0 2.50
10000
20000
30000
40000
50000
1.5 2.0 2.5
0
10000
20000
30000
40000
50000
1.5 2.0 2.50
10000
20000
30000
40000
50000
1.5 2.0 2.50
10000
20000
30000
40000
50000(a) Extract (non-spiked)
(c) post-spiked(b) standard
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
High Sensitivity Analysis of Acrylamide in Potato Chips by LC/MS/MS with Modi�ed QuEChERS Sample Pre-treatment Procedure
References[1] Swedish National Food Administration. “Information about acrylamide in food, 24 April 2002”, http://www.slv.se[2] Mottram, D.S., & Wedzicha, B.L., Nature, 419 (2002), 448-449. [3] Ahn, J.S., Castle, J., Clarke, D.B., Lloyd, A.S., Philo, M.R., & Speck, D.R., Food Additives and Contaminants, 19 (2002),
1116-1124. [4] Mastovska, K., & Lehotary, S.J., J. Food Chem., 54 (2006), 7001-7998.
PO-CON1472E
Determination of Benzimidazole Residues in Animal Tissue by Ultra High PerformanceLiquid Chromatography Tandem Mass Spectrometry
ASMS 2014 TP 281
Yin Huo, Jinting Yao, Changkun Li, Taohong Huang,
Shin-ichi Kawano, Yuki Hashi
Shimadzu Global COE, Shimadzu (China) Co., Ltd., China
2
Determination of Benzimidazole Residues in Animal Tissue by Ultra High Performance Liquid Chromatography Tandem Mass Spectrometry
IntroductionBenzimidazoles are broad-spectrum, high ef�ciency, low toxicity anthelmintic. Because some benzimidazoles and their metabolites showed teratogenic and mutagenic effects in animal and target animal safety evaluation experiment, many countries have already put benzimidazoles and metabolites as the monitoring object.
This poster employed a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method to determinate 16 benzimidazole residues in animal tissue. The method is simple, rapid and high sensitivity, which meets the requirements for the analysis of veterinary drug residue in animal tissue.
Method
(1) Animal tissue samples were extracted with ethyl acetate-50% potassium hydroxide-1% BHT(2) The samples were treated with n-hexane for defatting and further cleaned-up on MCX solid phase (SPE) cartridge. (3) The separation of benzimidazoles and their metabolites was performed on LC-MS/MS instrument.
Sample Preparation
The analysis was performed on a Shimadzu Nexera UHPLC instrument (Kyoto, Japan) equipped with LC-30AD pumps, a CTO-30A column oven, a DGU-30A5 degasser, and an SIL-30AC autosampler. The separation was carried out on a Shim-pack XR-ODS III (2.0 mmI.D. x 50 mmL., 1.6 μm, Shimadzu) with the column temperature at 30 ºC. A triple quadrupole mass spectrometer (Shimadzu LCMS-8040, Kyoto, Japan) was connected to the UHPLC instrument via an ESI interface.
LC/MS/MS Analysis
Analytical Conditions
UHPLC (Nexera system)
Column : Shim-pack XR-ODS III (2.0 mmI.D. x 50 mmL., 1.6 μm)
Mobile phase A : water with 0.1% formic acid
Mobile phase B : acetonitrile
Gradient program : as in Table 1
Flow rate : 0.4 mL/min
Column temperature : 30 ºC
Injection volume : 20 µL
Table 1 Time program
Time (min) Module Command Value
0.01
3.50
4.00
4.01
6.00
Pumps
Pumps
Pumps
Pumps
Controller
Pump B Conc.
Pump B Conc.
Pump B Conc.
Pump B Conc.
Stop
5
80
80
5
3
Determination of Benzimidazole Residues in Animal Tissue by Ultra High Performance Liquid Chromatography Tandem Mass Spectrometry
MS/MS (LCMS-8040 triple quadrupole mass spectrometer)
Ionization : ESI
Polarity : Positive
Ionization voltage : +4.5 kV
Nebulizing gas �ow : 3.0 L/min
Heating gas pressure : 15.0 L/min
DL temperature : 200 ºC
Heat block temperature : 350 ºC
Mode : MRM
Table 2 MRM parameters of 16 benzimidazoles (*: for quantitation)
CompoundPrecursor
m/z
300.10
282.00
202.00
218.00
316.20
266.30
240.30
298.30
296.30
238.30
298.30
314.30
256.30
303.20
250.30
332.20
Productm/z
268.05*
159.05
240.10*
208.05
175.10*
131.15
191.05*
147.10
159.15*
191.15
234.10*
191.10
133.20*
198.10
159.10*
224.05
264.15*
105.25
105.20*
133.20
266.10*
160.15
282.15*
123.15
123.20*
95.20
217.15*
261.10
218.15*
176.15
300.10*
159.05
Dwell Time(ms)
50
50
10
10
10
10
50
50
20
20
8
8
50
50
20
20
10
10
10
10
10
10
10
10
10
10
5
5
5
5
10
10
Q1 Pre Bias(V)
-15.0
-15.0
-14.0
-14.0
-30.0
-30.0
-30.0
-30.0
-11.0
-11.0
-30.0
-30.0
-15.0
-15.0
-13.0
-13.0
-13.0
-13.0
-15.0
-15.0
-30.0
-30.0
-14.0
-14.0
-16.0
-16.0
-30.0
-30.0
-30.0
-30.0
-15.0
-15.0
Q3 Pre Bias(V)
-18.0
-30.0
-17.0
-22.0
-18.0
-25.0
-13.0
-27.0
-30.0
-20.0
-25.0
-20.0
-24.0
-21.0
-30.0
-23.0
-27.0
-19.0
-20.0
-25.0
-18.0
-30.0
-19.0
-24.0
-22.0
-18.0
-23.0
-28.0
-23.0
-18.0
-21.0
-30.0
CE (V)
-21.0
-36.0
-12.0
-23.0
-24.0
-31.0
-23.0
-32.0
-34.0
-22.0
-19.0
-33.0
-27.0
-18.0
-37.0
-27.0
-21.0
-35.0
-26.0
-36.0
-22.0
-35.0
-22.0
-35.0
-26.0
-41.0
-28.0
-17.0
-17.0
-27.0
-22.0
-39.0
Fenbendazole
Albendazole sulfoxide
Thiabendazole
Thiabendazole-5-hydroxy
Oxfendazole
Albendazole
Albendazole -2-aminosulfone
Albendazole sulfone
Mebendazole
Mebendazole-amine
5-Hydroxymebendazole
Flubendazole
2-Amino�ubendazole
Cambendazole
Oxibendazole
Oxfendazole
4
Determination of Benzimidazole Residues in Animal Tissue by Ultra High Performance Liquid Chromatography Tandem Mass Spectrometry
Results and DiscussionA liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method has been developed to identify and quantify trace levels of 16 benzimidazoles residue (fenbendazole, albendazole sulfoxide, thiabendazole, thiabendazole- 5-hydroxy, oxfendazole, albendazole, albendazole-2-aminosulfone, albendazole sulfone, mebendazole, mebendazole-amine, 5-hydroxymebendazole, �ubendazole, 2-amino�ubendazole, cambendazole, oxibendazole, oxfendazole) in animal tissue. The MRM chromatograms of
16 drugs mixture are presented in Fig.1. The correlation coef�cients for 16 drugs (0.5 – 50 ng/mL) were found to 0.9993~0.9999. MRM chromatograms of pork samples and pork samples spiked with standards are shown in Fig.2. By analyzing 16 drugs at three levels including 0.5 ng/mL, 5 ng/mL, 50 ng/mL, excellent repeatability was demonstrated with the %RSD being better than 5% for all the compound within six injections as shown in Table 3. Results of recovery test were good as shown in Table 4.
Figure 1 MRM chromatograms of standard 16 drugs (1 ng/mL)(1: Thiabendazole-5-hydroxy; 2: Albendazole -2-Aminosulfone; 3: Thiabendazole;
4: Mebendazole-amine; 5: 2-Amino�ubendazole;6: 5-Hydroxymebendazole;7: Albendazole Sulfoxide; 8: Cambendazole; 9: Oxibendazole; 10: Oxfendazole;11: Albendazole sulfone; 12: Albendazole; 13: Mebendazole; 14: Oxfendazole;
15: Flubendazole; 16: Fenbendazole)
0.0 1.0 2.0 3.0 4.0 min
0
10000
20000
30000
40000
50000
60000
70000
16:300.10>268.05(+)15:314.30>282.15(+)14:332.20>300.10(+)(2.00)13:296.30>264.15(+)12:266.30>234.10(+)11:298.30>159.10(+)(2.00)10:316.20>159.15(+)(2.00)9:250.30>218.15(+)8:303.20>217.15(+)7:282.00>240.10(+)6:298.30>266.10(+)5:256.30>123.20(+)(2.00)4:238.30>105.20(+)(3.00)3:202.00>175.10(+)2:240.30>133.20(+)(2.00)1:218.00>191.05(+)(10.00)
151413
12
1110
98
76
54
32
1
16
Determination of Benzimidazole Residues in Animal Tissue by Ultra High Performance Liquid Chromatography Tandem Mass Spectrometry
5
Figure 2 MRM chromatograms of pork sample (left) and spiked pork sample (right) (1: Thiabendazole-5-hydroxy; 2: Albendazole -2-Aminosulfone; 3: Thiabendazole;
4: Mebendazole-amine; 5: 2-Amino�ubendazole;6: 5-Hydroxymebendazole;7: Albendazole Sulfoxide; 8: Cambendazole; 9: Oxibendazole; 10: Oxfendazole;11: Albendazole sulfone; 12: Albendazole; 13: Mebendazole; 14: Oxfendazole;
15: Flubendazole; 16: Fenbendazole)
Table 3 Repeatability of 16 drugs in pork sample (n=6)
CompoundArea
3.01
4.26
4.52
4.44
2.71
2.07
4.36
3.95
4.95
3.95
2.31
4.22
4.30
4.90
3.46
3.23
%RSD (0.5 ng/mL) %RSD (5.0 ng/mL) %RSD (50 ng/mL)
R.T.
0.059
0.202
0.272
0.526
0.121
0.073
0.392
0.103
0.093
0.363
0.091
0.107
0.339
0.150
0.091
0.170
R.T.
0.064
0.084
0.180
0.249
0.089
0.090
0.162
0.126
0.095
0.149
0.099
0.058
0.177
0.123
0.108
0.044
Area
1.48
2.86
2.85
3.91
2.91
1.29
2.08
0.63
1.69
2.72
0.79
1.52
2.53
3.38
1.31
3.09
Area
0.34
0.92
2.58
1.41
0.97
0.92
1.72
0.64
0.74
0.94
1.17
1.00
1.43
1.87
1.20
0.80
R.T.
0.082
0.153
0.132
0.158
0.105
0.099
0.177
0.113
0.094
0.243
0.140
0.091
0.166
0.121
0.125
0.084
Fenbendazole
Albendazole Sulfoxide
Thiabendazole
Thiabendazole-5-hydroxy
Oxfendazole
Albendazole
Albendazole -2-Aminosulfone
Albendazole sulfone
Mebendazole
Mebendazole-amine
5-Hydroxymebendazole
Flubendazole
2-Amino�ubendazole
Cambendazole
Oxibendazole
Oxfendazole
0.0 1.0 2.0 3.0 4.0 min0
10000
20000
30000
40000
50000
16:300.10>268.05(+)15:314.30>282.15(+)14:332.20>300.10(+)13:296.30>264.15(+)12:266.30>234.10(+)11:298.30>159.10(+)10:316.20>159.15(+)9:250.30>218.15(+)8:303.20>217.15(+)7:282.00>240.10(+)6:298.30>266.10(+)5:256.30>123.20(+)4:238.30>105.20(+)3:202.00>175.10(+)2:240.30>133.20(+)1:218.00>191.05(+)
0.0 1.0 2.0 3.0 4.0 min0
10000
20000
30000
40000
50000
16:300.10>268.05(+)15:314.30>282.15(+)14:332.20>300.10(+)13:296.30>264.15(+)12:266.30>234.10(+)11:298.30>159.10(+)10:316.20>159.15(+)9:250.30>218.15(+)8:303.20>217.15(+)7:282.00>240.10(+)6:298.30>266.10(+)5:256.30>123.20(+)4:238.30>105.20(+)3:202.00>175.10(+)2:240.30>133.20(+)1:218.00>191.05(+)(10.00)
15
1413
12
1110
98
76
54
321
16
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Determination of Benzimidazole Residues in Animal Tissue by Ultra High Performance Liquid Chromatography Tandem Mass Spectrometry
ConclusionThe sensitive and reliable LC/MS/MS technique was successfully applied for determination of 16 benzimidazoles residue. The calibration curves of 16 benzimidazoles ranging from 0.5 to 50 ng/mL were established and the correlation coef�cients were
0.9993~0.9999. The LODs of the 16 benzimidazoles were 1 -2.2 µg/kg. The recoveries were in the range of 80.9%~118.5% for pork samples, with relative standard deviations less than 5%.
Table 4 Recovery of 16 drugs in pork sample
CompoundSpike Conc.
(µg/kg)
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
Sample Conc.(µg/kg)
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
Measured Conc.(µg/kg)
9.5
8.1
9.8
10.0
11.4
9.6
9.6
11.8
11.3
11.8
9.8
10.4
9.3
10.8
9.6
9.1
Recovery(%)
94.5
80.9
98.2
99.8
113.8
96.3
96.1
118.5
112.8
118.3
97.8
103.6
92.6
107.8
96.1
90.7
Fenbendazole
Albendazole Sulfoxide
Thiabendazole
Thiabendazole-5-hydroxy
Oxfendazole
Albendazole
Albendazole -2-Aminosulfone
Albendazole sulfone
Mebendazole
Mebendazole-amine
5-Hydroxymebendazole
Flubendazole
2-Amino�ubendazole
Cambendazole
Oxibendazole
Oxfendazole
PO-CON1459E
High Sensitivity Quantitation Method of Dicyandiamide and Melamine in Milk Powders by Liquid ChromatographyTandem Mass Spectrometry
ASMS 2014 TP275
Zhi Wei Edwin Ting1, Jing Cheng Ng2*,
Jie Xing1 & Zhaoqi Zhan1
1 Customer Support Centre, Shimadzu (Asia Paci�c)
Pte Ltd, 79 Science Park Drive, #02-01/08, SINTECH IV,
Singapore Science Park 1, Singapore 1182642 Department of Chemistry, Faculty of Science,
National University of Singapore, 21 Lower Kent
Ridge Road, Singapore 119077, *Student
2
High Sensitivity Quantitation Method of Dicyandiamide and Melamine in Milk Powders by Liquid Chromatography Tandem Mass Spectrometry
IntroductionMelamine was found to be used as a protein-rich adulterant �rst in pet-food in 2007, and then in infant formula in 2008 in China [1]. The outbreak of the melamine scandal that killed many dogs and cats as well as led to death of six infants and illness of many had caused panic in publics and great concerns in food safety worldwide. Melamine was added into raw milk because of its high nitrogen content (66%) and the limitation of the Kjeldahl method for determination of protein level indirectly by measuring the nitrogen content. In fact, in addition to melamine and its analogues (cyanuric acid etc), a number of other nitrogen-rich compounds was reported
also to be potentially used as protein-rich adulterants, including amidinourea, biuret, cyromazine, dicyandiamide, triuret and urea [2]. Recently, low levels of dicyandiamide (DCD) residues were found in milk products from New Zealand [3]. Instead of addition directly as an adulterant, the trace DCD found in milk products was explained to be relating to the grass “contaminated by DCD”. Dicyandiamide has been used to promote the growth of pastures for cows grazing. We report here an LC/MS/MS method for sensitive detection and quanti�cation of both dicyandiamide (DCD) and melamine in infant milk powder samples.
ExperimentalHigh purity dicyandiamide (DCD) and melamine were obtained from Sigma Aldrich. Amicon Ultra-4 (MWCO 5K) centrifuge �ltration tube (15 mL) obtained from Millipore was used in sample pre-tretment. The milk powder sample was pre-treated according to a FDA method [1] with some
modi�cation as illustrated in Figure 1. The �nal clear sample solution was injected into LC/MS/MS for analysis. Stock solutions of DCD and melamine were prepared in pure water.
Table 1: Analytical conditions of DCD and melamine in milk powders on LCMS-8040
Fig 1: Sample pre-treatment work�ow
LC conditions
Alltima HP HILIC 3µ, 150 x 2.10mm
0.2 mL/min
A: 0.1 % formic acid in H2O/ACN (5:95 v/v)B: 20mM Ammonium Formate in H2O/ACN (50:50 v/v)
Gradient elution: 5% (0.01 to 3.0 min) → 95% (3.5 to 5.0 min) → 5% (5.5 to 9.0 min)
40ºC
5 µL
Column
Flow Rate
Mobile Phase
Elution Mode
Oven Temperature
Injection Volume
MS conditions
ESI
Positive
400ºC
300ºC
Ar (230kPa)
N2, 2.0L/min
N2, 15.0L/min
Interface
MS mode
Block Temperature
DL Temperature
CID Gas
Nebulizing Gas Flow
Drying Gas Flow
Weigh 2.0g of milk powder sample
Add 14mL of 2.5% formic acid
(1) Sonicate for 1hr
(2) Centrifuge at 6000rpm for 10min
Transfer 4mL of supernatant to Amicon Ultra-4(MWCO 5K) centrifuge �ltration tube (15mL)
Filter the �ltrate by a 0.2um PTFE syringe �lter
Collect clear �ltrate
To 50uL of �ltrate added 950uL of ACN
Further 10x dilution with ACN
LC/MS/MS analysis
Centrifuge at 7500rpm for 10min
3
High Sensitivity Quantitation Method of Dicyandiamide and Melamine in Milk Powders by Liquid Chromatography Tandem Mass Spectrometry
An LCMS-8040 triple quadrupole LC/MS/MS (Shimadzu Corporation, Japan) was used in this work. The system is consisted of a high pressure binary gradient Nexera UHPLC coupled with a LCMS-8040 MS system. An Alltima HP HILIC column was used for separation of DCD and
melamine with a gradient program developed (Table 1). The details of the LC and MS conditions are shown in Table 1. A set of calibrants (0.5, 1.0, 2.5, 5 and 10 ppb) was prepared from the stock solutions using of ACN/water (90/10) as diluent.
Results and Discussion
MRM optimization of DCD and melamine were performed using an automated MRM optimization program of the LabSolutions. The precursors were the protonated ions of DCD and melamine. Two optimized MRM transitions of each compound were selected and used for quantitation and confirmation. The MRM transitions and parameters are shown in Table 2.
MRM optimization
A LC/MS/MS method was developed for quantitation of DCD and melamine based on the MRM transitions in Table 2. Under the HILIC separation conditions (Table 1), DCD and melamine eluted at 2.55 min and 6.29 min as sharp peaks (see Figures 4 & 5). Figures 2 and 3 show the
calibration curves of DCD and melamine standard in neat solutions and in milk matrix solutions (spiked). The linearity with correlation coefficient (R2) greater than 0.997 across the calibration range of 0.5~10.0 ng/mL was obtained for both compounds in both neat solution and matrix (spiked).
Method Development
Figure 2: Calibration curves of DCD and melamine in neat solution
Table 2: MRM transitions and optimized parameters
Name Transition (m/z)Q1 Pre Bias Q3 Pre Bias
DCD
MEL
RT (min)
2.55
6.29
85.1 > 68.1
85.1 > 43.0
127.1 > 85.1
127.1 > 68.1
-15
-15
-26
-26
Voltage (V)
CE
-21
-17
-20
-27
-26
-17
-17
-26
0.0 2.5 5.0 7.5 Conc.0.0
2.5
5.0
7.5
Area (x10,000)
0.0 2.5 5.0 7.5 Conc.0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Area(x100,000)
DCD (85.1>68.1)R2 = 0.997
Melamine (127.1>85.1)R2 = 0.999
4
High Sensitivity Quantitation Method of Dicyandiamide and Melamine in Milk Powders by Liquid Chromatography Tandem Mass Spectrometry
Figure 3: Calibration curves of DCD and melamine spiked in milk powder matrix
Figure 4: Overlapping of six MRM peaks of 0.5 ng/mL DCD and melamine in neat solution
Figure 5: Overlapping of six MRM peaks of 0.5 ng/mL DCD and melamine in milk powder matrix
The repeatability of the method was evaluated at the levels of 0.5 ng/mL and 1.0 ng/mL. Figures 4 & 5 show the MRM chromatograms of DCD and melamine of six consecutive
injections of 0.5 ng/mL level with and without matrix. The peak area %RSD for the two analytes were lower than 9.2% (see Table 3).
Performance Evaluation
5.5 6.0 6.5 min0.0
1.0
2.0
3.0
4.0
5.0
(x1,000)
2.00 2.25 2.50 2.75 min
0.00
0.25
0.50
0.75
1.00
(x1,000)
DCD(85.1>68.1)
Melamine (127.1>85.1)
2.00 2.25 2.50 2.75 min
0.0
1.0
2.0
3.0
4.0
5.0
6.0(x100)
5.5 6.0 6.5 min0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5(x1,000)
DCD (85.1>68.1)
Melamine (127.1>85.1)
0.0 2.5 5.0 7.5 Conc.0.0
1.0
2.0
3.0
4.0
5.0
Area(x10,000)
0.0 2.5 5.0 7.5 Conc.0.0
0.5
1.0
1.5
2.0
2.5Area(x100,000)
Melamine (127.1>85.1)
R2 = 0.997
DCD (85.1>68.1)
R2 = 0.998
Table 3: Results of repeatability and sensitivity evaluation of DCD and melamine (n=6)
Sample %RSD LOD (ng/mL) LOQ (ng/mL)
In solvent
In matrix
Compd.
DCD
MEL
DCD
MEL
Conc. (ng/mL)
0.5
1.0
0.5
1.0
0.5
1.0
0.5
1.0
5.9
5.3
5.5
2.6
5.9
8.2
9.2
2.4
0.03
0.03
0.05
0.05
0.10
0.09
0.16
0.15
5
High Sensitivity Quantitation Method of Dicyandiamide and Melamine in Milk Powders by Liquid Chromatography Tandem Mass Spectrometry
The LOD and LOQ were estimated from the results of 0.5 ng/mL in both neat and matrix solution. The LOD and LOQ results were summarized in Table 3. The method achieved LOQs (in matrix) of 0.16 and 0.15 ng/mL (ppb) for DCD and melamine, respectively. Tables 4 & 5 show the results of matrix effect and recovery of the method. The matrix effects for DCD and melamine in the whole concentration ranges were at 64%~70%
and 62%~73%, respectively. The recovery was determined by comparing the results of pre-spiked and post-spiked mixed samples of DCD and melamine in the milk powder matrix (2.5 ng/mL each compound). The chromatograms of these samples are shown in Figure 6. The recovery of DCD and melamine were determined to be 103% and 105% respectively.
Figure 6: MRM peaks of DCD and melamine in pre- and post-spiked samples of 2.5 ng/mL (each). DCD and melamine were not detected in blank matrix of milk powder.
Table 4: Matrix effect (%) of DCD and melamine in milk powder matrix
Conc. (ng/mL) 2.5 5 10
DCD
MEL
66.9
73.1
1
65.4
62.5
0.5
70.4
62.2
64.8
68.9
66.6
68.0
Table 5: Recovery of DCD and melamine determined with spiked sample of 2.5 ng/mL
Compound Pre-spiked Area Post-spiked Area Recovery (%)
DCD
MEL
14,393
65,555
13,987
62,659
102.9
104.6
Melamine Pre-spiked
Melamine Post-spiked
DCD Post-spiked
DCD Pre-spiked
2.00 2.25 2.50 2.75 3.00
0
1000
2000
3000
4000
5000
6000
7000 1:85.10>43.00(+)1:85.10>68.05(+)
Dic
yand
iam
ide
2.00 2.25 2.50 2.75 3.00
0
1000
2000
3000
4000
5000
6000
7000 1:85.10>43.00(+)1:85.10>68.05(+)
6.00 6.25 6.50 6.75
0
2500
5000
7500
10000
12500
15000
175002:127.10>68.05(+)2:127.10>85.10(+)
6.00 6.25 6.50 6.75
0
2500
5000
7500
10000
12500
15000
175002:127.10>68.05(+)2:127.10>85.10(+)
Mel
amin
e
6.00 6.25 6.50 6.75
0
2500
5000
7500
10000
12500
15000
175002:127.10>68.05(+)2:127.10>85.10(+)
Mel
amin
e
2.00 2.25 2.50 2.75 3.00
0
1000
2000
3000
4000
5000
6000
7000 1:85.10>43.00(+)1:85.10>68.05(+)
Dic
yand
iam
ide
Blank matrix of milk powder
Blank matrix of milk powder
High Sensitivity Quantitation Method of Dicyandiamide and Melamine in Milk Powders by Liquid Chromatography Tandem Mass Spectrometry
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
ConclusionsA high sensitivity LC/MS/MS method was developed on LCMS-8040 for detection and quantitation of dicyandiamide (DCD) and melamine in milk powders. The method performance was evaluated using infant milk powders as the matrix. The method achieved LOQ of 0.16
ng/mL for both compounds in the matrix, allowing its application in simultaneous analysis of melamine, a protein adulterant in relatively high concentration, and dicyandiamide residue in trace level in milk powders samples.
References1. S. Turnipseed, C. Casey, C. Nochetto, D. N. Heller, FDA Food, LIB No. 4421, Volume 24, October 2008.2. S. MachMahon, T. H. Begley, G. W. Diachenko, S. A. Stromgren, Journal of Chromatography A, 1220, 101-107 (2012).3. http://www.naturalnews.com/041834_Fonterra_milk_powder_dicyandiamide.html
PO-CON1465E
Multiresidue pesticide analysis fromdried chili powder using LC/MS/MS
ASMS 2014 WP350
Deepti Bhandarkar, Shruti Raju, Rashi Kochhar,
Shailesh Damale, Shailendra Rane, Ajit Datar,
Jitendra Kelkar, Pratap Rasam
Shimadzu Analytical (India) Pvt. Ltd., 1 A/B Rushabh
Chambers, Makwana Road, Marol, Andheri (E),
Mumbai-400059, Maharashtra, India.
2
Multiresidue pesticide analysis from dried chili powder using LC/MS/MS
IntroductionPesticide residues in foodstuffs can cause serious health problems when consumed. LC/MS/MS methods have been increasingly employed in sensitive quanti�cation of pesticide residues in foods and agriculture products. However, matrix effect is a phenomenon seen in Electro Spray Ionization (ESI) LC/MS/MS analysis that impacts the data quality of the pesticide analysis, especially for complex matrix like spice/herb.Chili powder is one such complex matrix that can exhibit matrix effect (either ion suppression or enhancement). A calibration curve based on matrix matched standards can demonstrate true sensitivity of analyte in presence of
matrix. Therefore, this approach was used to obtain more reliable and accurate data as compared to quantitation against neat (solvent) standards[1].Multiresidue, trace level analysis in complex matrices is challenging and tedious. Feature of automatic MRM optimization in LCMS-8040 makes method development process less tedious. In addition, the lowest dwell time and pause time along with ultra fast polarity switching (UFswitching) enables accurate, reliable and high sensitive quantitation. UFsweeperTM II technology in the system ensures least crosstalk, which is very crucial for multiresidue pesticide analysis.
Method of Analysis
Commercially available red chili was powdered using mixer grinder. To 1 g of this chili powder, 20 mL water:methanol (1:1 v/v) was added and the mixture was sonicated for 10 mins. The mixture was centrifuged and supernatant was collected. This supernatant was used as diluent to prepare
pesticide matrix matched standards at concentration levels of 0.01 ppb, 0.02 ppb, 0.05 ppb, 0.1 ppb, 0.2 ppb, 0.5 ppb, 1 ppb, 2 ppb, 5 ppb, 10 ppb and 20 ppb. Each concentration level was then filtered through 0.2 µ nylon filter and used for the analysis.
Sample Preparation
Pesticides were analyzed using Ultra High Performance Liquid Chromatography (UHPLC) Nexera coupled with LCMS-8040 triple quadrupole system (Shimadzu
Corporation, Japan), shown in Figure 1. The details of analytical conditions are given in Table 1.
LC/MS/MS Analytical Conditions
Table 1. LC/MS/MS analytical conditions
• Column : Shim-pack XR-ODS (75 mm L x 3 mm I.D.; 2.2 µm)
• Guard column : Phenomenex SecurityGuard ULTRA Cartridge
• Mobile phase : A: 5 mM ammonium formate in water:methanol (80:20 v/v)
B: 5 mM ammonium formate in water:methanol (10:90 v/v)
• Flow rate : 0.2 mL/min
• Oven temperature : 40 ºC
• Gradient program (B%) : 0.0–1.0 min → 45 (%); 1.0–13.0 min → 45-100 (%);
13.0–18.0 min → 100 (%); 18.0–19.0 min → 100-45 (%);
19.0–23.0 min → 45 (%)
• Injection volume : 15 µL
• MS interface : ESI
• Polarity : Positive and negative
• Nitrogen gas �ow : Nebulizing gas 2 L/min; Drying gas 15 L/min
• MS temperature : Desolvation line 250 ºC; Heat block 400 ºC
• MS analysis mode : Staggered MRM
3
Multiresidue pesticide analysis from dried chili powder using LC/MS/MS
ResultsLC/MS/MS method was developed for analysis of 80 pesticides belonging to different classes like carbamate, organophosphate, urea, triazines etc. in a single run[2]. LOQ was determined for each pesticide based on the following criteria – (1) % RSD for area < 16 % (n=3), (2) % Accuracy between 80-120 % and (3) Signal to noise ratio (S/N) > 10.
LOQ achieved for 80 pesticides have been summarized in Table 2 and results for LOQ and linearity for each pesticide have been given in Table 3. Representative MRM chromatogram of pesticide mixture at 1 ppb level is shown in Figure 2. Representative MRM chromatograms at LOQ level for different classes of pesticides are shown in Figure 3.
Figure 1. Nexera with LCMS-8040 triple quadrupole system by Shimadzu
Table 2: Summary of LOQ achieved
LOQ (ppb)
Number of pesticides
0.01
1
0.02
1
0.05
3
0.1
8
0.2
17
0.5
24
1
26
Table 3. Results of LOQ and linearity for pesticide analysis
MRM Transition Polarity LOQ (ppb) Linearity (R2)
746.20>142.10
421.90>366.10
301.00>198.00
732.20>142.10
371.00>273.10
222.90>126.00
221.70>123.00
229.80>198.90
387.90>301.00
387.90>301.00
207.00>72.10
305.70>108.00
408.90>186.00
Name of compound
Spinosyn D
Fenpyroximate
Bifenazate
Spinosyn A
Spiromesifen
Acetamiprid
Carbofuran
Dimethoate
Dimethomorph I
Dimethomorph II
Isoproturon
Pirimiphos methyl
Tri�oxystrobin
Sr. No.
1
2
3
4
5
6
7
8
9
10
11
12
13
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
0.01
0.02
0.05
0.05
0.05
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.9987
0.9915
0.9947
0.9974
0.9957
0.9910
0.9971
0.9970
0.9991
0.9992
0.9984
0.9997
0.9989
4
Multiresidue pesticide analysis from dried chili powder using LC/MS/MS
MRM Transition Polarity LOQ (ppb) Linearity (R2)
367.70>198.85
215.90>174.00
235.90>143.00
324.85>108.10
310.60>111.00
384.70>198.80
434.70>330.00
248.80>159.90
283.90>252.00
267.90>174.90
367.80>181.90
299.90>173.90
252.90>126.00
257.90>125.10
354.90>88.00
293.90>196.90
189.90>162.90
208.10>116.05
411.10>190.10
338.00>99.10
305.70>201.00
349.90>266.00
483.75>452.90
357.90>280.80
363.70>193.90
315.90>247.00
313.90>70.10
352.90>227.90
507.70>167.00
288.70>205.00
314.90>99.00
330.90>284.90
411.90>356.20
280.00>220.10
221.70>150.00
162.90>88.00
362.15>303.00
283.90>70.10
260.80>75.00
276.80>96.90
342.90>151.00
890.30>305.10
333.70>139.00
Name of compound
Anilophos
Atrazine
Carboxin
Cyazofamid
Edifenphos
Ethion
Fipronil
Linuron
Metolachlor
Oxycarboxin
Phosalone
Phosphamidon
Thiacloprid
Thiobencarb
Thiodicarb
Triadimefon
Tricyclazole
Aldicarb
Benfuracarb
Bitertanol
Buprofezin
Clodinafop propargyl
Chlorantraniliprole
Diclofop methyl
Flufenacet
Flusilazole
Hexaconazole
Hexythiazox
Iodosulfuron methyl
Iprobenfos
Malaoxon
Malathion
Mandipropamid
Metalaxyl
Methabenzthiazuron
Methomyl
Oxadiazon
Penconazole
Phorate
Phorate sulfoxide
Thiophanate methyl
Avermectin B1a
Carpropamid
Sr. No.
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
Positive
Positive
Positive
Positive
Positive
Positive
Negative
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
1
1
0.9974
0.9985
0.9952
0.9971
0.9997
0.9957
0.9973
0.9945
0.9966
0.9995
0.9987
0.9997
0.9976
0.9977
0.9906
0.9994
0.9977
0.9962
0.9981
0.9935
0.9933
0.9978
0.9994
0.9976
0.9997
0.9983
0.9996
0.9909
0.9971
0.9981
0.9996
0.9997
0.9952
0.9996
0.9957
0.9988
0.9963
0.9992
0.9987
0.9991
0.9996
0.9990
0.9985
5
Multiresidue pesticide analysis from dried chili powder using LC/MS/MS
Figure 2. MRM chromatogram of pesticide mixture at 1 ppb level
MRM Transition Polarity LOQ (ppb) Linearity (R2)
241.90>127.00
415.30>186.00
198.90>128.10
385.00>329.10
310.80>158.00
228.10>60.00
886.30>158.10
311.90>236.10
330.70>268.00
306.95>57.10
229.90>202.70
680.90>254.05
247.90>129.00
331.00>116.00
293.90>70.10
328.90>125.00
281.90>212.10
372.70>302.70
368.00>231.10
209.90>110.90
414.90>182.00
321.90>96.10
201.90>103.90
246.80>89.10
Name of compound
Clomazone
Clorimuron ethyl
Cymoxanil
Diafenthiuron
Di�ubenzuron
Dodine
Emamectin benzoate
Fenamidone
Fenarimol
Fenazaquin
Flonicamid
Flubendiamide
Forchlorfenuron
Kresoxim methyl
Paclobutrazol
Pencycuron
Pendimethalin
Profenofos
Propargite
Propoxur
Pyrazosulfuron ethyl
Pyriproxyfen
Simazine
Thiomethon
Sr. No.
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Negative
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0.9967
0.9965
0.9949
0.9961
0.9982
0.9980
0.9983
0.9997
0.9900
0.9992
0.9971
0.9993
0.9956
0.9996
0.9974
0.9943
0.9932
0.9966
0.9950
0.9987
0.9992
0.9975
0.9992
0.9989
5.0 10.0 15.0 min
0
10000
20000
30000
40000
50000
Multiresidue pesticide analysis from dried chili powder using LC/MS/MS
6
Figure 3. Representative MRM chromatograms at LOQ level from different classes of pesticides
Conclusion• A highly sensitive method was developed for analysis of 80 pesticides belonging to different classes, from dried chili
powder in a single run.• Ultra high sensitivity, ultra fast polarity switching (UFswitching), low pause time and dwell time along with UFsweeperTM
II technology enabled sensitive, selective, accurate and reproducible multiresidue pesticide analysis from complex matrix like dried chili powder.
10.0 11.0 12.0 13.0
0
2500
5000
7500 70:283.90>252.00(+)
Met
olac
hlor
Chloroacetanilide
11.0 12.0 13.0 14.0
0
1000
2000
3000
4000
5000
600080:283.90>70.10(+)
Penc
onaz
ole
Azole
10.0 11.0 12.0 13.0
0
1000
2000
3000
4000
5000126:680.90>254.05(-)
Flub
endi
amid
eAnthranilicDiamide
15.0 16.0 17.0 18.0
0
250
500
750
1000
1250
121:421.90>366.10(+)
Fenp
yrao
xim
atePyrazole
16.0 17.0 18.0 19.0
0
100
200
300
115:746.20>142.10(+)
Spin
osyn
D
MacrocyclicLactone
6.0 7.0 8.0 9.0
1000
2000
3000
4000 42:215.90>174.00(+)
Atr
azin
e
Triazine
7.0 8.0 9.0 10.0
0
1000
2000
3000
400044:207.00>72.10(+)
Isop
rotu
ron
Urea
2.0 3.0 4.0 5.0
0
250
500
750
1000
15:229.80>198.90(+)
Dim
etho
ate
Organophosphorus N
5.0 6.0 7.0 8.0
1000
2000
3000
4000
33:221.70>123.00(+)
Car
bofu
ran
-Methyl Carbamate
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Multiresidue pesticide analysis from dried chili powder using LC/MS/MS
References[1] Kwon H, Lehotay SJ, Geis-Asteggiante L., Journal of Chromatography A, Volume 1270, (2012), 235–245.[2] Banerjee K, Oulkar DP et al., Journal of Chromatography A, Volume 1173, (2007), 98-109.
PO-CON1463E
Multi pesticide residue analysis in tobacco by GCMS/MS using QuEChERSas an extraction method
ASMS 2014 TP762
Durvesh Sawant(1), Dheeraj Handique(1), Ankush Bhone(1),
Prashant Hase(1), Sanket Chiplunkar(1), Ajit Datar(1),
Jitendra Kelkar(1), Pratap Rasam(1), Kaushik Banerjee(2),
Zareen Khan(2)
(1) Shimadzu Analytical (India) Pvt. Ltd., 1 A/B Rushabh
Chambers, Makwana Road, Marol, Andheri (E),
Mumbai-400059, Maharashtra, India.
(2) National Referral Laboratory, National Research
Centre for Grapes, P.O. Manjri Farm, Pune-412307,
Maharashtra, India.
2
Multi pesticide residue analysis in tobacco by GCMS/MS using QuEChERS as an extraction method
IntroductionIndia is the world’s second largest producer (after China) and consumer (after Brazil) of tobacco with nearly $ 1001.54 million revenue generated annually from its export.[1] In countries like India, with tropical-humid climate, the incidences of insect attacks and disease infestations are frequent and application of pesticides for their management is almost obligatory. Like any other crop, tobacco (Nicotiana tabacum Linn.), one of the world’s leading high-value crops, is also prone to pest attacks, and the farmers do apply various pesticides as a control measure. The residues of pesticides applied on tobacco during its cultivation may remain in the leaves at harvest that may even sustain post harvest processing treatments and could appear in the �nal product. Thus, monitoring of pesticide residues in tobacco is an important issue of critical concern from public health and safety point of view demanding implementation of stringent regulatory policies.[2] To protect the consumers by controlling pesticide residue
levels in tobacco, the Guidance Residue Levels (GRL) of 118 pesticides have been issued by the Agro-Chemical Advisory Committee (ACAC) of the Cooperation Center for Scienti�c Research Relative to Tobacco (CORESTA). Tobacco is a complex matrix and hence requires selective extraction and extensive cleanup such as QuEChERS (Quick Easy Cheap Effective Rugged Safe) to ensure trace level detection with adequate precision and accuracy. The objective of the present study was to develop an effective, sensitive and economical multi-pesticide residue analysis method for 203 pesticides in tobacco as listed in Table 1.
Figure 1. Dried tobacco
Method of Analysis
Extraction of pesticides was done using QuEChERS method, as described below.[3]
Extraction of pesticides from tobacco
Take 2 g of dry powdered tobacco leaves (Figure 1). Add 18 mL of water containing 0.5 % acetic acid. Homogenize the sample and Keep it for 30 min.
Add 10 mL ethyl acetate. Immediately, put 10 g sodium sulfate.
Homogenize it thoroughly at 15000 rpm for 2 min.
Centrifuge at 5000 rpm for 5 min for phase separation.
Draw 3 mL of ethyl acetate upper layer from the extract for further cleanup.
3
Multi pesticide residue analysis in tobacco by GCMS/MS using QuEChERS as an extraction method
Figure 2. GCMS-TQ8030 Triple quadrupole system by Shimadzu
• ASSP™ (Advanced Scanning Speed Protocol) enables high-speed scan and data acquisition for accurate quantitation at 20,000 u/sec
• Capable of performing simultaneous Scan/MRM• UFsweeper® technology efficiently sweeps residual ions from the collision cell for fast, efficient ion transport ensuring no
cross-talk• Two overdrive lenses reduce random noise from helium, high-speed electrons and other factors to improve S/N ratio• Flexible platform with EI (Electron Ionization), CI (Chemical Ionization), and NCI (Negative Chemical Ionization)
techniques• Full complement of acquisition modes including MRM, Scan/MRM, Precursor Ion, Product Ion and Neutral Loss Scan
Key Features of GCMS-TQ8030
Add 1 mL toluene to it and vortex for 0.5 min.
Add cleanup mixture [PSA (150 mg), C18 (150 mg), GCB (75 mg) and anhydrous MgSO4 (300 mg)] and vortex for 2 min.
Centrifuge the mixture at 7000 rpm for 7 min.
Collect the supernatant and �lter through a 0.2 µm PTFE membrane �lter.
Inject 2.0 µL of the clean extract into GCMS-TQ8030 (Figure 2).
4
Multi pesticide residue analysis in tobacco by GCMS/MS using QuEChERS as an extraction method
Table 1. List of pesticides
Pesticide
2,6-Dichlorobenzamide
2-Phenylphenol
3,4-Dichloraniline
3-Chloroaniline
4-Bromo 2-Chloro phenol
4,4-Dichlorobenzophenone
Acetochlor
Acrinathrin
Alachlor
Aldrin
Azinphos-ethyl
Azinphos-methyl
Azoxystrobin
Barban
Be�ubutamid
Ben�uralin
Benoxacor
Beta-endosulfan
Bifenox
Bifenthrin
Bitertanol
Boscalid
Bromacil
Bromophos-ethyl
Bromopropylate
Bromuconazole-1
Bromuconazole-2
Butralin
Butylate
Carbaryl
Carbofuran
Carfentrazone
Chlordane-trans
Chlordecone
Chlorfenvinphos
Chlormephos
Chlorobenzilate
Chloroneb
Chlorothalonil
Chlorpyriphos-ethyl
Chlorpyriphos-methyl
Chlorpyriphos-oxon
Chlorthal-dimethyl
Cinidon-ethyl
Cis-1,2,3,6 tetrahydrophthalimide
Clodinafop propargyl
Clomazone
Crimidine
Cyanophos
Cy�uthrin-1
Cy�uthrin-2
Pesticide
Cy�uthrin-3
Cy�uthrin-4
Cyhalofop-butyl
Cypermethrin-2
Cypermethrin-3
Cypermethrin-4
Cyprodinil
Delta-HCH
Demeton-s-methyl
Demeton-S-methyl sulphone
Dialifos
Diazinon
Dichlobenil
Dichlo�uanid
Diclofop
Dicloran
Dieldrin
Diethofencarb
Difenoconazole-1
Difenoconazole-2
Di�ubenzuron
Di�ufenican
Dimethipin
Dimethomorph-1
Dimethomorph-2
Dimoxystrobin
Diniconazole
Dinoseb
Dinoterb
Dioxathion
Edifenfos
Endosulfan sulphate
Endrin
Epoxiconazole
Ethal�uralin
Ethoprophos
Etoxazole
Etridiazole
Etrimfos
Famoxadone
Fenamidone
Fenarimol
Fenbuconazole
Fenchlorphos
Fenchlorphos oxon
Fenhexamid
Fenobucarb
Fenoxycarb
Fenthion sulphoxide
Fenvalerate
Fipronil
Pesticide
Fipronil sulphone
Flucythrinate-1
Flucythrinate-2
Flufenacet
Flumoixazine
Fluquinconazole
Flurochloridone-1
Flurochloridone-2
Flutolanil
Flutriafol
Fluxapyoxad
Folpet
Fuberidazole
Heptachlor
Hexaconazole
Iprobenfos
Isoprocarb
Isoprothiolane
Isopyrazam
Isoxaben
Lactofen
Lambda-cyhalothrin
Malaoxon
Malathion
Mepanipyrim
Mepronil
Metalaxyl
Metalaxyl M
Metazachlor
Metconazole
Methabenzthiazuron
Methacrifos
Methidathion
Methiocarb
Metholachlor-s
Methoxychlor
Metribuzin
Mevinphos
Monolinuron
Myclobutanyl
Napropamide
Nitrapyrin
Oxadiargyl
Oxadiazon
Oxycarboxin
p,p-DDE
Parathion-ethyl
Parathion-methyl
Penconazole
Pencycuron (Deg.)
Pendimethalin
Pesticide
Permethrin-1
Permethrin-2
Pethoxamid
Phosalone
Phosmet
Pirimicarb
Pretilachlor
Procymidone
Profenofos
Propanil
Propaquizafop
Propazine
Propham
Propiconazole-1
Propisoclor
Propyzamide
Proquinazid
Pyra�ufen-ethyl
Pyrazophos
Pyrimethanil
Pyriprooxyfen
Pyroquilon
Quinoxyfen
Simazine
Spirodiclofen
Sulfotep
Swep
Tebufenpyrad
Tebupirimfos
Tebuthiuron
Te�uthrin
Terbacil
Tetraconazole
Tetradifon
Thiobencarb
Tolyl�uanid
Tralkoxydim
Triadimefon
Tri-allate
Triazophos
Tricyclazole
Tri�oxystrobin
Tri�umizole
Tri�umuron
Tri�uralin
Tri�usulfuron
Triticonazole
Valifenalate
Vinclozolin
Zoxamide (Deg.)
Sr. No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
Sr. No.
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
Sr. No.
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
Sr. No.
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
5
Multi pesticide residue analysis in tobacco by GCMS/MS using QuEChERS as an extraction method
The analysis was carried out on Shimadzu GCMS-TQ8030 as per the conditions given below.
GCMS/MS Analytical Conditions
Chromatographic parameters
• Column : Rxi-5Sil MS (30 m L x 0.25 mm I.D.; 0.25 µm)
• Injection Mode : Splitless
• Sampling Time : 2.0 min
• Split Ratio : 5.0
• Carrier Gas : Helium
• Flow Control Mode : Linear Velocity
• Linear Velocity : 40.2 cm/sec
• Column Flow : 1.2 mL/min
• Injection Volume : 2.0 µL
• Injection Type : High Pressure Injection
• Total Program Time : 41.87 min
• Column Temp. Program : Rate (ºC /min) Temperature (ºC) Hold time (min)
70.0 2.00
25.00 150.0 0.00
3.00 200.0 0.00
8.00 280.0 10.00
Mass Spectrometry parameters
• Ion Source Temp. : 230.0 ºC
• Interface Temp. : 280.0 ºC
• Ionization Mode : EI
• Acquisition Mode : MRM
ResultsFor MRM optimisation, well resolved pesticides were grouped together. Standard solution mixture of approximately 1 ppm concentration was prepared and analyzed in Q3 scan mode to determine the precursor ion for individual pesticides. Selected precursor ions were allowed to pass through Q1 & enter Q2, also called as Collision cell. In Collision cell, each precursor ion was bombarded with collision gas (Argon) at different energies (called as Collision Energy-CE) to produce fragments (product ions). These product ions were further scanned in Q3 to obtain their mass to charge ratio. For each precursor ion, product ion with highest intensity and its
corresponding CE value was selected, thereby assigning a characteristic MRM transition to every pesticide. Based on MRM transitions, the mixture of 203 pesticides was analyzed in a single run (Figure 3). Method was partly validated for each pesticide with respect to linearity (0.5 to 25 ppb), reproducibility, LOQ and recovery. The validation summary for two pesticides namely Mevinphos and Parathion-ethyl (Sr. Nos.140 and 149 in Table 1) is shown in Figures 4 and 5. The summary data of linearity and LOQ for 203 pesticides is given in Table 2 and 3 respectively.
Multi pesticide residue analysis in tobacco by GCMS/MS using QuEChERS as an extraction method
6
Figure 3. MRM Chromatogram for 203 pesticides mixture
Figure 4. Summary data for mevinphos
Calibration overlay Linearity curve Recovery overlay
Linearity (R2)
0.9999
LOD (ppb)
0.3
LOQ (ppb)
1
S/N at LOQ
173
% RSD at LOQ(n=6)
6.93
% Recoveryat LOQ
89.28
Figure 5. Summary data for parathion-ethyl
Calibration overlay Linearity curve Recovery overlay
Linearity (R2)
0.9993
LOD (ppb)
1.5
LOQ (ppb)
5
S/N at LOQ
93
% RSD at LOQ(n=6)
4.05
% Recoveryat LOQ
109.10
10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 min-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
(x100,000)
15.0 15.5 16.0 16.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5(x10,000)
15.0 15.5 16.0 16.5
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0 (x1,000)
0.0 5.0 10.0 15.0 20.0 Conc.0.00
0.25
0.50
0.75
1.00
1.25
1.50Area (x100,000)
min min
7.25 7.50 7.75 8.00 8.25 8.50
0.00
0.25
0.50
0.75
1.00
(x10,000)
0.0 5.0 10.0 15.0 20.0 Conc.0.0
0.5
1.0
1.5
2.0
2.5Area (x100,000)
7.0 7.5 8.0 8.5 9.0
0.0
1.0
2.0
3.0
4.0
5.0
(x10,000)
min min
MRM : 192.00>127.00 MRM : 192.00>127.00
MRM : 291.10>137.00 MRM : 291.10>137.00
Post extraction spikePre extraction spike
Post extraction spikePre extraction spike
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Multi pesticide residue analysis in tobacco by GCMS/MS using QuEChERS as an extraction method
Conclusion• A highly sensitive method was developed for quantitation of 203 pesticides in complex tobacco matrix by using
Shimadzu GCMS-TQ8030. • The MRM method developed for 203 pesticides can be used for screening of pesticides in various food commodities. For
90 % of the pesticides, the LOQ of 10 ppb or below was achieved. • Ultra Fast scanning, UFsweeper® and ASSP™ features enabled sensitive, selective, fast, reproducible, linear and accurate
method of analysis.
Reference[1] Tobacco Board (Ministry of Commerce and Industry, Government of India), Exports performance during 2013-14,
(2014), 1. http://tobaccoboard.com/admin/statistics�les/Exp_Perf_Currentyear.pdf
[2] CORESTA GUIDE Nº 1, The concept and implementation of cpa guidance residue levels, (2013), 4. http://www.Coresta.org/Guides/Guide-No01-GRLs%283rd-Issue-July13%29.pdf
[3] Zareen S Khan, Kaushik Banerjee, Rushali Girame, Sagar C Utture et al., Journal of Chromatography A, Volume 1343, (2014), 3.
Table 2. Linearity Summary
Linearity (R2)
0.9950 - 1.0000
0.9880 - 0.9950
Sr. No.
1
2
Number ofpesticides
193
10
Sr. No.
1
2
3
4
LOQ (ppb)
1
5
10
25
Number ofpesticides
15
18
158
12
% RSD range(n=6)
6 – 15
3 – 15
0.95 – 15
1 – 10
S/N Ratiorange
16 – 181
19 – 502
10 – 14255
19 – 660
% Recoveryrange
70 – 130
Table 3. LOQ Summary
PO-CON1453E
Simultaneous quantitative analysis of20 amino acids in food samples withoutderivatization using LC-MS/MS
ASMS 2014 TP 510
Keiko Matsumoto1; Jun Watanabe1; Itaru Yazawa2
1 Shimadzu Corporation, Kyoto, Japan;
2 Imtakt Corporation, Kyoto, Japan
2
Simultaneous quantitative analysis of 20 amino acids in food samples without derivatization using LC-MS/MS
IntroductionIn order to detect many kinds of amino acids with high selectivity in food samples, the LC/MS analysis have been used widely. Amino acids are high polar compound, so they are hard to be retained to reverse-phased column such as ODS (typical method in LC/MS analysis). It needs their derivartization or addition of ion pair reagent in mobile phase to retain them. For easier analysis of amino
acids, it is expected to develop the method without using reagents mentioned above.This time, we tried to develop a simultaneous high sensitive analysis method of 20 amino acids by LC/MS/MS with mix-mode column (ion exchange, normal-phase) and the typical volatile mobile phase suitable for LC/MS analysis.
Methods and MaterialsAmino acid standard regents and food samples were purchased from the market. Standards of 20 kinds of amino acids were optimized on each compound-dependent parameter and MRM transition. As an LC-MS/MS system, HPLC was coupled to triple
quadrupole mass spectrometer (Nexera with LCMS-8050, Shimadzu Corporation, Kyoto, Japan). Sample was eluted with a binary gradient system and LC-MS/MS with electrospray ionization was operated in multiple-reaction-monitoring (MRM) mode.
Result
First, MRM method of 20 amino acids was optimized. As a result, all compounds were able to be detected high sensitively and were detected in positive MRM transitions. As the setting temperature of ESI heating gas was found to affected on the sensitivity of amino acids, it was also
optimized. Even though amino acids were not derivartized and ion-pairing reagent wasn’t used, 20 amino acids were retained by using a mixed-mode stationary phase structure and separated excellently on the below-mentioned condition.
Method development
Figure 1 LCMS-8050 triple quadrupole mass spectrometer
High Speed Mass Spectrometer
UF-MRM High-Speed MRM at 555ch/sec
UFswitching High-Speed Polarity Switching 5msec
3
Simultaneous quantitative analysis of 20 amino acids in food samples without derivatization using LC-MS/MS
Figure 2 Mass Chromatograms of 20 Amino acids (concentration of each compound : 10nmol/mL)
HPLC conditions (Nexera system)
Column : Intrada Amino Acid (3.0mmI.D. x 50mm, 3um, Imtakt Corporation, Kyoto, Japan)
Mobile phase
Case1
A : Acetonitrile / Formic acid = 100 / 0.1
B : 100mM Ammonium formate
Time program : B conc.14%(0-3 min) -100%(10min) - 14%(10.01-15min)
Case2 (High Resolution condition)
A : Acetonitrile / Tetrahydrofuran / 25mM Ammonium formate / formic acid = 9 / 75 / 16 / 0.3
B : 100mM Ammonium formate / Acetonitrile = 80 / 20
Time program : B conc.0%(0-2 min) - 5%(3min) - 30%(6.5min) - 100%(12min)
- 0%(12.01-17min)
Flow rate : 0.6 mL/min
Injection volume : 2 uL
Column temperature : 40 °C
MS conditions (LCMS-8050)
Ionization : ESI, Positive MRM mode
MRM transition are shown in Table 1.
Case1
Asn
Phe
Trp
Ile
Met
Pro
Tyr
Val
Ara
Thr
Glu (Cys)2
ArgLys
His
Gln
GlyAspSer
Mobile PhaseA: Acetonitrile / Formic acid = 100 / 0.1
B: 100mM Ammonium formate
2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min
4.4 4.5 4.6 4.7 4.8 4.9 5.0 5.1 5.2 min
Leu
4
Simultaneous quantitative analysis of 20 amino acids in food samples without derivatization using LC-MS/MS
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 min
PheTrpLeu
Met
Pro
TyrVal
ThrGlu (Cys)2
LysHis
Gln
Arg
Asn
AraGly
Asp Ser
Thr
4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5.0 5.1 5.2 min
Ile
In this study, two conditions of mobile phase were investigated. It was found that 20 amino acids were separated with higher resolution in case2. As the mobile phase condition of case1 is more simple and
the result of case1 was sufficiently well, case1 analytical condition was used for quantitative analysis. The dilution series of these compounds were analyzed. All amino acids were detected with good linearity and repeatability (Table1).
Figure 3 Mass Chromatograms of 20 Amino acids (concentration of each compound : 10nmol/mL)
Case2 (High Resolution condition)
Mobile PhaseA: Acetonitrile / Tetrahydrofuran / 25mM Ammonium formate / formic acid = 9 / 75 / 16 / 0.3
B: 100mM Ammonium formate / Acetonitrile = 80 / 20
Simultaneous quantitative analysis of 20 amino acids in food samples without derivatization using LC-MS/MS
5
Table1 Linearity and Repeatability of 20 amino acids
Trp
Phe
Tyr
Met
Lue, Lle
Val
Glu
Pro
Asp
Thr
Ala
Ser
Gln
Gly
Asn
(Cys)2
His
Lys
Arg
Range (nmol/mL)MRM Transition
205.10>188.10
166.10>120.10
182.10>136.00
150.10>56.10
132.10>86.15
118.10>72.05
148.10>84.10
116.10>70.10
134.20>74.10
120.10>74.00
90.10>44.10
106.10>60.20
147.10>84.10
76.20>29.90
133.10>74.05
241.00>151.95
156.10>110.10
147.10>84.10
175.10>70.10
Linearity
0.01-100
0.01-100
0.05-100
0.05-200
0.01-100
0.05-100
0.05-10
0.01-50
0.5-500
0.1-50
0.5-500
0.5-500
0.05-1
5-200
0.05-20
0.05-20
0.05-200
0.05-5
0.01-100
Coef�cient (r2)
0.9950
0.9971
0.9900
0.9963
0.9955
0.9991
0.9965
0.9933
0.9953
0.9923
0.9989
0.9988
0.9959
0.9974
0.9939
0.9909
0.9983
0.9908
0.9956
Repeatability*
%RSD
1.4
1.2
1.7
0.1
0.7
1.9
4.5
1.5
1.4
4.5
16.2
6.5
3.9
11.0
6.1
2.3
1.7
0.9
0.5
The analysis of the amino acids contained in sports beverage on the market was carried out. In the case of sports beverage, all amino acids written in the package were detected.
The analysis of 20amino acids in food samples
Figure 4 Mass Chromatograms of Sports Beverage (100 fold dilution with 0.1N HCl)
*@ 0.5nmol/mL : except for Gly, 5nmol/mL : for Gly
2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min
Phe
TrpIle
Met
Pro
Tyr
Val
Ara
ThrGlu
Arg
Lys
His
Gly
AspSer
4.3 4.4 4.5 4.6 4.7 4.8 4.9 5.0 5.1 min
Thr
Leu
Sports Beverage
Simultaneous quantitative analysis of 20 amino acids in food samples without derivatization using LC-MS/MS
6
Furthermore, Japanese Sake, Beer and sweet cooking rice wine (Mirin) were analyzed using this method. Japanese Sake and Beer were diluted with 0.1N HCl. Sweet cooking rice wine was diluted in the same way after a deproteinizing
preparation. These were filtered through a 0.2um filter and then analyzed. MRM chromatograms of each food samples are shown in Figure 5,6,7. Amino acids of each sample were detected with high sensitivity.
Figure 5 Mass Chromatograms of Japanese Sake (100 fold dilution with 0.1N HCl)
Figure 6 Mass Chromatograms of Beer (10 fold dilution with 0.1N HCl)
Figure7 Mass Chromatograms of Sweet Cooking Rice Wine (100 fold dilution with 0.1N HCl)
2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min
4.4 4.5 4.6 4.7 4.8 4.9 5.0 5.1 5.2min
Phe
Trp IleMet
Pro
Tyr
Val
Thr
Glu(Cys)2
LysHis
Gln
Arg
Asn
Ala
Ala
Gly Ser
2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min
4.5 4.6 4.7 4.8 4.9 5.0 5.1 min
Phe
Trp
Ile
Met
Pro
Tyr
ValGlu
Gln
Ala
Thr
Gly
Ser
Asn
Asp
(Cys)2LysHis
Arg
2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min
4.5 4.6 4.7 4.8 4.9 5.0 5.1 minPhe
Trp
IleMet
Pro
Tyr
Val
AlaThr Gly
Asn
Ser
Glu Gln
Asp
(Cys)2Lys
HisArg
Leu
Leu
Leu
Sweet Cooking Rice Wine
Beer
Japanese Sake
Simultaneous quantitative analysis of 20 amino acids in food samples without derivatization using LC-MS/MS
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Conclusions• 20 amino acids could be separated without derivatization using a typical volatile mobile phase suitable for LC/MS analysis
and detected with high sensitivity.• This methods was able to be applied to the analysis of amino acids in various food samples.
Environment
• Page 170
Rapid screening and confirmation
analysis of polycyclic aromatic
hydrocarbons (PAHs) with DART mass
spectrometry
• Page 176
Fast and highly sensitive analysis of
multiple drugs in ground-, surface- and
wastewater
• Page 182
Multi-residue analysis of pyrethroids in soil
and sediment using QuEChERS by LC/MS/MS
PO-CON1455E
Rapid Screening and con�rmationanalysis of polycyclic aromatichydrocarbons (PAHs) with DARTmass spectrometry
ASMS 2014 MP 551
Yu Takabayashi1, Jun Watanabe2, Motoshi Sakakura3,
Teruhisa Shiota3
1 SHIMADZU TECHNO-RESEARCH, INC., Tokyo, Japan;
2 Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan;
3 AMR Inc., Meguro-ku, Tokyo, Japan
2
Rapid Screening and con�rmation analysis of polycyclic aromatic hydrocarbons (PAHs) with DART mass spectrometry
IntroductionRecently, the regulation of the content of the polycyclic aromatic hydrocarbons (PAHs) in goods which may put into a mouth or may contact is advanced and the technologies of measuring PAHs quickly are being developed. The ionizing principle of DART (Direct Analysis in Real Time) using the excitation helium gas is able to widely ionize the wide-range compounds and it may also be able to ionize
the compounds which are not ionized by ESI. Since PAHs is ionizable by DART, PAHs can be quickly screened by holding up a sample directly to DART. In this research, the technique detected by DART-MS was developed coupling with LC and DART analysis after carrying out LC separation was performed.
Methods and MaterialsCommercial PAHs were used for the sample. The samples were applied to DART MS with the solution formed in suitable concentration or the powder formed. Small amount of the samples were picked up and held in the DART ionization gas stream using glass capillaries. In LC-DART MS analysis, the mixed-solution of PAHs standard was prepared and applied to HPLC. After carrying out chromatogram separation using a reverse phased column,
LC-DART MS analysis was conducted by loading an eluate to a DART ionization area continuously. DART OS ion source and single/triple quadrupole type mass spectrometer were used for this experiment. PAHs measured in the detection mode which performs a full scan mode with positive and negative simultaneous ionization.
Figure 1 DART-OS ion source & LCMS-2020
High Speed Mass Spectrometer
Ufswitching High-Speed Polarity Switching 15msec Ufscanning High-Speed Scanning 15,000u/sec
MS condition (LCMS-2020; Shimadzu Corporation)
Ionization : DART (Direct Analysis in Real Time)
Heater Temperature (DART) : 300°C to 500°C
Measuring mode (MS) : Positive/Negative scanning simultaneously
3
Rapid Screening and con�rmation analysis of polycyclic aromatic hydrocarbons (PAHs) with DART mass spectrometry
ResultFirst, in order to verify whether PAHs ionizes in DART, PAH standard reagents were analyzed in DART-MS. Benzo[a]anthracene, acenaphthene, anthracene, etc. were used as typical PAHs. When benzo[a]anthracene was analyzed, in the positive spectrum, the signal at m/z 229 which is equivalent to [M+H]+ was detected. Moreover, in the negative spectrum, the signal at m/z 243 which is equivalent to [M+O-H]- was detected. Similarly, acenaphthene and anthracene could also be ionized by DART-MS and were able to be assigned as molecular related ion. Additionally pyrene and �uoranthene were also examined. As each of these is structural isomers mutually in structural-formula C16H10, in the negative spectrum, the signal of [M+O-H]- is detected by m/z 217 in each other, and either was not able to identify whether the detected signal is pyrene or �uoranthene in analysis by DART-MS without chromatogram separation.
Figure 2 DART mass chromatogram and mass spectrum of Benzo[a]anthraceneA: positive mass chromatogram, B: negative mass chromatogram (The area with the orange dashed line is the time when sample was held in DART.)
C: positive mass spectrum, D: negative mass spectrum
100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 m/z0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0Inten. (x1,000,000)
229.3
245.2
261.3
100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 m/z0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5Inten. (x100,000)
243.2
259.3 275.1 291.2277.1125.0 179.3 220.6
0
2500000
5000000
7500000
10000000
12500000 1:BPC(+)
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 min0
250000
500000
7500002:BPC(-)
[M+H]+
M+
[M+O-H]-
Positive
Negative
A
B
C
D
Benzo[a]anthracene
C18H12Fw 228
Positive TIC m/z 100-300
Negative TIC m/z 100-300
4
Rapid Screening and con�rmation analysis of polycyclic aromatic hydrocarbons (PAHs) with DART mass spectrometry
Figure 3 DART mass spectra of acenaphthene (positive), anthracene (positive), pyrene (positive/negative) and �uoranthene (positive/negative)
100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 m/z0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0Inten.(x100,000)
154.2
155.2
171.2
202.3220.2 253.3
102.3
130.2 187.3142.3 268.9
Positive
100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 m/z0.0
0.5
1.0
1.5
2.0
2.5
3.0
Inten.(x1,000,000)
179.2
195.2
211.2158.3 225.1
[M+H]+
M+
[M+H]+M+
100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 m/z0.00
0.25
0.50
0.75
1.00
Inten.(x10,000,000)
204.2
193.1 218.2
AcenaphtheneC12H10Fw 154
AnthraceneC14H10Fw 178
PyreneC16H10Fw 202
100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 m/z0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
Inten.(x100,000)
217.2
190.3
233.3179.2253.3 269.3226.3165.2
298.1
205.2
101.1
255.6 287.3115.5 137.1
[M+O-H]-
100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 m/z0.0
0.5
1.0
1.5
2.0
2.5
Inten.(x1,000,000)
208.3
194.2
122.3
220.3169.2
222.3183.2136.3108.2236.3
100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 m/z0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0Inten.(x100,000)
217.1
167.1233.3
208.3 247.0194.2 222.7181.1 256.2 270.9165.8 283.0
�uorantheneC16H10Fw 202
Positive
Positive
Negative
[M+O-H]-
Positive
Negative
Rapid Screening and con�rmation analysis of polycyclic aromatic hydrocarbons (PAHs) with DART mass spectrometry
5
Then, it examined the sample applied to DART separating with LC in order to perform chromatogram separation. As the suitable flow rate for DART ionization was thought to be approximately 10uL/min, the splitter
located between column and DART ionization stage. Furthermore, the closed interface was adopted for sensitivity improvement.
Figure 4 DART devices integrated with HPLC (AMR Inc.)
Analytical Condition
Column : Unison UK-C8 (2.0mmI.D. x 100mm, 3um, Imtakt Corporation, Kyoto, Japan)
Mobile phase : 1mM Ammonium formate / Acetonitrile=75/25
Flow rate : 0.2mL/min (to DART: 0.01mL/min)
DART heater temperature : 500°C
Ionization : Positive/Negative SIM mode
splitter
Pump
Injector
Column
Mobile phase
Rapid Screening and con�rmation analysis of polycyclic aromatic hydrocarbons (PAHs) with DART mass spectrometry
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Figure 4 LC-DART mass chromatogram(a) Typical compound for DART; Quinine
(b) PAH mixture (4 compounds)
ConclusionsDART mass spectrometer coupled with HPLC was valuable for confirmation analysis of polycyclic aromatic hydrocarbons (PAHs)
As a result, by measurement of each PAHs standard reagent, each retention time was able to be confirmed and also each PAH was able to be detected in each retention time in the measurement using a PAH mixed
sample. The conclusion of this examination was understood that DART MS is effective in quick screening, and also LC-DART MS is effective in the confirmation analysis of detected PAHs in analysis of PAHs.
SIM 325(+) Quinine
SIM 202(+) pyrene
0
25000
50000
3:202.00(+)
4000
5000
6000
70004:217.00(-)
5000
7500
10000
12500 3:228.00(+)
3000
4000
5000 3:154.00(+)
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min
5000
10000
15000
20000
25000 3:178.00(+)
0.0 2.5 5.0 7.5 10.0 12.5 min0
10000
20000
30000
40000
50000
600002:325.00(+)
SIM 217(-) pyrene
SIM 228(+)benzo[a]anthracene
SIM 154(+) acenaphthene
SIM 178(+) anthracene
(a)
(b)
PO-CON1448E
Fast and highly sensitive analysisof multiple drugs in ground-, surface- and wastewater
ASMS 2014 TP 583
Klaus Bollig1; Sven Vedder2, Anja Grüning2
1 Shimadzu Deutschland GmbH, Duisburg, Germany; 2 Shimadzu Europe GmbH, Duisburg, Germany
2
Fast and highly sensitive analysis of multiple drugs in ground-, surface- and wastewater
IntroductionMany pharmaceuticals from medical treatments are metabolized or partially degraded in the body. An even larger amount of these compounds is excreted intact and pollutes the aquatic environment. Relevant classes of drugs are human or veterinary antibiotics, antiepileptics, analgetics and lipid lowering drugs or radio-opaque substances. The extent of damage caused to the environment and the resulting health risk for humans or animals should not be underestimated, even though it is
not understood in detail so far. The requirement for universal, reliable and fast methods for drug determination in water is steadily increasing. Highly sensitive triple-quad-MS systems are suitable tools for the analysis of residues in ground-, surface- and wastewater, but development of a simple, rapid and robust method for simultaneous measurement of trace levels of various different classes of analytes in complex matrices is a challenge.
MethodThis study describes a fast LC-MS/MS method for the determination of trace levels of different classes of drugs in water. With online SPE no further sample pretreatment is necessary. The quaternary system with low pressure gradient eluent (LPGE) and solvent blending functionality renders addition of a third LC-Pump unnecessary. The
solvent blending function was further used for method development. High speed values for MRM recording and the fastest polarity switching time of 5 ms are essential physical parameters for a maximum of data points on various classes of analytes in different polarities during one single analysis.
One of the first steps during this automated process is the precursor ion selection, followed by m/z adjustment of the precursor. The collision energy is optimized for the most abundant fragments and finally the fragment m/z is
adjusted. Six optimization steps were performed via flow injection analysis, each taking 30 seconds (Figure 2). The result of these automated steps was the automatic generation of a final MRM method (Table 1).
LC-MS/MS Method Optimization
Figure 1. LCMS-8050 triple quadrupole mass spectrometer
3
Fast and highly sensitive analysis of multiple drugs in ground-, surface- and wastewater
Figure 2. Automated MRM Optimization of the drug Sulfamethazin
1st Step: m/z Precursor adjustment
5th Step: Setting Q3 Prerod Bias
2nd Step: Setting Q1 Prerod Bias
4th Step: m/z Product Ion adjustment
3rd Step: Product Ion / CE selection
6th Step: CE �ne tuning
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 min
-0.25
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
2.50
2.75
3.00
3.25
3.50
3.75
4.00
4.25
4.50
4.75
5.00
5.25
5.50
5.75
6.00
6.25(x1,000,000)
1:Sulfamethazin 279.70(+)1:Sulfamethazin 279.60(+)1:Sulfamethazin 279.50(+)1:Sulfamethazin 279.40(+)1:Sulfamethazin 279.30(+)1:Sulfamethazin 279.20(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.00(+)1:Sulfamethazin 278.90(+)1:Sulfamethazin 278.80(+)1:Sulfamethazin 278.70(+)
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 min
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
2.50
2.75
3.00
3.25
3.50
3.75
4.00
4.25
4.50
4.75
5.00
5.25
5.50
5.75
6.00
6.25
6.50
6.75
7.00
7.25(x1,000,000)
1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)1:Sulfamethazin 279.10(+)
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 min
-0.05
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
1.05
1.10
1.15
1.20
1.25
1.30
1.35
1.40
1.45
(x1,000,000)
3:Sulfamethazin 279.10>65.50(+) CE: -50.03:Sulfamethazin 279.10>65.40(+) CE: -50.03:Sulfamethazin 279.10>65.30(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.10(+) CE: -50.03:Sulfamethazin 279.10>65.00(+) CE: -50.03:Sulfamethazin 279.10>64.90(+) CE: -50.03:Sulfamethazin 279.10>64.80(+) CE: -50.03:Sulfamethazin 279.10>64.70(+) CE: -50.03:Sulfamethazin 279.10>64.60(+) CE: -50.03:Sulfamethazin 279.10>64.50(+) CE: -50.02:Sulfamethazin 279.10>186.50(+) CE: -18.02:Sulfamethazin 279.10>186.40(+) CE: -18.02:Sulfamethazin 279.10>186.30(+) CE: -18.02:Sulfamethazin 279.10>186.20(+) CE: -18.02:Sulfamethazin 279.10>186.10(+) CE: -18.02:Sulfamethazin 279.10>186.00(+) CE: -18.02:Sulfamethazin 279.10>185.90(+) CE: -18.02:Sulfamethazin 279.10>185.80(+) CE: -18.02:Sulfamethazin 279.10>185.70(+) CE: -18.02:Sulfamethazin 279.10>185.60(+) CE: -18.02:Sulfamethazin 279.10>185.50(+) CE: -18.01:Sulfamethazin 279.10>92.50(+) CE: -35.01:Sulfamethazin 279.10>92.40(+) CE: -35.01:Sulfamethazin 279.10>92.30(+) CE: -35.01:Sulfamethazin 279.10>92.20(+) CE: -35.01:Sulfamethazin 279.10>92.10(+) CE: -35.01:Sulfamethazin 279.10>92.00(+) CE: -35.01:Sulfamethazin 279.10>91.90(+) CE: -35.01:Sulfamethazin 279.10>91.80(+) CE: -35.01:Sulfamethazin 279.10>91.70(+) CE: -35.01:Sulfamethazin 279.10>91.60(+) CE: -35.01:Sulfamethazin 279.10>91.50(+) CE: -35.0
0.000 0.025 0.050 0.075 0.100 0.125 0.150 0.175 0.200 0.225 0.250 0.275 0.300 0.325 min
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2
1.3
1.4
1.5
(x1,000,000)
3:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -50.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -15.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -30.0
0.000 0.025 0.050 0.075 0.100 0.125 0.150 0.175 0.200 0.225 0.250 0.275 0.300 0.325 min
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2
1.3
1.4
1.5
1.6
1.7(x1,000,000)
3:Sulfamethazin 279.10>65.20(+) CE: -45.03:Sulfamethazin 279.10>65.20(+) CE: -46.03:Sulfamethazin 279.10>65.20(+) CE: -47.03:Sulfamethazin 279.10>65.20(+) CE: -48.03:Sulfamethazin 279.10>65.20(+) CE: -49.03:Sulfamethazin 279.10>65.20(+) CE: -50.03:Sulfamethazin 279.10>65.20(+) CE: -51.03:Sulfamethazin 279.10>65.20(+) CE: -52.03:Sulfamethazin 279.10>65.20(+) CE: -53.03:Sulfamethazin 279.10>65.20(+) CE: -54.03:Sulfamethazin 279.10>65.20(+) CE: -55.02:Sulfamethazin 279.10>186.10(+) CE: -10.02:Sulfamethazin 279.10>186.10(+) CE: -11.02:Sulfamethazin 279.10>186.10(+) CE: -12.02:Sulfamethazin 279.10>186.10(+) CE: -13.02:Sulfamethazin 279.10>186.10(+) CE: -14.02:Sulfamethazin 279.10>186.10(+) CE: -15.02:Sulfamethazin 279.10>186.10(+) CE: -16.02:Sulfamethazin 279.10>186.10(+) CE: -17.02:Sulfamethazin 279.10>186.10(+) CE: -18.02:Sulfamethazin 279.10>186.10(+) CE: -19.02:Sulfamethazin 279.10>186.10(+) CE: -20.01:Sulfamethazin 279.10>92.20(+) CE: -25.01:Sulfamethazin 279.10>92.20(+) CE: -26.01:Sulfamethazin 279.10>92.20(+) CE: -27.01:Sulfamethazin 279.10>92.20(+) CE: -28.01:Sulfamethazin 279.10>92.20(+) CE: -29.01:Sulfamethazin 279.10>92.20(+) CE: -30.01:Sulfamethazin 279.10>92.20(+) CE: -31.01:Sulfamethazin 279.10>92.20(+) CE: -32.01:Sulfamethazin 279.10>92.20(+) CE: -33.01:Sulfamethazin 279.10>92.20(+) CE: -34.01:Sulfamethazin 279.10>92.20(+) CE: -35.0
50.0 75.0 100.0 125.0 150.0 175.0 200.0 225.0 250.0 m/z0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Inten. (x100,000)
186.2
148.9
205.5124.292.3
186.2
156.1
124.2
108.2
186.1
124.2
156.1
108.2
92.2
213.2
124.2
92.2
108.2
186.1
156.1
213.3
204.1
124.2
92.2
108.2
186.1
65.2 213.2156.0
92.2
124.2108.2
65.2
149.480.0 201.2
92.2
124.265.1 108.2
80.1
92.265.2
124.3107.280.2
190.853.2 168.2
65.2
92.380.1 108.1124.253.2 197.4143.3
4
Fast and highly sensitive analysis of multiple drugs in ground-, surface- and wastewater
Table 1. Optimized MRM transitions of 9 drugs
Compound Mode MRM transitions Collision energy (kV)
Sulfamethazin
Sulfamethoxazol
Beza�brat
Carbamazepine
Diclofenac
Clo�bric acid
Ibuprofen
Iopamidol
Iopromid
ESI positive
ESI positive
ESI positive
ESI positive
ESI positive
ESI negative
ESI negative
ESI negative
ESI negative
279,10>186,10 / 279,10>92,20
253,90>92,20 / 253,90>156,15
362,10>139,15 / 362,10>316,25
237,10>194,20 / 237,10>179,20
296,00>214,15 / 296,00>215,15
213,00>127,00 / 213,00>85,00
205,10>161,30
775,80>126,95
790,00>127,00
-17 / -31
-26 / -15
-25 / -15
-19 / -34
-34 / -19
15 / 15
7
22
26
The solvent blending functionality entails automated mobile phase preparation on a LPGE (low pressure gradient) unit which is integrated in the binary LC pumps. The blending function eliminates the need of mobile phase pre-mixing, as necessary with ordinary binary pumps.Mobile phase composition can simply be changed in the
method without physically changing the solvents. Therefore solvent blending is a powerful tool for easy and efficient elucidation of the SPE, the gradient and the starting conditions. During this study the solvent blending function was used for optimization of the SPE conditions. A second LPGE unit was used for the analytical gradient.
Solvent Blending
Figure 3. Solvent blending functionality
1: prepare 5 mmol/L Ammonium formate (pH 8.5)
2: prepare H2O
5: prepare mobile phase A (SPE loading condition); different conditions tested !
200 mL 800 mL
6: prepare mobile phase B1 and B2 (analytical condition and gradient)
200 mL 800 mL
Traditional methodStep 1 Step 2 Step 3
Set these to system
Mobile phase blending function
Only step 1!
Set these to system
3: prepare MeOH
4: prepare 0,0025%NH4OH
LPGE Unit:Mobile phase composition for SPE loading, solvent blending allows to change conditions automatically
2nd LPGE Unit:Gradient for SPE release and separation
1: prepare 5 mmol/L Ammonium formate (pH 8.5)
2: prepare H2O
3: prepare MeOH
4: prepare 0,0025%NH4OH
5
Fast and highly sensitive analysis of multiple drugs in ground-, surface- and wastewater
Final methodSPE Conditions
Injection volume : 250 µl
SPE-column : Strata-X , 25 µm , 20*2 mm
SPE-�ow rate : 1 ml/min
SPE-loading buffer : 1 mmol/L ammonium formate (LPGE Pump B)
Analytical Conditions (LPGE Pump A)
Column : Kinetex C8, 2.6 µm, 100*2.1 mm
Flow rate : 0.5 ml/min
Solvent A : 0.0025% NH4OH
Solvent B : MeOH 1 min – 2.5 min analytical separation
Gradient : 0 min : 30% B
: 1 min : 30% B
: 1.5 min : 95% B
: 4.5 min : 95% B
: 4.51 min : 30% B
: 6 min : 30% B (Stop)
LCMS Conditions
Interface : ESI
Nebulizing Gas Flow : 2.2 L/min
Heating Gas Flow : 12 L/min
Interface Temperature : 400 ºC
Desolvation Line Temperature : 150 ºC
Heat Block Temperature : 400 ºC
Drying Gas Flow : 6 L/min
Polaritiy Switching Time : 5 ms
Figure 4. Scheme of online-SPE extraction (A) and analytical separation (B)
HPLC/MS Work�ow
Pump 1
Autosampler
Waste
SPE-Column
Analytical-Column+ LCMS 8050
Pump 2
A Pump 1
Autosampler
Waste
SPE-Column
Analytical-Column+ LCMS 8050
Pump 2
B
Fast and highly sensitive analysis of multiple drugs in ground-, surface- and wastewater
ResultsIn this study we developed a fast method for direct online SPE LC-MS/MS analysis of 9 different drugs in water with a minimal LC con�guration of two binary pumps equipped with LPGE units. The solvent blending function was used for method development of the SPE extraction. The second LPGE unit was used for SPE release and analytical gradient
separation. Each compound was quanti�ed in a concentration range from 0.05 ng/ml up to 2 ng/ml. Measurements were performed on Shimadzu’s LCMS-8050 Triple Quad MS System. The calibration curves and lowest calibration point is shown in �gure 5.
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Figure 5. Calibration curve and lowest calibration point at 0.05 ng/ml of each compound
PO-CON1444E
Multi-residue analysis of pyrethroidsin soil and sediment using QuEChERSby LC/MS/MS
ASMS 2014 TP 560
Yuka Fujito1, Kiyomi Arakawa1, Yoshihiro Hayakawa1
1 Shimadzu Corporation. 1, Nishinokyo-Kuwabaracho
Nakagyo-ku, Kyoto 604–8511, Japan
2
Multi-residue analysis of pyrethroids in soil and sediment using QuEChERS by LC/MS/MS
IntroductionPytrethroids are one of the most widely used commercial household insecticides in agricultural or non-agricultural application sites. Synthetic pyrethroids are poorly water-soluble, but are strongly adsorbed to soil, therefore these compounds are increasingly being found in soil or sediments. Recently, soil and sediment contamination by pyrethroids has been detected in both urban and agricultural area, and it’s becoming a global concern due
to the in�uence on the insects and aquatic invertebrates. Therefore, quick, high-sensitive and universal analysis methods are required. The analysis of pyrethroids is typically performed by GC or GC/MS because of their hydrophobicity. In this study, we report the development of a simultaneous analysis technique for trace amounts of pyrethroids by LC/MS/MS.
Sample preparationSample preparation was carried out by the use of the QuEChERS method. In case of the soil samples, hydration of sample with water before acetonitrile extraction is required to improve the recovery and operability. Result of several different extraction methods that changed the
amount of the soil and water added, we �nally adopted a combination of 5 g soil (or 10 g sediment) and 5 mL water, and the following procedures were based on the original QuEChERS method.
Figure 1 Chemical structure of pyrethroids
Materials and MethodsMaterials
Sampling point
Residential garden (Kyoto, Japan)
Lake Biwa (Shiga, Japan)
Sample
Soil
Sediment
Permethrin
Pyrethrin
I : R=CH3
II : R=CO2CH3
Cyhalothrin
Te�uthrin
Esfenvalerate
3
Multi-residue analysis of pyrethroids in soil and sediment using QuEChERS by LC/MS/MS
LC/MS/MS analsisHPLC conditions (Nexera UHPLC system, Shimadzu)
Column : Phenomenex Kinetex 2.6 µm PFP 100Å (100 mm x 2.1 mm I.D.)
Mobile phase : A 5mM ammonium acetate - water
: B Methanol
Gradient program : 40 % B (0 min.) → 100 % B (10 -12 min.) → 40 % B (12.01-15 min.)
Flow rate : 0.2 mL / min.
Column temperature : 40 ºC
Injection volume : 1 μL
MS conditions (LCMS-8050, Shimadzu)
Ionization : ESI (positive / negative)
Interface temperature : 100 ºC
DL temperature : 100 ºC
Heat block temperature : 400 ºC
Nebulizing gas : 3.0 L / min.
Drying gas : 15.0 L / min.
Heating gas : 3.0 L / min.
Weigh 5 g soil / 10 g sediment
(Add STDs solution)
Add 5mL water
Add 10mL acetonitrile
Add salt mixture*1 • 4g MgSO4
• 1g NaCl• 1g Trisodium citrate dehydrate• 0.5g Disodium hydrogencitrate sesquihydrate
Shake vigorously by hand 1min.
Centrifuge for 10min. (Extract 1)
Step 1 : Acetonitrile extraction
*1 : Q-sep QuEChERS Extraction Salts (RESTEK) *2 : Q-sep QuEChERS dSPE Tubes (RESTEK)
Step 2 : Clean-up
Transfer 6mL Extract 1 into dSPE tube*2
• 900 mg MgSO4
• 150 mg PSA• 45 mg GCB
Shake vigorously by hand 1min.
Centrifuge for 5min.
Transfer the supernatant into a vial
Filtration using disposable �lter
LC/MS/MS analysis
4
Multi-residue analysis of pyrethroids in soil and sediment using QuEChERS by LC/MS/MS
Figure 2 LCMS-8050 triple quadrupole mass spectrometer
Table 1 MRM transitions of pyrethroids
Compounds Polarity Quantitative ion (m/z) Con�rmation ion (m/z)
pyrethrin-I
pyrethrin-II
fenpropathrin
cycloprothrin
deltamethrin
esfenvalrate
cypermethrin
cy�uthrin
ethofenprox
permethrin
cyhalothrin
bifenthrin
acrinathrin
acrinathrin
sila�uofen
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
329.20>161.20
373.20>161.20
367.20>125.20
498.90>181.10
522.80>280.90
437.10>167.30
433.10>191.10
450.90>191.00
394.20>177.30
408.10>183.30
467.10>225.10
440.00>181.20
559.00>208.20
540.10>372.20
426.20>287.10
329.20>105.20
373.20>105.20
367.20>181.20
498.90>229.20
522.80>181.10
437.10>125.30
433.10>181.20
450.90>206.10
394.20>107.20
408.10>355.20
467.10>141.10
440.00>166.10
559.00>181.10
540.10>345.30
426.20>168.20
High Speed Mass Spectrometer
Ultra Fast Scanning - 30,000 u / sec. Ultra Fast Polarity Switching - 5 msec. Ultra Fast MRM - Max. 555 transitions / sec
Result
In this study, we selected and evaluated 15 pyrethroids (pyrethrin, fenpropathrin, cycloprothrin, deltamethrin, esfenvarelate, cypermethrin, cyfluthrin, ethofenprox, permethrin, cyhalothrin, bifenthrin, acrinathrin, tefluthrin, silafruofen) which are the most widely used for household or
agrocultural insecticides worldwide. Except for tefluthrin, which was not ionized by LC/MS under conditions tested, all other 14 compounds were successfully detected in ESI positive mode or in both positive and negative mode.
MRM of pyrethroid standards
5
Multi-residue analysis of pyrethroids in soil and sediment using QuEChERS by LC/MS/MS
Figure 3 MRM chromatograms
Figure 4 MRM chromatograms of the LOQs of typical pyrethroids
Table 2 Calibration curves
compounds min. conc. max. conc. r2
pyrethrin I
pyrethrin II
fenpropathrin
cycloprothrin
deltamethrin
esfenvalerate
cypermethrin
cy�uthrin
ethofenprox
trans-permethrin
cis-permethrin
cyhalothrin
bifenthrin
acrinathrin (+)
acrinathrin (-)
sila�uofen
0.5
0.5
0.02
0.5
0.05
0.5
0.05
0.5
0.01
0.02
0.02
0.1
0.02
0.1
0.5
0.01
500
500
100
100
100
100
100
100
100
100
100
100
100
100
500
100
0.9996
0.9997
0.9993
0.9991
0.9992
0.9990
0.9986
0.9976
0.9993
0.9996
0.9994
0.9993
0.9995
0.9987
0.9993
0.9999
(ppb)7.0 8.0 9.0 10.0 min
0
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
1300000
1400000
1500000
pyrethrin-II
pyrethrin-I
fentropathrin
cycloprothrindeltamethrinesfenvalrate
cypermethrin
ethofenprox
permethrin
cyhalothrin
bifenthrin
acrinathrinsila�uofen
cy�uthrin
permethrin0.02 ppb
trans-
cis-
9.5 10.0
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
(x1,000)
fenpropathrin0.02 ppb
9.0 9.5
0.00
0.25
0.50
0.75
1.00
1.25(x1,000)
bifenthrin0.02 ppb
9.5 10.0 10.5
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
(x1,000)
10.0 10.5 11.0
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
2.50
(x1,000)
sila�uofen0.01 ppb
Multi-residue analysis of pyrethroids in soil and sediment using QuEChERS by LC/MS/MS
6
Figure 5 Recovery of 14 pyrethroids from soil and sediment matrices (10 ppb spiked)
soil (residential garden)
Reco
very
(%)
ethofenprox
Figure 6 Chromatograms of prethroids in the soil
permethrin
sediment (lake)
Table 3 Result of quantitative analysis in the soil and sediment
pyrethrin-I
pyrethrin-II
fenpropathrin
cycloprothrin
deltamethrin
esfenvalrate
cypermethrin
cy�uthrin
ethofenprox
permethrin
cyhalothrin
bifenthrin
acrinathrin
sila�uofen
soil
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0.01 ppb*
0.03 ppb
n.d.
n.d.
n.d.
n.d.
sediment
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
All target compounds showed good recoveries from soil and sediment matrices in the range 70-120% by the QuEChERS
method. Neither matrix effect (Ion suppression or enhancement) nor sample preparation losses were observed.
Recovery from soil and sediment matrices
The quantitative analysis of the soil and sediment sample was performed. Ethofenprox and permethrin was detected
from the soil sample at approximately 0.02 and 0.06 μg / kg, respectively.
Quantitative analysis of soil and sediment
n.d. : not detected* : <LOQ
0
20
40
60
80
100
120
140
Pyre
thrin
-2
Pyre
thrin
-1
Fenpro
pathrin
Cyclo
proth
rin
Deltam
ethrin
Esfe
nvalra
te
Cyper
met
hrin
Cy�uth
rin
Ethofe
nprox
trans-P
erm
ethrin
cis-P
erm
ethrin
Cyhalo
thrin
Bifenth
rin
Acrinat
hrin
Sila�
uofen
0
20
40
60
80
100
120
140
Pyre
thrin
-2
Pyre
thrin
-1
Fenpro
pathrin
Cyclo
proth
rin
Deltam
ethrin
Esfe
nvalra
te
Cyper
met
hrin
Cy�uth
rin
Ethofe
nprox
trans-P
erm
ethrin
cis-P
erm
ethrin
Cyhalo
thrin
Bifenth
rin
Acrinat
hrin
Sila�
uofen
STDs spiked after prep STDs spiked before prep
soil blank
solvent blank
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Multi-residue analysis of pyrethroids in soil and sediment using QuEChERS by LC/MS/MS
Conclusions• A method for quanti�cation of 14 pyrethroids in soil and sediment at ppt-level concentrations was developed by
LC/MS/MS.• In this study, neither matrix effect nor sample preparation losses were observed in the recovery test, demonstrating the
applicability of QuEChERS method to sample preparation of soil and sediment.
Metabolism
• Page 197High sensitivity analysis of metabolites in serum using simultaneous SIM and MRM modes in a triple quadrupole GC/MS/MS
• Page 202Analysis of D- and L-amino acids using auto- mated pre-column derivatization and liquid chromatography-electrospray ionization mass spectrometry
• Page 208Characterization of metabolites in microsomal metabolism of aconitine by high-performance liquid chromatography/quadrupole ion trap/time-of-flight mass spectrometry
• Page 213Simultaneous analysis of primary metabolites by triple quadrupole LC/MS/MS using penta- fluorophenylpropyl column
PO-CON1443E
High Sensitivity Analysis ofMetabolites in Serum UsingSimultaneous SIM and MRM Modesin a Triple Quadrupole GC/MS/MS
ASMS 2014 ThP 641
Shuichi Kawana1, Yukihiko Kudo2, Kenichi Obayashi2,
Laura Chambers3, Haruhiko Miyagawa2
1 Shimadzu, Osaka, Japan, 2 Shimadzu, Kyoto, Japan,
3 Shimadzu Scienti�c Instruments, Columbia, MD
2
High Sensitivity Analysis of Metabolites in Serum Using Simultaneous SIM and MRM Modes in a Triple Quadrupole GC/MS/MS
IntroductionGas chromatography / mass spectrometry (GC–MS) and a gas chromatography-tandem mass spectrometry (GC-MS/MS) are highly suitable techniques for metabolomics because of the chromatographic separation, reproducible retention times and sensitive mass detection.
Sample• Human serum
MRM measurement modeSome compounds with low CID ef�ciency produce insuf�cient product ions for MRM transitions, and the MRM mode is consequently less sensitive than SIM for these compounds.
Our suggestionSIM, MRM, and simultaneous SIM/MRM modes are evaluated for analysis of metabolites in human serum.
Materials and MethodSample and Sample preparation
Sample Preparation1)
Instrumentation
Freeze-dry
Residue
Sample
Add 40 µL methoxyamine solution (20 mg/mL, pyridine)
Heat at 30 ºC for 90 min
Add 20 µL MSTFA
Heat at 37 ºC for 30 min
1) Nishiumi S et. al. Metabolomics. 2010 Nov;6(4):518-528
Supernatant 250 µL
Add 250 µL water / methanol / chloroform (1 / 2.5 / 1)
Add internal standard (2-Isopropylmalic acid)
Stir, then centrifuge
Extraction solution 225 µL
Add 200 µL Milli-Q water
Stir, then centrifuge
50uL serum
GC-MS : GCMS-TQ8040 (SHIMADZU)
Data analysis : GCMSsolution Ver.4.2
Database : GC/MS Metabolite Database Ver.2 (SHIMADZU)
Column : 30m x 0.25mm I.D., df=1.00µm (5%-Phenyl)-methylpolysiloxane
3
High Sensitivity Analysis of Metabolites in Serum Using Simultaneous SIM and MRM Modes in a Triple Quadrupole GC/MS/MS
Simultaneous SIM and MRM modes in GC/MS/MSFigure 1 shows the theory of Simultaneous SIM and MRM modes. This analysis mode can measure SIM and MRM data in a single analysis.
Method Creation using Database and SmartMRMFigure 3 shows the GC/MS Metabolites Database Ver.2. This database involves conditions of SIM and MRM in 186 metabolites and a method creation function we call SmartMRM. SmartMRM creates MRM, SIM, SIM/MRM methods from Database automatically.
• Select the MRM, SIM and SIM/MRM conditions of 186 TMS derivatization metabolites from GC/MS Metabolites Database Ver.2.
• Select the two transitions (or ions) each metabolite.
Poor sensitivity of MRM in some compounds because of low CID ef�ciency
Figure 1 The concept of simultaneous SIM and MRM analysis mode.
Figure 3 GC/MS Metabolites Database Ver.2
Figure 2 Mass Spectrum of Precursor (or SIM) and Product ion
SIM
MRMSIM
MRM
Q1 Q3Collision Cell
SIMSIM CID
100 200 300 400 0
25
50
75
100 %
361
73
217 147 437 103 271
243 319 191
100 200 300 0
25
50
75
100
%
169
103 73
243 361
Precursor ion (or SIM) Product ion
CID
4
High Sensitivity Analysis of Metabolites in Serum Using Simultaneous SIM and MRM Modes in a Triple Quadrupole GC/MS/MS
A number of Identi�cation metabolites in serum Table 1 shows the identi�cation results of metabolites in human serum using SIM, MRM and simultaneous SIM/MRM analysis modes in GC/MS/MS. In SIM/MRM, the metabolites, which were insuf�cient sensitivity in MRM, were measured by SIM and the other metabolites were measured by MRM.
ResultsComparison of the chromatogram between SIM and MRM in human serum
Detected the peak in MRM because of high selectivity
Peak was not detected in MRM because of low CID ef�ciency.
SIM MRM
SIM MRM
0.5
1.0
1.5
2.0
2.5
3.0
3.5
2.5
5.0
7.5
0.25
0.50
0.75
1.00
1.25
1.50
1.75
(x100,000)333.10160.10
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
(x100,000)238.10218.10
(x10,000)218.10>73.00
(x100)238.10>91.00
238.10>91.00
(x10,000)333.10>143.10333.10>171.10
21.00 21.25 21.00 21.25
21.25 21.50 21.2521.00 21.50
21.2521.00 21.500.250.500.751.001.251.501.75
a) Glucuronic acid-meto-5TMS(2)
b) S-Benzyl-Cysteine-4TMS
Table 1 The number of identi�ed metabolites each analysis mode
note) A:Target and Con�rmation ions were detected.; B: Either Target or Con�rmation ion was detected. Another one was overlapped by contaminants.; C: Either Target or Con�rmation ion was detected.
Modes
SIM
MRM
SIM/MRM
A
57
131
133
B
51
14
22
C
8
1
1
Total
116
146
156
High Sensitivity Analysis of Metabolites in Serum Using Simultaneous SIM and MRM Modes in a Triple Quadrupole GC/MS/MS
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Fig.4 shows a number of metabolites in each mode can be measured. In metabolites with low CID ef�ciency, SIM are superior to MRM if there are no interfering substances to the target metabolites.
Figure 4 Detected metabolites in human serum each analysis mode.
Conclusions• Analytical results from the SIM and MRM modes identi�ed 116 and 146 metabolites, respectively.• In metabolites with poor CID ef�ciency, the sensitivity of SIM is more than 10 times higher than MRM.• Simultaneous SIM and MRM modes in a single analysis (SIM/MRM) improves the sensitivity and reproducibility for
analysis of metabolites in human serum compared to MRM alone. • A novel SIM/MRM expands the utility of a triple quadrupole GC/MS/MS
The reproducibility(n=6) in MRM and SIM/MRMTable 2 Comparison of the reproducibility results from MRM and SIM/MRM analysis. A number of detected metabolites involves A, B and C in Table 1.
%RSD
- 4.99%
5 - 9.99%
10 - 14.99%
15 - 19.99%
> 20%
MRM
73
26
8
9
30
146
SIM/MRM
76
30
10
10
30
156
Improvement
+3
+4
+2
+1
0
+10
SIM MRM
10 40 106
Metabolites with low CIDef�ciency in MRM
Metabolites withinterference in SIM
PO-CON1451E
Analysis of D- and L-amino acids usingautomated pre-column derivatizationand liquid chromatography-electrosprayionization mass spectrometry
ASMS 2014 MP739
Kenichiro Tanaka1; Hidetoshi Terada2; Yoshiko Hirao2;
Kiyomi Arakawa2; Yoshihiro Hayakawa2
1. Shimadzu Scienti�c Instruments, Inc., Columbia, MD;
2. Shimadzu Corporation, Kyoto, Japan
2
Analysis of D- and L-amino acids using automated pre-column derivatization and liquid chromatography-electrospray ionization mass spectrometry
IntroductionRecently, several species of D- amino acids have been found in mammals including humans and their physiological functions have been elucidated. Quantitating each enantiomer of amino acids is indispensable for such studies. In order to diagnose diseases, it is desirable that D- and L-amino acid can be separately quantitated and applied to metabolic analysis. Pre-column derivatization with o-phthalaldehyde (OPA) and N-acetyl-L-cysteine(NAC) is widely utilized for the analysis of D- and L- amino acids since the method can be performed with a rapid reversed phase separation on a relatively simple hardware (U)HPLC con�guration with
good reliability. One of the drawbacks of pre-column derivatization is less reproducibility due to the tedious manual procedure and human errors. We have launched an autosampler for a UHPLC system equipped with an automated pretreatment function that allows overlapping injections in which the next derivatization proceeds during the current analysis for saving total analytical time. We have applied this autosampler and its function to fully automate pre-column derivatization for the determination of amino acids. In this study, we developed a methodology which enabled the automated procedure of pre-column chiral derivatization of D- and L- amino acids.
Experimental
The system used was a SHIMADZU UHPLC Nexera pre-column Amino Acids (AAs) system consisting of LC-30AD solvent delivery pump, DGU-20A5R degassing unit, SIL-30AC autosampler, CTO-30A column oven, and SHIMADZU triple quadrupole mass spectrometer LCMS-8040. The software is integrated in the LC/MS/MS
workstation (LabSolutions, Shimadzu Corporation, Japan) so that selected conditions can be seamlessly translated into method �les and registered to a batch queue, ready for instant analysis. A 1.9um YMC-Triart C8 column (2.0 mm x 150 mm L.) was used for the analysis.
Instruments
Derivatizing solutions: 0.1 mol/L boric acid buffer was prepared by dissolving 6.18 g of boric acid and 2.00 g of sodium hydroxide in 1 L of water. 10 mmol/L NAC solution was prepared by dissolving 16.3 mg of N-acetyl-L-cysteine in 10 mL of the 0.1 mol/L boric acid buffer. 10 mmol/L OPA solution was prepared by dissolving 6.7
mg of o-phthalaldehyde in 0.3 mL of ethanol, adding 0.7 mL of the 0.1 mol/L boric acid buffer and 4 mL of water.Fig.1 shows the schematic procedure for amino acids derivatization with the SIL-30AC.Samples, including the derivatized amino acids, were injected onto the UHPLC and separated under the conditions shown in Table 1.
Derivatization Method
3
Analysis of D- and L-amino acids using automated pre-column derivatization and liquid chromatography-electrospray ionization mass spectrometry
Fig.1 Schematic procedure of automated pre-column derivatization
Table 1 UHPLC and MS analytical conditions
Mobile Phase : A : 10 mmol/L Ammonium Bicarbonate solution
B : Acetonitrile/Methanol = 1/1(v/v)
Initial B Conc. : 0%
Flow Rate : 0.4 mL/min
Column Temperature : 40 ºC
Injection Volume : 1 μL
LC Time Program : 0 -> 5%(0.01min), 5%(0.01-1.00min), 5 ->20%(1.00 - 15.00min),
20 - 25%(15.00 - 24.00min), 25 – 90%(24.00 - 24.50min),
90%(24.50 - 27.50min), 90 - 0% (27.50 – 28.50min)
Ionization Mode : ESI
Nebulizing Gas Flow Rate : 3 L/min
Drying Gas Flow Rate : 15 L/min
DL Temperature : 300 ºC
Heating Block Temperature : 450 ºC
Result
A standard solution containing 27 amino acids was prepared at 1 mmol/L concentration each in 0.1 mol/L HCl solution. The MS conditions such as ESI positive and negative ionization modes were optimized in parallel with the column separation, and compound dependent parameters such as CID and pre-bias voltage were adjusted
using the function for automatic MRM optimization. The transition that provided the highest intensity was used for quanti�cation. Table 2 shows the MRM transition of each derivatized amino acid. The MRM chromatogram is illustrated in Fig.2.
Analysis of Standard Solution
(1)
Take 20 μL of 10 mmol/L NAC solution
Supply 1 μL ofsample solution to the vial for mixing
(3)
Take 20μL of 10 mmol/L OPA solution
Mix the sample solutionand derivatizing solutions
Inject 1μL of the mixed solution to the column
Supply 20 μL of NAC solution to thevial for mixing
Supply 20 μL of10 mmol/L OPA solution to the vial for mixing
(5)
Take 1 μL of sample solution
Wait for 3min untilthe derivatization ends
Take 1μL of the mixedsolution
(2) (4)
(6) (7) (8) (10)(9)
4
Analysis of D- and L-amino acids using automated pre-column derivatization and liquid chromatography-electrospray ionization mass spectrometry
Fig. 2 Chromatogram of a 27 amino acid standard solution
Compound
Aspartic acid
Glutamic acid
Serine
Glutamine
Glycine
Histidine
Threonine
Arginine
Tyrosine
Valine
Tryptophan
Isoleucine
Phenylalanine
Polarity
+
+
+
+
+
+
+
+
+
+
+
+
+
Precursor m/z
395.00
409.10
367.00
408.20
337.00
417.10
381.20
436.10
443.00
379.10
466.20
393.00
427.20
Product m/z
130.00
130.05
130.00
130.05
130.00
244.05
130.05
263.10
130.05
250.05
337.10
264.05
298.05
Table 2 Compounds, Ionization polarity and MRM transition
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min
0
25000
50000
75000
100000
125000
150000
175000
200000
225000
250000
12
3 45
6
78
9
10
11
12 13
14
1516
17
18 1921
22
20
23
24
2526
27
■Peaks
1. D-Aspartic acid, 2. L-Aspartic acid, 3. L-Glutamic acid, 4. D-Glutamic acid, 5. D-Serine, 6. L-Serine, 7. L-Glutamine8. D-Glutamine, 9. Glycine, 10. L-Histidine, 11. D-Histidine ,12. D-Threonine, 13. L-Threonine, 14. L-Arginine15. D-Arginine, 16. D-Alanine, 17. L-Alanine, 18. D-tyrosine, 19. L-Tyrosine, 20. L-Valine, 21. D-Valine22. L-Tryptophan, 23. D-Tryptophan, 24. L-Isoleucine, 25. D-Phenylalanine, 26. L-Phenylalanine, 27.D-Isoleucine
5
Analysis of D- and L-amino acids using automated pre-column derivatization and liquid chromatography-electrospray ionization mass spectrometry
CompoundRepeatability (%RSD)
D-Aspartic acid
D-Glutamic acid
D-Serine
D-Glutamine
D-Histidine
D-Threonine
D-Arginine
D-Alanine
D-Tyrosine
D-Valine
D-Tryptophan
D-Isoleucine
D-Phenylalanine
5 μmol/L
3.5
3.7
4.8
4.1
4.3
3.8
3.4
4.0
3.2
3.3
3.9
3.1
3.5
25 μmol/L
2.5
3.1
3.0
3.4
1.8
2.6
1.7
2.3
2.9
2.2
3.2
2.9
1.8
Table 3 Reproducibility
Compound
D-Asparic acid
D-Glutamic acid
D-Serine
D-Glutamine
D-Histidine
D-Threonine
D-Arginine
D-Alanine
D-Tyrosine
D-Valine
D-Tryptophan
D-Isoleucine
D-Phenylalanine
Cali.F
Y = (44661.8)X + (1829.61)
Y = (12191.8)X + (10390.7)
Y = (22319.5)X + (-2869.30)
Y = (3458.60)X + (1521.83)
Y = (5778.33)X + (-341.182)
Y = (10800.6)X + (-1874.07)
Y = (10535.7)X + (-1298.12)
Y = (15349.1)X + (-4719.98)
Y = (17098.7)X + (-1812.69)
Y = (23707.7)X + (772.548)
Y = (18089.1)X + (-3620.41)
Y = (44017.1)X + (67903.1)
Y = (22426.0)X + (-736.090)
r2
0.998
0.999
0.999
0.999
0.998
0.999
0.998
0.999
0.999
0.999
0.998
0.999
0.999
Table 4 Linearity
Reproducibility and linearity in this analysis were evaluated with a plasma spiked standard solution. As a result, less than 5% relative standard deviation of peak areas were obtained. Table 3 shows the reproducibility of repeated analysis of spiked sample (n=6). Five different levels of
spiked sample concentration from 1 to 100 μmol/L standard solution were used for the linearity evaluation. The coef�cients of determination (r2) were approximately 0.999. Table 4 shows the summary for the linearity results.
Method Validation
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Analysis of D- and L-amino acids using automated pre-column derivatization and liquid chromatography-electrospray ionization mass spectrometry
Considering the frequency of amino acids analysis in physiological samples, the recovery of spiked samples were con�rmed. In addition, the results indicated that the recovery ratio of most amino acids are around 100%.Table 5 shows the summarized results for the recovery of each amino acid.
Conclusions• The combination of Shimadzu triple quadrupole mass spectrometer and Nexera UHPLC provides reliable pre-column
derivatized AAs analysis with enhanced productivity.• An established method was successfully applied to the separation of D- and L- amino acids with excellent reliability.
CompoundRecovery (100%)
D-Asparic acid
D-Glutamic acid
D-Serine
D-Glutamine
D-Histidine
D-Threonine
D-Arginine
D-Alanine
D-Tyrosine
D-Valine
D-Tryptophan
D-Isoleucine
D-Phenylalanine
5 μmol/L
100.3
92.8
97.9
103.2
104.8
101.1
102.4
93.5
98.1
101.0
97.8
98.8
104.5
25 μmol/L
107.1
97.8
100.6
104.3
100.4
98.8
99.6
99.5
101.0
99.2
100.4
102.4
100.9
Table 5 Recovery
PO-CON1476E
Characterization of metabolites in microsomal metabolism of aconitineby high-performance liquid chromatography/quadrupole ion trap/time-of-�ight mass spectrometry
ASMS 2014 WP 739
Cuiping Yang1, Changkun Li2, Tianhong Zhang1,
Qian Sun2, Yueqi Li2, Guixiang Yang2, Taohong Huang2,
Shin-ichi Kawano2, Yuki Hashi2, Zhenqing Zhang1,* 1Beijing Institute of Pharmacology & Toxicology, 2Shimadzu Global COE, Shimadzu (China) Co., Ltd., China
2
Characterization of metabolites in microsomal metabolism of aconitine by high-performance liquid chromatography/quadrupole ion trap/time-of-�ight mass spectrometry
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5
0.0
2.5
5.0
7.5(x1,000,000)
Introduction
Results
Aconitine (AC) is a bioactive alkaloid from plants of the genus Aconitum, some of which have been widely used as medicinal herbs for thousands of years. AC is also well known for its high toxicity that induces severe arrhythmias leading to death. Although numerous studies have raised on its pharmacology and toxicity, data on the identi�cation
metabolites of AC in liver microsomes are limited. The study of metabolic pathways is very important for ef�cacy of therapy and evaluation of toxicity for those with narrow therapy window. The aim of our work was to obtain the metabolic pathways of AC by the human liver microsomes.
Methods and Materials
The typical reaction mixture incubation contained 10 μmol/L aconitine and was preincubated at 37 ºC for 3 min. Reactions were initiated by adding 50 μL of NADPH (20 mmol/L), then incubated at 37 ºC in a waterbath shaker for
60 min. The reactions were terminated by adding 3-volume of ice-cold acetonitrile, then vortexed and centrifuged to remove precipitated protein.
Sample Preparation
Instrument : LCMS-IT-TOF (Shimadzu Corporation, Japan);
UFLCXR system (Shimadzu Corporation, Japan);
Column : Shim-pack XR-ODS II (2.0 mmI.D. x 75 mmL.,2.2 μm)
Mobile phase : A: water (0.1% formic acid+5 mmol ammonium formate),
B: acetonitrile
Gradient program : 30%B (0-4 min)-80%B (8 min)-80%B (8-11 min)-30%B (11.01-17 min)
Flow rate : 0.3 mL/min
M11M1
M0
M2M3
M4
M5
M6
M7
M8M9
M10
M12
M13 M14M16
M15
B
Fig.1 TIC chromatogram (A) and mass chromatograms of the metabolites of AC in the microsomal incubation mixture of human (B)
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.00.0
2.5
5.0
7.5(x1,000,000)
1:TIC (1.00)
A
3
Characterization of metabolites in microsomal metabolism of aconitine by high-performance liquid chromatography/quadrupole ion trap/time-of-�ight mass spectrometry
Fig. 3 Proposed metabolic pro�le of AC in the human liver microsomes
Fig. 2 Proposed fragmentation pathway of AC
OH+
OH
OO
O
O
N
O
OH
H
OH
O
O
C34H48NO11+
Exact Mass: 646.3227C32H44NO9
+
Exact Mass: 586.3016
C31H40NO8+
Exact Mass: 554.2754
C25H34NO8+
Exact Mass: 476.2284
C29H36NO8+
Exact Mass: 526.2441
C25H36NO9+
Exact Mass: 494.2390 C22H26NO4+
Exact Mass 368.1862 C21H25NO4+
Exact Mass 354.1705
O
OH
O
+
O
O
N
O
OH
H
OH
O
OH
O
+
O
O
N
O
OH
H
OH
O
OH
O
+
O
O
HN
O
OH
H
OH
O
OH
O
+
O
O
N
O
OH
H
OH
O
OH
O
+
O
O
N
O
OH
H
OH
OOH
O
O
HN
OH
OHH+
O
O
HN
HOH
OHH+
O
HO
HO
O
O
O
N
O
OH
H
OH
O
O
OH
HO
HO
O
O
O
N
O
OH
H
OH
O
O
OH
HO
O
O
O
OH
N
O
OH
H
OH
O
O
O
HO
O
O
O
O
N
HOH2C
O
OH
H
OH
O
O
O
OH
O
OH
O
O
N
O
OH
H
OH
O
O
OH
O
O
O
O
N
HOH2C
O
OH
H
OH
O
O
O
OH
O
O
O
O
N
O
OH
H
OH
O
O
O
OH
O
O
O
OH
N
O
OH
H
OH
O
O
OH
OH
O
O
O
O
N
O
OH
H
OH
O
O
O
OH
O
O
O
N
O
OH
H
OH
O O
O
O
O
N
O
OH
H
OH
O
O
O
OH
O
O
O
O
HN
O
OH
H
OH
O
OO
O
OH
O
O
N
O
OH
H
OH
O
O
OH
OH
O
O
O
O
N
O
OH
H
OH
O
O
M0
M13
M15
M11
M8M9M2
M10
M7
M16
M12
M3 M1
M5
O
HO
HO
O
O
O
N
O
OH
H
OH
O
O
M6O
HO
O
O
O
O
N
O
OH
H
OH
O
O
M4
O
HO
O
O
O
O
N
HOH2C
O
OH
H
OH
O
O
M14
4
Characterization of metabolites in microsomal metabolism of aconitine by high-performance liquid chromatography/quadrupole ion trap/time-of-�ight mass spectrometry
No.
M0
M1
M2
M3
M4
M5
M6
M7
M8
M9
M10
M11
M12
M13
M14
M15
M16
RT(min)
22.3
10.5
11.2
11.3
11.8
12.2
13.3
13.5
13.7
13.8
14.1
15.0
15.1
16.0
17.3
17.6
17.9
Meas.MW(m/z)
646.3230
618.2922
616.2754
604.3140
630.2930
586.3005
616.2769
632.3035
648.3016
618.2935
618.2890
662.3179
602.2948
632.3054
662.3209
632.3068
584.2826
Pred.MW(m/z)
646.3222
618.2909
616.2752
604.3116
630.2909
586.3011
616.2752
632.3065
648.3015
618.2909
618.2909
662.3171
602.2960
632.3065
662.3171
632.3065
584.2854
mDaerror
0.8
1.3
0.2
2.4
2.1
0.6
2.3
3.0
0.1
3.0
1.5
0.8
1.6
1.1
3.8
0.3
2.8
MS2 data
586.3000, 554.2752, 526.2785, 494.2536, 476.2431, 404.2432, 368.1847, 354.1687
558.2710, 498.2469, 480.2378, 436.2093, 354.1725
556.2510, 554.2335, 494.2106, 478.2321, 434.1908, 402.1682
554.2744, 522.2398, 434.1898
570.2686, 552.2576, 510.2457, 492.2381
568.2938, 554.2705, 522.2537, 466.2168, 434.1922
584.2477, 524.2316, 434.1941
572.2866, 512.2638, 494.2468, 480.2283, 462.2214, 290.2236, 354.1652, 340.1871
588.2702, 570.2654, 528.2566, 510.2434, 406.2161
558.2714, 494.2109, 476.2400, 340.1548
558.2722, 494.2127, 476.2009, 354.1635
602.2964, 570.2654, 542.2750, 510.2434, 420.2416
584.2533, 524.2249, 510.2179, 406.1582
572.2853, 512.2661, 480.2368, 476.2445, 436.2082, 368.1812
602.2947, 570.2654, 542.2766, 510.2434, 478.2187
586.2973, 526.2738, 508.2273, 494.2490
552.2669, 492.2111, 460.2063
Formula
C34H47NO11
C32H43NO11
C32H41NO11
C32H45NO10
C33H43NO11
C32H43NO9
C32H41NO11
C33H45NO11
C33H45NO12
C32H43NO11
C32H43NO11
C34H47NO12
C32H43NO10
C33H45NO11
C34H47NO12
C33H45NO11
C32H41NO9
Biotransformation
Parent
deethylation
bidemethylation+dehydrogenation
deacetylation
demethylation+dehydrogenation
deacetylation+dehydration
bidemethylation+dehydrogenation
demethylation
oxidation+demethylation
bidemethylation
bidemethylation
oxidation
deacetylation+dehydrogenation
demethylation
oxidation
demethylation
deacetylation+dehydration+dehydrogenation
ppmerror
1.26
2.10
0.26
3.94
3.35
0.96
3.68
4.81
0.23
4.88
2.43
1.21
2.66
1.80
5.74
0.42
4.82
Table1 Mass data for characterization of metabolites in of AC in the microsomalincubation mixture of human
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Characterization of metabolites in microsomal metabolism of aconitine by high-performance liquid chromatography/quadrupole ion trap/time-of-�ight mass spectrometry
Conclusions In this study, totaling 16 metabolites were found and characterized in the humam liver microsomes incubation mixture, including O-demethylation, oxidation, bidemethylation, dehydrogenation, N-deethylation, deacetylation, dehydration and besides M1, M3, M4, M9, M13 and M15, all the left ten of them were �rst identi�ed and reported. Collectively, these data provide a foundation for the clinical use of AC and contributes to a wider understanding of xenobiotic metabolism and toxicity evaluation.
PO-CON1447E
Simultaneous analysis of primarymetabolites by triple quadrupole LC/MS/MSusing penta�uorophenylpropyl column
ASMS 2014 WP 613
Tsuyoshi Nakanishi1, Takako Hishiki2, Makoto Suematsu2,3
1 Shimadzu Corporation, Kyoto, Japan,
2 Department of Biochemistry, School of Medicine,
Keio University, Tokyo, Japan,
3 Japan Science and Technology Agency,
Exploratory Research for Advanced Technology,
Suematsu Gas Biology Project, Tokyo, Japan
2
Simultaneous analysis of primary metabolites by triple quadrupole LC/MS/MS using penta�uorophenylpropyl column
IntroductionVarious metabolic pathways are controlled to keep a biological function in the cell and to monitor the rapid and slight changes of these metabolism, a simple simultaneous analysis is required for quanti�cation of primary metabolites. A typical LC/MS system with an ODS column is not effective to measure primary metabolites because of low af�nity of ODS column to hydrophilic metabolites. Here we report the
simultaneous measurement of 97 metabolites by triple quadrupole LC/MS/MS using penta�uorophenylpropyl column. In this experiment, MRM transitions of these metabolites were optimized and this method was applied to biological samples. Furthermore, to evaluate the accuracy of developed method for quanti�cation, simultaneous analysis by PFPP column was compared to measurement of ion-paring chromatography.
Commercially available compounds were used as standards to optimize MRM transition and LC condition for separation. Mixed standard solutions were diluted to a range of 10 nM~10000 nM for a calibration curve and an aliquot of 3 µL was subjected to LC/MS/MS measurement.Mice were sacri�ced under anesthesia and the isolated heart/liver tissues were rapidly frozen in liquid nitrogen. Frozen liver or heart tissues (>50 mg) from mice were homogenized in 0.5 mL methanol including L-methionine sulfone and 2-morpholinoethanesulfonic
acid (MES) as internal standards. After a general chloroform/methanol extraction, upper aqueous layer �ltered through 5-kDa cutoff �lter. The �ltrate was dried up and dissolved in 0.1 mL puri�ed water. Further, the solution was diluted to 20-100 folds in puri�ed water. An aliquot of 3 µL was analyzed to measure primary metabolites by LC/MS instrument, Nexera UHPLC system and LCMS-8030/LCMS-8040 triple quadrupole mass spectrometer. The following is detailed conditions of LC/MS mesurement.
Methods and materials
3
Simultaneous analysis of primary metabolites by triple quadrupole LC/MS/MS using penta�uorophenylpropyl column
UHPLC conditions (Nexera system using a PFPP column)
Column : Discovery HS F5 150 mm×2.1 mm, 3.0 µm
Mobile phase A : 0.1% Formate/water
B : 0.1% Formate/acetonitrile
Flow rate : 0.25 mL/min
Time program : B conc.0%(0-2.0 min) - 25%(5.0 min) - 35%(11.0 min)
- 95%(15.0.-20.0 min) - 0%(20.1-25.0 min)
Injection vol. : 3 µL
Column temperature : 40°C
MS conditions (LCMS-8030/LCMS-8040)
Ionization : Positive/Negative, MRM mode
DL Temp. : 250°C
HB Temp : 400°C
Drying Gas : 10 L/min
Nebulizing Gas : 2.0 L/min
Result
The MRM transitions for 97 standard compounds were optimized on both positive and negative mode by flow injection analysis (FIA). The MRM transitions of the 97 metabolites were determined as described in Table 1. Subsequently, LC condition was investigated to separate the 97 metabolites with a good resolution. As a consequence, the 97 metabolites were eluted from a PFPP column with a gradient of acetonitrile for <15 min in the
condition described in Figure 1. The linearity of this method was also confirmed by the simultaneous analysis of a serial of diluted calibration curve.
Figure 1 shows the MRM chromatogram of 97 metabolites at a concentration of 5 µM. In this figure, we can see the peak from all metabolites with a good separation.
Optimization of MRM transition
4
Simultaneous analysis of primary metabolites by triple quadrupole LC/MS/MS using penta�uorophenylpropyl column
Table 1 MRM transition of 97 metabolites
Name Product ion Precursor ion Linearity (R2)
2-Aminobutyrate
Acetylcarnitine
Acetylcholine
Adenine
Adenosine
Adenylsuccinate
ADMA
Ala
AMP
Arg
Argininosuccinate
Asn
Asp
cAMP
Carnitine
Carnosine
cCMP
cGMP
Choline
Citicoline
Citrulline
CMP
Creatine
Creatinine
Cys
Cystathionine
Cysteamine
Cystine
Cytidine
Cytosine
Dimethylglycine
DOPA
Dopamine
Epinephrine
FAD
GABA
gamma-Glu-Cys
Gln
Glu
Gly
GMP
GSH
Guanosine
His
Histamine
Homocysteine
Homocystine
Hydroxyproline
Hypoxanthine
Ile
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
104.10
204.10
147.10
136.00
268.10
464.10
203.10
89.90
348.00
175.10
291.00
133.10
134.00
330.00
162.10
227.10
306.00
346.00
104.10
489.10
176.10
324.00
132.10
114.10
122.00
223.00
78.10
241.00
244.10
112.00
104.10
198.10
154.10
184.10
786.15
104.10
251.10
147.10
147.90
75.90
364.00
308.00
284.00
155.90
112.10
136.00
269.00
132.10
137.00
132.10
58.05
85.05
87.05
119.05
136.05
252.10
70.10
44.10
136.05
70.10
70.10
87.15
74.05
136.05
103.05
110.05
112.10
152.05
60.05
184.10
70.05
112.05
44.05
44.05
76.05
88.05
61.05
151.95
112.05
95.10
58.05
152.10
91.05
166.10
136.10
87.05
84.10
84.15
84.10
30.15
152.05
179.10
152.00
110.10
95.05
90.10
136.05
86.05
55.05
86.20
Polarity
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
0.99
0.99
0.99
0.98
0.99
0.99
0.99
0.99
0.99
0.99
0.99
0.99
0.99*
0.99
0.99
0.99*
0.99
0.99
0.99
0.99*
0.99
0.99
0.99
0.99
0.99*
0.99
0.98*
0.99
0.99
0.99
0.99
0.99*
0.99*
0.99
0.99*
0.99
0.99*
0.99
0.99*
0.99*
0.99
0.99*
0.99
0.99
0.99*
0.99*
0.99
0.99
0.98*
0.99
Name Product ion Precursor ion Linearity (R2)
Inosine
Kynurenine
Leu
L-Norepinephrine
Lys
Met
Methionine-sulfoxide
Nicotinamide
Nicotinic acid
Ophthalmic acid
Ornitine
Pantothenate
Phe
Pro
SAH
SAM
SDMA
Ser
Serotonin
Thr/Homoserine
Thymidine
Thymine
TMP
Trp
Tyr
Uracil
Uridine
Val
2-Oxoglutarate
Allantoin
Cholate
cis-Aconitate
Citrate
FMN
Fumarate
GSSG
Guanine
Isocitrate
Lactate
Malate
NAD
Orotic acid
Pyruvate
Succinate
Taurocholate
Uric acid
Xanthine
No.
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
269.10
209.10
132.10
170.10
147.10
149.90
166.00
123.10
124.05
290.10
133.10
220.10
166.10
115.90
385.10
399.10
203.10
105.90
177.10
120.10
243.10
127.10
322.90
205.10
182.10
113.00
245.00
118.10
144.90
157.00
407.20
172.90
191.20
455.00
115.10
611.10
150.00
191.20
89.30
133.10
663.10
155.00
86.90
117.30
514.20
167.10
151.00
137.05
192.05
86.05
152.15
84.10
56.10
74.10
80.05
80.05
58.10
70.10
90.15
120.10
70.10
134.00
250.05
70.15
60.10
160.10
74.15
127.10
54.05
81.10
188.15
136.10
70.00
113.05
72.15
101.10
97.10
343.15
85.05
111.10
97.00
71.00
306.00
133.00
111.10
89.05
114.95
541.05
111.10
87.05
73.00
107.10
123.95
108.00
Polarity
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
0.99
0.99
0.99
0.99
0.99
0.99
0.99
0.99
0.99
0.99
0.99
0.99
0.99
0.99
0.98
0.99*
0.99
0.99*
0.99
0.99
0.99
0.99*
0.99*
0.99
0.99
0.99*
0.99
0.99
0.98*
0.98*
0.99**
0.99
0.99*
0.99
0.99**
0.99*
0.99*
0.99*
0.97*
0.99*
0.99*
0.99
0.99*
0.99*
0.99*
0.99*
0.99*
Calibration curve was obtained at a range of concentration from 10 nM to 10000 nM.* Calibration curve was obtained at a range of concentration from 100 nM to 10000 nM.** Calibration curve was obtained at a range of concentration from 1000 nM to 10000 nM.
5
Simultaneous analysis of primary metabolites by triple quadrupole LC/MS/MS using penta�uorophenylpropyl column
Figure 1 MRM chromatogram of 97 compounds
Simultaneous analysis of 99 compounds was performed for heart / liver tissue extracts as biological samples. Figure 2 shows MRM chromatograms of 99 compounds from tissue extracts (liver/heart). In this measurement, 83/97 metabolites were detected from liver tissue extracts and 88/97 metabolites were confirmed from heart tissue extracts. These results show this method is also effective to
simultaneous analysis of biological samples. As shown in the resulting MRM chromatogram, some major peaks were derived from the metabolites which were known to be characteristic to each tissue. Furthermore, this characteristic difference in each tissue was also confirmed in some faint peaks (e.g., cholate, cystine and homocysteine).
Application to tissue extracts as biological samples
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
Simultaneous analysis of primary metabolites by triple quadrupole LC/MS/MS using penta�uorophenylpropyl column
6
We have previously reported simultaneous analysis of 55 metabolites which were related to central carbon metabolic pathway by using ion pairing chromatography at ASMS conference 2013. To evaluate the accuracy of this simultaneous method using PFPP column, we compared the resulting peak area of 25 metabolites, which were covered as targets in both methods. The 25 metabolites are Lysine, Arginine, Histidine, Glycine, Serine, Asparagine, Alanine, Glutamine, Threonine, Methionine, Tyrosine, Glutamate, Aspartae, Phenylalanine, Tryptophan, Cysteine, CMP, NAD, GMP, TMP, AMP, cGMP, cAMP, MES and L-Methionine sulfone as internal standards. Heart tissue extracts were prepared from mice (n=9) according to the
method described above and the aliquots were measured by the simultaneous method using either ion pairing chromatography or PFPP separation system. As a result, we could see the similar trend of elevation/decrease of peak area in metabolites of 20/25 between nine samples. The peak areas between 9 samples of representative metabolites are shown in Figure 3. This result shows that a ratio of areas between 9 samples is kept in both methods. The four metabolites (TMP, cGMP, cAMP and Cysteine) could be hardly detected on simultaneous analysis by alternately ion-paring chromatography or PFPP column. Tryptophan had a faint peak in this experiment and led to the low similarity.
Correlation between PFPP and ion pairing Methods
Figure 2 MRM chromatogram of liver/heart tissue extracts
Liver Tissue
Heart Tissue
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0
0
5000000
10000000
15000000
20000000
25000000
30000000
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0
0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
9000000
10000000
Acetylcarnitine
Creatine
Ophtalmic acid
GSSG
Guanosine
S-Adenosylhomocysteine
GSH
AMP
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Simultaneous analysis of primary metabolites by triple quadrupole LC/MS/MS using penta�uorophenylpropyl column
Conclusions• The 97 metabolites were separated by PFPP column with high resolution and this method was applied to biological
samples.• The utility of this simultaneous analysis using PFPP column was con�rmed by comparing between PFPP and ion paring
chromatography.
0.0E+00
5.0E+05
1.0E+06
1.5E+06
1 2 3 4 5 6 7 8 9
MES
0.0E+00
5.0E+05
1.0E+06
1.5E+06
2.0E+06
1 2 3 4 5 6 7 8 9
Serine
0.0E+00
1.0E+05
2.0E+05
3.0E+05
4.0E+05
5.0E+05
1 2 3 4 5 6 7 8 9
Threonine
0.0E+00
1.0E+05
2.0E+05
3.0E+05
4.0E+05
5.0E+05
1 2 3 4 5 6 7 8 9
L-Methionine sulfone
PFPP
0.0E+00
2.0E+05
4.0E+05
6.0E+05
8.0E+05
1.0E+06
1 2 3 4 5 6 7 8 9
MES
0.0E+00
5.0E+03
1.0E+04
1.5E+04
2.0E+04
2.5E+04
1 2 3 4 5 6 7 8 9
Serine
0.0E+00
1.0E+04
2.0E+04
3.0E+04
4.0E+04
1 2 3 4 5 6 7 8 9
Threonine
0.0E+00
2.0E+05
4.0E+05
6.0E+05
8.0E+05
1.0E+06
1 2 3 4 5 6 7 8 9
L-Methionine sulfone
Ion pairing
0.0E+00
2.0E+06
4.0E+06
6.0E+06
8.0E+06
1 2 3 4 5 6 7 8 9
Aspartate
0.0E+005.0E+041.0E+051.5E+052.0E+052.5E+053.0E+05
1 2 3 4 5 6 7 8 9
GMP
0.0E+00
5.0E+06
1.0E+07
1.5E+07
1 2 3 4 5 6 7 8 9
AMP
0.0E+00
1.0E+06
2.0E+06
3.0E+06
4.0E+06
1 2 3 4 5 6 7 8 9
Phenylalanine
PFPP
0.0E+00
1.0E+05
2.0E+05
3.0E+05
4.0E+05
5.0E+05
1 2 3 4 5 6 7 8 9
Aspartate
0.0E+001.0E+042.0E+043.0E+044.0E+045.0E+046.0E+04
1 2 3 4 5 6 7 8 9
GMP
0.0E+001.0E+052.0E+053.0E+054.0E+055.0E+056.0E+05
1 2 3 4 5 6 7 8 9
AMP
0.0E+00
1.0E+04
2.0E+04
3.0E+04
4.0E+04
1 2 3 4 5 6 7 8 9
Phenylalanine
Ion pairing
Figure 3 Correlation of peak areas between PFPP and ion-pairing method
Life Science
• Page 222Surface analysis of permanent wave processing hair using DART-MS
• Page 229Analysis of allergens found in cosmetics using MDGC-GCMS (Multi-Dimensional Gas Chromato-graph Mass Spectrometer)
PO-CON1454E
Surface analysis of permanent waveprocessing hair using DART-MS
ASMS 2014 MP 476
Shoji Takigami1, Erika Ikeda1, Yuta Takagi1,
Jun Watanabe2, Teruhisa Shiota3
1 Gunma University, Kiryu, Japan;
2 Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan;
3 AMR Inc., Meguro-ku, Tokyo, Japan
2
Surface analysis of permanent wave processing hair using DART-MS
IntroductionPermanent wave processing of hair is carried out at two processes as follows; (A) Reducing agent (permanent wave 1 agent) makes the bridge construction between the keratin protein molecular chains of hair, especially disul�de (S-S) bond of cystine residue cleaved to thiol (-SH) group and hear results a wave and curl.(B) Oxidizing agent (permanent wave 2 agent) makes -SH group oxidized to be reproduced S-S bond. As reducing agents used for permanent wave 1 agent, the thing of cosmetics approval, such as cysteamine hydrochloride and a butyrolactone thiol (brand name Spiera, other than quasi drugs, such as ammonium thioglycolate, acetyl cystein, and thiolactic acid, are used.
After hair is applying permanent wave processing and coloring repeatedly, the chemical structure of a keratin molecule and �ne structure in the hair have been damaged and it resulted as damage hair. It is thought that hair becomes dryness and twining if the cuticle which covers hair is damaged, so it is important to investigate the surface structure of hair and its chemical structure changing.DART (Direct Analysis in Real Time), a direct atmospheric pressure ionization source, is capable of analyzing samples directly with little or no sample preparation. Here, analysis of the ingredient which has deposited on the permanent wave processing hair surface was tried using this DART combined with a mass spectrometer.
The chemical state and property were investigated in the surface of the hair which repeated permanent wave processing with these reducing agents.
Figure 1 DART-OS ion source & LCMS-2020
High Speed Mass Spectrometer
Ufswitching High-Speed Polarity Switching 15msec Ufscanning High-Speed Scanning 15,000u/sec
TGA(thioglycolate)
CA(cysteamine hydrochloride)
BLT(butyrolactone thiol)
SH
O
HOO
O
SH
Fw 92 Fw 113 Fw 118
H2NSH
HCl
Wave ef�ciency is good in a weak alkaline (pH 8 - 9.5)
Wave ef�ciency is good in a weak alkaline (pH 8 - 9.5)
Wave ef�ciency is good in a weak acid (pH 6)
3
Surface analysis of permanent wave processing hair using DART-MS
Methods and MaterialsThe Chinese virgin hair purchased from the market was washed with the 0.5% non-ionic surfactant containing saturated EDTA solution, and then it was considered as untreated hair sample. Permanent wave processing of hair was prepared as following; the 0.6M TGA solution and 0.6M CA solution which were adjusted to pH8.5 with aqueous ammonia and the 0.6M BLT solution adjusted to pH6.0 with arginine water, which were used as a reducing
agent. After hair sample was reduced for 15 minutes at 35°C using each solvent, it was carried out oxidation treatment at 35°C by being immersed in 8% sodium bromate solution (pH7.2) for 15 minutes. LCMS-2020 (Shimadzu) was coupled with DART-OS ion source (IonSense) and hear samples were held onto DART gas �ow directly, then their surface analyzed.
MS condition (LCMS-2020; Shimadzu Corporation)
Ionization : DART (Direct Analysis in Real Time)
Heater Temperature (DART) : 350°C
Measuring mode (MS) : Positive/Negative scanning simultaneously
Chinese Virgin Hair
0.5% Laureth - 9 solution - EDTA saturated 35°C 1h
Water washing and air drying
Untreated Permanent wave processing by agent 1 & 2 at 0.6M each
Analyzed by DART-MS
permanent wave 1 agent : TGA or CA (pH 8.5; aqueous ammonium) BLT (pH 6; arginine) 35°C 15min
Water washing
Water washing
Britton - Robinson buffer (pH 4.6) 35°C 15min
Water washing
permanent wave 2 agent : 8% NaBrO3 solution (pH 7.2) 35°C 15min
Air drying
Repeat6 times
4
Surface analysis of permanent wave processing hair using DART-MS
ResultAfter repeating operation of permanent wave processing 1-6 times using TGA (thioglycollic acid), CA (cysteamine), and BLT (Butyrolactonethiol), hair was immersed for 15 minutes at 35°C and with a �ush and air-drying, then permanent wave processing hair was prepared. In order to investigate the ingredient which has deposited on the permanent wave processing hair surface, DART-MS analysis
was performed. DART-MS analysis was conducted in order of #1 Untreated (woman hair), #2 control; ammonia treatment (pH 8.5), #3 0.6M thioglycolic acid (TGA) processing, #4 0.6M butyrolactone thiol (BLT) processing, #5 0.6M cysteamine hydrochloride (CA) processing and #6 control; arginine processing (pH 6).
In the DART mass spectra of #1 untreated and #6 control, many signals considered as triglyceride and diglyceride were detected in both positive and negative spectra obtained by DART-MS. In #3 0.6M thioglycolic acid (TGA) processing spectra, the signal in particular of TGA origin was not detected. In #4 BLT processing spectra (Figure 3), the signals considered to be oxidized BLT (3, 3'-dithiobis (tetrahydrofuran2-one), molecular weight 234) were detected at m/z 235 and 252 in the positive mode. The signal m/z 235 is equivalent to [M+H]+ and m/z 252, [M+NH4]+. In the negative mode, the signals, m/z 115,
231 were detected. They were considered the signal equivalent to [M-H]- and [2M-H]- of BLT oxide compound (C4H4O2S, molecular weight 116) in which two hydrogen atoms were removed from BLT. Carrying out permanent wave processing by BLT, it was found that the dimer of BLT accumulated on the cuticle surface.In #5 CA processing spectrum (Figure 5), the signal considered to be the dimer (Fw152) origin in which CA carried out S-S bond in the positive mode was detected at m/z 153. This is equivalent to [M+H]+.
Figure 2 TIC chromatogram of each sample analyzing with DART
0
25000000
50000000
75000000 2:TIC(+)
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 min
5000000
10000000
150000004:TIC(-)
#1 #2 #3 #4 #5 #6
Positive TIC m/z 30-2000
Negative TIC m/z 30-2000
Surface analysis of permanent wave processing hair using DART-MS
5
Figure 3 DART-MS spectra of #4 BLT processingThe BLT-related signals were detected from the positive and the negative spectra.
Figure 4 DART-MS spectra of #5 CA processingThe CA-related signal was detected from the positive spectrum
100 200 300 400 500 600 700 800 900 1000 1100 m/z0.00
0.25
0.50
0.75
1.00
1.25
1.50
Inten. (x10,000,000)
252
486282 368 424 516
100 200 300 400 500 600 700 800 900 1000 1100 m/z0.0
1.0
2.0
3.0
4.0
5.0Inten. (x100,000)
179
115
231
321347
411 501 579
235
Positive
Negative
[M+H]+[M+NH4]+
[M-H]-
[2M-H]-
100 200 300 400 500 600 700 800 900 1000 1100 m/z0.00
0.25
0.50
0.75
1.00
1.25
1.50
Inten. (x1,000,000)
282124
391
252
468424 563
184600102 644 691 769 851 922
153
Positive
[2M+H]+
Surface analysis of permanent wave processing hair using DART-MS
6
In order to indicate clearly the signals specifically detected in each sample, the extraction chromatograms (XIC) were shown (Figure 5). It turned out that BLT-related signals were detected only in #4 and the CA-related signal in #5. Moreover, although the signal intensity was weak, the signal at negative m/z 325 was detected from all samples. Negative m/z 325 is equivalent to [M-H]- of 18
methyl eicosanoic acid (18MEA, molecular weight 326). 18MEA is one of lipid components which protect a cuticle. There is no significant difference of this signal in the hair between treated hair and untreated hair. We would like to inquire so that intensity difference can be found out by further verifying the detection technique in the future.
Figure 5 XIC chromatorgam of each sample analyzing with DART
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 min
0
500000
1000000 2:152.85(+)
0
5000000
10000000 2:234.70(+)
0
5000000
100000002:251.75(+)
0
250000
4:114.95(-)
0
500000
4:230.90(-)
0
500000
1000000
1500000 2:123.85(+)
0
50000
1000004:325.15(-)
Positive XIC m/z 153
Positive XIC m/z 252
Negative XIC m/z 231
Negative XIC m/z 115
#1
Positive XIC m/z 124
#2 #3 #4 #5 #6
Positive XIC m/z 235
Negative XIC m/z 325
Surface analysis of permanent wave processing hair using DART-MS
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
ConclusionsBy direct analysis of the hair by DART-MS, the chemical structure change in the surfaces of hair, such as permanent wave processing, was able to be observed.
PO-CON1469E
Analysis of allergens found in cosmetics using MDGC-GCMS (Multi-Dimensional Gas Chromatograph Mass Spectrometer)
ASMS 2014 TP761
Sanket Chiplunkar, Prashant Hase, Dheeraj Handique,
Ankush Bhone, Durvesh Sawant, Ajit Datar,
Jitendra Kelkar, Pratap Rasam
Shimadzu Analytical (India) Pvt. Ltd., 1 A/B Rushabh
Chambers, Makwana Road, Marol, Andheri (E),
Mumbai-400059, Maharashtra, India.
2
Analysis of allergens found in cosmetics using MDGC-GCMS (Multi-Dimensional Gas Chromatograph Mass Spectrometer)
IntroductionCosmetics, fragrances and toiletries (Figure 1) are used safely by millions of people worldwide. Although many people have no problems, irritant and allergic reactions may occur. Irritant and allergic skin reactions are the types of contact dermatitis. Essential oils present in fragrance contain some natural and synthetic compounds, which may cause allergic reactions to the end user after application. There are 26 potential allergens listed by
European Directive (EU) 2003/15/EC and International Fragrance Association (IFRA)[1] labeled on cosmetics. Shimadzu MDGC-GCMS technology facilitates the identi�cation and quanti�cation of these allergens to comply with the threshold limits of 100 ppm for rinse-off products.Co-eluting peaks were resolved completely with the help of MDGC-GCMS heart-cut technique.
Figure 1. Cosmetics, fragrances and toiletries
Method of Analysis
Shampoo samples were collected from local market. Standard solutions of 23 allergens were procured from ACCU Standard and dilutions were carried out in Ethanol/Acetonitrile to yield 1000 ppm concentration. Further dilutions were made in methanol.MDGC-GCMS technique was effectively used to minimize matrix effect. Co-eluting peaks were resolved with heart-cut technique using two columns of different
polarities. In MDGC-GCMS, 1st instrument was GC-2010 Plus equipped with FID as a detector and 2nd instrument was GCMS-QP2010 Ultra with MS as a detector. Columns in both the instruments were connected with Deans switch. Allergens in shampoo samples were determined by using this technique. For sample preparation, following methodology was adopted.
Extraction of allergens from shampoo sample
Part method validation was carried out by performing system precision, sample precision, linearity and recovery study. For validation, solutions of different concentrations
were prepared using 40 ppm (actual concentration) standard stock solution mixture of allergens.
1) Blank Solution : 10 mL of methanol was transferred in 20 mL centrifuge tube and vortexed for 5 minutes. The mixture was then centrifuged for 5 minutes at 3000 rpm. This solution was filtered through 0.2 µm nylon syringe filter. Initial 2 mL was discarded and remaining filtrate was collected.
2) Sample Solution : 1 g of shampoo sample was weighed in 10 mL volumetric flask and diluted up to the mark with methanol. Above mixture was transferred in 20 mL centrifuge tube. Further processing was done as mentioned in blank solution.
3) Spike Sample Solution : For recovery study, 1 g of sample was spiked with different volumes of standard stock solution. The above procedure was repeated for preparing different concentration levels of allergens in samples. These spiked samples were treated as mentioned in sample solution.
3
Analysis of allergens found in cosmetics using MDGC-GCMS (Multi-Dimensional Gas Chromatograph Mass Spectrometer)
MDGC-GCMS Analytical ConditionsThe instrument con�guration used is shown in Figure 2. Samples were analyzed using Multi-Dimensional GC/GCMS as per the conditions given below.
Table 1. Method validation parameters
Figure 2. Multi-Dimensional GC/GCMS System by Shimadzu
Figure 3. Schematic diagram of multi-Deans switch in MDGC-GCMS
Parameter Concentration
System Precision
Sample Precision
Linearity
Accuracy / Recovery
10 ppm
10 % in Methanol
2.5, 5, 7.5, 10, 15 (ppm)
5, 10, 15 (ppm)
4
Analysis of allergens found in cosmetics using MDGC-GCMS (Multi-Dimensional Gas Chromatograph Mass Spectrometer)
MDGC-GCMS analytical parametersChromatographic parameters (1st GC : GC-2010 Plus)
• Column : Stabilwax (30 m L x 0.25 mm I.D.; 0.25 μm)
• Injection Mode : Split
• Split Ratio : 5.0
• Carrier Gas : Helium
• Column Flow : 2.27 mL/min
• Detector : FID
• APC Pressure : 200 kPa (For switching)
• Column Oven Temp. : Rate (ºC /min) Temperature (ºC) Hold time (min)
50.0 0.00
15.00 100.0 0.00
5.00 240.0 43.67
Chromatographic parameters (2nd GCMS : GCMS-QP2010 Ultra)
• Column : Rxi-1ms (30 m L x 0.25 mm I.D.; 0.25 μm)
• Detector : Mass spectrometer
• Ion Source Temp. : 200 ºC
• Interface Temp. : 240 ºC
• Ionization Mode : EI
• Event Time : 0.30 sec
• Mode : SIM and SCAN
• Column Oven Temp. : Rate (ºC /min) Temperature (ºC) Hold time (min)
80.0 13.00
3.00 180.0 0.00
10.00 260.0 20.67
• Total Program Time : 75.00 min
Results
MDGC-GCMS technique was used to avoid matrix interference from sample. Using multi-Deans switch and heart-cut technique (Figure 3), co-eluted components from the 1st column were transferred to the 2nd column with different polarity.
Sample analysis using MDGC-GCMS
5
Analysis of allergens found in cosmetics using MDGC-GCMS (Multi-Dimensional Gas Chromatograph Mass Spectrometer)
Figure 5. Chromatogram with 1st column (FID)
Figure 4. Chromatogram of spiked sample solution before switching
Table 2. Summary of results for precision on GC and GCMS
Figure 6. SIM chromatogram with 2nd column (MS)
5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 min
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
uV (x100,000) Chromatogram
Sam
ple
- 1
Lim
onen
e
Lina
lool
Met
hyl h
eptin
e ca
rbon
ate
Sam
ple
- 2
Sam
ple
- 3 Citr
al -
1
Citr
al -
2 Citr
onel
lol
Ger
anio
lBe
nzyl
Alc
ohol
Hyd
roxy
-citr
onel
lal
Cin
nam
al
Euge
nol
Am
yl c
inna
mal
Ani
syl a
lcoh
olC
inna
myl
alc
ohol
Fern
esol
- 1
Isoe
ugen
olFe
rnes
ol -
2Fe
rnes
ol -
2H
exyl
cin
nam
ald
ehyd
e
Cou
mar
in
Am
ylci
n na
myl
alc
ohol
Sam
ple
- 5
Benz
yl b
enzo
ate
Sam
ple
- 6
Benz
yl s
alic
ylat
e
Benz
yl C
inna
mat
e
12.0 13.0 14.0 15.0 16.0 17.0 min
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
uV (x10,000) Chromatogram
Met
hyl h
eptin
e ca
rbon
ate
Sam
ple
- 2
Sam
ple
- 3
Citr
al -
1
Citr
al -
2
Citr
onel
lol
Ger
anio
l Benz
yl A
lcoh
ol
25.0 25.5 26.0 26.5 27.0 27.5 28.0 28.5 min
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0uV (x10,000) Chromatogram
Am
yl c
inna
mal
Ani
syl a
lcoh
olC
inna
myl
alc
ohol
Fern
esol
- 1
Isoe
ugen
ol Fern
esol
- 2
Fern
esol
- 2
Hex
yl c
inna
m a
ldeh
yde
26.5 27.0 27.5 28.0 min0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
2.50
2.75
3.00
3.25
3.50uV (x10,000) Chromatogram
26.2
56
26.4
91
28.1
05
Target compound - Isoeugenol
27.0 27.5 28.0 28.5 29.0 29.5-1.0
0.0
1.0
2.0
3.0
4.0
5.0
6.0
(x100,000)
134.00 (100.00)115.00 (100.00)92.00 (100.00)137.00 (100.00)109.00 (100.00)138.00 (100.00)103.00 (100.00)149.00 (100.00)164.00 (100.00)
Target compound - Isoeugenol
Summary of results
Result
% RSD for area (n=6) < 2.0
% RSD for area (n=6) < 2.0
Concentration
10 ppm
Unknown
Sample name
23 Allergens mixture
Shampoo
Type of sample
Standard
Cosmetic
Sr. No.
1
2
Analysis of allergens found in cosmetics using MDGC-GCMS (Multi-Dimensional Gas Chromatograph Mass Spectrometer)
6
Figure 7. Linearity graph for linalool
Table 3. Linearity by GC
For the quantitation studies, the shampoo sample was spiked with allergens standard to achieve 5, 10 and 15 ppm concentrations. Recovery studies were performed on 13 allergens, having co-elution or matrix interference, using heart-cut technique. The quantitation of these allergens was carried out using 2nd detector (MS) in SIM mode.
In below recovery study, some allergens had recovery value out side the acceptance limit (70-130 %). Optimization can be done by means of change in sample clean up procedure and filtration study.
Quantitation of allergens in shampoo sample
Name of allergen
Linalool
Methyl heptine carbonate
Citronellol
Geraniol
Hydroxy citronellal
Cinnamal
Amyl Cinnamal
Coumarin
Amylcin namyl alcohol
Benzyl benzoate
Sr. No.
1
2
3
4
5
6
7
8
9
10
Linearity (R2)
0.9945
0.9949
0.9965
0.9962
0.9973
0.9959
0.9976
0.9971
0.9983
0.9979 0.0 2.5 5.0 7.5 10.0 12.5 Conc.0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
Area (x10,000)
Figure 8. Linearity graph for benzyl cinnamate
Table 4. Linearity by GCMS
Name of allergen
Limonene
Benzyl alcohol
Citral - 1
Citral - 2
Eugenol
Anisyl alcohol
Cinnamyl alcohol
Isoeugenol
Farnesol - 1
Farnesol - 2
Hexyl cinnam aldehyde
Benzyl salicylate
Benzyl cinnamate
Sr. No.
1
2
3
4
5
6
7
8
9
10
11
12
13
Linearity (R2)
0.9945
0.9871
0.9889
0.9902
0.9894
0.9916
0.9937
0.9902
0.9919
0.9929
0.9932
0.9853
0.9927
0.0 2.5 5.0 7.5 10.0 12.5 Conc.0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
Area(x10,000)
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Analysis of allergens found in cosmetics using MDGC-GCMS (Multi-Dimensional Gas Chromatograph Mass Spectrometer)
Conclusion• MDGC-GCMS method was developed for quantitation of allergens present in cosmetics. Part method validation was
performed as per ICH guidelines.[2] Results obtained for reproducibility, linearity and recovery studies were well within acceptable limits.
• Simultaneous SCAN/SIM and high-speed scan rate 20,000 u/sec are the characteristic features of GCMS-QP2010 Ultra, which enables quantitation of allergens at very low concentration level.
• Matrix effect from cosmetics was selectively eliminated using MDGC-GCMS with multi-Deans switching unit and heart-cut technique.
• MDGC-GCMS was found to be very useful technique for simultaneous identi�cation and quantitation of components from complex matrix.
Reference[1] IFRA guidelines (International Fragrance Association), GC/MS Quanti�cation of potential fragrance allergens, Version 2,
(2006), 6.[2] ICH guidelines, Validation of Analytical Procedures: Text And Methodology Q2(R1), Version 4, (2005).
Figure 9. Overlay SIM chromatogram of unspiked and spiked sample
Table 5. Quantitation of allergens – Recovery Study
Name of allergen
Limonene
Benzyl alcohol
Citral - 1
Citral - 2
Eugenol
Anisyl alcohol
Cinnamyl alcohol
Isoeugenol
Farnesol - 1
Farnesol - 2
Hexyl cinnam aldehyde
Benzyl salicylate
Benzyl cinnamate
Sr. No.
1
2
3
4
5
6
7
8
9
10
11
12
13
Level -15 ppm
127
114
101
97
96
94
98
103
83
84
121
63
66
Level -210 ppm
% Recovery
126
114
106
103
105
105
106
108
95
95
122
47
61
Level -315 ppm
129
123
114
112
116
116
115
118
107
106
130
32
5625.0 27.5 30.0 32.5
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
2.50
2.75
3.00
(x1,000)
Farnesol-1
min
Farnesol-2
Spiked
Unspiked
m/z : 69.00
Technical Applications
�•�Page�238Applications�of�desorption�corona�beam� ionization-mass�spectrometry
�•�Page�243Rapid�analysis�of�carbon�fiber�reinforced�plastic�using�DART-MS
•�Page�249Analysis�of�styrene�leached�from�polystyrene�cups�using�GCMS�coupled�with�Headspace�(HS)�sampler
PO-CON1474E
Applications of Desorption Corona Beam Ionization-Mass Spectrometry
ASMS 2014 WP 393
Yuki Hashi1, Shin-ichi Kawano1, Changkun Li1, Qian Sun1,
Taohong Huang1, Tomoomi Hoshi2, Wenjian Sun3
1Shimadzu (China) Co., Ltd., Shanghai, China 2Shimadzu Corporation, Kyoto, Japan 3Shimadzu Research Laboratory (Shanghai) Co., Ltd.,
Shanghai, China
2
Applications of Desorption Corona Beam Ionization-Mass Spectrometry
IntroductionNumerous ambient ionization mass spectrometric techniques have been developed for high throughput analysis of various compounds with minimum sample pretreatment.(1) Desorption corona beam ionization (DCBI) is a more recent technique.(2) In DCBI, helium is used as discharge gas and heating of the gas is required for sample
desorption. A visible thin corona beam is formed by using hollow needle/ring electrode structure. This feature facilitates localizing sampling areas and obtaining good reproducibility of data. Details of DCBI hardware are shown in Figs. 1 and 2. In this study, DCBI was applied for analysis of various samples.
Figure 2 DCBI interface
Figure 1 Schematic diagram of DCBI
+
-
HVDC
LVDC
MS inlet
Counter electrode
Heated thin wall tubing
Helium �ow
Sample and stage
Dischargeneedle
Sampling capillary
Corona beam
MS Inlet
DCBI probe
Manual liquidsampler
3
Applications of Desorption Corona Beam Ionization-Mass Spectrometry
Results and DiscussionIn this experiment, all compounds with variety of polarity from non- to high-polar gave protonated molecules (Figs. 3-8). Methomyl gave also fragment ions (m/z 106) by
cleavage at methylcarbamoyl group, while fragment ions with signi�cant intensity were not observed for other compounds. Analysis time was less than 1 minute.
Method
Samples (melamine, saturated hydrocarbon mixture, polyaromatic hydrocarbon mixture, testosterone, pirimicarb, and methomyl) were dissolved in methanol or acetonitrile.
Sample Preparation
Samples were analyzed using a DCBI system coupled to a LCMS-2020 quadrupole mass spectrometer (Shimadzu Corporation, Japan). The system was operated with the DCBI control software and LabSolutions for LCMS version 5.42.
DCBI-MS Analysis
Analytical Conditions
Figure 3 Mass spectrum of melamine (0.5 mg/mL)
DCBI
Flow rate : 0.6 L/min
HV discharge : +2.0-3.0 kV
He gas temperature : 350 ºC
Sample volume : 1, or 2 µL
MS (LCMS-2020 quadrupole mass spectrometer)
Polarity : Positive
DL temperature : 250 ºC
BH temperature : 400 ºC
Mass range : m/z 100-500
100.0 105.0 110.0 115.0 120.0 125.0 130.0 135.0 140.0 145.0 m/z0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
Inten. (x1,000)
127.1
136.0 148.6
4
Applications of Desorption Corona Beam Ionization-Mass Spectrometry
Figure 4 Mass spectrum of saturated hydrocarbon mixture (1 mg/mL)
Figure 5 Mass spectrum of polyaromatic hydrocarbon mixture (2 mg/mL)
Figure 6 Mass spectrum of testosterone (1 mg/mL)
Compound MWC10H22 142C11H24 156C12H26 170C13H28 184C14H30 198C15H32 212C16H34 226C17H36 240C18H38 254C19H40 268C20H42 282C21H44 296C22H46 310C23H48 324C24H50 338C25H52 352
100 150 200 250 300 350 m/z0.00
0.25
0.50
0.75
1.00
1.25
1.50Inten. (x100,000)
241.3213.2
255.3269.3
199.2
283.3
297.3185.2
311.3
171.2 325.3
339.3157.2
115.1 143.2 367.4
100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 m/z0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5Inten. (x10,000)
153.1
155.2
179.1
203.1167.2
209.1195.1129.1
141.2 235.1115.1 276.2
Compound MWNaphthalene 128Acenaphthylene 152Acenaphthene 154Fluorene 166Anthracene 178Phenanthrene 178Pyrene 202Fluoranthene 202Chrysene 228Benzo[a]anthracene 228
150 200 250 300 350 400 450 m/z0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
Inten. (x10,000)
289.2
331.2 461.4112.1
424.5
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Applications of Desorption Corona Beam Ionization-Mass Spectrometry
ConclusionThe DCBI system was successfully applied for analysis of samples with various polarity.Mass spectra were quickly obtained after sample introduction to the DCBI probe.The method is useful for fast identi�cation of various compounds.
References(1) Monge ME et al, Chem. Rev. 113 (2013), 2269-2308(2) Hua W et al, Analyst 135 (2010), 688-695
Figure 7 Mass spectrum of pirimicarb (0.5 mg/mL) Figure 8 Mass spectrum of methomyl (0.5 mg/mL)
100 150 200 250 300 350 400 450 m/z0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
Inten. (x100,000)
239.2
182.2
100 150 200 250 300 350 400 450 m/z0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2
Inten. (x100,000)
163.0
105.9
194.0121.9 252.0 354.1 394.3208.0
PO-CON1456E
Rapid analysis of carbon �berreinforced plastic using DART-MS
ASMS 2014 TP 782
Hideaki Kusano1, Jun Watanabe1, Yuki Kudo2,
Teruhisa Shiota3
1 Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan;
2 Bio Chromato, Inc., Fujisawa, Japan;
3 AMR Inc., Meguro-ku, Tokyo, Japan
2
Rapid analysis of carbon �ber reinforced plastic using DART-MS
IntroductionDART (Direct Analysis in Real Time) can ionize and analyze samples directly under atmospheric pressure, independent of the sample forms. Then it is also possible to measure in form as it is, without sample preparation. Qualitative analysis of target compounds can be conducted very fast and easily by combining DART with LCMS-2020/8030 which have ultra high-speed scanning and ultra high-speed polarity switching. Carbon-�ber-reinforced plastics, CFRP is the �ber-reinforced plastic which used carbon �ber for the reinforced material, which is only called carbon resin or
carbon in many cases. An epoxy resin is mainly used for a base material in CFRP. While CFRP is widely used taking advantage of strength and lightness, most approaches which measure CFRP with analytical instruments were not tried, triggered by the dif�culty of the preparation.DART (Direct Analysis in Real Time), a direct atmospheric pressure ionization source, is capable of analyzing samples with little or no sample preparation. Here, rapid analysis of carbon �ber reinforced plastic was carried out using DART combined with a mass spectrometer.
Methods and MaterialsThermosetting polyimide (carbon-�ber-reinforced plastics) and thermoplastic polyimide (control sample) were privately manufactured. After cutting a sample in a suitable size, it applied DART-MS analysis. They were introduced to the DART gas using tweezers. The DART-OS ion source (IonSense, MA, USA) was interfaced onto the single quadrupole mass spectrometer LCMS-8030 (Shimadzu,
Kyoto Japan). Ultra-fast polarity switching was utilized on the mass spectrometer to collect full scan data. LCMS-8030 can achieve the polarity switching time of 15msec and the scanning speed of up to 15,000u/sec, therefore the loop time can be set at less than 1 second despite the relatively large scanning range of 50-1,000u.
Figure 1 CFRP:carbon-�ber-reinforced plastic
MS condition (LCMS-8030; Shimadzu Corporation)
Ionization : DART (Direct Analysis in Real Time)
3
Rapid analysis of carbon �ber reinforced plastic using DART-MS
Result3 CFRP samples were analyzed by DART-MS. Mass chromatograms of each sample were shown in Figure 3 and mass spectra in Figure 4.
Figure 2 DART-OS ion source (IonSense) & triple quadrupole LCMS (Shimadzu)
Figure 3 TIC chromatogram of CFRP samples #1, #2, #3
High Speed Mass Spectrometer
UFswitching High-Speed Polarity Switching 15msec UFscanning High-Speed Scanning 15,000u/sec
Sample
#1 thermoplastic polyimide (control)
#2 thermosetting polyimide (molded; dried)
#3 thermosetting polyimide (immediately after molded; wet state with solvent)
Analytical Condition
Heater Temperature (DART) : 300ºC
Measuring mode (MS) : Positive/Negative scanning simultaneously
0
25000000
50000000
1:MIC1(+)
7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 min
0
1000000
2000000
3000000
4000000
5000000
6000000 2:MIC1(-)
#1 #2 #3
Positive TIC m/z 50-500
Negative TIC m/z 50-500
4
Rapid analysis of carbon �ber reinforced plastic using DART-MS
Since the thermosetting polyimide used for this measurement was molded using the organic solvent (N-methyl pyrrolidone, C5H9NO, molecular weight 99), molecular related ions of N-methyl pyrrolidone, [M+H]+ (m/z 100) and [2M+H]+ (m/z 199), were detected very strongly in the mass spectrum of #1. The mass spectrum of #2 also showed the same ions that intensity was intentionally detected strongly compared with #3 although intensity was weak compared with #1. Even if
it raised the heating gas temperature of DART to high temperature (up to 500°C), MS signal considered to originate in the structural information of CFRP was not able to be obtained.Then, the optional heating mechanism, ionRocket (Bio Chromato, Inc.; Figure 5), in which a sample could be heated directly was developed to the sample stage of DART, and analysis of CFRP was verified by heating the sample directly up to 600°C.
Figure 4 DART-MS spectra of each sample
Sample
#4 thermosetting polyimide (molded; dried)
#5 thermoplastic polyimide (control)
Analytical Condition
Heater Temperature (DART) : 400°C
Temperature control (ionRocket) : 0-1min room temp., 4min 600°C
Measuring mode (MS) : Positive scanning
50 100 150 200 250 m/z0.0
2.5
5.0
7.5
Inten. (x1,000,000)
199.1100.1 282.2228.3172.1
Positive, m/z 50-300#1
50 100 150 200 250 m/z0.0
2.5
5.0
7.5
Inten. (x1,000,000)
199.1100.1
Positive, m/z 50-300 #3
50 100 150 200 250 m/z0.0
2.5
5.0
7.5
Inten. (x1,000,000)
199.1100.1172.2 282.3
[M+H]+ [2M+H]+
Positive, m/z 50-300#2 N-methyl pyrrolidoneC5H9NOMw 99
Rapid analysis of carbon �ber reinforced plastic using DART-MS
5
When heating temperature was set to 600ºC, the rudder shape signals of 28u (C2H4) interval was appeared around m/z 900. This signal was more notably detected with the thermosetting polyimide sample than the thermoplastic sample. Since the sample was heated at
high temperature, it was considered that the thermal decomposition of resin started, the thermal decomposition ingredient of polyimide clustered, and possibly the structures of the rudder signals of equal interval were generated.
Figure 5 DART-MS system integrated with ionRocket
r.t.
600°C
time[min]1 4
excitation helium
DART ion source
evaporated ingredient
small heating furnace
sample pot
heater
MSspectrometer
Rapid analysis of carbon �ber reinforced plastic using DART-MS
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Figure 6 DART-MS with ionRocket spectra of each sample
AcknowledgmentWe are deeply grateful to Mr. Yuichi Ishida, Japan Aerospace Exploration Agency (JAXA), offered the CFRP sample used for this experiment.
ConclusionsThe result of having analyzed the carbon fiber plastic CFRP (thermosetting polyimide and thermoplastic polyimide) using DART-MS,
a. residue of the solvent used in fabrication was able to be checked by direct analysis of CFRP by DART. b. analyzing CFRP by DART and the heating option ionRocket, the difference between thermosetting polyimide and thermoplastic polyimide was able to be found out.
Zoom
#4
#4 thermosetting polyimide
#5 thermoplastic polyimide
#5
PO-CON1464E
Analysis of styrene leached from polystyrene cups using GCMS coupledwith Headspace (HS) sampler
ASMS 2014 TP763
Ankush Bhone(1), Dheeraj Handique(1), Prashant Hase(1),
Sanket Chiplunkar(1), Durvesh Sawant(1), Ajit Datar(1),
Jitendra Kelkar(1), Pratap Rasam(1), Nivedita Subhedar(2)
(1) Shimadzu Analytical (India) Pvt. Ltd., 1 A/B Rushabh
Chambers, Makwana Road, Marol, Andheri (E),
Mumbai-400059, Maharashtra, India.
(2) Ramnarain Ruia College, L. Nappo Road,
Matunga (E), Mumbai-400019, Maharashtra, India.
2
Analysis of styrene leached from polystyrene cups using GCMS coupled with Headspace (HS) sampler
IntroductionWorldwide studies have revealed the negative impacts of household disposable polystyrene cups (Figure 1) on human health and environment.Molecular structure of styrene is shown in Figure 2. Styrene is considered as a possible human carcinogen by the WHO and International Agency for Research on Cancer (IARC).[1] Migration of styrene from polystyrene cups containing beverages has been observed.[2] Styrene enters into our body through the food we take, mimics estrogens in the
body and can therefore disrupt normal hormonal functions. This could also lead to breast and prostate cancer.The objective of this study is to develop a sensitive, selective, accurate and reliable method for styrene determination using low carryover headspace sampler, HS-20 coupled with Ultra Fast Scan Speed 20,000 u/sec, GCMS-QP2010 Ultra to assess the risk involved in using polystyrene cups.
Figure 1. Polystyrene cup Figure 2. Structure of styrene
Method of Analysis
This study was carried out by extracting styrene from commercially available polystyrene cups and recoveries were established by spiking polystyrene cups with standard solution of styrene. Solutions were prepared as follows,
Extraction of styrene from polystyrene cups
Method was partly validated to support the findings by performing reproducibility, linearity, LOD, LOQ and recovery studies. For validation, solutions of different concentrations were prepared using standard stock solution of styrene (1000 ppm) as mentioned in Table 1.
1) Standard Stock Solution: 1000 ppm of styrene standard stock solution in DMF: Water-50:50 (v/v) was prepared. It was further diluted with water to make 100 ppm and 1 ppm of standard styrene solutions.
2) Calibration Curve: Calibration curve was plotted using standard styrene solutions in the concentration range of 1 to 50 ppb with water as a diluent. 5 mL of each standard styrene solution was transferred in separate 20 mL headspace vials and crimped with automated crimper.
3) Sample Preparation: 150 mL of boiling water (around 100 ºC)[1] was poured into polystyrene cups. The cup was covered with aluminium foil and kept at room temperature for 1 hour. After an hour, 5 mL of sample from the cup was transferred into the 20 mL headspace vial and crimped with automated crimper.
3
Analysis of styrene leached from polystyrene cups using GCMS coupled with Headspace (HS) sampler
HS-GCMS analytical parametersHeadspace parameters
• Sampling Mode : Loop
• Oven Temp. : 80.0 ºC
• Sample Line Temp. : 130.0 ºC
• Transfer Line Temp. : 140.0 ºC
• Equilibrating Time : 20.00 min
• Pressurizing Time : 0.50 min
• Pressure Equilib. Time : 0.10 min
• Load Time : 0.50 min
• Load Equilib. Time : 0.10 min
• Injection Time : 1.00 min
• Needle Flush Time : 10.00 min
• GC Cycle Time : 23.00 min
HS-GCMS Analytical ConditionsFigure 3 shows the analytical instrument, HS-20 coupled with GCMS-QP2010 Ultra on which samples were analyzed with following instrument parameter.
Table 1. Method validation parameters
Figure 3. HS-20 coupled with GCMS-QP2010 Ultra by Shimadzu
Parameter Concentration (ppb)
Linearity
Accuracy / Recovery
Precision at LOQ level
Reproducibility
1, 2.5, 5, 10, 20, 50
2.5, 10, 50
1
50
4
Analysis of styrene leached from polystyrene cups using GCMS coupled with Headspace (HS) sampler
Chromatographic parameters
• Column : Rxi-5Sil MS (30 m L x 0.25 mm I.D., 0.25 μm)
• Injection Mode : Split
• Split Ratio : 10.0
• Carrier Gas : Helium
• Flow Control Mode : Linear Velocity
• Linear Velocity : 36.3 cm/sec
• Pressure : 53.5 kPa
• Column Flow : 1.00 mL/min
• Total Flow : 14.0 mL/min
• Total Program Time : 12.42 min
• Column Oven Temp. : Rate (ºC /min) Temperature (ºC) Hold time (min)
50.0 0.00
40.00 200.0 1.00
30.00 280.0 5.00
Mass Spectrometry parameters
• Ion Source Temp. : 200 ºC
• Interface Temp. : 230 ºC
• Ionization Mode : EI
• Event Time : 0.20 sec
• Mode : SIM
• m/z : 104,103 and 78
• Start Time : 1.00 min
• End Time : 5.00 min
Results
Mass spectrum of styrene is shown in Figure 4. From the mass spectrum, base peak of m/z 104 was used for quantitation where as m/z 103 and 78 were used as reference ions. SIM chromatogram of 50 ppb standard styrene solution
with m/z 104, 103 and 78 is shown in Figure 5.Method validation data is summarized in Table 2. Figures 6 and 7 show overlay of SIM chromatograms for m/z 104 at linearity levels and calibration curve respectively.
Fragmentation of styrene
5
Analysis of styrene leached from polystyrene cups using GCMS coupled with Headspace (HS) sampler
Figure 4. Mass spectrum of styrene
Figure 5. SIM chromatogram of 50 ppb standard styrene solution
Table 2. Validation summary
Summary of validation results
Result
% RSD : 1.74 (n=6)
R2 : 0.9996
LOD : 0.2 ppb
LOQ : 1 ppb
S/N ratio : 38 (n=6)
% RSD : 3.2 (n=6)
Concentration in ppb
50
1 – 50
1 – 50
1
Parameter
Reproducibility (% RSD)
Linearity* (R2)
LOD
LOQ
Precision at LOQ
Compound Name
Styrene
Sr. No.
1
2
3
4
5
45.0 50.0 55.0 60.0 65.0 70.0 75.0 80.0 85.0 90.0 95.0 100.0 105.0 m/z
0
25
50
75
100
Inten.104
103
78
51
52 63 74 8965 985844
85
2.325 2.350 2.375 2.400 2.425 2.450 2.475 2.500 2.525
0.0
2.5
5.0
7.5
(x1,000,000)
78.00 (10.00)103.00 (10.00)104.00 (10.00)
min
* Linearity levels – 1, 2.5, 5, 10, 20 and 50 ppb.
Analysis of styrene leached from polystyrene cups using GCMS coupled with Headspace (HS) sampler
6
Figure 7. Calibration curve for StyreneFigure 6. Overlay of SIM chromatograms for m/z 104 at linearity levels
Figure 8. Overlay SIM chromatograms of spiked and unspiked samples
Table 3. Summary of results for sample analysis
Analysis of leachable styrene from polystyrene cups was done as per method described earlier. Recovery studies were carried out by spiking 2.5, 10 and 50 ppb of standard
styrene solutions in polystyrene cups. Figure 8 shows overlay SIM chromatogram of spiked and unspiked samples. Table 3 shows the summary of results.
Quantitation of styrene in polystyrene cup sample
Sample Name
Unspiked sample
Spiked polystyrene cups
Sr. No.
1
2
Observed Concentration
in ppb
9.8
12.0
18.5
55.9
Parameter
Precision
Recovery
Spiked Concentration
in ppb
NA
2.5
10
50
% Recovery
NA
88.0
87.0
92.2
0 10 20 30 40 Conc.0
250000
500000
750000
1000000
1250000
Area
R2 = 0.9996
2.2 2.3 2.4 2.5 2.6
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
(x1,000,000)
1 ppb
2.5 ppb
5 ppb
10 ppb
20 ppb
50 ppb
min
m/z : 104.00
2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8
0.0
2.5
5.0
7.5
(x100,000)
Spiked
Unspiked
m/z : 104.00
min
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© Shimadzu Corporation, 2014
First Edition: June, 2014
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Analysis of styrene leached from polystyrene cups using GCMS coupled with Headspace (HS) sampler
Conclusion• HS-GCMS method was developed for quantitation of styrene leached from polystyrene cup. Part method validation was
performed. Results obtained for reproducibility, linearity, LOQ and recovery studies were within acceptable criteria.• With low carryover, the characteristic feature of HS-20 headspace, reproducibility even at very low concentration level
could be achieved easily.• Ultra Fast Scan Speed 20,000 u/sec is the characteristic feature of GCMS-QP2010 Ultra mass spectrometer, useful for
quantitation of styrene at very low level (ppb level) with high sensitivity.
References[1] Maqbool Ahmad, Ahmad S. Bajahlan, Journal of Environmental Sciences, Volume 19, (2007), 422, 424.[2] M. S. Taw�ka; A. Huyghebaerta, Journal of Food Additives and Contaminants, Volume 15, (1998), 595.
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