Direct LCMS Analysis
description
Transcript of Direct LCMS Analysis
Direct LC/MS Analysis of Basic
Drugs in Plasma by On-line
pretreatment with Weak Cation-
Exchange Pretreatment Column
Masatoshi Takahashi, William Hedgepeth, Shimadzu Scientific
Instruments Inc, Columbia, MD, USA
Introduction
The Shim-pack MAYI-column series has been developed to enable on-line pretreatment of bio-
samples for HPLC. The outer surfaces of the silica particles are coated with methyl cellulose
polymer, and the inner surface of pores are chemically bonded with stationary phase such as C18
(ODS type), C8, strong cation group (SCX) and so on. The Shim-pack MAYI columns show the
high ability for protein extraction from plasma samples due to the size exclusion function and the
hydrophilic surface of the particles. Bio-samples can be pretreated automatically by a column-
switching HPLC (Co-Sense for BA system, Shimadzu) with the MAYI pretreatment column.
The Shim-pack MAYI-SCX has strong ion exchange groups as the stationary phase to trap basic
compounds efficiently. However, the column requires a high amount of salts in the mobile phase
for eluting drugs retained by ion exchange interactions; the high concentration of salts in the
mobile phase may produce some problems with the use of mass spectrometry.
A new pretreatment column, Shim-pack MAYI-WCX (prototype), which has weak cation-exchange
groups as stationary phase, has been developed for trapping of basic compounds in
consideration of LCMS use. It is expected that the WCX stationary phase allows elution of basic
drugs under weak acidic mobile phase that is moderate for MS spec. The retention ability and
pretreatment effect of the Shim-pack MAYI-WCX was evaluated for desipramine, dibucaine,
lidocaine, verapamil and amitriptyline by using a column-switching system (Co-Sense for BA
system, Shimadzu).
Experimental
System Configuration
The HPLC was a Shimadzu Prominence system with a SIL-20A autosampler, LC-20AD pump
systems, a DGU-20A5 degasser, a CTO-20AC oven and a FCV-12AH six-port flow changeover valve
(see Figure 1). A single-Q mass spectrometer, LCMS-2010EV (Shimadzu), was used for detection.
Shim-pack MAYI-WCX (prototype, 10 × 4.6 mm, Shimadzu) was used as the pretreatment column.
Quantitative analysis
On-line pretreatment conditions
Column: Shim-pack MAYI-WCX (prototype, 4.6mmI.D., 10mmL)
Mobile phase (C1)*1: 5mM ammonium acetate/acetonitrile=96/4
Mobile phase (C2)*2: water/acetonitrile =1/4
*1) For trapping of test drugs, *2) For washing
Mobile phase (D)*3: Water
*3) For on-line dilution
Total flow rate: 3.0mL/min (C:0.3mL/min, D:2.7mL/min)
Dilution factor: 10 fold
Extraction time: 2min
Column temp: 40℃
Analytical conditions
Column: Shim-pack XR-ODS (3.0mmI.D., 50mmL)
Mobile phase (A): A :0.1% formic acid in water
Mobile phase (B): B :0.1% formic acid in acetonitrile
Gradient: 12%B (0min-2.00min) → 30%B (9.00min)
→80%B (9.02min-10min) → 12%B(10.02min) → stop(13min)
Flow rate: 0.7mL/min
Column temp: 40℃Detection: ESI(+)
Figure 1: System Configuration
Analytical
Column
Autosampler MAYI
WCXPump
MS spectrometer
C2
A
B
D
Pump
Pump
Degasser
Degasser
4 5
32
61
C1
Mix
er
Tee
Oven
Degasser
Pump
|||LCMS-2010EVLIQUID CHROMATOGR APH -M ASS SPECTROMETER
SHIMADZU
Results
1. The elution curve of plasma proteins from the Shim-pack MAYI-WCX was monitored
at UV 280nm to measure protein extraction efficiency. The time required for protein
extraction is listed in Figure 2. We regarded the time at which the elution curve fell to
a level of 5mAbs as the extraction time. The Shim-pack MAYI-WCX required 0.3 to
1.2 minutes for protein extraction when the injection volume was from 2 to 50 L.
2. The retention performance of basic drugs on the Shim-pack MAYI-WCX was
investigated for the purpose of controlling trap/elution of basic drugs (Table 1). The
basic drugs could be sufficiently trapped by use of neutral pH mobile phases. Acidic
mobile phases facilitated elution of the basic drugs from MAYI-WCX.
3. The test drugs were analyzed with good linearity and reproducibility by using
optimized conditions (Figures 3, 4).
4. The Shim-pack MAYI-WCX provided different selectivity from that of the Shim-pack
MAYI-ODS and showed good effect on removal of matrices in the plasma sample
(Figure 5).
5. When the Shim-pack MAYI-WCX was coupled to an analytical ODS column in the
column-switching system, the drugs were concentrated at the top of the analytical
ODS column at eluting conditions for the Shim-pack MAYI-WCX. As a result, the
combination provided very sharp peak shape (Figure 6).
Figure 2: Time for extraction of plasma proteins from Shim-pack MAYI-WCX,
dilution factor: 10 fold (Cflow/Dflow =1/7), loading solution: 10mM ammonium
acetate/acetonitrile=95/5, flow rate: 3.0mL/min
Injection volume Extraction time
2 L 0.37 min
5 L 0.51 min
10 L 0.62 min
20 L 0.69 min
50 L 1.21 min
0.0 0.5 1.0 1.5 2.0 2.5 min0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
uV (x1,000,000)
Injection volume
2 L, 5 L,10 L, 20 L, 50 L
Are
a
Table 1: Retention ability for the test basic drugs on MAYI-WCX column;
Retention time means elution time at peak initial point. Flow rate: 3.0mL/min
(total).
Mobile phaseRetention time (min)
Dibucaine Verapamil Lidocaine Desipramine Amitriptyline
0.02%Formic Acid/ACN=95/5 0.23 0.23 0.14 0.18 0.24
0.02%Acetic Acid/ACN=95/5 0.64 0.70 0.30 0.38 0.50
0.02%Acetic Acid 6.02 4.71 0.64 0.89 2.41
10mM Ammonium
Acetate/ACN=95/5
4.0 4.0 0.9 1.6 2.8
10mM Ammonium Acetate >10.00 >10.00 >10.00 >10.00 >10.00
Water/ACN=95/5 >10.00 >10.00 2.79 6.51 >10.00
Water >10.00 >10.00 >10.00 >10.00 >10.00
10mM Ammonium
Acetate/ACN=95/5 (Online dilution
with water)
(Dilution factor :10)
>10.00 >10.00 >10.00 >10.00 >10.00
Figure 3: MS chromatogram of 5 basic drugs spiked in human plasma; each
concentration: 20ng/mL, Injection volume: 20 L.
1: Desipramine
2: Dibucaine
3: Lidocaine
4: Verapamil
5: Amitriptyline
1
23
4
5
0
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
0 50 100 150 200 250
Lidocaine
Verapamil
Amitriptyline
Lidocaine Verapamil Amitriptyline
n=1 711696 1757291 1087215
n=2 713181 1742033 1107764
n=3 703238 1711805 1112317
n=4 719508 1721872 1095871
n=5 719461 1718240 1092197
S.D. 6713.043177 18872.6241 10595.7699
Average 713416.8 1730248.2 1099072.8
%RSD 0.940970717 1.09074664 0.96406443
Conc. (ng/mL)
Figure 4: Calibration curves (each 2, 10, 20, 100, 200ng/mL, 20 L injection)
and reproducibility (each 200ng/mL, 20 L injection) of 3 drugs in spiked
human plasma, lidocaine: r=0.9975, verapamil: r= 0.9991, amitriptyline:
r=0.9992
MAYI-WCX
MAYI-ODS
Figure 5: Comparison of MS TIC chromatogram of human plasma sample
pretreated by MAYI-WCX or MAYI-ODS
1: Desipramine
2: Dibucaine
3: Lidocaine
4: Verapamil
5: Amitriptyline
MAYI-WCX
MAYI-ODS
1
23
4
5
1
23
45
Figure 6: Comparison of MS chromatogram of 5 basic drugs in spiked human
plasma sample pretreated by MAYI-WCX or MAYI-ODS; each concentration:
20ng/mL, Injection volume: 20 L.
Conclusions
1. The Shim-pack MAYI-WCX column provided the good advantage of
matrices clean-up in bio-samples.
2. Basic drugs trapped on the Shim-pack MAYI-WCX could be eluted at
conditions of weak acid mobile phase, and the column didn’t require a high
amount of cations in the mobile phase. This means the Shim-pack MAYI-
WCX has high compatibility with MS spec.
3. The Shim-pack MAYI-WCX showed excellent ability for trapping amine
compounds in plasma samples.
4. The Shim-pack MAYI-WCX has a high potential ability to realize high-
resolution analysis with a combination of smaller particle ODS columns and
an analytical column.
AcknowledgmentWe would like to express our thanks to Yoshiaki Sato, Naoki Asakawa, the co-
developers of the system and Shim-pack MAYI-WCX at Eisai Co., Ltd.