Glipizide (500 ng) Glimepiride (500 ng) Memory effects in...

min0 1 2 3 4 5

mAU

0

50

100

150

200

250

DAD (230 nm) Glipizide (500 ng)

Glimepiride (500 ng)

0 1 2 3 4 5 Tim e [min]0

1

2

3

4 x106 Intens.

Glipizide (25 ng) (m/z) 446 321 Da

Glimepiride (25 ng)(m/z) 491 352 Da

MS2 (APCI/ITD)

Memory effects in the interface

generate additional peak tailing!

Consequence:Enhance on

chromatographic resolution; more often increased resolution

means longer chromatographic runs !

Chromolith Performance RP-18e (100 x 4.6 mm), 25 oC; aq. 0.1% HCOOH / MeOH (30/70), 1 mL/min.

APCI/ Dry gas: Temp. 300 oC, 4 L/min; Vap. temp. 350 oC; Nebulizer gas press. 60 psi; Corona 4000 V; Vcap. 3500 V;

4x10

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

6.5

7

7.5

Abundance vs. Acquisition Time (min)0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2

+ MRM (130.2 -> 60.2) Test04.d

1 1 2

4x10

00.25

0.50.75

11.25

1.51.75

22.25

2.52.75

33.25

3.53.75

44.25

4.54.75

Abundance vs. Acquisition Time (min)0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8

+ MRM (130.2 -> 71.2) Test09.d

1 1 2

Target compound: Metformin – positive;

IS: 1-methylbiguanide– positive;

Column: Zorbax CN, 150 mm L x 4.6 mm i.d.; 5 µm d.p.; Agilent Technologies; Cat. No.:

883952-705; Cyanopropyl chemically modified, ultra-pure silica (>99.999%), end-

capped; S.N.: USJ0003915;

Column temperature: 25°C;

Mobile phase components:

Solvent A: Aqueous 10mM CH3COONH4 at pH= 3.5 with CH3COOH;

Solvent B: Acetonitrile;

Elution: isocratic;

Composition: Solvent A / Solvent B = 50 / 50;

Flow rate: 0.8 mL/min.

Injection volume: 5 µL (1 ng in column, each).

ESI IONIZATION PARAMETERS:

Drying gas temperature: 350°C;

Drying gas flow: 10 L/min;

Pressure of the nebulizer gas: 60 psi.;

Vcap: 1500 V.

Dwell: 50

Fragmentor: 100

Collision energy: 10

Delta EMV: 600

WORKING MODE: MS2 / MRM

Target compound: m/z = 130.2 to 60.2

IS: m/z = 116.2 to 60.2

Target compound: Metformin – positive;

IS: 1-methylbiguanide– positive;

Column: Zorbax Eclipse XDB-CN, 150 mm L x 4.6 mm i.d.; 3.5 µm d.p.; Agilent

Technologies; Cat. No.: 963967-905; Cyanopropyl chemically modified, ultra-pure

silica (>99.999%), end-capped; S.N.: USBAA01195;

Column temperature: 25°C;

Mobile phase components:

Solvent A: Aqueous 10mM CH3COONH4 at pH= 3.5 with CH3COOH;

Solvent B: Acetonitrile;

Elution: isocratic;

Composition: Solvent A / Solvent B = 20 / 80;

Flow rate: 0.8 mL/min.

Injection volume: 5 µL (1 ng in column, each).

ESI IONIZATION PARAMETERS:



Pressure of the nebulizer gas: 60 psi.;

Vcap: 1500 V.

Dwell: 50

Fragmentor: 100

Collision energy: 10

Delta EMV: 600

WORKING MODE: MS2 / MRM

Target compound: m/z = 130.2 to 60.2

IS: m/z = 116.2 to 60.2

IONIZATION YELDS

Depending upon M.Ph. composition!

Poor reproducibility with respect to other spectrometric detectors!

0 100 200 300 400 500 600 700350

400

450

500

550

600

650

700

750

800

850

900

950

1000

Average

Average - 2*s (for RCV=2.46%)

Average + 2*s (for RCV=2.46%)

Average - 2*s (for RCV=15%)

Average + 2*s (for RCV=15%)

IS p

eak

area

(L.U

.)

Sample number

rxy = -0.2

0 100 200 300 400 500 6008

10

12

14

16

18

20

22

IS p

eak

area

(mAu

)Sample number

Average + 2*s (for RCV=15%)

Average + 2*s (for RCV=6.64%)

Average - 2*s (for RCV=6.64%)

Average - 2*s (for RCV=15%)

Average

rxy= 0.2

Trends for I.S. peak area values

Gliquidone BE study I.S. – Fluoranthene; 644 samples real plasma samples Zorbax SB-C18 50 mm x 4.6 mm x 1.8 m; 60 oC Vinj = 50 L; Aq. 0.1% H3PO4 / ACN (70/30); gradient elution to 100% ACN; Flow rate: 2 mL/min; Ex. 230 nm; Em. 415 nm; duration – 1.8 min.

Tenoxicam BE study I.S. – Piroxicam; 560 samples real plasma samples Zorbax SB-C18 50 mm x 4.6 mm x 1.8 m; 60 oC Vinj = 50 L; Aq. 0.1% H3PO4 / ACN (70/30); gradient elution to 100% ACN; Flow rate: 2 mL/min; UV - 368 nm (resp. time 0.1 s); duration – 4 min.

0 100 200 300 400 500 600 70040000

50000

60000

70000

80000

90000

100000

110000

120000

130000

Average - 2*s (for RSD=15%)

Average + 2*s (for RSD=15%)

Average - 2*s (for RSD=5.63%)

Average + 2*s (for RSD=5.63%)

IS p

eak

area

Sample number

Averagerxy = 0.81


0 200 400 600 80060000

80000

100000

120000

140000

160000

180000

200000

Average





IS p

eak

area

Sample number

rxy = -0.8

Bromazepam BE study I.S. – Nitrazepam; 644 samples real plasma samples Zorbax SB-C18 Rapid Resolution 30 mm x 2.1 mm x 3.5 m; 25 oCVinj = 5 L Aq. 0.1% HCOOH / MeOH (60/40); isocratic; Flow rate: 0.8 mL/min(+)AP-ESI/MS2 (QQQ): Nitrazepam (m/z) 282316 Da; Bromazepam (m/z) 282316 Da Dry gas temp.: 350 oC; flow 10 L/min; Nebulizer gas press. 60 psi, V cap. 4000 V.

Metformin BE study I.S. – 1-methylbiguanide; 832 samples real plasma samples Zorbax CN 150 mm x 4.6 mm x 5 m; 25 oC Vinj = 5 L; Aq. 0.1% 10 mM acetate buffer pH=3.5 / ACN (50/50); isocratic; Flow rate: 0.8 mL/min (+)AP-ESI/MS2 (QQQ): I.S. (m/z) 11660 Da; Metformin (m/z) 13060 Da Dry gas temp.: 325 oC; flow 10 L/min; Nebulizer gas press. 60 psi, V cap. 1500 V.

0 200 400 600 800 1000160000

180000

200000

220000

240000

260000

280000

300000

320000

340000

360000

Average

Average - 2*s (for RSD=13.2%)

Average + 2*s (for RSD=13.2%)



IS p

eak

area

Sample number

rxy= -0.92

source cleaning


Meloxicam BE studyI.S. – 4-hydroxy-2-methyl-N-(pyridine-2-yl)-2-H-1,2-benzothiazine-3-carboxamide-1,1-dioxide ; 936 samples real plasma samples Zorbax Eclipse XDB-C18 15 mm x 4.6 mm x 5.5 m; 25 oC Vinj = 5 L; Aq. 0.2% HCOOH / ACN (30/70); isocratic; Flow rate: 0.8 mL/min (+)AP-ESI/MS2 (QQQ): I.S. (m/z) 33295 Da; Meloxicam (m/z)352115 Da Dry gas temp.: 350 oC; flow 10 L/min; Nebulizer gas press. 60 psi, V cap. 3000 V.

0 100 200 300 400 500 600 7001000000

2000000

3000000

4000000

5000000

6000000

7000000

8000000

9000000

10000000

11000000

Average

Average - 2*s (for a RSD=36,3%)

Average + 2*s (for a RSD=36,3%)

Average - 2*s (for a RSD=15%)

I.S. p

eak

area

Sample number

Average + 2*s (for a RSD=15%)

cleaning source

Glibenclamide BE studyI.S. – gliquidone ; 624 samples real plasma samples Chromolith Performance RP-18e, 10 mm x 4.6 mm; 40 oC Vinj = 50 L; Aq. 0.1% HCOOH / ACN (42/58); isocratic; Flow rate: 1.0 mL/min (+)APCI/MS2 (ITD): I.S. (m/z) 528403 Da; Glibenclamide (m/z) 494369 Da Dry gas temp.: 300 oC; flow 5 L/min; Nebulizer gas press. 60 psi, Corona 5000 V; V cap. 4500 V.

Consequence: response stability will directly influence on the frequency of running calibrations and QC samples sets!

LUOL Calibration

y = 0.1226x + 0.029R2 = 0.9999

0.0000

2.0000

4.0000

6.0000

8.0000

10.0000

12.0000

14.0000

0 20 40 60 80 100 120

Concentration (ppb)

Ana

lyte

/I.S.

pea

k ar

ea ra

tio

Concentration (ppb) Analyte/I.S. peak ratio RSD%(n=6) Backinterpolated

Concentration (ppb) %Bias

0 0.0000 - - -0.5 0.0814 2.76 0.43 -14.6

1 0.1426 4.59 0.9 -7.42.5 0.3231 0.32 2.4 -4.1

5 0.6053 0.61 4.7 -6.010 1.2646 1.26 10.1 0.825 3.2175 3.22 26.0 4.050 6.1437 6.14 49.9 -0.3

100 12.2664 12.27 99.8 -0.2

N

NHOH

H

O

H

CH3

CH3

Non-linear response functions!

Consequence:Find ways of

making linearisation !

Nicergoline BE study

I.S.–1,3’-(carboxypropyl)-3,7-dimethyl xanthine;

Also analyzed: metabolites LUOL and MELUOL

Zorbax Eclipse XDB-C8, 150 mm x 4.6 mm x 3.5 m; 40 oCVinj = 300 L; Aq. 0.1% HCOOH / 0.1% HCOOH in MeOH (74/26); gradient elution; Flow rate: 0.8 mL/min

(+)APCI/MS2 (ITD): LUOL (m/z) 287255+269 DaDry gas temp.: 250 oC; flow 5 L/min; Vaporizer temp. 350 oC; Nebulizer gas press. 60 psi, Corona 5000 V; V cap. 4500 V.

12 14

0.0

1.0

1.5

2.0x105

0 2 4 6 8 10 [min]

0.5

Intens.

DAcDLTZ

IS

DMeDLTZ

DLTZ

N

S

NH+

H

O

OO

O

NH+

SH

O

OO

O

NH+

S

O

OO

S

C+

O

N

O

S+

O

N

S+

C

N

OH

O

S

O

N

CH

+

NSH

+

O

C+

O

SH

N

C+

SHN

C6H4 OMe

- HN(CH3)2

m/z=415 m/z=370

- CH3COOH

m/z=310

m/z=178

- CO

m/z=150

-

Diltiazem BE study

I.S. – diastereoisomer of DLTZ; Also analyzed: metabolites Desacetyldiltiazem (DAcDLTZ) and Desmethyldiltiazem (DMeDLTZ) Zorbax Eclipse XDB-C8, 150 mm x 4.6 mm x 5 m; 45 oC Vinj = 500 L; Aq. 0.2% TEA at pH=3.5 with HCOOH / ACN (75/25); isocratic; Flow rate: 0.8 mL/min (+)ESI/MS2 (ITD): IS = DLTZ: (m/z) 415370, 310, 178, 150 Da (isolation window 408 ± 10 Da) DAcDLTZ: (m/z) 373328 + 223 + 178 + 150 Da (isolation window 373 ± 2 Da) DMeDLTZ: (m/z) 401370, 341, 310, 178, 150 Da (isolation window 408 ± 10 Da) Dry gas temp.: 365 oC; flow 12 L/min; Nebulizer gas press. 65 psi.; V cap. 1500 V.

Limited resolution (IS, DMeDLTZ, DLTZ) single isolation window broad isolation one (± 10 Da) no CID fragmentation Product ions Precursor

ions !

DLTZ

y = 0.0073x + 0.1059R2 = 0.9822

y = -9E-06x2 + 0.0105x + 0.0341R2 = 0.9985

0

0.5

1

1.5

2

2.5

3

3.5

0 50 100 150 200 250 300 350 400 450

Concentration (ppb)

Ana

lyte

/I.S

. pea

k ar

ea ra

tio

Con

cent

ratio

n (p

pb)

Ana

lyte

/I.S.

pea

k ar

ea

ratio

Bac

kint

erpo

late

d co

ncen

trat

ion

valu

es

(LIN

EAR

)

% B

ias

Bac

kint

erpo

late

d co

ncen

trat

ion

valu

es

(BIN

OM

IAL)

% B

ias

0.5 0.01813 -12.1 -2519.3 -1.5 -403.8 1 0.02464 -11.2 -1219.8 -0.9 -190.0 5 0.08249 -3.2 -164.4 4.6 -7.5

10 0.16973 8.8 -11.9 13.1 30.6 25 0.30335 27.2 8.9 26.2 4.9 50 0.56244 63.0 25.9 52.7 5.4

100 1.05993 131.6 31.6 107.6 7.6 200 1.72370 223.1 11.5 192.8 -3.6 400 2.85453 379.0 -5.3 419.3 4.8

Non – linear calibration !Linear versus Binomial

DLTZ

y = 0.7848x - 1.5799R2 = 0.9982

-2.000

-1.500

-1.000

-0.500

0.000

0.500

1.000

-0.50 0.00 0.50 1.00 1.50 2.00 2.50 3.00

Log(concentration)

Log(

anal

yte/

I.S. p

eak

area

ratio

)

Con

cent

ratio

n (p

pb)

Log

(con

cent

ratio

n)

Ana

lyte

/I.S.

pea

k ar

ea ra

tio

Log

(Ana

lyte

/I.S.

pe

ak a

rea

ratio

)

Bac

k in

terp

olat

ed

conc

entr

atio

n va

lues

% B

ias

0.5 -0.30 0.017 -1.766 0.6 12.8 1 0.00 0.025 -1.608 0.9 -10.1 5 0.70 0.083 -1.078 4.3 -14.2

10 1.00 0.170 -0.770 10.7 6.5 25 1.40 0.303 -0.518 22.4 -10.3 50 1.70 0.562 -0.250 49.5 -1.1

100 2.00 1.060 0.025 111.4 11.4 200 2.30 1.724 0.236 207.7 3.9 400 2.60 2.855 0.456 396.5 -0.9

Non – linear calibration !Log – Log representation

Non – linear calibration ! Log – Log representationCarbamazepina - 8 Levels, 8 Levels Used, 8 Points, 8 Points Used, 6 QCs

Concentration (ppb)

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

6.5

0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000

y = -1.9875E-008 * x ^ 2 + 8.1426E-004 * x + 0.0202R^2 = 0.99973470

Calibration

y = 0,91256x - 2,82874R2 = 0,99938

-1,500

-1,000

-0,500

0,000

0,500

1,000

1,500 2,000 2,500 3,000 3,500 4,000 4,500

log Carbamazepine concentration (ng/mL)

log

Peak

are

a ra

tio C

arba

maz

epin

e / I

.S.

Internal Standard: 7-nitro-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one– positive;

Target compound 1: Carbamazepine- 5H-Dibenzo[b,f]azepine-5-carboxamide– positive;

Target compound 2: Carbamazepine 10,11-epoxide- positive;

Column: Zorbax Eclipse XDB-C18, Rapid Resolution, 50 mm L x 4.6 mm i.d.; 1.8 µm

d.p.; Octadecyl chemically modified, ultra-pure silica (>99.995%), double end-

capped; S.N.:USWDY04740; Agilent Technologies; Cat. No.:927975-902;

Column temperature : 25°C;

Solvent A: 0.1% HCOOH;

Solvent B: Acetonitril;

Elution: isocratic; Composition of the mobile phase: Solvent A / Solvent B = 70/ 30;

Flow rate : 0.8 mL/min.

Injection volume : 5 µL.

(+) ESI



Pressure of the nebulizer gas : 55 psi;

Vcap: 1500 V.

Collision Energy: 25 V.

Fragmentor: 90 V.

ΔEMV: 600 V.

MRM

IS: m/z = 282.0 to 236.0 (qual. 254.0);

Target compound 1: m/z = 237.0 to 194.0 (qual. 192.0);

Target compound 2: m/z = 253.0 to 180.0 (qual. 210.0).

Concentrations (ng/mL):

I.S.: 1000 ;

Target compound 1: Concentrations (ng/mL):1) 100; 2) 200; 3) 500; 4) 1000; 5) 2000; 6)

3500; 7) 5000; 8) 10000.

Non – linear calibration (Log – Log representation) – Results!

Real samples for 27 volunteers

0

0.0002

0.0004

0.0006

0.0008

0.001

0.0012

0 200 400 600 800 1000 1200 1400

Sample number

Res

pons

e to

con

cent

ratio

n ra

tio

(nor

mal

ized

ratio

)

P1

P2

MSD

waste

C1

C2

Inj NH2

CH3

OH

Cathine [(1S,2S)-2-amino-1-phenylpropane-1-ol] CAS 492-39-7

Study of Ephedrines in Urine Target analyte: Cathine; I.S.: 4-aminoantipyrine Also analyzed: norephedrine, ephedrine, pseudoephedrine, N-methylephedrine C1 – Oasis HLB (polyvinylpyrrolidone) 20 mm x 3 mm x 5 m; 25 oC; Vinj = 100 L; Direct injection of filtered urine. C2 – Purosphere Star C18, 125 mm x 4 mm x 5 m; 25 oC M. Ph. 1: Aq. 0.015% HCOOH at pH 10.5 with TEA; isocratic elution; M. Ph. 2: Aq. 0.015% HCOOH at pH 10.5 with TEA / MeOH (55/45); isocratic; Flow rate: 1.0 mL/min (+)ESI/MS2 (QQQ): Cathine (m/z) 134117; 91 Da; NO MOLECULAR ION IN THE ESI SOURCE! Dry gas temp.: 350 oC; flow 10 L/min; Nebulizer gas press. 60 psi.; V cap. 3500 V.

OH2+

CH3

NH2CH

+

CH3

NH2

CH

C+

CH3

CH

CH3

NH3+

C+

CH2

CH2+152134

117

- NH3- H2

115

- C2H2

91

Non – linear calibrations ! Analysis of Ephedrines in Urine(+) ionisation despite alkaline mobile phase character; Fragmentation within the source.

CATHINE

y = 0.0139x2 + 0.0059x + 0.0044R2 = 0.9992

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 2 4 6 8 10 12Concentrations (ppm)

Ana

lyte

/I.S.

pea

k ar

ea ra

tio

Con

cent

ratio

n (p

pm)

Ana

lyte

/I.S.

pea

k ar

ea ra

tio

Bac

k in

terp

olat

ed

conc

entr

atio

n va

lues

% B

ias

0 0 - - 0.625 0.0158 0.7 14.9 1.25 0.0398 1.4 11.8

2.5 0.1104 2.6 2.3 5 0.3568 4.8 -3.5

7.5 0.8558 7.6 1.6 10 1.4465 10.0 -0.2

Non – linear calibration !Binomial representation

n

xB

n

yA

n

xx

n

yxyx

B

n

ii

n

ii

n

i

n

ii

i

n

i

n

ii

n

ii

ii

11

1

2

12

1

11

LINEAR

Con

cent

ratio

n (p

pb)

Ana

lyte

/I.S.

pea

k ar

ea

ratio

Bac

k in

terp

olat

ed

conc

entr

atio

n va

lues

% B

ias

25 0.0574 -42.4 -269.7 50 0.1105 -9.2 -118.5

100 0.2088 52.2 -47.8 500 1.0657 587.7 17.5

1000 1.9468 1138.4 13.8 1500 2.7224 1623.2 8.2 2000 3.3371 2007.3 0.4 2500 4.0217 2435.3 -2.6

METFORMIN

y = 0.0016x + 0.1253R2 = 0.992

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 500 1000 1500 2000 2500 3000

Concentration (ppb)

Ana

lyte

/I.S.

pea

k ar

ea r

atio

Metformin BE study

I.S. – 1-methylbiguanide; Zorbax CN 150 mm x 4.6 mm x 5 m; 25 oC Vinj = 5 L; Aq. 0.1% 10 mM acetate buffer pH=3.5 / ACN (50/50); isocratic; Flow rate: 0.8 mL/min

(+)AP-ESI/MS2 (QQQ): I.S. (m/z) 11660 Da; Metformin (m/z) 13060 Da Dry gas temp.: 325 oC; flow 10 L/min; Nebulizer gas press. 60 psi, V cap. 1500 V.

Non-linearCalibrations

Weighing by 1/x or 1/x2!

Increasing linearity domain!

ii

n

ii

n

iii

n

ii

n

iii

n

in

ii

n

iii

ii

n

in

ii

n

iii

n

iii

iii

xw

w

wxB

w

wyA

w

wxwx

w

wywxwyx

B

1

1

1

1

1

1

1

2

12

1

1

11

LINEAR - Weighing 1/x Concentration (ppb) Analyte/I.S. peak area ratio Back interpolated concentration values % Bias

25 0.0574 18.2 -27.450 0.1105 48.9 -2.2

100 0.2088 105.8 5.8500 1.0657 601.6 20.3

1000 1.9468 1111.5 11.21500 2.7224 1560.4 4.02000 3.3371 1916.1 -4.22500 4.0217 2312.3 -7.5

Metformin weighted 1/x

y = 0.001728x + 0.026043R^2 = 0.9923

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

0 500 1000 1500 2000 2500 3000Concentration (ppb)

Ana

lyte

/I.S.

pea

k ar

ea ra

tio

ii

n

ii

n

iii

n

ii

n

iii

n

in

ii

n

iii

ii

n

in

ii

n

iii

n

iii

iii

xw

w

wxB

w

wyA

w

wxwx

w

wywxwyx

B

1

1

1

1

1

1

1

2

12

1

1

11

Metformin weighted 1/x2

y = 0.001846x + 0.013402R^2 = 0.9893

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

0 500 1000 1500 2000 2500 3000Concentration (ppb)

Ana

lyte

/I.S.

pea

k ar

ea ra

tio

LINEAR - Weighing 1/x2 Concentration (ppb) Analyte/I.S. peak area ratio Back interpolated concentration values % Bias

25 0.0574 23.8 -4.6 50 0.1105 52.6 5.2

100 0.2088 105.9 5.9 500 1.0657 570.0 14.0

1000 1.9468 1047.3 4.7 1500 2.7224 1467.5 -2.2 2000 3.3371 1800.5 -10.0 2500 4.0217 2171.4 -13.1

Amlodipine BE study

I.S. – (3-Ethyl-5-methyl-(4-R,S)-2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-6-methyl- 1.4-dihydropyridine-3,5-dicarboxilate); Zorbax Eclipse XDB-C18, 150 mm x 4.6 mm x 3.5 m; 40 oC Vinj = 200 L; Aq. 0.1% HCOOH / ACN (60/40); isocratic elution; Flow rate: 0.8 mL/min (+)ESI/MS2 (QQQ): Amlodipine (m/z) 409 238 Da; I.S. (m/z) 321 167 Da Dry gas temp.: 350 oC; flow 10 L/min; Nebulizer gas press. 60 psi; V cap. 4000 V.

x102

0

1

2

3

4

Abundance vs. Acquisition Time (min)

0.5 1 1.5 2 2.5 3 3.5

+ EIC MRM (** -> 238.0) test419.d 3.2301 2 2

LLOQ sample

Residual peak in blank samples

%20100samples LLOQin AreaPeak

samplesBlank in areaPeak Residual LLOQ Confirm.

Sometimes, too high sensitivity

is obtained !

Consequence:Residual peak areas in blank

samples limitatemethod LLOQ.

20 ppb amlodipine in water

20 ppb amlodipine spiked to deproteinized blank plasma

Yield ~ 30 %

20 ppb amlodipine spiked to plasma and then deproteinized

Yield ~ 30 %

x104

0

1

2

3

4

5

Abundance vs. Acquisition Time (min)

0.5 1 1.5 2 2.5 3 3.5

+ EIC MRM (** -> 238.0) 2.8921 1 2 2

Sam

ple

20 n

g/m

L (w

ater

)

20 n

g/m

L (d

epro

tein

ized

bla

nk

plas

ma)

20 n

g/m

L (b

lank

pl

asm

a)

Peak Areas 1 202,864 72,348 84,040 2 235,531 74,147 80,329 3 261,237 76,714 77,511 4 294,921 83,399 81,939 5 285,747 84,433 82,091 6 333,760 85,658 83,603

Average 274,010 79,450 81,586 s 49,910 5,745 2,394

RSD % 14.21 7.23 2.93 Recovery within samples in water and plasma matrix (%) 29.8

Recovery within samples with plasma matrix (%) 102.7

Signal suppression due to co-extracted plasma matrix !Consequences: Capacity factors for target compounds higher than 5 are strongly recommended.

Sometimes, signal enhancement may be observed. In such cases, “addition method” may be considered as a practical solution.

# Type of

anticoagulation agent in plasma

sample Am

lodi

pine

co

nc. (

ng/m

L)

Analyte / I.S. peak area ratio

Cal

cula

ted

conc

.

Mea

n co

nc.

RSD

%

% B

ias

1. 1.0387 8.7 2. 1.1093 9.3 3. 1.1182 9.3 4. 1.0382 8.7 5. 1.2087 10.1 6.

Li-Heparin 7.5

1.0814 9.0

9.2 5.8 22.4

7. 0.9523 8.0 8. 0.9357 7.8 9. 0.9856 8.2 10. 1.0142 8.5 11. 1.0272 8.6 12.

K-EDTA 7.5

1.0234 8.6

8.3 3.9 10.2

13. 0.8297 6.9 14. 0.8400 7.0 15. 0.8245 6.9 16. 0.8456 7.1 17. 0.8655 7.2 18.

Citrate 7.5

0.8663 7.2

7.1 2.1 - 5.8

Influence of the anticoagulation agent should be

carefully studied !

Enhancement / supressioneffects may

arrise!

min0 2.5 5 7.5 10 12.5 15 17.5 20

mAU

0

500

1000

1500

2000

2500 MWD1 B, Sig=238,4 Ref=480,20

min0 2.5 5 7.5 10 12.5 15 17.5 20 22.5

mAU

-75

-50

-25

0

25

50

75

100 MWD1 B, Sig=238,4 Ref=480,20

UV trace on C1

UV trace on C2

P1

P2

MSD

waste

C1

C2

Inj

Felodipine BE Study Target analyte: Felodipine; I.S.: Impurity C of Felodipine C1 – LiChrosphere ADS RP-18 (shielded stationary phase) 30 mm x 4.6 mm x 5 m; 35 oC; Vinj = 500 L; Direct injection of filtered plasma.C2 – Zorbax Eclipse XDB C18, 150 mm x 4.6 mm x 3.5 m; M. Ph. 1: Aq. 0.1% H3PO4 at pH 5 with TEA / MeOH (98/2); Flow rate: 0.8 mL/min; gradient elution (composition + flow rate); M. Ph. 2: Aq. 0.1% HCOOH / MeOH (27.5/72.5); Isocratic elution; Flow rate: 1.0 mL/min (+)APCI/MS2 (ITD): Felodipine (m/z) 384 356 + 354 + 352 + 340 + 338 Da; I.S. (m/z) 398 370 + 368 + 352 + 342 + 340 Da; Dry gas temp.: 300 oC; flow 5 L/min; Vaporizer temperature 350 oC; Nebulizer gas press. 60 psi.; Corona 6000 V; V cap. 3500 V.

Less SamplePreparation

means a serious

improvementof the

process overall

reprodu-cibility!

Volunteer

0.00

0.50

1.00

1.50

2.00

2.50

3.00

0 10 20 30 40 50

Time (hours)

Felo

dipi

ne C

once

ntra

tion

(ng/

mL)

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 Time [min]

0.0

0.5

1.0

1.5

x106 Intens.

I.S.

Felodipine

Blank Plasma Sample

Spiked Plasma Sample (10 ng/mL)

High sensitivity (tenths of ppt) and required reproducibility is reached with a less performant mass analyzer (such as IT)!

Consequences: Costs are traduced in longer separation times and complicated experimental set-up.

1x10

0

1

2

3

4

Abundance vs. Acquisition Time (min)0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5

+ MRM (609.3 -> 195.1) ReserpineCheckout001.d Smooth (1) Noise (ASTM) = 2.17; SNR (1.497min) = 22.6

1.4971 1

1x10

0

1

2

3

4

5


1.5071 1

1x10

00.5

11.5

22.5

33.5

4


1.4971 1

1x10

00.5

11.5

22.5

33.5

4


1.5171 1

1x10

00.5

1

1.52

2.53

3.5


1.5071 1

1 2

3

4

5

6

0 1 2 3 4 5 6 7 8

Time [min]

0

1

2

3

4

5

x104Intens. 5 pg of reserpine in the interface

5 fg of reserpine in the interface

RSD% = 14.3% RSD% = 7.1%RSD% <= 15%

QQQ IT

The inherent sensitivity and reproducibility of the mass analyzer !

180

160

140

120

100

80

60

40

20200 180 160 140 120 100 80 60 40 20

Conc. (ng/mL) on ESI / ITD

Con

c. (n

g/m

L) o

n A

PCI /

ITD

Conc. (ng/mL) on ESI / QQQ (1)

Conc. (ng/m

L) on ESI / QQ

Q (2)

rxy = 0.8279

rxy = 0.8279 rxy = 0.8731

rxy = 0.9958

20

40

60

80

100

120

140

160

180

200

20 40 60 80 100 120 140 160 180 200

BROMAZEPAM BE Study Analyte: Bromazepam; I.S. Nitrazepam

Sample preparation: Protein precipitation with ACN (Plasma / ACN = 1 / 2) Samples from 1 volunteer (all range of concentrations); 4 aliquots for each sample; 2 aliquots by ESI/QQQ; 1 aliquot by

ESI/ITD; 1 aliquot by APCI/ITD; for each series of data one calibration and QC sets (n=6) have been considered. Zorbax SB-C18; 30 mm x 2.1 mm x 3.5 mm; 25 oC Purospher Star C-18; 125 mm x 4 mm x 5 mm; 25 oC Vinj = 5 mL Vinj = 200 mL Aq. 0.1% HCOOH / MeOH (60/40), Flow 0.8 mL/min (k’ = 7.2 for Bromazepam, 11.2 for I.S.).

Aq. 0.1% HCOOH / MeOH (56/44), Flow 0.8 mL/min, in order to obtain same k’

(+) ESI/MS2 (QQQ); (+) ESI/MS2 (ITD); (+) APCI/MS2 (ITD) Dry gas temp.: 350 oC; flow 10 L/min; Nebulizer gas press. 60 psi.; V cap. 4000 V.

Dry gas temp.: 350 oC; flow 10 L/min; Nebulizer gas press. 60 psi.; V cap. 2535 V. Dry gas temp.: 350 oC; flow 10 L/min; Vaporizer temperature: 350 oC Nebulizer gas press. 60 psi.; Corona 4000 V; V cap. 3000 V.

Analyte: m/z (316/318) 182 Da; I.S.: m/z (282) 180 Da;

Different ion sources!

Different mass analyzers!

Apparently, IP separation mechanism is useless with MSD! In practice, perfluorurated IPA may be used!

Analysis of Ephedrines in Urine Samples Target analytes: Cathine; Norephedrine; Ephedrine; Pseudoephedrine; N-Methylephedrine;I.S.: 4-aminoantipyrine

Luna C18(2) 250 mm x 4.6 mm x 5 m; 25 oC; Vinj = 5 L; Non-extractive sample preparation technique.M. Ph.: Aq. 0.15% HEPTAFLUOROBUTYRIC ACID (HFBA) / MeOH / I-PrOH (70/24/6); Flow rate: 0.8 mL/min; isocratic elution.

(+)ESI/MS2 (QQQ): Cathine + Norephedrine (m/z) 134 91 (cant.); 115 (cal.) Da;Ephedrine + Pseudoephedrine (m/z) 166 148 (cant.); 117 (cal.) Da;N-Methylephedrine (m/z) 180 162 (cant.); 147 (cal.) Da;I.S. (m/z) 204 56 (cant.); 94 (cal.) Da;

Dry gas temp.: 350 oC; flow 10 L/min; Nebulizer gas press. 60 psi.; V cap. 3500 V.

I.S.

Cathine

Norephedrine

Ephedrine

Pseudoephedrine Methylephedrine

TRENDS FOR CATHINE PEAK AREA (HFBA)

y = -381.2x + 66055R2 = 0.9203

45000

50000

55000

60000

65000

70000

75000

0 10 20 30 40 50 60

Sample No.

Cat

hine

Pea

k A

rea

TRENDS FOR CATHINE PEAK AREA (HCOOH)

y = -15.975x + 12403R2 = 0.1395

0

5000

10000

15000

20000

25000

0 5 10 15 20 25 30 35 40

Sample numberC

athi

ne P

eak

Are

a

But….

Problems always may be solved … “chromatographically” !

I.S.Cathine

Pseudoephedrine

Ephedrine

Methylephedrine

Norephedrine

Analysis of Ephedrines in Urine Samples Target analytes: Cathine; Norephedrine; Ephedrine; Pseudoephedrine; N-Methylephedrine;I.S.: 4-aminoantipyrine

Betasil Phenyl 250 mm x 4.6 mm x 5 m; 25 oC; Vinj = 5 L; Non-extractive sample preparation technique.M. Ph.: Aq. 0.1% HCOOH / MeOH (78/22); Flow rate: 0.8 mL/min; isocratic elution.

(+)ESI/MS2 (QQQ): Cathine + Norephedrine (m/z) 134 91 (cant.); 115 (cal.) Da;Ephedrine + Pseudoephedrine(m/z) 166 148 (cant.); 117 (cal.) Da;N-Methylephedrine (m/z) 180 162 (cant.); 147 (cal.) Da;I.S. (m/z) 204 56 (cant.); 94 (cal.) Da;


0 8

x10

4 10 12 Time [min]

0.00

0.25

0.50

1.00

Intens.

6

0.75

1.25

8

2

(+) APCI / MS2(+) APCI / MS (EIC)

(+) ESI / MS (EIC)

(+) ESI / MS2

(-) ESI / MS2

Lova

stat

ic A

cid

Sim

vast

atic

Aci

d

Lova

stat

in

Sim

vast

atin

Analyte Decomposition in the Ion Source !

Agreement between the ionization pattern and the type of the mass analyzer!

60.5

60 80 100 120 140 160 180 m/z0.0

0.5

1.0

1.5

6 x10

Intens

NH3+

NH

NCH3

CH3

NH NH

MS

60.4

85.1

113.1

130.1

60 80 100 120 140 160 180 m/z0.0

0.5

1.0

1.5

2.0

2.5

3.0 4 x10

Intens.

NH2 NH2

NH2+

130.1

(MS)2

[M+H+]

Metformin BE Study in Metformin / Glibenclamide combinations (only ITD available to date)Target analytes: Metformin; Glibenclamide; I.S.: Metformin Impurity D; Gliquidone Zorbax CN, 150 mm x 4.6 mm x 5 m; 25 oC; Vinj = 50 L; Sample preparation based on protein precipitation. M. Ph.: Aq. 10 mM NH4OOCCH3 at pH 3.5 with CH3COOH / ACN (50/50); Flow rate: 1.0 mL/min; Isocratic elution; (+)APCI/MS2 (ITD): Metformin TIC (m/z) 130 Da (EIC); I.S. TIC (m/z) 127 Da (EIC); Dry gas temp.: 300 oC; flow 5 L/min; Vaporizer temperature 350 oC; Nebulizer gas press. 60 psi.; Corona 4000 V; V cap. 2336 V.

0 1 2 30.0

0.2

0.4

0.6

0.8

1.0

7x10

Intens

Time [min] 4 5

I.S. 1

Met

form

in

I.S. 2

Glib

encl

amid

e

(+) MS

(+) MS2

50 ng

SIMVASTATIN

Precursor ion: m/z = 441.0; Product ions: m/z = 325.0

LOVASTATIN

Precursor ion: m/z = 405.0; Product ions: m/z = 199.0; 225.0; 243.0; 267.0; 285.0; 303.0.

282.3

419.1441.2

0.0

0.5

1.0

1.5

2.0

2.5 x106 Intens.

200 250 300 350 400 450 m/z

325.2

441.2

0.0

0.5

1.0

1.5

x106Intens.

200 250 300 350 400 450 m/z

199.1 267.1

285.1

303.1 387.1

405.1

200 225 250 275 300 325 350 375 400 425 m/z0

1

2

3

4

5 x105

Intens. 199.1

225.1243.1

267.1

285.1

303.2

200 225 250 275 300 325 350 375 400 425 m/z0.0

0.2

0.4

0.6

0.8

1.0 x105Intens.

O

H CH3

O

H

O

CH3CH3 CH3

H

O

H

OHHHCH3 H

O

H CH3

OH

O

CH3CH3

H

O

H

OHHHCH3 H

H

Cluster formation !

System dependent

phenomena!

In MS2,the choice of the right precursor

ion is essential for

the reproduci-

bility of quantitative

data.

Simvastatin BE Study

Target analytes: Simvastatin + Simvastatic Acid; I.S. 1: Lovastatin; ; I.S. 2: Lovastatic Acid

Zorbax Eclipse XDB-C18, 150 mm x 4.6 mm x 3.5 m; 25 oC; Vinj = 50 L; M. Ph.: Aq. 0.1% HCOOH / ACN (35/65); Flow rate: 0.8 mL/min; Isocratic elution;

ESI/MS2 (ITD): Simvastatin (m/z) 441 325 Da (+);I.S. 1 (m/z) 405 199 + 225 + 243 + 267 + 285 + 303 Da (+);Simvastatic Acid

(m/z) 435 319 Da (-);I.S. 2

(m/z) 421 319 Da (-);


Reproducibility = f(precursor ion)

0

10000000

20000000

30000000

40000000

50000000

60000000

70000000

80000000

0 5 10 15 20 25 30

Sample no.

Peak

Are

a

Molecular ions are precursorsNa - adducts ions are precursors

Lovastatin RSD%=16.6%

Lovastatin RSD% = 4.32%

Simvastatin RSD% = 33.1%

Simvastatin RSD%=4.7%

Signal reproducibility is better for

Simvastatin when the Na adduct ion is

chosen as precursor ion while for Lovastatin

the molecular [M+H]+

precursor ion provides better results.

And all this long story, only for … a methyl

group!

Conclusions:

1. In LC/MS no advantage is taken for free. Everytime you had to pay for it!

2. LC/MS methods should be attentively validated before a large scale application. During a large scale application, periodic checkings for linearity and accuracy are mandatory (don’t forget to justify frequency)!

3. MSD imposes simple and direct sample preparation methods. Otherwise, closely follows on the reproducibility of results.

4. Is better to focus on understating phenomena. In depth systemoperating abilities are necessary, but this comes after!

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