method development for chiral lc/ms/ms analysis of acidic...

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878

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Method Development for Chiral LC/MS/MS Analysis of Acidic Stereoisomeric Pharmaceutical Compounds with Polysaccharide-based Stationary Phases

Liming Peng, Swapna Jayapalan, and Tivadar Farkas

Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA

Developing simple and straightforward reversed phase (RP) chiral LC separations coupled with highly sensitive MS detection is required for conducting drug metabolism and pharmacokinetic studies of stereoisomers and a challenging part of the drug discovery process. We have presented our successful results on using polysaccharide-based stationary phases coupled with API-MS/MS detection for the analysis of various basic and neutral pharmaceutical compounds in reversed

phase elution using NH4HCO3 or NH4Ac as buffer salts and acetonitrile or methanol as organic modifiers.1 This investigation is extended to the effectiveness of using an acidic mobile phase for the separation and detection of acidic stereoisomers by ESI or APCI LC/MS/MS and to method development of Chiral LC/MS/MS analysis using polysaccharide-based chiral stationary phase (CSPs).

Introduction

Figure 1. Structures of Chiral Phases

Lux® Cellulose-2 or-4 Cellulose tris (3-chloro-4-methylphenylcarbamate) or

(4-chloro-3-methylphenylcarbamate)

Lux® Cellulose-1 Cellulose tris (3,5-dimethylphenylcarbamate)

Lux® Amylose-2 Amylose tris (5-chloro-2-methylphenylcarbamate)

Lux® Cellulose-3Cellulose tris (4-methylbenzoate)

OO

OCONH

Cl

Cl

OCONH

Cl

HNOCO CH3

CH3

CH3

OO

OCONH

Cl

Cl

OCONH

Cl

HNOCOMe MeCH3

MeCH3

O

O

OCONH

OCONH

HNOCO

Cl

Cl

CH3

CH3

Cl

CH3

CH3

CH3

OO

OCONH

OCONHHNOCO

CH3

CH3

CH3

CH3

O

O

O

O

O

C

O

Me

MeMe CC

O

O

Figure 2. Molecular Structures of Acidic Racemates

OHOOO

N

O

H3C HCH3

O

OH

OH

O NH

N

O

O

OH

O

F

S

OH

O

O

OH OH

O

N

O

OH

Cl

H

O

OH

O

HO

O

OHO

Cl

N

NS S

CF3

O

O NH2

H

O O

H

O

N

O

OH

S

O

O

N

B

OH

OH

H

O O

H

N

NS S

O

O NH2

Cl

Cl

Cl

OH

N

O

O

Ibuprofen Flurbiprofen Suprofen Fenoprofen Carprofen Indoprofen

Proglumide Abscisic Acid

Warfarin Bendroflumethiaze Trichlormethiazide

Mecoprop Ketorolac 1-(Phenylsulfonyl)-3-indoleboronic acid

Etodolac

Table 1. MRM Transitions and Concentrations of Acidic Racemates

Compound IS and MRM Conc.* Compounds IS and MRM Conc.*Ibuprofen ESI-205.1/160.1 100 Abscisic acid APCI-263.0/152.7 50

Flurbiprofen ESI-243.0/198.7 50 Mecoprop ESI-214.1/141.7 50

Suprofen ESI+261.1/111.0 50 Ketorolac ESI-254.0/209.8

ESI+256.2/105.0

50

Fenoprofen APCI-241.0/196.8 100 Etodolac ESI-286.1/242.0 50

Carprofen APCI-272.8/228.8 100 Warfarin ESI-307.1/160.9

ESI+309.2/163.1

50

Indoprofen ESI+282.1/236.1 50 Bendroflumethiaze ESI-420.1/77.9 50

Proglumide ESI-333.1/120.9 50 Trichlormethiazide ESI-377.8/241.6 50

1-(Phenylsulfonyl)-3-indoleboronic acid

ESI-300.0/209.8 100 * Conc. (ng/mL)

InstrumentationHPLC System: Agilent® 1200 series

Pump: G1312B (Binary Pump)

Autosampler: G1337C HP-ALS-SL

MS Detector: AB SCIEX™ API 4000™ LC/MS/MS -Turbo V™ Source with ESI or APCI probe

MS Detection TurbolonSpray® —ESI or APCI in Positive or Negative Ion Mode; MRM transition

HPLC ConditionsFlow Rate: 0.2 mL/min (3 µm, 150 x 2.0 mm) or 1.0 mL/min (5 µm, 250 x 4.6 mm), flow split to

0.25 mL/min into MS/MSInjection Volume: 5 µL (150 x 2.0 mm) or 20 µL (250 x 4.6 mm)

Columns: Lux® Cellulose-1 3 µm 150 x 2.0 mm Lux® Cellulose-2 3 µm 150 x 2.0 mm Lux® Amylose-2 3 µm 150 x 2.0 mmLux® Cellulose-4 5 µm 250 x 4.6 mm Lux® Cellulose-3 5 µm 250 x 4.6 mm Kinetex® 2.6 µm C18 50 x 2.1 mm (used for achiral analysis)

Mobile Phases: 1. Acetonitrile/Methanol: 0.1 % Formic acid (FA)2. Acetonitrile/Methanol: 5 mM NH4HCO3 – achiral analysis3. Acetonitrile/Methanol: 5 mM HCOONH4 (NH4FA) – achiral analysis4. Acetonitrile/Methanol: 5 mM CH3COONH4(NH4AC)- achiral analysis

Experimental Conditions

Figure 3. LC/MS/MS Responses of Acidic Racemates in ESI- with Different Additives and Methanol

0.1 % FA:MeOH5 mM NH4FA:MeOH 5 mM NH4HCO3:MeOH5 mM NH4HCO3:MeOH

1500000

1250000

1000000

750000

500000

250000

0

0.1 % FA:MeOH5 mM NH4FA:MeOH 5 mM NH4HCO3:MeOH5 mM NH4HCO3:MeOH

WarfarinEtodolac

Ibuprofen

Fenoprofen

Flurbiprofen

Proglumide

Bendroflumethiaze

1-(PS)-3-indoleboronic

KetorolacMecoprop

Carprofen

Figure 4. LC/MS/MS Responses of Acidic Racemates in ESI- with Different Additives and Acetonitrile

5 mM NH4HCO3:CH3CN

5 mM NH4FA:CH3CN

0.1 % FA:CH3CN

5 mM NH4AC:CH3CN

5 mM NH4HCO3:CH3CN

5 mM NH4FA:CH3CN0.1 % FA:CH3CN5 mM NH4AC:CH3CN

Figure 5. The Effect of Acidic Additives on the Enantioseparation of Acidic Racemates

in RP

min0 2 4 6 8 10 12 14 16 18

mAU

0

10

20

30

40

DAD1 D, Sig=220,8 Ref=off (F:\PHEN4157\HPCHEM\1\DATA\LP0509\LP050507.D)

14.642 15.595

min0 2 4 6 8 10 12 14 16 18

mAU

0

10

20

30

40

50

60


14.915 15.889

min0 2 4 6 8 10 12 14 16 18

mAU

0

5

10

15

20

25

30


14.954 15.932

min0 2 4 6 8 10 12 14

mAU

0

50

100

150

200

250

300

350

VWD1 A, Wavelength=220 nm (F:\PHEN4228\HPCHEM\1\DATA\LP0310\LP 03-22-10 2010 -2\LP 03-22-10 2010-03-22 20-12-32\LP-03-

9.047

10.061

min0 2 4 6 8 10 12 14

mAU

0

100

200

300

400

500

600

VWD1 A, Wavelength=220 nm (LP0310\LP 03-22-10-2 2010-03-23 10-52-16\LP-03-22-10-015.D)

3.256

4.950

5.904

min0 2 4 6 8 10 12 14

mAU

0

100

200

300

400

500

600

700

VWD1 A, Wavelength=220 nm (LP0310\LP 03-22-10-3 2010-03-23 11-47-19\LP-03-22-10-016.D)

4.628

5.581

A. CH3CN / 0.1 % HAc (40:60) Rs: 1.64

B. CH3CN / 0.1 % FA (40:60) Rs: 1.65

C. CH3CN / 0.1 % TFA (40:60) Rs: 1.64

E. CH3CN / 0.1 % FA (60:40) Rs: 4.69

F. CH3CN / 0.1 % TFA (60:40) Rs: 4.94

D. CH3CN / 0.1 % HAc (60:40) Rs: 2.72

Flurbiprofen (pKa 4.33 ) Lux® 5 µm Amylose-2 250 x 4.6 mm

BendroflumethiazeLux® 5 µm Cellulose-1 250 x 4.6 mm

Conc.: 250 µg/mL; Flow Rate: 1.0 mL/min; Injection: 10 µL; Detection: UV @ 220 nm HAc - Acetic Acid TFA - Trifluoroacetic Acid

Figure 6. The Effect of Organic Modifier on the Enantioseparation of Acidic Racemates in RP

Ibuprofen Lux® 5 µm Cellulose-3 250 x 4.6 mm

ProglumideLux® 3 µm Cellulose-1 150 x 2.0 mm

FA - Formic Acid

X I C o f -M R M ( 1 2 p a i rs ): 3 3 3 .0 5 2 /1 2 0 .9 0 0 D a f r o m S a m .. . M a x . 1 .1 e 5 cp s .

2 4 6 8 1 0

T im e , m i n

0 .0 0

2 .0 0 e 4

4 .0 0 e 4

6 .0 0 e 4

8 .0 0 e 4

1 .0 0 e 5

1 .1 5 e 52 . 4 2

X IC o f - M R M (1 2 p a ir s ) : 2 0 5 .1 4 1 /1 6 0 .9 0 0 D a f r o m S a m .. . M a x . 2 .4 e 4 c p s .

2 4 6 8 1 0 1 2 1 4

T im e , m i n

5 0 0 0 .0

1 .0 e 4

1 .5 e 4

2 .0 e 4

iX I C o f -M R M ( 1 2 p a i rs ): 3 3 3 .0 5 2 /1 2 0 .9 0 0 D a f r o m S a m .. . M a x . 3 .6 e 4 cp s .

2 4 6 8 1 0

T im e , m i n

0 .0

1 .0 e 4

2 .0 e 4

3 .0 e 4

3 .6 e 4

X IC o f - M R M (1 2 p a ir s ) : 2 0 5 .1 4 1 /1 6 0 .9 0 0 D a f r o m S a .. . M a x . 6 3 5 9 .0 c p s .

2 4 6 8 1 0 1 2 1 4

T im e , m i n

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

5 0 0 0

6 0 0 0

i

.9

X I C o f -M R M ( 1 2 p a i rs ): 3 3 3 .0 5 2 /1 2 0 .9 0 0 D a f r o m S a m .. . M a x . 1 .6 e 4 cp s .

2 4 6 8 1 0

T im e , m i n

0 .0

5 0 0 0 .0

1 .0 e 4

1 .5 e 4

7 .2 1

X IC o f - M R M (1 2 p a ir s ) : 2 0 5 .1 4 1 /1 6 0 .9 0 0 D a f r o m S a .. . M a x . 4 1 7 1 .2 c p s .

2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4

T im e , m i n

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

s

2 0 .8 4

CH3CN / 0.1 % FA (30:70) CH3CN / 0.1 % FA (40:60)

CH3CN / 0.1 % FA (60:40) CH3CN / 0.1 % FA (60:40)

CH3CN / 0.1 % FA (50:50)CH3CN / 0.1 % FA (40:60)

Figure 7. Enantioseparations on Lux Cellulose-2 with Acetonitrile or Methanol as Modifier

X I C o f -M R M ( 6 p a i rs ): 3 3 3 .0 5 2 /1 2 0 .9 0 0 D a f ro m S .. . M a x . 2 .3 e 4 c p s .

2 4 6 8 1 0 1 2

T im e , m i n

0 .0

2 .0 e 4

X IC o f -M R M ( 1 2 p a ir s ): 3 3 3 .0 5 2 /1 2 0 .9 0 0 D a f ro m . . . M a x . 1 .4 e 4 cp s .

2 4 6 8 1 0 1 2 1 4

T im e , m i n

1 .0 e 4

1 .4 e 4

n

t

e

n

p

.2

X I C o f -M R M ( 6 p a i rs ): 2 5 3 .9 4 7 /2 0 9 .8 0 0 D a f ro m . . . M a x . 5 7 8 5 .9 c p s .

2 4 6 8 1 0 1 2

T im e , m i n

2 0 0 0

4 0 0 0

5 7 8 6


2 4 6 8 1 0 1 2 1 4

T im e , m i n

1 .0 0 e 4

1 .2 4 e 4

I

p

.

X I C o f -M R M ( 6 p a i rs ): 4 2 0 .0 4 8 /7 7 .9 0 0 D a f ro m S a .. . M a x . 1 .3 e 4 c p s .

2 4 6 8 1 0 1 2

T im e , m i n

5 0 0 0 .0 0

1 .0 0 e 4

6 .8 4

X IC o f -M R M ( 1 2 p a ir s ): 4 2 0 .0 4 8 /7 7 .9 0 0 D a f ro m . . . M a x. 3 4 0 5 .0 cp s.

2 4 6 8 1 0 1 2 1 4

T im e , m i n

0

3 4 0 5

t

p

3 .1 4

X I C o f -M R M ( 6 p a i rs ): 2 9 9 .9 9 4 /2 0 9 .8 0 0 D a f ro m . . . M a x . 1 3 9 6 .8 c p s .

2 4 6 8 1 0 1 2

T im e , m i n

5 0 0

1 0 0 0

1 3 9 7 .

X IC o f -M R M ( 1 2 p a ir s ): 2 9 9 .9 9 4 /2 0 9 .8 0 0 D a f ro m . . . M a x. 2 2 0 0 .1 cp s.

2 4 6 8 1 0 1 2 1 4

T im e , m i n

1 5 0 0

2 0 0 0

.

X I C o f -M R M ( 6 p a i rs ): 2 8 6 .1 0 1 /2 4 2 .0 0 0 D a f ro m S .. . M a x . 9 .2 e 4 c p s .

2 4 6 8 1 0 1 2

T im e , m i n

5 .0 e 4

9 .2 e 4 . .


2 4 6 8 1 0 1 2 1 4

T im e , m i n

1 .0 0 e 5

1 .5 0 e 5

,

c

p

FA - Formic Acid

Ketorolac CH3CN / 0.1 % FA (40:60)

Proglumide CH3CN / 0.1 % FA (40:60)

1-(Phenylsulfonyl)-3-indoleboronic acid CH3CN / 0.1 % FA (40:60)

Etodolac CH3CN / 0.1 % FA (40:60)

Etodolac Methanol / 0.1 % FA (70:30)

Bendroflumethiaze Methanol / 0.1 % FA (70:30)

Ketorolac Methanol / 0.1 % Formic Acid (70:30)

Proglumide Methanol / 0.1 % FA (70:30)

Bendroflumethiaze CH3CN / 0.1 % FA (40:60)

1-(Phenylsulfonyl)-3-indoleboronic acid Methanol / 0.1 % FA (70:30)

Figure 8. Enantioseparations in RP on Lux Cellulose-1 with Acetonitrile

X I C o f -M R M ( 5 p a i rs ): 2 8 6 .1 0 1 /2 4 2 .0 0 0 D a f ro m S a m .. . M a x . 3 .0 e 5 c p s .

2 4 6 8 1 0

T im e , m in

0 .0

1 .0 e 5

2 .0 e 5

3 .0 e 5

X IC o f - M R M ( 5 p a i rs ) : 3 3 3 .0 5 2 /1 2 0 .9 0 0 D a f r o m S a m .. . M a x . 2 .8 e 4 cp s .

2 4 6 8 1 0

T im e , m in

0 .0

1 .0 e 4

2 .0 e 4

2 .8 e 4

s

s


2 4 6 1 0

T im e , m in

0 .0

5 .0 e 4

1 .0 e 5

1 .4 e 5

X IC o f - M R M ( 2 p a i rs ) : 2 7 2 .7 6 1 /2 2 8 .8 0 0 D a f r o m S a .. . M a x . 8 3 6 3 .8 cp s .

2 4 6 8 1 0

T im e , m in

0

5 0 0 0

8 3 6 4

sX I C o f -M R M ( 2 p a i rs ): 3 7 7 .7 7 4 /2 4 1 .6 0 0 D a f ro m S a m .. . M a x . 1 .5 e 5 c p s .

2 4 6 1 0

T im e , m in

0 .0

5 .0 e 4

1 .0 e 5

1 .5 e 5

X IC o f - M R M ( 5 p a i rs ) : 4 2 0 .0 4 8 /7 7 .9 0 0 D a f r o m S a m p .. . M a x . 1 .1 e 4 cp s .

2 4 6 8 1 0 1 2

T im e , m in

0 .0 0

5 0 0 0 .0 0

1 .0 0 e 4

sX I C o f -M R M ( 1 2 p a i rs ): 2 9 9 .9 9 4 /2 0 9 .8 0 0 D a f r o m S .. . M a x . 1 0 8 8 .2 cp s .

5 1 0 1 5 2 0

T im e , m in

5 0 0

1 0 0 0

X IC o f - M R M ( 1 2 p a i rs ) : 2 5 3 .9 4 7 /2 0 9 . 8 0 0 D a f r o m S .. . M a x . 4 3 9 9 .5 cp s .

5 1 0 1 5 2 0

T im e , m in

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

c

p

s

Warfarin CH3CN / 0.1 % FA (60:40)

Trichlormethiazide CH3CN / 0.1 % FA (30:70)


Carprofen APCI- CH3CN / 0.1 % FA (60:40)





FA - Formic Acid

X I C o f -M R M ( 6 p a i rs ): 2 8 6 .1 0 1 /2 4 2 .0 0 0 D a fro m S a m p l.. . M a x . 1 .3 e 6 cp s .

2 4 6 8 1 0

T im e , m i n

5 .0 0 e 5

1 .0 0 e 6

1 .3 0 e 61

X IC o f - M R M (6 p a ir s ) : 3 0 7 .1 3 4 /1 6 0 .9 0 0 D a fr o m S a m p l . .. M a x. 1 .8 e 5 c p s.

2 4 6 8 1 0

T im e , m i n

0 .0

5 .0 e 4

1 .0 e 5

1 .5 e 5

t

X I C o f -M R M ( 6 p a i rs ): 2 9 9 .9 9 4 /2 0 9 .8 0 0 D a fro m S a m .. . M a x . 1 5 7 7 .1 cp s .

2 4 6 8 1 0 1 2 1 4

T im e , m i n

0

5 0 0

1 0 0 0

1 5 0 0.

X IC o f - M R M (6 p a ir s ) : 2 5 3 .9 4 7 /2 0 9 .8 0 0 D a fr o m S a m .. . M a x. 4 5 5 6 .1 c p s.

2 4 6 8 1 0 1 2 1 4

T im e , m i n

0

2 0 0 0

4 0 0 0

t

s

.

X I C o f -M R M ( 6 p a i rs ): 3 3 3 .0 5 2 /1 2 0 .9 0 0 D a fro m S a m p l.. . M a x . 2 .7 e 4 cp s .

2 4 6 8 1 0

T im e , m i n

0 .0

1 .0 e 4

2 .0 e 4

2 .7 e 48

X IC o f - M R M (2 p a ir s ) : 3 7 7 .7 7 4 /2 4 1 .6 0 0 D a fr o m S a m p l . .. M a x. 9 .3 e 4 c p s.

2 4 6 8 1 0 1 2 1 4

T im e , m i n

0 .0

5 .0 e 4

9 .3 e 4.

X I C o f -M R M ( 6 p a i rs ): 4 2 0 .0 4 8 /7 7 .9 0 0 D a fro m S a m p le .. . M a x . 1 .2 e 4 cp s .

2 4 6 8 1 0

T im e , m i n

0 .0 0

5 0 0 0 .0 0

1 .0 0 e 4

.

X IC o f - M R M (6 p a ir s ) : 4 2 0 .0 4 8 /7 7 .9 0 0 D a fr o m S a m p l e . .. M a x. 1 .5 e 4 c p s.

2 4 6 8 1 0

T im e , m i n

0 .0

5 0 0 0 .0

1 .0 e 4

1 .5 e 4

.7

Figure 9. Enantioseparations in RP on Lux Cellulose-4 with Acetonitrile

Etodolac

CH3CN / 0.1% FA (50:50)




Trichlormethiazide CH3CN / 0.1 % FA (30:70)


Warfarin CH3CN / 0.1 % FA (60:40)


X I C o f -M R M ( 4 p a i rs ): 2 5 3 .9 4 7 /2 0 9 .8 0 0 D a fro m S a m p l.. . M a x. 2 .4 e 4 cp s .

1 .0 2 .0 3 .0 4 .0 5 .0 6 .0 7 .0 8 .0

T im e , m i n

5 0 0 0 .0

1 .0 e 4

1 .5 e 4

2 .0 e 4

4 .1 9

X IC o f + M R M ( 5 p a i rs ) : 2 5 6 .1 8 9 /1 0 5 .0 0 0 D a f r o m S a m p l .. . M a x. 2 .2 e 5 c p s .

1 .0 2 .0 3 .0 4 .0 5 .0 6 .0 7 . 0 8 .0

T im e , m i n

0 .0

5 .0 e 4

1 .0 e 5

1 .5 e 5

2 .0 e 5

4 .1 9

X I C o f -M R M ( 4 p a i rs ): 3 0 7 .1 3 4 /1 6 0 .9 0 0 D a fro m S a m p l.. . M a x. 3 .7 e 4 cp s .

1 .0 2 .0 3 .0 4 .0 5 .0 6 .0 7 .0 8 .0

T im e , m i n

0 .0

1 .0 e 4

2 .0 e 4

3 .0 e 4

3 .7 e 4

X IC o f + M R M ( 5 p a i rs ) : 3 0 9 .1 7 8 /1 6 3 .1 0 0 D a f r o m S a m p l .. . M a x. 7 .0 e 4 c p s .

1 .0 2 .0 3 .0 4 .0 5 .0 6 .0 7 . 0 8 .0

T im e , m i n

2 .0 e 4

4 .0 e 4

6 .0 e 4

7 .0 e 4

n

.

X I C o f + M R M ( 5 p a ir s ) : 2 6 1 .1 2 0 /1 1 1 .0 0 0 D a f ro m S a m p ... a x . 1 .3 e 5 cp s .

1 .0 2 .0 3 .0 4 .0 5 .0 6 .0 7 .0 8 .0

T im e , m i n

0 .0 0

5 .0 0 e 4

1 .0 0 e 5

1 .2 7 e 5

X IC o f - M R M (4 p a ir s ) : 2 4 2 .9 5 4 /1 9 8 .7 0 0 D a fr o m S a m p l .. . M a x. 3 .5 e 4 c p s .

1 .0 2 .0 3 .0 4 .0 5 .0 6 .0 7 .0 8 .0

T im e , m i n

5 0 0 0 .0

1 .0 e 4

1 .5 e 4

2 .0 e 4

2 .5 e 4

3 .0 e 4

3 .5 e 4

.

Figure 10. Enantioseparations in RP on Lux Amylose-2 with Acetonitrile

Ketorolac 253.95/209.8 (ESI-) CH3CN / 0.1 % FA (60:40)

Warfarin 309.18/163.10 (ESI+) CH3CN / 0.1 % FA (60:40)

Warfarin 307.13/160.90 (ESI-) CH3CN / 0.1 % FA (60:40)

Suprofen 261.12/111.00 (ESI+) CH3CN / 0.1 % FA (60:40)

Ketorolac 256.19/105.00 (ESI+) CH3CN / 0.1 % FA (60:40)

Flurbiprofen 242.95/198.70 (ESI-) CH3CN / 0.1 % FA (60:40)

Figure 11. Enantioseparations in RP on Lux Cellulose-3 with Acetonitrile Modifier


2 4 6 8 1 0 1 2 1 4 1 6

T i m e , m in

0 .0

5 .0 e 4

6 .8 e 4.

X IC o f - M R M ( 9 p a i rs ) : 2 4 0 .9 7 3 /1 9 6 .8 0 0 D a fr o m S a m ... M a x . 1 .6 e 5 cp s .

5 1 0 1 5 2 0 2 5

T i m e , m in

1 .0 e 5

1 .6 e 5

.

X I C o f + M R M ( 5 p a ir s ) : 2 6 1 .1 2 0 /1 1 1 .0 0 0 D a f ro m S a .. . M a x . 1 .1 e 5 c p s .

2 4 6 8 1 0 1 2

T i m e , m in

0 .0 0

1 .0 0 e 5

X IC o f - M R M ( 9 p a i rs ) : 2 5 3 .9 4 7 /2 0 9 .8 0 0 D a fr o m S a .. . M a x . 3 8 0 2 .9 cp s .

2 4 6 8 1 0 1 2

T im e , m in

0

3 8 0 3


2 4 6 8 1 0 1 2

T i m e , m in

0 .0

5 .0 e 5

9 .3 e 5

X IC o f + M R M ( 2 p a i rs ): 2 8 2 .0 9 4 /2 3 6 .1 0 0 D a f ro m S a . .. M a x . 5 .9 e 4 cp s .

5 1 0 1 5

T im e , m in

0 .0

5 .0 e 4

X I C o f -M R M ( 1 2 p a i rs ): 2 0 5 .1 4 1 /1 6 0 .9 0 0 D a fr o m S a . .. M a x . 3 .9 e 4 c p s .

2 4 6 8 1 0 1 2

T i m e , m in

0 .0

3 .9 e 4

X IC o f - M R M ( 1 2 p a i rs ) : 3 3 3 .0 5 2 /1 2 0 . 9 0 0 D a fr o m S a .. . M a x . 3 .1 e 4 cp s .

2 4 6 8 1 0

T im e , m in

0 .0

3 .1 e 4

X I C o f -M R M ( 2 p a i rs ): 2 4 0 .9 7 3 /9 2 .9 0 0 D a f ro m S a m .. . M a x . 6 9 0 5 .7 cp s .

5 1 0 1 5 2 0 2 5

T im e , m in

0

5 0 0 0

6 9 0 6 .

X IC o f - M R M ( 3 p a i rs ) : 2 6 2 .9 6 8 /1 5 2 .7 0 0 D a fr o m S a m ... M a x . 2 .4 e 4 cp s .

2 4 6 8 1 0 1 2 1 4

T im e , m in

0 .0

2 .0 e 4

,

c

p

s

Mecoprop CH3CN / 0.1 % FA (40:60)

Suprofen (ESI+) CH3CN / 0.1 % FA (40:60)



Indoprofen CH3CN / 0.1 % FA (80:20)


Ibuprofen CH3CN / 0.1 % FA (95:5)

Fenoprofen (APCI-) CH3CN / 0.1 % FA (40:60)

Abscisic acid (APCI-) CH3CN / 0.1 % FA (60:40)

Flurbiprofen CH3CN / 0.1 % FA (40:60)

Chiral LC/MS/MS experiments

Five different polysaccharide-based chiral stationary phases – Lux® Cellulose-1, Lux Cellulose-2, Lux Cellulose-3, Lux Cellulose-4, and Lux Amylose-2 (Figure 1) were explored in the reversed phase elution mode for the separation of a variety of acidic compounds of pharmaceutical interest in mobile phases made of 0.1 % formic acid (FA) with acetonitrile or methanol as organic modifier and MS/MS detection.

Selection of mobile phase additives

As acidic analyte presents as anion ion at the neutral pH, which is not suitable for retaining on polysaccharide-based CSPs and resulting in early elution without enantiseparation, the acidic additive is often used as a counter ion in mobile phase for suppression of dissociation of acidic analyte to increase the elution time and enantioselectivity of acidic racemates on CSPs. Three acids - trifluoroacetic acid (TFA)- pKa 0.59; formic acid (FA) - pKa 3.75; acetic acid HAc) - pKa 4.76 were evaluated on Lux Cellulose-1 and Lux Amylose-2 CSPs as acidic additives. In general, these additives provide similar enantioresolution for weakly acidic racemates (Figure 5, a, b, c) while the stronger acidic additive (TFA) performs better for the stronger acidic racemates (Figure 5, d, e, f). Formic acid provides comparable enantioseparations and peak shapes to TFA. Considering the “memory effect” of TFA on polysaccharide-based CSPs and its ion suppressing tendency in MS detection, formic acid is preferred over TFA in RP MPs for the chiral separation and MS/MS detection of acidic racemates.

Effect of mobile phase additives

The LC/MS/MS responses of acidic racemates with ESI in negative mode in 5 mM HCOONH4, 5 mM CH3COONH4 and 5 mM NH4HCO3 containing mobile phases with either acetonitrile or methanol as organic modifier using an achiral column – Kinetex® C18 – were compared to responses in 0.1 % FA (Figure 3 and 4). The results

show that MS/MS responses are comparable in 0.1 % FA CH3CN or MeOH to the responses of analytes in other additives considerd here in the ESI- mode. This proves that 0.1 % FA as acidic additive in mobile phases is compatible for MS/MS detection and favored for enantioseparation of acidic racemates, except for carprofen which showed very poor responses in all of the additives at ESI- mode. This proves that 0.1 % FA as acidic mobile phase additive is fully compatible with MS/MS detection of acidic racemates and can be implemented as the first choice.

Effect of organic modifier on chiral resolution

Acetonitrile or methanol was used in chiral RP HPLC separations as organic modifiers. Decreasing the eluting strength of the mobile phase by decreasing the portion of acetonitrile or methanol in the mobile phase will increase retention and resolution (Figure 6). However, once enantiomers elute later than 10 minutes with only partial resolution, baseline separation can rarely be achieved by further decreasing the % organic modifier in the mobile phase. In our study, acetonitrile was more successful in providing chiral resolution on Lux CSP in RP mode compared to methanol (Figure 7).

Chiral LC/MS/MS applications

Figures 8-11 demonstrate 15 chiral separations on Lux® CSPs. APCI- source was employed for the MS/MS detection of carprofen, abscisic acid, and fenoprofen as APCI- provides much better MS/MS signals than ESI- for these three acidic racemates. ESI+ mode was used for the MS/MS detection of suprofen. Most compounds evaluated here eluted in less than 10 min with baseline resolution in mobile phases of various eluting strength. The results show that Lux® Cellulose-3 was most successful in separating acidic racemates (10 racemates), especially non-steroidal anti-inflammatory racemates.

Results and Discussion

Conclusions

Fifteen successful chiral LC/MS/MS analyses are demonstrated for acidic racemic compounds on the polysaccharide-based CSPs Lux Cellulose-1, Lux Cellulose-2, Lux Cellulose-3, Lux Cellulose-4, and Lux Amylose-2 in reversed phase elution mode.

Formic acid as acidic additive in mobile phases is compatible for MS/MS detection and favored for

enantioseparation of acidic racemates in RP.

Increasing the volume fraction of organic modifier (acetonitrile or methanol) in the RP mobile phase has the expected effect: it decreases retention and enantioselectivity; adjusting the organic modifier content of the mobile phase is essential to optimizing chiral resolution.

References

1. Liming P, Tivadar F, Swapna J, Chirality 2011 poster2. H.R. Buser, M.D. Müller, Anal. Chem. 64 (1992) 31683. M.L. de la Puente, J. Chromatogr. A 1055 (2004) 554. S.M.R. Stanley, W.K. Wee, B.H. Lim, H.C. Foo, J. Chromatogr. B 848 (2007) 292

TrademarksLux and Kinetex are registered trademarks of Phenomenex, Inc. Agilent is a registered trademark of Agilent Technologies.API 4000 and Turbo V are trademarks of AB SCIEx Pte. Ltd. TurboIonSpray is a registered trademark of AB SCIEx Pte. Ltd. AB SCIEx™ is being used under license.

DisclaimerPhenomenex, Inc. is not affiliated with Agilent Technologies or AB SCIEx.Comparative separations may not be representative of all applications.

© 2011 Phenomenex, Inc. All rights reserved.

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