Method Development for Chiral LC/MS/MS Analysis ... - Phenomenex

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
PO87820611_L_1

Introduction
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).

Figure 1. Structures of Chiral Phases
Lux ® Cellulose-2 or-4
Cellulose tris (3-chloro-4-methylphenylcarbamate) or
(4-chloro-3-methylphenylcarbamate)
Cl
Lux ® Amylose-2
Amylose tris (5-chloro-2-methylphenylcarbamate)
Cl
OCONH
CH 3
O CONH
CH 3
Cl
O
O
Cl
HNOCO OCONH
CH 3
CH 3
Cl
HNOCO
O
CH 3
O
O CONH
Cl
Lux ® Cellulose-1
Cellulose tris (3,5-dimethylphenylcarbamate)
CH 3
CH 3
HNOCO
OCONH
O
O
OCONH
CH 3
CH 3
CH 3
CH 3
Me
Cl
Me
OCONH
Lux ® Cellulose-3
O
Cellulose tris (4-methylbenzoate)
O
CH 3
Cl
MeCH3
O
HNOCO
OCONH
C
Me
O
O
O
C
O
O
O
C
Me
O
Cl
MeCH3

Figure 2. Molecular Structures of Acidic Racemates
OH
O
S
OH
Cl
OH
O
F
N
H
O
O
N
O
O
O
OH
OH
HO
Ibuprofen Flurbiprofen Suprofen Fenoprofen Carprofen Indoprofen
O
O
OH
NH
N
O
HO
O
HO
O
O
OH
O
N
O
O
S
O
N
OH
B
OH
H 3
C
Proglumide
H
N
Etodolac
CH 3
O
O
OH
O
Abscisic Acid
OHOOO
H
N
N
H S
O O
Cl
OH
Mecoprop Ketorolac 1-(Phenylsulfonyl)-3-
indoleboronic acid
Cl H
N Cl
Cl
CF 3
O
N
H S
S
O NH 2
O O
O
S
O NH 2
Warfarin Bendroflumethiaze Trichlormethiazide

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 50
ESI + 256.2/105.0
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 50
ESI + 309.2/163.1
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)

Experimental Conditions
Instrumentation
HPLC 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 Conditions
Flow 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/MS
Injection 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 mm
Lux ® 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 NH 4HCO 3 – achiral analysis
3. Acetonitrile/Methanol: 5 mM HCOONH 4 (NH 4FA) – achiral analysis
4. Acetonitrile/Methanol: 5 mM CH 3COONH 4(NH 4AC)- achiral analysis

Figure 3. LC/MS/MS Responses of Acidic
Racemates in ESI - with Different Additives
and Methanol
1500000
0.1 % FA:MeOH
5 mM NH4FA:MeOH
5 mM NH4HCO3:MeOH
5 mM NH4HCO3:MeOH
1250000
1000000
0.1 % FA:MeOH
5 mM NH4FA:MeOH
5 mM NH4HCO3:MeOH
5 mM NH4HCO3:MeOH
750000
500000
250000
0
Warfarin
Etodolac
Ibuprofen
Fenoprofen
Flurbiprofen
Proglumide
Bendroflumethiaze
1-(PS)-3-indoleboronic
Ketorolac
Mecoprop
Carprofen

Figure 4. LC/MS/MS Responses of Acidic
Racemates in ESI- with Different Additives
and Acetonitrile
5 mM NH 4 HCO 3 :CH 3 CN
5 mM NH 4 FA:CH 3 CN
0.1 % FA:CH 3 CN
5 mM NH 4 AC:CH 3 CN
5 mM NH 4 HCO 3 :CH 3 CN
5 mM NH 4 FA:CH 3 CN
0.1 % FA:CH 3 CN
5 mM NH 4 AC:CH 3 CN

Figure 5. The Effect of Acidic Additives on the
Enantioseparation of Acidic Racemates
in RP
Bendroflumethiaze
Lux ® 5 µm Cellulose-1 250 x 4.6 mm
Flurbiprofen (pK a
4.33 )
Lux ® 5 µm Amylose-2 250 x 4.6 mm
mAU
40
DAD1D, Sig=220,8 Ref=off (F:\PHEN4157\HPCHEM\1\DATA\LP0509\LP050507.D)
14.642
VWD1A, Wavelength=220nm(F:\PHEN4228\HPCHEM\1\DATA\LP0310\LP03-22-102010-2\LP03-22-102010-03-2220-12-32\LP-03-
mAU
350
9.047
300
10.061
15.595
min
30
20
10
A. CH 3CN / 0.1 % HAc (40:60)
Rs: 1.64
250
200
150
100
50
D. CH 3CN / 0.1 % HAc (60:40)
Rs: 2.72
0
0
0 2 4 6 8 10 12 14 16 18 min 0 2 4 6 8 10 12 14 min
DAD1D, Sig=220,8 Ref=off (F:\PHEN4157\HPCHEM\1\DATA\LP0509\LP050504.D)
VWD1A, Wavelength=220nm(LP0310\LP03-22-10-22010-03-2310-52-16\LP-03-22-10-015.D)
mAU
mAU
600
4.950
60
14.915
500
5.904
50
B. CH 3CN / 0.1 % FA (40:60)
E. CH
400
3CN / 0.1 % FA (60:40)
40
Rs: 1.65
30
300
Rs: 4.69
20
200
10
100
3.256
0
0
0 2 4 6 8 10 12 14 16 18
0 2 4 6 8 10 12 14 min
DAD1D, Sig=220,8 Ref=off (F:\PHEN4157\HPCHEM\1\DATA\LP0509\LP050511.D)
VWD1A, Wavelength=220nm(LP0310\LP03-22-10-32010-03-2311-47-19\LP-03-22-10-016.D)
mAU
mAU
700
30
4.628
14.954
600
25
15.932 F. CH
500
5.581 3CN / 0.1 % TFA (60:40)
20
15
C. CH 3CN / 0.1 % TFA (40:60)
Rs: 1.64
400
300
Rs: 4.94
15.889
min
10
5
0
0 2 4 6 8 10 12 14 16 18
200
100
0
0 2 4 6 8 10 12 14
min
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
Proglumide
Lux ® 3 µm Cellulose-1 150 x 2.0 mm
Ibuprofen
Lux ® 5 µm Cellulose-3 250 x 4.6 mm
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 fr o m S a m ... M a x . 1 .1 e 5 c p s .
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 fr o m S a m ... M a x . 2 .4 e 4 c p s .
1 .1 5 e 5
1 .0 0 e 5
2 . 4 2
CH 3CN / 0.1 % FA (60:40)
2 .0 e 4
CH 3CN / 0.1 % FA (60:40)
8 .0 0 e 4
1 .5 e 4
6 .0 0 e 4
4 .0 0 e 4
1 .0 e 4
2 .0 0 e 4
5 0 0 0 .0
0 .0 0
2 4 6 8 1 0
T im e , m i n
i
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 fr o m S a m ... M a x . 3 .6 e 4 c p s .
2 4 6 8 1 0 1 2 1 4
T im e , m i n
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 fr o m S a ... M a x . 6 3 5 9 .0 c p s .
3 .6 e 4
3 .0 e 4
CH 3CN / 0.1 % FA (40:60)
6 0 0 0
5 0 0 0
CH 3CN / 0.1 % FA (50:50)
.9
2 .0 e 4
4 0 0 0
3 0 0 0
1 .0 e 4
2 0 0 0
0 .0
2 4 6 8 1 0
T im e , m i n
i
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 fr o m S a m ... M a x . 1 .6 e 4 c p s .
1 0 0 0
2 4 6 8 1 0 1 2 1 4
T im e , m i n
.1 0 0 a fr o m S a 1 7 1 X IC o f - M R M (1 2 p a ir s ) : 2 0 5 4 1 /1 6 0 .9 D ... M a x . 4 .2 c p s .
1 .5 e 4
7 .2 1
4 0 0 0
CH 3CN / 0.1 % FA (30:70) CH 3CN / 0.1 % FA (40:60)
2 0 .8 4
1 .0 e 4
3 0 0 0
5 0 0 0 .0
2 0 0 0
1 0 0 0
0 .0
2 4 6 8 1 0
T im e , m i n
FA ‐ Formic Acid
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
s

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 fro m S ... M a x . 2 .3 e 4 c p s .
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 c p s .
2 .0 e 4
Proglumide
CH 3CN / 0.1 % FA (40:60)
1 .4 e 4
1 .0 e 4
.2
Proglumide
Methanol / 0.1 % FA (70:30)
0 .0
n
2 4 6 8 t 1 0 1 2
T im e , m i n
e
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 fro m . .. n M a x . 5 7 8 5 .9 c p s .
2 4 6 8 1 0 1 2 1 4
T im e , m i n
X IC o f -M R M ( 1 2 p a ir s ): 2 5 3 .9 4 7 /2 0 9 .8 0 0 D a f ro m ... M a x . 1 .2 e 4 c p s .
5 7 8 6
4 0 0 0
2 0 0 0
Ketorolac
CH 3CN / 0.1 % FA (40:60)
I
1 .2 4 e 4
1 .0 0 e 4
Ketorolac
Methanol / 0.1 % Formic Acid (70:30)
.
2 4 6 8 1 0 1 2
T im e , m i n
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 ...
p
M a x . 1 .3 e 4 c p s .
2 4 6 8 1 0 1 2 1 4
T im e , m i n
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 c p s.
1 .0 0 e 4
5 0 0 0 .0 0
Bendroflumethiaze
CH3CN / 0.1 % FA (40:60)
6 .8 4
3 4 0 5
3 .1 4
Bendroflumethiaze
Methanol / 0.1 % FA (70:30)
2 4 6 8 1 0 1 2
T im e , m i n
p
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 . .. M a x . 1 3 9 6 .8 c p s .
0
2 4 6 8 1 0 1 2 1 4
T im e , m i n
2 9 .8 0 D a f ro m 0 0 X IC o f -M R M ( 1 p a ir s ): 2 9 .9 9 4 /2 0 9 0 ... M a x. 2 2 .1 c p s.
1 3 9 7
1 0 0 0
5 0 0
1-(Phenylsulfonyl)-3-indoleboronic acid
CH 3CN / 0.1 % FA (40:60)
t
.
2 0 0 0
1 5 0 0
.
1-(Phenylsulfonyl)-3-indoleboronic acid
Methanol / 0.1 % FA (70:30)
2 4 6 8 1 0 1 2
T im e , m i n
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 ...
p
M a x . 9 .2 e 4 c p s .
2 4 6 8 1 0 1 2 1 4
T im e , m i n
X IC o f -M R M ( 1 2 p a ir s ): 2 8 6 .1 0 1 /2 4 2 .0 0 0 D a f ro m ... M a x . 1 .7 e 5 c p s .
9 .2 e 4
5 .0 e 4
Etodolac
CH 3CN / 0.1 % FA (40:60)
.
.
1 .5 0 e 5
1 .0 0 e 5
Etodolac
Methanol / 0.1 % FA (70:30)
FA ‐ Formic Acid
2 4 6 8 1 0 1 2
T im e , m i n
2 4 6 8 1 0 1 2 1 4
T im e , m i n
,
c
p

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 fro m S a m ... M a x. 3 .0 e 5 c p s .
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 fr o m S a m ... M a x . 2 .8 e 4 c p s .
3 .0 e 5
2 .0 e 5
Etodolac
CH 3CN / 0.1 % FA (60:40)
2 .8 e 4
2 .0 e 4
Proglumide
CH 3CN / 0.1 % FA (40:60)
1 .0 e 5
1 .0 e 4
0 .0
2 4 6 8 1 0
T i m e , m in
X I C o f -M R M ( 5 p a i rs ): 3 0 7 .1 3 4 /1 6 0 .9 0 0 D a fro m S a m ... s M a x. 1 .4 e 5 c p s .
0 .0
2 4 6 8 1 0
T im e , m in
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 fr o m S a ... M a x . 8 3 6 3 .8 c p s .
1 .4 e 5
1 .0 e 5
Warfarin
CH 3CN / 0.1 % FA (60:40)
8 3 6 4
5 0 0 0
Carprofen APCI -
CH 3CN / 0.1 % FA (60:40)
5 .0 e 4
0 .0
s
2 4 6 1 0
T i m e , m in
X 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 fro m S a m ... s M a x. 1 .5 e 5 c p s .
0
2 4 6 8 1 0
T im e , m in
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 fr o m S a m p ... M a x . 1 .1 e 4 c p s .
1 .5 e 5
1 .0 e 5
Trichlormethiazide
CH 3CN / 0.1 % FA (30:70)
1 .0 0 e 4
Bendroflumethiaze
CH 3CN / 0.1 % FA (40:60)
5 .0 e 4
5 0 0 0 .0 0
0 .0
2 4 6 1 0
T i m e , m in
X 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 fr o m S ... s M a x. 1 0 8 8 .2 cp s .
0 .0 0
2 4 6 8 1 0 1 2
T im e , m in
p a 4 D a fr o m S 4 3 X IC o f - M R M ( 1 2 i rs ) : 2 5 3 .9 7 /2 0 9 . 8 0 0 ... M a x . 9 9 .5 c p s .
1 0 0 0
5 0 0
1-(Phenylsulfonyl)-3-indoleboronic acid
CH 3CN / 0.1 % FA (30:70)
4 0 0 0
3 0 0 0
2 0 0 0
Ketorolac
CH 3CN / 0.1 % FA (30:70)
1 0 0 0
5 1 0 1 5 2 0
5 1 0 1 5 2 0
T i m e , m in
T im e , m in
FA ‐ Formic Acid
c
p
s

Figure 9. Enantioseparations in RP on Lux
Cellulose-4 with Acetonitrile
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 .
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.
1 .3 0 e 6
1 .0 0 e 6
Etodolac
CH 3CN / 0.1% FA (50:50)
1
1 .5 e 5
1 .0 e 5
Warfarin
CH 3CN / 0.1 % FA (60:40)
5 .0 0 e 5
5 .0 e 4
t
2 4 6 8 1 0
T im e , m i n
fro m S a m 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 ... M a x . 1 5 7 7 .1 cp s .
0 .0
2 4 6 8 1 0
T im e , m i n
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.
1 5 0 0
1 0 0 0
1-(Phenylsulfonyl)-3-indoleboronic acid
CH 3CN / 0.1 % FA (40:60)
.
4 0 0 0
Ketorolac
CH 3CN / 0.1 % FA (40:60)
.
5 0 0
2 0 0 0
t
0
2 4 6 8 1 0 1 2 1 4
T im e , m i n
p a .0 0 fro m S a m s
.7 e X I C o f -M R M ( 6 i rs ): 3 3 3 5 2 /1 2 0 .9 0 D a p l... M a x . 2 4 cp s .
0
2 4 6 8 1 0 1 2 1 4
T im e , m i n
p .7 7 0 fr o m S a m 9 .3 X IC o f - M R M (2 a ir s ) : 3 7 7 4 /2 4 1 .6 0 D a p l ... M a x. e 4 c p s.
2 .7 e 4
2 .0 e 4
Proglumide
CH 3CN / 0.1 % FA (40:60)
8
9 .3 e 4
5 .0 e 4
Trichlormethiazide
CH 3CN / 0.1 % FA (30:70)
.
1 .0 e 4
0 .0
2 4 6 8 1 0
T im e , m i n
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 .
0 .0
2 4 6 8 1 0 1 2 1 4
T im e , m i n
p .0 4 D fr o m S a m 1 .5 X IC o f - M R M (6 a ir s ) : 4 2 0 8 /7 7 .9 0 0 a p l e ... M a x. e 4 c p s.
1 .0 0 e 4
Bendroflumethiaze
CH 3CN / 0.1 % FA (50:50)
.
1 .5 e 4
1 .0 e 4
.7
Bendroflumethiaze
CH3CN / 0.1 % FA (80:20)
5 0 0 0 .0 0
5 0 0 0 .0
0 .0 0
2 4 6 8 1 0
0 .0
2 4 6 8 1 0
T im e , m i n
T im e , m i n

Figure 10. Enantioseparations in RP on Lux
Amylose-2 with Acetonitrile
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 .
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 fr o m S a m p l ... M a x. 2 .2 e 5 c p s .
2 .0 e 4
1 .5 e 4
Ketorolac
253.95/209.8 (ESI - )
CH 3CN / 0.1 % FA (60:40)
4 .1 9
2 .0 e 5
1 .5 e 5
Ketorolac
256.19/105.00 (ESI + )
CH 3CN / 0.1 % FA (60:40)
4 .1 9
1 .0 e 4
1 .0 e 5
5 0 0 0 .0
5 .0 e 4
1 .0 2 .0 3 .0 4 .0 5 .0 6 .0 7 .0 8 .0
T im e , m i n
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 .
0 .0
1 .0 2 .0 3 .0 4 .0 5 .0 6 .0 7 . 0 8 .0
T im e , m i n
5 p 0 9 .1 .1 0 a fr o m S a m ... M a x. c p s X IC o f + M R M ( a i rs ) : 3 7 8 /1 6 3 0 D p l 7 .0 e 4 .
3 .7 e 4
3 .0 e 4
2 .0 e 4
Warfarin
307.13/160.90 (ESI - )
CH 3CN / 0.1 % FA (60:40)
7 .0 e 4
6 .0 e 4
4 .0 e 4
Warfarin
309.18/163.10 (ESI + )
CH 3CN / 0.1 % FA (60:40)
.
1 .0 e 4
2 .0 e 4
0 .0
n
1 .0 2 .0 3 .0 4 .0 5 .0 6 .0 7 .0 8 .0
T im e , m i 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
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 .2 7 e 5
1 .0 0 e 5
Suprofen
261.12/111.00 (ESI + )
CH 3CN / 0.1 % FA
(60:40)
3 .5 e 4
3 .0 e 4
2 .5 e 4
2 .0 e 4
Flurbiprofen
242.95/198.70 (ESI - )
CH 3CN / 0.1 % FA (60:40)
.
5 .0 0 e 4
1 .5 e 4
1 .0 e 4
0 .0 0
1 .0 2 .0 3 .0 4 .0 5 .0 6 .0 7 .0 8 .0
5 0 0 0 .0
1 .0 2 .0 3 .0 4 .0 5 .0 6 .0 7 .0 8 .0
T im e , m i n
T im e , m i n

Figure 11. Enantioseparations in RP on Lux
Cellulose-3 with Acetonitrile Modifier
X I C o f -M R M ( 9 p a i rs ): 2 1 4 .1 0 2 /1 4 1 .7 0 0 D a fro m S a m ... M a x . 6 .8 e 4 c p s .
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 c p s .
6 .8 e 4
5 .0 e 4
0 .0
Mecoprop
CH 3CN / 0.1 % FA (40:60)
2 4 6 8 1 0 1 2 1 4 1 6
T i m e , m in
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 .
.
1 .6 e 5
1 .0 e 5
Flurbiprofen
CH 3CN / 0.1 % FA (40:60)
5 1 0 1 5 2 0 2 5
T i m e , m in
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 c p s .
.
1 .0 0 e 5
Suprofen (ESI + )
CH 3CN / 0.1 % FA (40:60)
3 8 0 3
Ketorolac
CH 3CN / 0.1 % FA (40:60)
0 .0 0
2 4 6 8 1 0 1 2
T i m e , m in
i rs /2 4 a fro m S a c p X I C o f -M R M ( 9 p a ): 2 8 6 .1 0 1 2 .0 0 0 D m ... M a x . 9 .3 e 5 s .
0
2 4 6 8 1 0 1 2
T im e , m in
a i /2 D a f ro m S c p X IC o f + M R M ( 2 p rs ): 2 8 2 .0 9 4 3 6 .1 0 0 a ... M a x . 5 .9 e 4 s .
9 .3 e 5
5 .0 e 5
Etodolac
CH 3CN / 0.1 % FA (40:60)
5 .0 e 4
Indoprofen
CH 3CN / 0.1 % FA (80:20)
0 .0
2 4 6 8 1 0 1 2
T i m e , m in
a /1 D a fr o m S c p X I C o f -M R M ( 1 2 p i rs ): 2 0 5 .1 4 1 6 0 .9 0 0 a ... M a x . 3 .9 e 4 s .
0 .0
5 1 0 1 5
T im e , m in
.0 D a fr o m S M X IC o f - M R M ( 1 2 p a i rs ) : 3 3 3 5 2 /1 2 0 . 9 0 0 a ... a x . 3 .1 e 4 c p s .
3 .9 e 4
Ibuprofen
CH 3CN / 0.1 % FA (95:5)
3 .1 e 4
Proglumide
CH 3CN / 0.1 % FA (80:20)
0 .0
2 4 6 8 1 0 1 2
T i m e , m in
i rs /9 2 fro m S a m M c p X I C o f -M R M ( 2 p a ): 2 4 0 .9 7 3 .9 0 0 D a ... a x . 6 9 0 5 .7 s .
0 .0
2 4 6 8 1 0
T im e , m in
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 c p s .
6 9 0 6 .
5 0 0 0
Fenoprofen (APCI - )
CH 3CN / 0.1 % FA (40:60)
2 .0 e 4
Abscisic acid (APCI - )
CH 3CN / 0.1 % FA (60:40)
0
5 1 0 1 5 2 0 2 5
0 .0
2 4 6 8 1 0 1 2 1 4
T im e , m in
T im e , m in
,
c
p
s

Results and Discussion
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 polysaccharidebased
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)- pK a
0.59; formic
acid (FA) - pK a
3.75; acetic acid HAc) - pK a
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 CH 3
CN 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.

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 poster
2. H.R. Buser, M.D. Müller, Anal. Chem. 64 (1992) 3168
3. M.L. de la Puente, J. Chromatogr. A 1055 (2004) 55
4. S.M.R. Stanley, W.K. Wee, B.H. Lim, H.C. Foo, J. Chromatogr. B 848
(2007) 292
Trademarks
Lux 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.
Disclaimer
Phenomenex, 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.