A reverse phase HPLC method for the separation of two stereo isomers of 2-[4-(methylsulfonyl)phenyl]-3-(3(r)-oxocyclopentyl)propanoic acid

Article abstract of DOI:10.1002/chir.20915

This study describes successful method development and separation of two stereo isomers of 2-[4-(methylsulfonyl)phenyl]-3-(3(R)-oxocyclopentyl)propanoic acid by reverse phase high-performance liquid chromatography. Baseline resolution was achieved on a J'sphere-ODS-H80 (150 mm × 4.6 mm, 4 μm) column using mobile phase consisting of 0.05% triflouroacetic acid in water-acetonitrile (85:15, v/v) at a flow rate of 1.0 ml/min. The detection was carried out at 228 nm. The title compound, in turn, can be obtained by C-alkylation of methyl 2-[4-(methylthio)phenyl]acetate with 2(S)-iodomethyl-8,8- dimethyl-6,10-dioxaspiro[4.5]decane followed by consecutive hydrolysis and oxidation. The partially validated analytical method (system suitability, peak homogeneity, linearity, precision, robustness, and solution stability) has limit of detection and limit of quantification, 0.15 and 0.50 μg/ml respectively. Alternatively, the new method is being routinely utilized to monitor epimerization of α-carbon of the propanoic acid in the title compound by crystallization-induced dynamic resolution. Copyright

Full text of DOI:10.1002/chir.20915

CHIRALITY 23:277–280 (2011)
A Reverse Phase HPLC Method for the Separation of Two Stereo
Isomers of 2-[4-(Methylsulfonyl)phenyl]-3-(3(R)-
oxocyclopentyl)propanoic Acid
1
1
2
3
4
PRAKASH M. DAVADRA,1 TIMIR K. PATEL, JIGNESH C. CHAUHAN, RAJENDRA K. KHARUL, BIPIN PANDEY, MUKUL R. JAIN,
*
AND ATUL H. BAPODRA5
1Department of Biopharmaceutics, Zydus Research Centre, Sarkhej-Bavla N. H. No. 8A, Moraiya, Ahmedabad 382 213, Gujarat, India
2Department of Medicinal Chemistry, Zydus Research Centre, Sarkhej-Bavla N. H. No. 8A, Moraiya, Ahmedabad 382 213, Gujarat, India
3Department of CMC, Zydus Research Centre, Sarkhej-Bavla N. H. No. 8A, Moraiya, Ahmedabad 382 213, Gujarat, India
4Department of Pharmacology and Toxicology, Zydus Research Centre, Sarkhej-Bavla N. H. No. 8A, Moraiya, Ahmedabad 382 213, Gujarat, India
5Department of Chemistry, M. D. Science College, Porbandar 360 575, Gujarat, India
ABSTRACT
This study describes successful method development and separation of two
stereo isomers of 2-[4-(methylsulfonyl)phenyl]-3-(3(R)-oxocyclopentyl)propanoic acid by
reverse phase high-performance liquid chromatography. Baseline resolution was achieved on a
J’sphere-ODS-H80 (150 mm 3 4.6 mm, 4 lm) column using mobile phase consisting of 0.05%
triflouroacetic acid in water-acetonitrile (85:15, v/v) at a flow rate of 1.0 ml/min. The detection
was carried out at 228 nm. The title compound, in turn, can be obtained by C-alkylation of
methyl 2-[4-(methylthio)phenyl]acetate with 2(S)-iodomethyl-8,8-dimethyl-6,10-dioxaspiro[4.5]-
decane followed by consecutive hydrolysis and oxidation. The partially validated analytical
method (system suitability, peak homogeneity, linearity, precision, robustness, and solution sta-
bility) has limit of detection and limit of quantification, 0.15 and 0.50 lg/ml respectively. Alter-
natively, the new method is being routinely utilized to monitor epimerization of a-carbon of the
propanoic acid in the title compound by crystallization-induced dynamic resolution. Chirality
C
VV
23:277–280, 2011.
2010 Wiley-Liss, Inc.
KEY WORDS: chiral separation; RP-HPLC; validation; achiral column; column temperature
INTRODUCTION
and precise analytical method for the separation of two Chiral
isomers, which has been used to check performance of epi-
merization process.
Approximately, 25% of commercially available chiral drugs
are either racemates or diastereomers. The biological activity
of chiral molecule substantially differs from its racemates or
its stereoisomers is well documented.1,2 Thus, the separation
of each isomer in such mixtures is usually critical to both the
pharmaceutical industry as well as the regulatory agencies to
assure the safety, quality, and efficacy of drug.3,4
The method development involved selection and optimi-
zation of the stationary phase, mobile phase, column tem-
perature, so forth. The method was validated for the pa-
rameters, such as system suitability, peak homogeneity,
precision, limit of detection, limit of quantification, linear-
ity, robustness, and finally solution stability. The method
was able to detect 0.15 lg/ml of each chiral isomer, after
5 ll injection.
The high-performance liquid chromatography (HPLC) is a
powerful and widely used technique for the separation and
quantitation of diastereomers.5–12 The use of chiral columns
to separate chiral isomers using HPLC is very common, such
as cylodextrin,8 3,5-dimethylphenylcarbamates of cellulose
and amylase,9 nucleosil chiral-2,7 and Chiral-AGP.12 Gopal
et al.10 used quinine as an additive to the mobile phase to
enhance separation of diastereomers of a pharmaceutical
compound on a diol column. In light of frequent use of chiral
column, there are reports5–7,11 describing the use of achiral
column in reversed phase mode to separate diastereomers.
The objective of this work was to develop HPLC method
for the separation of new set of chiral isomers (diaster-
eomers). The title compound, 2-[4-(methylsulfonyl)phenyl]-3-
(3(R)-oxocyclopentyl)propanoic acid (Fig. 1) has novel skele-
ton and its derivatives exhibit interesting and biologically
useful properties, during pharmacological evaluations.
Although target molecule has two chiral centers, where one
is with fixed (R)-configuration in cyclopentanone ring, and
the other chiral center is prone to racemization leading to
two diastereomers (S,R) and (R,R). (S,R) Chiral isomer
(S,R) has been epimerized to (R,R) by crystallization-
induced dynamic resolution. This study reports a suitable
MATERIALS AND METHODS
Chemicals
HPLC grade Trifluoroacetic acid was purchased from Merck
(Germany), whereas acetonitrile was purchased from s. d. Fine Chem-
icals (India). Water used was from Barnstead (USA) water purification
system.
The compound, 2-[4-(methylsulfonyl)phenyl]-3-(3(R)-oxocyclopentyl)-
propanoic acid was synthesized by literature procedure (Scheme 1).13–15
The mixture of two chiral isomers (Fig. 1) needed for this study was
obtained by deprotection of the compound 6 with 2 N HCl and acetone
*Correspondence to: Prakash Madhavjibhai Davadra, Department of Biophar-
maceutics, Zydus Research Centre, Sarkhej-Bavla N. H. No. 8A, Moraiya,
Ahmedabad 382 213, Gujarat, India. E-mail: prakashdavadra@zyduscadila.
com or prakashdavadra@yahoo.co.in
Received for publication 31 January 2010; Accepted 9 July 2010
DOI: 10.1002/chir.20915
Published online 13 October 2010 in Wiley Online Library
(wileyonlinelibrary.com).
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VV
2010 Wiley-Liss, Inc.

278
DAVADRA ET AL.
Sample Preparation
Stock solution of diastereomeric mixture of the title compound (1000
lg/ml) was prepared by dissolving an appropriate amount of the sub-
stance in mobile phase. The analyte concentration of each diastereomer
was fixed as 100 lg/ml. Working solutions for method validation were
prepared by subsequence dilution of the stock solution in mobile phase.
Chromatographic Conditions
The chromatographic conditions were optimized using a J’sphere-ODS-
H80 (150 mm 3 4.6 mm, 4 lm, YMC make, Japan) HPLC column. The mo-
bile phase was 0.05% trilouroacetic acid in water-acetonitrile (85:15, v/v).
The flow rate was set at 1.0 ml/min. The column was maintained at 308C,
and the detection was carried out at 228 nm. The injection volume was 5 ll.
Fig. 1. Chemical structure of 2-[4-(methylsulfonyl)phenyl]-3-(3(R)-oxocy-
clopentyl)propanoic acid.
at 268C with 98.8% chemical purity. It has been confirmed by spectro-
scopic techniques. The characterization data are as below.
Method Validation
1H NMR (DMSO-d6, 400 MHz) ( ppm: 1.79–1.94 (m, 4 H), 2.03–2.19
(m, 4 H), 2.23–2.31 (m, 1 H), 3.19 (s, 3 H), 3.81–3.91 (m, 1 H), 7.61 (d, J
The system suitability parameters, such as resolution (Rs), symmetry
(S), retention factor (k), separation factor (a), and efficiencies (N) were
determined and results are presented in Table 1 (See, result of 308C).
Peak homogeneity of both diastereomers was confirmed using PDA and
MS detector. The precision of the method was checked by analyzing six
replicate samples of both diastereomers at the analyte concentration
(100 lg/ml) and calculating the percentage relative standard deviation
of retention time and peak area.
Limit of detection (LOD) and Limit of quantification (LOQ) were
determined at a signal-to-noise ratio of 3 and 10.16 LOD and LOQ were
achieved by injecting a series of dilute solutions. The precision at LOQ
level was checked by analyzing six test solutions and calculating the per-
centage relative standard deviation of retention time and peak area. De-
tector response linearity was evaluated by determining eight working
solutions from 0.5 (LOQ) to 200 lg/ml, (0.5, 1, 2, 5, 10, 50, 100, and 200
lg/ml), prepared in mobile phase. Peak area and concentration of dia-
stereomers were subjected to regression analysis to calculate calibration
equation and correlation coefficient.
To determine robustness of the method, experimental conditions were
purposely altered and chromatographic resolution between the two dia-
stereomers was evaluated. To study the effect of flow rate on the resolu-
tion of diastereomers, it was changed by 0.1 units; from 0.9 to 1.1 ml/
min. The effect of change in percent acetonitrile (varying 6 1%) and con-
centration of triflouroacetic acid (varying 6 0.01% in water) on resolution
was studied, whereas other mobile phase components were held con-
stant, as stated under chromatographic conditions. To study the effect of
column temperature on the resolution of diastereomers, it was main-
tained at 25 and 358C instead of 308C.
5 8.4 Hz, 2 H), 7.88 (d, J 5 8.4 Hz, 2 H), 12,71 (bs, 1 H); IR (KBr) cm21
:
3431 (br), 2926, 1732, 1705, 1306, 1153.
C-alkylation of methyl 2-[4-(methylthio)phenyl]acetate (2) with 2(S)-
iodomethyl-8,8-dimethyl-6,10-dioxaspiro[4.5]decane (3) followed by con-
secutive hydrolysis and oxidation yielded two chiral isomers (diaster-
eomers) of 2-[4-(methylsulfonyl)phenyl]-3-(3(R)-oxocyclopentyl)propa-
noic acid.13 The iodo compound (3) was in turn synthesized from chiral
acid (8) ([a]D 5 116.68, c 5 1.5% in CH3OH) (Scheme 2), which was
synthesized and resolved according to the literature procedure.14,15
Additionally, chiral purity for iodo compound (3) was checked using chi-
ral GC (Column: BetDEXtm [225 1 120], 60 m, 0.25 mm, thickness 0.25
lm), which was found 100% chirally pure. Although, title compound
(Fig. 1) contains two chiral centers, which may have theoretically four
isomers. However, one of the chiral center of cyclopentanone ring
attached to 3-position of propanoic acid is fixed, and mechanistically,
there is no chance of recemization during various synthetic steps, there
is only one possibility of racemization at 2-position of propanoic acid.
Therefore, only two diastereomers (S,R) and (R,R) are possible.
Instrumentation
A 1100 series HPLC system from Agilent Technology (Germany) was
used. It was equipped with degasser, quaternary pump, auto sampler,
auto injector, thermostatic compartment, and photodiode array detector.
The output signal was monitored and processed using Agilent Chemsta-
tion software. Peak homogeneity was checked by Shimadzu LC-MS
2010A (Japan) using positive electron spray ionization mode.
Standard solution stability at analyte concentration was studied by
keeping the solution in tightly capped volumetric flask at room tempera-
Scheme 1. Reaction scheme for the synthesis and epimerization of 2-[4-(methylsulfonyl)phenyl]-3-(3(R)-oxocyclopentyl)propanoic acid.
Chirality DOI 10.1002/chir

RP-HPLC METHOD FOR SEPARATION OF CHIRAL ISOMERS
279
Scheme 2. Reaction scheme for the synthesis of 2(S)-iodomethyl-8,8-dimethyl-6,10-dioxaspiro[4.5]decane (Iodo compound).
TABLE 1. Effect of column temperature on system suitability
parameters
ODS-AQ column. This column had hydrophilic endcappings,
which could allow polar eluent to penetrate between the C18
chains to enhance solute-stationary phase interactions. Stron-
ger retention and often different selectivity are encountered
on ODS-AQ compared with conventionally endcapped
ODS.17 An initial attempt was made using phosphate buffer
(pH 7.0; 20 mM), but separation was not achieved by using
either methanol or acetonitrite as an organic modifier in dif-
ferent compositions.
In further attempt, acetate (pH 4.5; 20 mM) and phosphate
(pH 3.0; 20 mM) buffers were tried. The separation of dia-
stereomers was observed but it was not baseline separation.
To improve the separation, 0.05% triflouroacetic acid in
water-acetonitrile (85:15, v/v) was tried. The baseline separa-
tion was achieved with slight fronting in the peaks of both
the diastereomers using YMC ODS-AQ and Kromasil-C18 col-
umns. After these optimizations, the achiral column YMC
J’sphere-ODS-H80 (150 mm 3 4.6 mm, 4 lm) and mobile
phase consisting of 0.05% triflouroacetic acid in water-acetoni-
trile at different compositions were tried. Finally, (85:15, v/v)
composition was found to be appropriate allowing good base-
line resolution of both the diastereomers at a flow rate of
1.0 ml/min (Fig. 2).
Effect of the column temperature was also investigated on
the separation of diastereomers. Experiment were carried
out at 25, 30, 35, and 408C column temperature, whereas the
other mobile phase components were held constant as stated
under the chromatographic conditions. The influence of tem-
perature on different parameters, such as retention and sepa-
ration factors, peak symmetry, efficiencies, and resolution, is
summarized in Table 1. It was observed that an increase in
temperature leads to a general decrease in the retention fac-
tors and resolution. Therefore, 308C temperature seems suit-
able to achieve desirable diastereomeric separation.
System
suitability
parameters
258C
308C
358C
408C
Retention
factor
Resolution
k1 5 13.72 k1 5 11.92 k1 5 10.50
k2 5 14.91 k2 5 12.94 k2 5 11.40 k2 5 10.03
Rs 5 2.23 Rs 5 2.19 Rs 5 2.11 Rs 5 2.01
k1 5 9.25
Numbers of N1 5 13111 N1 5 12677 N1 5 12649 N1 5 12260
theoretical N2 5 13248 N2 5 13138 N2 5 12734 N2 5 12118
plates
Symmetry
S1 5 0.79
S2 5 0.80
a 5 1.09
S1 5 0.82
S2 5 0.83
a 5 1.08
S1 5 0.78
S2 5 0.79
a 5 1.08
S1 5 0.74
S2 5 0.75
a 5 1.08
Selectivity
ture on a laboratory bench for 2 days. Sample solution was analyzed ev-
ery 6 h interval up to the study period.
RESULTS AND DISCUSSION
Method Development
The objective of this study was to separate the two diaster-
eomers of the title compound, using an achiral column,
because diastereomers possess different physical properties.
To develop the suitable HPLC method for the separation of
diastereomers, different mobile phases and stationary phases
were used. For this, different columns were used, namely:
YMC ODS-AQ, Kromasil-C18, and YMC J’sphere-ODS-H80.
The compound of interest has a free carboxylic acid group.
Therefore, ionization of compound was expected to play an
important role in retention and in the separation of both the
diastereomers. To investigate effect of pH of the mobile
phase, couples of experiments were performed using YMC
Fig. 2. HPLC chromatogram and its corresponding LCMS line spectrum; Column: YMC J’shere-ODS-H80; Mobile phase: 0.05% trifloroacetic acid in water-ace-
tonitrile (85:15, v/v). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Chirality DOI 10.1002/chir

280
DAVADRA ET AL.
TABLE 2. Robustness of the developed HPLC method
J’sphere-ODS-H80 with resolution greater than 2.0 between
the two diastereomers. The method was validated with
respect to system suitability, precision, LOD, LOQ, linearity,
robustness, and solution stability. Satisfactory results were
observed for all the parameters. The proposed method has
been successfully used to check the epimerization of the iso-
mers at the 2-position in propanoic acid.
Parameter
Resolution between two diastereomers (n 5 3)
Flow rate (ml/min)
0.9
1.0
1.1
2.20
2.19
2.09
Acetonitrile percentage (%) in mobile phase
14
15
16
2.28
2.19
1.98
ACKNOWLEDGMENTS
The authors wish to thank the management of Zydus
Research Centre for providing us research facility and
encouragement. The authors would also like to thank our
colleagues for their cooperation in carrying out this work.
Concentration of trifloroacetic acid (%) in water
0.04
0.05
0.06
2.16
2.19
2.17
Column temperature (8C)
LITERATURE CITED
25
30
35
2.23
2.19
2.11
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The system suitability results (Table 1, See 308C) showed
that resolution between both the diastereomers was not less
than 2.1. Peak purity of both the diastereomers was not less
than 0.999/990 using PDA detector. The scanning (50–2000
mass unit) of both peaks using MS detector showed mass
peak of the title compound corresponding to M11(311),
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In the precision study, the percentage relative standard
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0.1% and 0.3% for retention time and peak area respectively,
indicating good precision of the method. The LOD and LOQ
concentrations were estimated to be 0.15 and 0.50 lg/ml for
both the diastereomers, when the signal-to-noise ratio of 3
and 10 were used as the criteria. The method precision for
diastereomer-1 and diastereomer-2 at LOQ was 1.8 and 1.4%
RSD, respectively.
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lg/ml for both the diastereomers. The equation of the cali-
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CONCLUSIONS
A simple, suitable, linear, precise, and economical HPLC
method was described for the diastereomeric separation of
two chiral isomers of the 2-[4-(methylsulfonyl)phenyl]-3-
(3(R)-oxocyclopentyl)propanoic acid (Fig. 1). The baseline
separation was achieved on an achiral column (YMC
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Chirality DOI 10.1002/chir