A validated normal phase LC method for enantiomeric separation of rasagiline mesylate and its (S)-enantiomer on cellulose derivative-based chiral stationary phase

Article abstract of DOI:10.1002/chir.22150

A simple, sensitive, and robust normal-phase isocratic HPLC-UV method was developed and validated for the enantiomeric separation of rasagiline mesylate and its (S)-enantiomer. The rasagiline and its (S)-enantiomer were resolved on a Chiralcel-OJ-H (4-methylbenzoate cellulose coated on silica) column using a mobile phase consisting of n-hexane:isopropyl alcohol:ethanol:diethyl amine (96:2:2:0.01) at a flow rate of 1.0 ml/min. The column temperature was maintained at 27 °C and elution was monitored at 215 nm. The resolution (R_s) between the enantiomers was found to be more than 2.0. The limit of detection and the limit of quantification of the (S)-enantiomer were found to be 0.35 and 1.05 μg/ml, respectively. The developed method was validated as per ICH guidelines with respect to linearity, limit of detection and quantification, accuracy, precision, and robustness - and satisfactory results were obtained. The sample solution and mobile phase were found to be stable up to 48 h. The method is useful for routine evaluation of the quality of rasagiline mesylate in bulk drug-manufacturing units.

Full text of DOI:10.1002/chir.22150

CHIRALITY 25:324–327 (2013)
A Validated Normal Phase LC Method for Enantiomeric Separation of
Rasagiline Mesylate and Its (S)-Enantiomer on Cellulose Derivative-
Based Chiral Stationary Phase
2
P. SUNIL REDDY,1,2 K. SUDHAKAR BABU, AND NAVNEET KUMAR1
*
1Dr. Reddy’s Laboratories Ltd., IPDO, Bachupally, Hyderabad–500072, A.P, India
2Department of Chemistry, S. K. University, Anantapur–515055, A.P., India
ABSTRACT
A simple, sensitive, and robust normal-phase isocratic HPLC-UV method
was developed and validated for the enantiomeric separation of rasagiline mesylate and its
(S)-enantiomer. The rasagiline and its (S)-enantiomer were resolved on a Chiralcel-OJ-H
(4-methylbenzoate cellulose coated on silica) column using a mobile phase consisting of
n-hexane:isopropyl alcohol:ethanol:diethyl amine (96:2:2:0.01) at a flow rate of 1.0 ml/min.
The column temperature was maintained at 27 ꢀC and elution was monitored at 215 nm.
The resolution (Rs) between the enantiomers was found to be more than 2.0. The limit of
detection and the limit of quantification of the (S)-enantiomer were found to be 0.35 and
1.05 mg/ml, respectively. The developed method was validated as per ICH guidelines with
respect to linearity, limit of detection and quantification, accuracy, precision, and robustness—
and satisfactory results were obtained. The sample solution and mobile phase were found to
be stable up to 48 h. The method is useful for routine evaluation of the quality of rasagiline
mesylate in bulk drug-manufacturing units. Chirality 25:324–327, 2013. © 2013 Wiley Periodicals, Inc.
KEY WORDS: rasagiline; validation; chiral HPLC; enantiomeric purity
INTRODUCTION
quantitative determination of the enantiomeric impurity of
rasagiline in bulk drugs. Therefore, it was felt necessary
to develop an accurate, precise, and robust enantioselective
normal-phase HPLC method for the separation and determi-
nation of the undesired (S)-enantiomer of rasagiline in
bulk drugs.
The present work deals with the development and validation
of a normal phase LC method to determine the enantiomeric
purity of rasagiline using a new commercialized chiral stationary
phase, namely, cellulose tris (4-methylbenzoate) coated on
silica. The developed LC method was validated with respect
to precision, accuracy, limits of detection and quantification,
linearity, and robustness. These studies were performed in
accordance with established ICH guidelines.
Enantiomers of racemic drugs often show different behaviors
in pharmacological action and metabolic process. In 1992, the
U.S. Food and Drug Administration issued a policy statement
for the development of new steroisomeric drugs that requires
acceptable manufacturing control of synthesis and impurities,
adequate pharmalogical and toxicological assessment,
proper characterization of metabolism and distribution,
and appropriate clinical evaluation.1
Rasagiline is a highly potent, selective, irreversible, second-
generation monoamine oxidase inhibitor with selectivity for type
B of the monoamine oxidase enzyme (MAO-B) and has been
used for the treatment of idiopathic Parkinson’s disease (PD).
Its chemical designation is (R)-N-(prop-2-ynyl)-2,3-dihydro-1H-
inden-1-amine methane sulfonate (Fig. 1).2 The recomended
dosage for initial monotherapy is 1 mg once daily. When
rasagiline is used as adjunctive therapy with levodopa, the
recommended initial dose is 0.5 mg/day and may be increased
to 1 mg/day if the desired clinical effect is not achieved.3,4
Rasagiline is produced as a single isomer, and (S)-enantiomer
could be present as a chiral impurity. In general, the (R)-isomer
(rasagiline) shows higher binding affinities than the corre-
sponding compound in the S configuration (S-isomer), and this
difference is more pronounced for MAO-B than for MAO-A.5
A literature survey reveals that few LC assay methods
are reported for determination of rasagiline in bulk drug and
pharmaceutical preparation.6,7 The assay method by HPLC8
describes the separation of degradation impurities from
rasagiline formed during forced degradation studies, but it
was out of our scope because it did not separate and determine
the enantiomeric impurities. Estimation of rasagiline in human
plasma by liquid chromatography-tandem mass spectrometry
has been performed.9,10 However, an extensive literature survey
revealed that no LC method has been reported, including in
major pharmacopoeias such as USP, EP and BP, for the
© 2013 Wiley Periodicals, Inc.
EXPERIMENTAL
Chemicals and Reagents
Rasagiline mesylate and its (S)-enantiomer (Fig. 1) were supplied by
Dr. Reddy’s Laboratories Ltd., India. HPLC-grade n-hexane and isopropyl
alcohol were purchased from Rankem, India. Ethanol and diethyl amine
were purchased from Merck, India.
Chromatographic Conditions
Samples were analyzed on a Waters Alliance 2695 separation
module (Waters Corporation, Milford, MA, USA) equipped with a
2489 UV/visible detector. The method was developed using
a
Chiralcel-OJ-H (250 mm  4.6 mm, 5 mm) column with mobile phase
consisting of n-hexane:isopropyl alcohol:ethanol:diethyl amine
(96:2:2:0.01). The flow rate of the mobile phase was 1.0 ml/min.
*Correspondence to: P. Sunil Reddy, Analytical R&D, Dr. Reddy’s Laboratories
Ltd., IPDO, Bachupally, Hyderabad-500072, A.P, India; E-mail: sunilpsr@yahoo.com
Received for publication 26 June 2012; Accepted 30 November 2012
DOI: 10.1002/chir.22150
Published online 9 May 2013 in Wiley Online Library
(wileyonlinelibrary.com).

NORMAL PHASE LC METHOD FOR ENANTIOMERIC SEPARATION OF RASAGILINE MESYLATE AND ITS (S)-ENANTIOMER
325
Linearity
Linearity test solutions were prepared by diluting the stock
solutions to the required concentrations. The solutions were
prepared at seven concentration levels from LOQ to 150% of
specification level (i.e., 1.05, 1.33, 2.63, 3.96, 5.25, 6.58, and
7.88 mg/ml). A calibration curve was plotted between the
responses of peak versus analyte concentration. The coefficient
correlation, slope and y-intercept of the calibration curve and
% bias are reported.
Fig. 1. Structures of (a) rasagiline and (b) its (S)-enantiomer.
The column temperature was maintained at 27 ꢀC and the detection
wavelength was 215 nm. The sample injection volume was 10 ml.
Accuracy
The accuracy of the method for (S)-enantiomer was evaluated
in triplicate, using four concentration levels from LOQ to 150%
(i.e., 1.05, 2.63, 5.25, and 7.88 mg/ml). The percentage recovery
of (S)-enantiomer was calculated at each level.
Preparation of System Suitability Solution
Isopropyl alcohol was used as the diluent. A system suitability solution
of rasagiline mesylate and its (S)-enantiomer was prepared by dissolving
1.0 mg of each substance in 2.5 ml of methanol and then diluting to 10 ml
to obtain a final concentration of 0.1 mg/ml.
Robustness
Preparation of Standard Solution
To determine the robustness of the developed method,
experimental conditions were deliberately altered and the
resolution between rasagiline and its (S)-enantiomer and
system suitability parameters for the rasagiline standard were
recorded. The variables evaluated in the study were column
temperature (+5 ꢀC), flow rate (+0.2 ml/min), and composition
of organic solvents (isopropyl alcohol and ethanol) in the
mobile phase (+10%).
A stock solution of rasagiline mesylate was prepared by dissolving 35.0
mg of the drug in 2.5 ml of methanol and then diluting to 10 ml (3.5 mg/ml).
A working standard solution was prepared by diluting the above stock
solution with diluent to obtain a final concentration of 3.5 mg/ml.
Preparation of Sample Solution
To prepare a sample solution, 35.0 mg of rasagiline mesylate was
dissolved in 2.5 ml of methanol with sonication and then diluting to 10 ml.
Solution Stability and Mobile Phase Stability
The solution stability of rasagiline mesylate spiked with its
(S)-enantiomer was determined by leaving the test solution
and standard solutions in tightly capped volumetric flasks at
room temperature for 48 h and then by measuring the
amount of rasagiline (S)-enantiomer at every 24-h interval
against a freshly prepared standard solution. The stability of
mobile phase was also determined by freshly prepared test
solutions of rasagiline mesylate spiked with its (S)-enantiomer
at 24-h intervals for 48 h. The mobile phase was not changed
during the study.
METHOD VALIDATION
The proposed method was validated as per ICH guide-
lines.11 The following validation characteristics were
addressed: accuracy, precision, limit of detection and quanti-
fication, linearity, range, and robustness.
System Suitability
System suitability was determined before sample analysis
from six replicate injections of the standard solution
containing 3.5 mg/ml of rasagiline mesylate. The acceptance
criteria were: less than 5% relative standard deviation (RSD)
for peak areas, the USP tailing factor less than 2.0 for the
rasagiline peak from standard solution and from system suit-
ability solution, and a minimum resolution of 2.5 between
rasagiline and its (S)-enantiomer.
RESULTS AND DISCUSSION
Method Development
The main aim of the chromatographic method development
was to achieve the separation of rasagiline and its (S)-enantiomer
and accurate quantification of the (S)-enantiomer. A racemic
mixture solution of rasagiline and (S)-enantiomer (0.1 mg/ml
each) was prepared in methanol and isopropyl alcohol. To
develop a robust and suitable HPLC method for the separation
of the two enantiomers, different stationary phases and mobile
phases were employed. The racemic mixture solution was
subjected to separation by normal-phase LC on the Chiralpak
IA (250 mm  4.6 mm, 5-mm column) with n-hexane and
isopropyl alcohol in a 96:4 ratio as a mobile phase. Broad peak
shapes were observed with resolution less than 1.0. To separate
both the components, the composition of the mobile phase was
changed to n-hexane, isopropyl alcohol, and ethanol in the ratio
of 96:2:2 and the Chiralcel-OJ-H (250 mm  4.6 mm, 5 mm)
column was selected for separation. Resolution was observed
between the enantiomers but peak tailing was also observed.
With the addition of 0.01% diethyl amine to mobile phase,
rasagiline and its (S)-enantiomer were eluted with superior
resolution and symmetrical peak shape. The final optimized
conditions are described as follows.
Precision
The precision of the method was verified by repeatability and
intermediate-precision studies. Repeatability was checked by
injecting six individual preparations of rasagiline mesylate
spiked with (S)-enantiomer at the 0.15% level. The %RSD of
area for the (S)-enantiomer was calculated. The intermediate
precision of the method was also evaluated using a different
analyst and a different instrument and performing the analysis
on different days.
Limits of Detection (LOD) and Quantification (LOQ)
The LOD and LOQ for the (S)-enantiomer were determined
at a signal-to-noise ratio of 3:1 and 10:1, respectively, by
injecting a series of dilute solutions with known concentrations.
A precision study was also carried out at the LOQ level by
injecting six individual preparations of (S)-enantiomer and
calculating the %RSD of the area.
Chirality DOI 10.1002/chir

326
SUNIL REDDY ET AL.
TABLE 1. System suitability test results
The separation was achieved using a Chiralcel-OJ-H
(250 mm  4.6 mm, 5 mm) column with mobile phase
consisting of n-hexane:isopropyl alcohol:ethanol:diethyl
amine (96:2:2:0.01). The flow rate of the mobile phase was
1.0 ml/min. The column temperature was maintained at
27 ꢀC and the detection wavelength was 215 nm. The sample
injection volume was 10 ml.
Observed values
Parameters
Specification
precision
intermediate precision
Resolutiona
% RSD
> 2.5
< 5.0%
< 2.0
5.8
1.7
1.3
5.4
1.4
1.3
USP Tailing
METHOD VALIDATION
aResolution between rasagiline and its (S)-enanatiomer.
System Suitability
System suitability shall be checked for the conformance of
the suitability and reproducibility of the chromatographic
system for analysis. The system suitability was evaluated on
the %RSD of the peak area and USP tailing factor for rasagiline
peak from standard solution; and resolution between rasagiline
and its (S)-enantiomer from system suitability solution (Fig. 2a).
All critical parameters tested met the acceptance criteria
(Table 1).
Limits of Detection (LOD) and Quantification (LOQ)
The limit of detection and limit of quantification for
(S)-enantiomer were found to be 0.35 and 1.05 mg/ml.
The %RSD value of precision at LOQ level was 2.2.
Accuracy
The percentage recovery of (S)-enantiomer in rasagiline
samples varied from 98.3 to 101.9%. The LC chromatogram
of rasagiline sample spiked with (S)-enantiomer at 0.15% level
is shown in Fig. 2b. The recovery values for (S)-enantiomer
are presented in Table 2.
Precision
The %RSD for the area of the (S)-enantiomer in the repeat-
ability study was within 0.7% and in the intermediate precision
study was within 1.3%, which confirms the good precision of
the method.
Linearity
The linearity calibration plot for the (S)-enantiomer
was obtained over the calibration ranges tested, i.e., LOQ
to 150% of specification level (Fig. 2c). The correlation coeffi-
cient obtained was greater than 0.999. The equation of the
calibration curve for the (S)-enantiomer was y = 16,848.4x –
1594.1. The % bias was found to be 1.8%. The above result
shows that an excellent correlation existed between peak
area and concentration of the (S)-enantiomer over a range
of 1.05 to 7.88 mg/ml.
Robustness
In all the deliberate varied chromatographic conditions
(flow rate, column temperature, and composition of organic
solvent), both analytes were adequately resolved and the
elution order remained unchanged. The resolution between
rasagiline and its (S)-enantiomer was greater than 2.5, the %
RSD for rasagiline peak from the standard solution was less
than 3.8, and the tailing factor was less than 1.4. The results
are reported in Table 3.
Solution Stability and Mobile Phase Stability
The variability in the estimation of (S)-enantiomer was
within +10% during solution stability. The results from the
solution stability and mobile phase stability experiments
confirmed that mobile phase, sample solution, and standard
solutions were stable up to 48 h.
TABLE 2. Recovery results for (S)-enantiomers (n = 3)
Spiked
levels
Added
(mg/ml)
Recovered
(mg/ml)
Recovery
(%)
RSD
(%)
Fig. 2. Typical chromatograms for the resolution of (a) a racemic mixture
of rasagiline and its (S)-enantiomer; (b) rasagiline spiked with its (S)-enantio-
mer at 0.15% level; and (c) a racemic mixture of rasagiline and its (S)-enantio-
mer at 0.15% concentration of the racemic solution. Chromatographic
conditions: Chiralcel-OJ-H (250 mm  4.6 mm, 5 mm) column at 27 ꢀC; mobile
phase: n-hexane:isopropyl alcohol:ethanol:diethyl amine (96:2:2:0.01); flow
rate, 1.0 ml/min; wavelength, 215 nm.
LOQ
50%
100%
150%
1.05
2.63
5.25
7.88
1.07
2.60
5.16
7.93
101.9
98.9
98.3
4.3
1.9
0.5
0.8
100.6
Chirality DOI 10.1002/chir

NORMAL PHASE LC METHOD FOR ENANTIOMERIC SEPARATION OF RASAGILINE MESYLATE AND ITS (S)-ENANTIOMER
TABLE 3. Robustness results of HPLC method
327
Observed system suitability parameters
Variation in chromatographic condition
resolutiona > 2.5
USP tailing < 2.0
% RSD < 5.0 (n = 6)
Column temperature 22 ꢀC
5.3
5.6
6.0
5.6
5.9
4.9
4.7
5.0
1.3
1.2
1.3
1.3
1.3
1.4
1.4
1.3
1.8
1.2
2.1
1.9
1.5
2.5
2.3
1.7
Column temperature 32 ꢀC
Flow rate 0.8 ml/min
Flow rate 1.2 ml/min
n-hexane: isopropyl alcohol: ethanol: diethyl amine (960:20:18:0.1 ml)
n-hexane: isopropyl alcohol: ethanol: diethyl amine (960:20:22:0.1 ml)
n-hexane: isopropyl alcohol: ethanol: diethyl amine (960:18:20:0.1 ml)
n-hexane: isopropyl alcohol: ethanol: diethyl amine (960:22:20:0.1 mL)
aResolution between rasagiline and its (S)-enanatiomer. Actual chromatographic conditions: Chiralcel-OJ-H (250 mm  4.6 mm, 5 mm) column at 27 ꢀC; mobile
phase: n-hexane:isopropyl alcohol:ethanol:diethyl amine (96:2:2:0.1 ml); 1.0 ml/min of flow rate; wavelength of 215 nm.
3. Finberg JP, Lamensdrof I, Commissiong J W, Youdim MB. Pharmacology
and neuroprotective properties of rasagiline. J Neural Transm Suppl
CONCLUSIONS
A simple, accurate, and efficient normal-phase chiral
HPLC method was developed and validated for quantitative
analysis of the (S)-enantiomer from rasagiline. A cellulose-
based chiral column, the Chiralcel OJ-H column, was
found to be selective for rasagiline and its (S)-enantiomer.
Satisfactory results were obtained from method validation
and the method found to be precise, accurate, linear, and
robust during validation. The developed method can be
conveniently used by quality control departments for the
quantitative determination of enantiomeric impurity in
rasagiline as a bulk drug.
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Parkinson’s disease. Clin Interv Aging 2010;5:149–156.
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A. Binding of rasagiline-related inhibitors to human monoamine oxidases:
a kinetic and crystallographic analysis. J Med Chem 2005;48:8148–8154.
6. Lakshmi MV, Rao JVLNS, Rao AL. Development and validation of RP-
HPLC method for the estimation of rasagiline tablet dosage forms.
Rasayan J Chem 2010;3:621–624.
7. Jayavarapu KR, Murugeasn J, Mantada PK. Validated RP-HPLC method
for the estimation of rasagiline in pure and tablet dosage form. J Pharm
Res 2011;4:1376–1377.
8. Kumar RN, Rao GN, Naidu PY. Stability-indicating RP-HPLC method for
determination of rasagiline mesylate in bulk and pharmaceutical dosage
forms. Int J Appl Bio Pharm Technol 2010;1:247–259.
9. Ma J, Chen X, Duan X, Deng P, Wang H, Zhong D. Validated LC-MS/MS
method for quantitative determination of rasagiline in human plasma and its
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sensitive liquid chromatography-tandem mass spectrometry: assay devel-
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ACKNOWLEDGMENT
The authors are thankful to the management of Dr. Reddy’s
Laboratories Ltd., Hyderabad for providing facilities to carry
out this work.
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Chirality DOI 10.1002/chir