Development and validation of a reversed-phase HPLC method for determination of lesatropane and enantiomeric impurity

Article abstract of DOI:10.1002/chir.20971

Lesatropane is a novel muscarinic receptor agonist and is currently being under preclinical development in China as a single enantiomer drug for the treatment of primary glaucoma. A reversed-phase chiral HPLC method for determination of lesatropane and enantiomeric impurity was developed. Enantiomeric separation of lesatropane from its enantiomer (desatropane) was achieved in normal-phase mode with Chiralpak AD-H and in reversed-phase mode with Chiralpak AS-RH. The conditions using a Chiralpak AS-RH column and mobile phase of K₂HPO₄-KH₂PO₄ (pH 7.0; 0.02 M)-acetonitrile (69:31, v/v) at a flow rate of 0.5 ml/min have been fully validated with satisfactory specificity, linearity, accuracy, and precision. The method was found to be suitable for the simultaneous quantitation of lesatropane and enantiomeric impurity desatropane. Copyright

Full text of DOI:10.1002/chir.20971

CHIRALITY 23:581–584 (2011)
Development and Validation of a Reversed-Phase HPLC Method for
Determination of Lesatropane and Enantiomeric Impurity
1
LI-MIN YANG,1 YI-FAN XIE,1 ZHOU-HUI GU,2 AI-LING WANG,2 HONG-ZHUAN CHEN,1 AND YANG LU
*
1Department of Pharmacy, Shanghai Jiao Tong University School of Medicine, Shanghai, China
2Shanghai PharmValley Corp, Shanghai, China
ABSTRACT
Lesatropane is a novel muscarinic receptor agonist and is currently being
under preclinical development in China as a single enantiomer drug for the treatment of pri-
mary glaucoma. A reversed-phase chiral HPLC method for determination of lesatropane and
enantiomeric impurity was developed. Enantiomeric separation of lesatropane from its enan-
tiomer (desatropane) was achieved in normal-phase mode with Chiralpak AD-H and in
reversed-phase mode with Chiralpak AS-RH. The conditions using a Chiralpak AS-RH column
and mobile phase of K2HPO4–KH2PO4 (pH 7.0; 0.02 M)–acetonitrile (69:31, v/v) at a flow rate
of 0.5 ml/min have been fully validated with satisfactory specificity, linearity, accuracy, and pre-
cision. The method was found to be suitable for the simultaneous quantitation of lesatropane
C
VV
and enantiomeric impurity desatropane. Chirality 23:581–584, 2011.
2011 Wiley-Liss, Inc.
KEY WORDS: enantiomeric separation; lesatropane; amylose-based chiral stationary phase;
muscarinic agonist
INTRODUCTION
dated for determining the linearity, limit of detection (LOD)
and limit of quantification (LOQ), precision, and accuracy.
The enantiomers of a racemic drug often differ in pharma-
cokinetic behavior or pharmacological action.1 To assure
clinical safety and efficacy, the pharmacological evaluation of
both enantiomers is an integral part of new drug develop-
ment.2 The preparation of a single enantiomer drug and con-
trolling its enantiomeric impurity are important not only to
avoid unwanted pharmaceutical or toxicological side effects
but also to assure its therapeutic efficacy.
Satropane, 3a-paramethylbenzenesulfonyloxy-6b-acetoxy-
tropane, could be resolved in 3S,6S-isomer named lesatro-
pane and 3R,6R-isomer named desatropane (Fig. 1). Interest-
ingly, although many tropane derivatives (atropine, scopola-
mine, etc.) serve as muscarinic antagonist, satropane and its
analogs were muscarinic agonist. The analogs of satropane
have been synthesized and resolved in our laboratory and
the pharmacological characteristics of the racemics and each
enantiomers of them as well as their structure–activity rela-
tionship have been studied systematically.3,4 The results
showed that the eliciting agonistic activity of 3S,6S-isomers
is more potent than that of both 3R,6R-isomers and racemic
ones on muscarinic receptors and in suppressing hyperten-
sive intraocular pressure.5,6 Currently, lesatropane as a novel
muscarinic agonist is being under preclinical development in
China as a single enantiomer drug for the treatment of pri-
mary glaucoma.
As the obvious impurity in lesatropane is its enantiomer
desatropane, the technique that can quickly assess the enan-
tiomeric purity of lesatropane as bulk drug and its prepara-
tion is necessary during its development and manufacturing
processes.
In this article, we report the development and validation of
the methods for separating lesatropane from desatropane,
using normal- and reversed-phase high-performance liquid
chromatography (HPLC) modes with amylose-based chiral
stationary phase (CSP). Lesatropane and desatropane could
be resolved on both Chiralpak AD-H and Chiralpak AS-RH.
The reversed-phase HPLC with Chiralpak AS-RH was vali-
MATERIALS AND METHODS
Chemicals and Materials
Lesatropane (>99.5%), desatropane (>99.0%), and satropane were pre-
pared by Institute of Drug Research, Shanghai Jiao Tong University
School of Medicine. Three batches of lesatropane hydrochloride bulk
products (080501, 080701, and 080801) were provided by Shanghai
PharmValley Corp. HPLC grade n-hexane, isopropanol, and phosphoric
acid were purchased from Dikma (Beijing, China), and acetonitrile from
Sigma-Aldrich (St. Louis). Diethylamine (DEA) and potassium hydrogen
phosphate were obtained from Shanghai Reagent (Shanghai, China).
Deionized water used throughout the study was taken from an Ultra
Water system (Sartorius).
Equipment and Chromatographic Conditions
Chromatographic studies were performed on Agilent 1100 HPLCs
(Agilent, Palo Alto, CA), equipped with an autosampler, thermostat-col-
umn device, a variable-wavelength UV detector and a data acquisition
system using the HP Chemstation software (Rev A. 10.02).
For normal-phase HPLC, separation of lesatropane and enantiomeric
impurity was carried out on Chiralpak AD-H column (250 mm 3 4.6 mm
i.d., 5 lm; Daicel Chemical Industries, Tokyo, Japan) using a mobile
phase (n-hexane:isopropanol:DEA, 80:20:0.1, v/v/v) at a flow rate of 0.7
ml/min. For reversed-phase mode, the enantiomeric separation was
achieved on Chiralpak AS-RH column (150 mm 3 4.6 mm i.d., 5 lm, Dai-
cel Chemical Industries) using a mobile phase consisting of potassium
Contract grant sponsor: National Natural Science Founding of China;
Contract grant numbers: 30672441 and 30873057; Contract grant sponsor:
Key Basic Project of Shanghai Municipal Science and Technology Commis-
sion; Contract grant number: 08JC1413600; Contract grant sponsor:
National Comprehensive Technology Platforms for Innovative Drug R&D;
Contract grant number: 2009ZX09301-007
*Correspondence to: Yang Lu, Shanghai Jiao Tong University School of Med-
icine, South Chongqing Road 280, Shanghai 200025, China.
E-mail: huaxue@shsmu.edu.cn
Li-Min Yang and Yi-Fan Xie contributed equally to this article.
Received for publication 14 October 2010; Accepted 26 April 2011
DOI: 10.1002/chir.20971
Published online 11 July 2011 in Wiley Online Library
(wileyonlinelibrary.com).
C
VV
2011 Wiley-Liss, Inc.

582
YANG ET AL.
Fig. 1. Structure of the enantiomers of satropane.
Fig. 2. Chromatogram of satropane on Chiralpak AD-H column, n-hex-
ane–isopropanol–DEA (80:20:0.1, v/v/v), 0.7 ml/min, 258C; UV detection at
227 nm. [Color figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
hydrogen phosphate buffer (pH 7.0; 0.02 M)–CH3CN (69:31, v/v) at a
flow rate of 0.5 ml/min. The sample injection volume was 10 ll. The
detection was carried out at 227 nm.
Fig. 4. The separation of lesatropane and desatropane on Chiralpak AS-
RH column, K2HPO4–KH2PO4 (pH 7.0; 0.02 M)–acetonitrile (69:31, v/v), 0.5
ml/min, 258C; UV detection at 227 nm. The concentrations of the reference
substance solutions are 0.2 mg/ml for lesatropane and desatropane, 1.0 mg/
ml for satropane. [Color figure can be viewed in the online issue, which is
available at wileyonlinelibrary.com.]
Method Validation
The linearity of the detector response for lesatropane and enantio-
meric impurity desatropane was assessed by preparing six calibration
standards solutions of lesatropane at 10–1000 lg/ml (0.05–5.0 times of
analyte concentration of 200 lg/ml) and desatropane at 0.5–10 lg/ml
(0.0025–0.05 times of the analyte concentration) in acetonitrile, respec-
tively. Three replicates were prepared per concentration level. Linear
regression curves were obtained by plotting peak area versus concentra-
tion, using the least squares method.
Method precision was determined by measuring the repeatability and
intermediate precision of peak areas for lesatropane and enantiomeric
impurity (desatropane). Repeatability was evaluated by analyzing lesatro-
pane bulk drug sample (200 lg/ml) six times. Intermediate precision
was evaluated by analyzing same sample using different instruments and
analysts on different days. The accuracy was evaluated by the standard
addition and recovery experiments. Known amounts of lesatropane were
added at 80%, 100%, and 120%( w/w) and desatropane at 2.5%, 4%, and 5%
(w/w) in lesatropane bulk drug (080501), relative to sample concentra-
tion (200 lg/ml). Each preparation was analyzed in triplicate and per-
cent recovery was calculated for lesatropane and desatropane. LOD was
established at a signal-to-noise ratio of 3 and LOQ at a signal-to-noise ra-
tio of 10. Standard solution stability at analyte concentration was studied
(stored at room temperature) over a period of 24 h with analysis at 0, 6,
12, and 24 h.
The sample for lesatropane analysis was prepared by dissolving an
accurately weighed amount of ꢀ10.00 mg of each batch of bulk lesatro-
TABLE 1. Chromatographic parameters for lesatropane and
desatropane on Chiralpak AS-RH
Analytes
t (min)
k0
N
T
a
Rs
Lesatropane
Desatropane
14.5
17.5
3.04
3.68
3316
3443
1.48
1.43
1.21
2.16
Fig. 3. Chromatograms concerning the resolution of satropane on amy-
lose-based CSPs, K2HPO4–KH2PO4 (pH 8.0) buffer–acetonitrile (70:30, v/v),
0.5 ml/min, 258C; UV detection at 227 nm. [Color figure can be viewed in the
online issue, which is available at wileyonlinelibrary.com.]
t, retention time; k0, capacity factor; N, efficiency; T, tailing; a, selectivity; Rs,
resolution.
Chirality DOI 10.1002/chir

RP-HPLC METHOD FOR DETERMINATION OF LESATROPANE
583
TABLE 2. Linearity parameters and values of lesatropane and desatropane
Linearity range
Linearity equationa
Correlation
LOD
(ng/ml)
LOQ
(ng/ml)
Analytes
(lg/ml)
(n 5 6)
coefficient (r2)
Lesatropane
Desatropane
10–1000
0.5–10
y 5 37,536x 1 22.24
y 5 37,744x 1 0.44
0.9998
0.9998
1.1
1.3
3.3
2.5
y, the enantiomer’s peak area; x, the enantiomer’s concentration.
pane in 50 ml of acetonitrile to obtain 0.2 mg/ml solution. 1 mg/ml sam-
ple solution for desatropane analysis was prepared by dissolving an accu-
rately weighed amount of 10.00 mg of each batch of bulk lesatropane in
10 ml of acetonitrile.
hence, the resolution of enantiomers occurred. Cellulose
with a linear structure contains b-glycosidic linkage, whereas
amylose with a helical structure contains a-glycosidic. Com-
paring the resolution capacity, amylose was better than cellu-
lose, which could be attributed to the fact that the amylose
CSPs are more helical in nature and possess well-defined
grooves.9–11 Carboxylate and sulfonate groups of satropane
effectively interact with the carbamate groups on the chiral
helical groove of amylose-based CSPs via hydrogen bonding.
It is the ‘‘ability’’ of one enantiomer (with respect to the
other) to reach a better steric fit in the chiral helical groove
that induces the separation. In contrast to the normal-phase
mode, the amylose tris[(S)-a-methylbenzylcarbamate] phase
(Chiralpak AS-RH) performed better than the amylose
tris(3,5-dimethylphenylcarbamate) phase (Chiralpak AD-RH,
IA) in enantiomeric separating satropane under reversed-
phase conditions, suggesting that the nature of the substitu-
ents on the phenyl groups affects the polarity of the carba-
mate residues and leads to the different chiral discrimina-
tions under normal- and reversed-phase modes. Besides the
polar interactions, p–p interactions between the aromatic
group of a CSP and those of the solute may play some role in
chiral recognition, particularly in reversed-phase HPLC.12
(S)-a-Methylbenzylcarbamate (AS) may have strong p–p
interactions with a chiral compound than 3,5-dimethylphenyl-
carbamate (AD).
RESULTS AND DISCUSSION
Enantiomeric Separation in Normal-Phase Mode
On the basis of our experience for enantiomeric separation
of racemic anisodamine,7 Chiralpak AD column consisting of
silica gel coated with amylose tris(3,5-dimethylphenyl carba-
mate) was selected at first in normal-phase mode to resolve
satropane and the enantiomeric separation was achieved
using the mobile phase system n-hexane–isopropanol–DEA
(Fig. 2). DEA added to the mobile phase could reduce the
peak tailing by masking the residual silanol groups of the
CSP.8 Working with mobile phases of n-hexane–isopropanol,
that is without DEA, led to only partial resolution or none at
all. The satisfactory separation was achieved when DEA con-
tent ranging from 0.05% to 0.4% (v/v) in the mobile phase.
The retention time of lesatropane and desatropane was 9.77
and 11.36 min, respectively, using a mobile phase (n-hexa-
ne:isopropanol:DEA, 80:20:0.1, v/v/v). The capacity factor
k’_1 of lesatropane, separation factor a, and resolution Rs
were 1.24, 1.29, and 3.48, respectively.
Enantiomeric Separation in Reversed-Phase Mode
We tried to direct our experiments aiming the develop-
ment of a method that could be easily used not only for the
quantitation of lesatropane and enantiomeric impurity in the
bulk drug but also further in its preparation (eye drops). Nor-
mal-phase mode is not applicable to analyze directly aqueous
samples. So the reversed-phase HPLC method was devel-
oped and validated.
With respect to normal-phase HPLC, the reversed-phase
mode is particularly advantageous in the pharmaceutical
analysis and quality control for lesatropane preparations in
saline-containing drops. To get optimum resolution for lesa-
tropane and desatropane in reversed-phase mode, diverse
polysaccharide-based CSPs were conducted. No separation
was observed on cellulose-based CSPs (Chiralcel OD-RH
and OJ-RH) using diverse mobile phases. However, the enan-
tiomeric separation of satropane was observed on amylose-
based CSPs (Chiralpak AD-RH, AS-RH and an immobilized
type IA CSP) using the mobile phase K2HPO4–KH2PO4
buffer (pH 8.0)–acetonitrile (Fig. 3, tested by Daicel Chemi-
cal Industries), and a good resolution (Rs 5 2.16) was
obtained using Chiralpak AS-RH. As lesatropane is unstable
in a solution pH ꢁ 8, the water or phosphate buffer (pH 6.5–
7.5) as aqueous part of the mobile phase was further studied
on Chiralpak AS-RH. The enantiomeric separation was poor
using the mobile phase consisted of water and acetonitrile,
and the optimal separation was obtained using the mobile
phase system K2HPO4–KH2PO4 (pH 7.0 6 0.1)–acetonitrile
(69:31, v/v) with good resolution and shorter analysis time
(Fig. 4 and Table 1).
Method Validation
The conditions of reversed-phase mode HPLC method for
the determination of lesatropane and desatropane have been
fully validated. Specificity has been demonstrated and is
shown in Figure 4. Good linearity was obtained for lesatro-
pane in 10–1000 lg/ml and for desatropane 0.5–10 lg/ml.
LOD and LOQ were determined to be 1.1 and 3.3 ng/ml for
lesatropane, 1.3 and 2.5 ng/ml for desatropane, respectively.
Linearity parameters can be found in Table 2. The linear
regression model was evaluated using a lack-of-fit test. Statis-
tical analyses were performed with SAS version 9.2 (SAS
TABLE 3. Recovery values for accuracy assays of lesatropane
and desatropane
These results suggest that the polysaccharide linkages
and three-dimensional structure may play a crucial role in
the enantiomeric separations of satropane. During chiral re-
solution, the enantiomers fit stereogenically in different fash-
ions into the chiral grooves of the CSP which is stabilized by
various types of bondings of different magnitudes and,
Concentration
added (lg/ml)
Analyte
Recovery (%)
RSD%
Lesatropane
Desatropane
158, 199, 245
4.05, 5.06, 6.07
99.32, 99.31, 99.47
98.98, 98.95, 96.16
1.11
0.96
Chirality DOI 10.1002/chir

584
YANG ET AL.
TABLE 4. The contents of lesatropane and desatropane in
three batches of lesatropane products
phase HPLC method with Chiralpak AS-RH was fully vali-
dated with satisfactory results obtained for specificity, linear-
ity, accuracy, precision, LOD, and LOQ. Thus, as the method
was convenient, it could be used in the quality control and
purity testing of lesatropane in bulk drug.
Sample
080501
080701
080801
Lesatropane (%, w/w)
Desatropane (%, w/w)
99.45
0.05
99.51
0.16
99.05
0.45
LITERATURE CITED
1. Sahajwalla CG. New drug development 141. New York: Marcel Dekker
Inc.; 2004. p 421–426.
Institute, Cary, NC). F-test for lack-of-fit: F 5 2.69 (lesatro-
pane), F 5 2.36 (desatropane), P > 0.05. The results
revealed that the regression model fits the calibration data
well. Method precision has a relative standard deviation
(RSD%) for area responses below 2.0% for repeatability (lesa-
tropane 0.29% and desatropane 1.20%) and for intermediate of
precision (lesatropane 0.17% and desatropane 0.89%). The ac-
curacy was evaluated using the standard addition and recov-
ery experiments for lesatropane and desatropane. Known
amounts of the analyte were added in bulk samples in tripli-
cate at 80%, 100%, and 120% of analyte concentration. Accepta-
ble recoveries are presented in Table 3. No significant
change in the peak area was observed during solution stabil-
ity experiments. Hence, sample solution of the analyte was
stable after preparation for at least 24 h.
2. De Camp WH. Chiral drugs: the FDA perspective on manufacturing and
control. J Pharm Biomed Anal 1993;11:1167–1172.
3. Pei XF, Gupta TH, Badio B, Padgett WL, Daly JW. 6b-Acetoxynortropane:
a potent muscarinic agonist with apparent selectivity toward M2-recep-
tors. J Med Chem 1998;41:2047–2055.
4. Niu YY, Yang LM, Liu HZ, Cui YY, Zhu L, Feng JM, Yao JH, Chen HZ,
Fan BT, Chen ZN, Lu Y. Activity and QSAR study of Baogongteng A and
its derivatives as muscarinic agonists. Bioorg Med Chem Lett. 2005;
15:4814–4818.
5. Yang LM, Zhu L, Niu YY, Chen HZ, Lu Y. (1R,3S,5R,6S)-6-Acetoxy-3-(4-
methylphenylsulfonyloxy)tropane. Acta Crystallogr E 2008;64:o2331.
6. Zhu L, Yang LM, Cui YY, Zheng PL, Niu YY, Wang H, Lu Y, Ren QS,
Wei PJ, Chen HZ. Stereoselectivity of satropane, a novel tropane analog,
on iris muscarinic receptor activation and intraocular hypotension. Acta
Pharmacol Sin 2008;29:177–184.
7. Yang LM, Xie YF, Chen HZ, Lu Y. Diastereomeric and enantiomeric
high-performance liquid chromatographic separation of synthetic anisod-
amine. J Pharm Biomed Anal 2007;43:905–909.
Sample Analysis
8. Ahuja S, Dong MW. Handbook of pharmaceutical analysis by
HPLC. Amsterdam: Elsevier Inc.; 2005. p 481.
The method afore-mentioned is also suitable for the quanti-
tative determination of lesatropane and its enantiomeric im-
purity in the bulk drug, which was applied for the quantita-
tion in samples of three batches of bulk lesatropane and the
results are recorded in Table 4. The peaks were identified by
injecting and comparing with the retention times of the indi-
vidual compounds.
9. Andersson ME, Aslan D, Clarke A, Roeraade J, Hagman G. Evaluation of
generic chiral liquid chromatography screens for pharmaceutical analy-
sis. J Chromatogr A 2003;1005:83–101.
10. Ali I, Kumerer K, Aboul-Enein HY. Mechanistic principles in chiral sepa-
rations using liquid chromatography and capillary electrophoresis. Chro-
matographia 2006;63:295–307.
11. Ali I, Saleem K, Hussain I, Gaitonde VD, Aboul-Enein HY. Polysaccha-
rides chiral stationary phases in liquid chromatography. Sep Purif Rev
2009;38:97–147.
CONCLUSION
The amylose-based CSPs (Chiralpak AD-RH and Chiralpak
AS-RH) were found to be successful in separating lesatro-
pane and its undesired enantiomer. The proposed reversed-
12. Yashima E. Polysaccharide-based chiral stationary phases for high-per-
formance liquid chromatographic enantioseparation. J Chromatogr A
2001;906:105–125.
Chirality DOI 10.1002/chir