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Identification and characterization of a principal oxidation impurity in clopidogrel drug substance and drug product

https://doi.org/10.1016/j.jpba.2007.12.021Get rights and content

Abstract

The focus of this study is identification, isolation and characterization of a principal oxidation impurity of clopidogrel which ranged from 0.05 to 0.12% using high performance liquid chromatography. This impurity is considered as principal oxidation impurity as it is observed in oxidative degradation (stress) study. Preparative HPLC with Xterra MS C18 ODB column was used to isolate the impurity. The isolated impurity was co-injected with the sample containing impurities and found the retention time match of the spiked impurities. A thorough study was undertaken to characterize this impurity and based on their spectral data (UV, MS, MSn 1H/13C, DEPT and 2D NMR) the structure was characterized as 5-[1-(2-chlorophenyl)-2-methoxy-2-oxoethyl]-6,7-dihydrothieno[3,2-c]pyridin-5-ium with a molecular weight 320 amu.

Introduction

Clopidogrel bisulfate, methyl (+)-(S)-α-(2-chlorophenyl)-6,7-dihydrothieno[3,2-c]pyridine-5(4H)-acetate sulfate (1:1), is a potent oral antiplatelet agent often used in the treatment of coronary artery disease, peripheral vascular disease and cerebrovascular disease. It is marketed by Bristol-Myers Squibb and Sanofi-Aventis under the trade name Plavix which is the world's second highest selling pharmaceutical with sales of US$5.9 billion. The mechanism of action of clopidogrel is irreversible blockade of the adenosine diphosphate (ADP) receptor P2Y12 and is important in platelet aggregation, the cross-linking of platelets by fibrin. The blockade of this receptor inhibits platelet aggregation by blocking activation of the glycoprotein IIb/IIIa pathway. Platelet inhibition can be demonstrated 2 h after a single dose of oral clopidogrel, but the onset of action is slow, so that a loading-dose of 300–600 mg is usually administered [1].

Four impurities of clopidogrel have been already identified and documented in the literature [2], [3], [4], [5], [6] and named as clopidogrel related compound A, positional stereo isomers of clopidogrel named as clopidogrel related compounds B1 and B2 and a chiral isomer of clopidogrel named as clopidogrel related compound C. It has also been established that these positional stereoisomers (B1 and B2) are process impurities and other impurities are formed during the process and also self-degradation. Marketed samples of Plavix and few batches of drug substances were analyzed using reported method [3]. An Ultron ES-OVM L 57, chiral specific column (Shinwa chemical industries, Japan) with dimensions of 150 mm × 4.6 mm i.d. packed with 5.0 μ particle size was employed for separation. Acetonitrile–potassium phosphate buffer (10 mM) (75:25, v/v) was used as mobile phase and flow rate was kept at 0.8 ml/min with the detection at 220 nm for 30 min. Relative retention time (RRT) of the related compounds A, B1, clopidogrel B2 and C were found, respectively, at 0.46, 0.93, 1.0, 1.1 and 2.10. Related compound D was found at about 2.0 min (RRT of 0.30). As the related compound D elutes in the void volume of the system, various buffer composition, pH, gradients were attempted and found unsuccessful. The main problems that occurred were peak shape, peak purity and blank interference due to peroxide. Hence, conventional, cost effective, new method was developed wherein good peak shape and peak purity were achieved. The new method involves Hypersil BDS C8 column (Thermo Electron Corporation) with gradient conditions for the separations.

During the analysis, it has been observed that the new impurity content in clopidogrel tablets, were in the range of 0.05–0.07% (by area percentage) and in drug substance it ranges from 0.08 to 0.12% (by area percentage). Typical chromatograms of clopidogrel drug substance, drug product and drug product spiked with the related compound D were shown in Fig. 1. It is mandatory requirement from regulatory authorities, to identify and characterize any unknown impurity present in it at a level as low as 0.05% [7], [8]. The presence of this impurity in tablets can have a significant impact on quality and safety of the important drug. The isolation of any impurity is required to find the response in an analytical method and also to validate the analytical procedure for its quantitative estimation. Even though the same impurity is formed during oxidation condition, there is no report on the isolation and characterization. Hence comprehensive study was undertaken to identify and characterize the oxidative impurity. The new oxidation impurity in this paper referred as related compound D. The chemical structures of clopidogrel, and related compound D were shown in Fig. 2.

Section snippets

Materials and methods

Clopidogrel bisulphate was purchased from Dr. Reddys Laboratories Ltd., Hyderabad, India and clopidogrel tablets of brand name Plavix manufactured by Sanofi Pharma Bristol Myers Squibb Inc. were used. Potassium phosphate and ammonium acetate, GR grade was obtained from E. Merck, India. Methanol, acetonitrile of HPLC grade were obtained from E. Merck, India. Purified water was collected through Milli-Q water purification system (Millipore, USA). Dimethylsulphoxide-d6 (DMSO-d6) was purchased from

Results and discussion

In forced oxidative degradation study 2 and 7% of related compound D was found in the drug substance and drug product (Plavix), respectively. Hence the experiment was used to enrich the impurity. It was also confirmed that related compounds A and C are the degradation products and related compounds B1 and B2 are process impurities based on their trend. The higher level of related compound D in the drug product reveals that the susceptibility is more in the drug product than that in drug

Conclusions

The major oxidative degradation product related compound D in clopidogrel drug substance as well as drug product was isolated by preparative LC and was characterized by using spectroscopic techniques namely NMR, MS and MSn.

Acknowledgements

The authors wish to thank the management of Torrent Pharmaceuticals Ltd. and Torrent Research Center for allowing us to carryout the present work. The authors are also grateful to the Director Dr. C. Dutt of Torrent Research Center for his constant encouragement. The authors also wish to thank friends and the other colleagues of Torrent Research Center for their cooperation.

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