Journal of Pharmaceutical and Biomedical Analysis 49 (2009) 55–63 Contents lists available at ScienceDirect Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba Study of forced decomposition behavior of lamivudine using LC, LC–MS/TOF and MS n Gaurav Bedse, Vijay Kumar, Saranjit Singh ∗ Department of Pharmaceutical Analysis, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, SAS Nagar 160062, Punjab, India article info Article history: Received 22 September 2008 Accepted 4 October 2008 Available online 15 October 2008 Keywords: Lamivudine Stress studies Stability-indicating assay method MS n LC–MS/TOF Degradation pathway Mechanism abstract Lamivudine was subjected to forced decomposition conditions of hydrolysis (neutral, acidic and alkaline), oxidation, photolysis and thermal stress, as suggested in the ICH guideline Q1A(R2). The drug showed instability in acid and alkali, while it remained stable in neutral conditions. It also degraded extensively under oxidative environment. It remained stable to light and thermal stress. In total, five degradation products were formed, which could be separated by LC on a C18 column using a gradient method. To characterize the products, first a complete fragmentation pathway of the drug was established by carrying out multi-stage (MS n ) and MS/TOF accurate mass studies. The same was compared to fragment pattern of the degradation products resulting from LC–MS/TOF studies. The accurate mass values obtained from LC–MS/TOF were used to obtain elemental compositions, and the total information helped in identification of the degradation products. Subsequently, degradation pathway of the drug was laid down, along with mechanisms of formation of the degradation products. There is no previous information on these aspects on the drug in the literature. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Lamivudine belongs to the class of dideoxynucleoside reverse transcriptase inhibitors, and is a potent inhibitor of human immun- odeficiency virus (HIV), the causative agent of the acquired immunodeficiency syndrome (AIDS) [1,2]. Intracellularly, lamivu- dine is phosphorylated to an active 5 ′ -triphosphate metabolite. Lamivudine triphosphate inhibits the activity of HIV-1 reverse transcriptase by DNA chain termination after incorporation of the nucleoside analogue into viral DNA [3]. Furthermore, lamivudine also shows activity against hepatitis B virus [4]. Chemically, lamivudine is (2R,5S)-4-amino-1-(2-hydroxy- methyl-1,3-oxathiolan-5-yl)-(1H)-pyrimidin-2-one (Fig. 1). In the literature, many LC and LC–MS methods have been reported for analysis of the drug and its metabolites in biological fluids [5–9]. However, very little is reported on the decomposition behavior of the drug. Kaul et al. carried out stress studies on the drug under acidic, basic, oxidative, thermal and photolytic conditions, and separated the products on a HPTLC plate [10]. But neither the extent of degradation, nor the nature/structures of degradation products, and the degradation pathway were reported. A monograph on the drug was finalized in 2006 by the World Health Organization ∗ Corresponding author. Tel.: +91 172 2214682; fax: +91 172 2214692. E-mail address: ssingh@niper.ac.in (S. Singh). (WHO) for inclusion in the International Pharmacopoeia (Ph. Int.), in which a list of 12 impurities and their structures were provided [11]. However, the listed impurities are not classified into process impurities and degradation products. So the intrinsic degradation profile of drug under prescribed stress conditions [12,13] is still unknown. Hence, an integral aim of the present study was to investigate the complete degradation behavior of the drug. It was done through a systematic investigation involving: (i) forced decomposition of the drug under a variety of stress conditions, (ii) resolution of prod- ucts employing a LC–MS compatible method, (iii) conduct of LC–MS studies to establish fragmentation profiles of the drug and the degradation products, (iv) elucidation of structures of degradation products through comparative study of mass data, and (v) ascer- taining degradation pathway and mechanism of decomposition of the drug based on the total information collected. 2. Experimental 2.1. Drug and reagents Pure lamivudine was obtained as gratis sample from Aurobindo Pharma Ltd. (Hyderabad, India) and it was used without fur- ther purification. Analytical reagent (AR) grade sodium hydroxide (NaOH) was purchased from Ranbaxy Laboratories (SAS Nagar, India), hydrochloric acid (HCl) from LOBA Chemie Pvt. Ltd. 0731-7085/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jpba.2008.10.002