Journal of Pharmaceutical and Biomedical Analysis 53 (2010) 833–842 Contents lists available at ScienceDirect Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba LC–MS/MS studies of ritonavir and its forced degradation products R. Nageswara Rao ∗ , B. Ramachandra, R. Mastan Vali, S. Satyanarayana Raju Analytical Chemistry Division, Discovery Laboratory, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500607, India article info Article history: Received 20 January 2010 Received in revised form 7 June 2010 Accepted 8 June 2010 Available online 16 June 2010 Keywords: Antiretroviral Ritonavir Forced degradation LC–APCI–MS/MS Fragment ions abstract Forced degradation of ritonavir (RTV), under the conditions of hydrolysis (acidic, basic and neutral), oxidation, photolysis and thermal stress as prescribed by ICH was studied using LC–MS/MS. Eight degra- dation products were formed and their separation was accomplished on Waters XTerra ® C 18 column (250 mm × 4.6 mm i.d., 5 m) using water:methanol:acetonitrile as (40:20:40, v/v/v) mobile phase in an isocratic elution mode by LC. The method was extended to LC–MS/MS for characterization of the degra- dation products and the pathways of decomposition were proposed. No previous reports were found in the literature regarding the characterization of degradation products of ritonavir. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Protease inhibitors are responsible for marked reductions in morbidity and mortality in patients with advanced human immun- odeficiency virus (HIV) infection [1]. Inhibition of HIV protease is one of the most important approaches for therapeutic intervention of acquired immunodeficiency syndrome (AIDS). Ritonavir (RTV) is a selective, competitive and reversible inhibitor of both HIV-1 and HIV-2 proteases. It is widely used in the treatment against AIDS and particularly to inhibit liver enzyme, viz., cytochrome P450-3A4 (CYP3A) [2]. It is not only used on its own as an antiretroviral but also a booster of other anti-HIV drugs to slow and/or prevent the onset of AIDS. Chemically, RTV is (5S,8S,10S,11S)-10-hydroxy- 2-methyl-5-(1-methylethyl)-1-[2-(1-methylethyl)-4-thiazolyl]- 3,6-dioxo-8,11-bis(phenylmethyl)-2,4,7,12-tetraazatridecan-13- oicacid,5-thiazolylmethyl ester (Fig. 1). Several HPLC methods were published for determination of antiretrovirals including RTV in different formulations [1–4]. HPTLC was used for simultaneous determination of ritonavir and lopinavir in capsules [5]. A few CE and LC–MS methods were reported for analysis of RTV and its metabolites in biological fluids [6–9]. Inter- national pharmacopoeia (Ph.Int.) describes a LC method to separate RTV and its impurities. However, no brand names of columns (L1) were mentioned in pharmacopoeial texts, due to which ana- lysts often face difficulty in selecting a suitable stationary phase. ∗ Corresponding author at: Analytical Chemistry Division, Discovery Laboratory, Indian Institute of Chemical Technology, D215, Discovery Laboratory, Tarnaka, Hyderabad 500607, India. Tel.: +91 40 27193193; fax: +91 40 27173387. E-mail addresses: rnrao55@yahoo.com, rnrao@iict.res.in (R.N. Rao). Recently, Yekkala et al. [2], have evaluated the pharmacopoeia method to check the suitability of a set of commercial columns and found that Hypersil BDS complies with system suitability requirement of Ph.Int. However, studies on degradation behavior of RTV were limited in the literature. A stability indicating assay of RTV in soft gelatin capsules was reported by Dias et al. [1]. In another report, Donato et al. [3], studied the forced degradation of ritonavir and lopinavir under hydrolytic (acid, base and neu- tral), oxidative and photolysis. However, in both cases, neither the characterization of degradation products nor the degradation pathways were reported. The International Conference on Harmo- nization (ICH) guidelines [10], suggest stress studies on a drug to establish its inherent stability characteristics not only for identifi- cation of degradation products but also understanding the stability of drug molecule. So, it is of great importance to know the com- plete degradation profile of RTV, which is not yet reported in the literature. The present manuscript describes the degradation behavior of RTV under hydrolysis (acidic, basic and neutral), oxidation, thermal and photolysis conditions. Optimization of LC conditions to separate the drug and its degradation products on a reverse phase C 18 column, method validation, characterization of degrada- tion products, and mechanism of degradation by LC–MS/MS were discussed. 2. Experimental 2.1. Chemicals and reagents Analytical grade reagents and HPLC grade solvents were used. Glass-distilled and deionized water NANOpure (Branstead, USA) 0731-7085/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jpba.2010.06.004