Journal of Chromatography B, 857 (2007) 224–230 Development and validation of an enantioselective HPLC–UV method using Chiralpak AD-H to quantify (+)- and (-)-torcetrapib enantiomers in hamster plasma—application to a pharmacokinetic study Ravi Kumar Trivedi a,b , P.K. Dubey b , Ramesh Mullangi a,∗ , Nuggehally R. Srinivas a,c a Drug Metabolism and Pharmacokinetics, Discovery Research, Dr. Reddy’s Laboratories Ltd., Miyapur, Hyderabad-500 049, India b Department of Chemistry, JNTU College of Engineering, Kukatpally, Hyderabad-500 085, India c Drug Development, Discovery Research, Dr. Reddy’s Laboratories Ltd., Miyapur, Hyderabad-500 049, India Received 9 May 2007; accepted 14 July 2007 Available online 6 August 2007 Abstract A chiral selective, accurate and reproducible high-performance liquid chromatographic (HPLC) method was developed and validated for direct separation of individual enantiomers of torcetrapib (TTB) [(+)-TTB and (-)-TTB]. TTB enantiomers and IS were extracted from a small aliquot of plasma (100 L) by simple liquid–liquid extraction using acetonitrile as extraction solvent. The enantiomers were resolved on Chiralpak AD- H ® (250 mm × 4.6 mm, 5 m) with the mobile phase consisting of n-hexane:isopropyl alcohol (IPA) in the ratio of 95:5 (v/v). The eluate was monitored using an UV detector set at 254 nm. Baseline separation of the TTB enantiomers and the internal standard (IS, DRL-17859), free from endogenous interferences was achieved. The resolution factor between the enantiomers was optimized and found to be not less than five. During method development, the IPA content in the mobile phase was optimized for separation of peaks of interest. Additionally, both flow rate and column temperature were optimized for an improved baseline separation of the enantiomers. Ratio of peak area of each enantiomer to IS was used for quantification of plasma samples. Nominal retention times of (+)-TTB, (-)-TTB and IS were 9.4, 13.8 and 17.5min, respectively. The standard curves for TTB enantiomers were linear (r 2 > 0.999) in the concentration range 0.1–10 g/mL for each enantiomer. Absolute recovery, when compared to neat standards, was 88.7–90.0% for TTB enantiomers and 100% for IS from the hamster plasma. The lower limit of quantification (LLOQ) for each enantiomer of TTB was 0.1 g/mL. The inter-day precisions were in the range of 4.57–6.32 and 5.66–11.0% for (+)-TTB and (-)-TTB, respectively. The intra-day precisions were in the range of 1.60–7.36 and 2.76–13.6% for (+)-TTB and (-)-TTB, respectively. Accuracy in the measurement of quality control (QC) samples was in the range of 95.6–109% and 92.7–108% for (+)-TTB and (-)-TTB, respectively. Both enantiomers were stable in a series of stability studies, viz. bench-top (up to 12 h), auto-sampler (up to 24 h) and freeze/thaw cycles (n = 3). Stability of TTB enantiomers was established in hamster plasma for 15 days at -80 ◦ C. The application of the assay to a pharmacokinetic study of (-)-TTB in hamsters is described. © 2007 Elsevier B.V. All rights reserved. Keywords: Torcetrapib; Enantioselective; Quantitation; Chiralpak AD-H ® ; Chirality; HPLC–UV; Hamsters; Pharmacokinetics 1. Introduction Despite on-going advancement in understanding and treatment, cardiovascular diseases continue to remain the leading cause of morbidity and mortality. Although significant reductions in cardiovascular risk can be achieved by effectively lowering low-density lipoprotein cholesterol (LDL-C), treated ∗ Corresponding author. Tel.: +91 40 23045439; fax: +91 40 23045438. E-mail address: mullangiramesh@drreddys.com (R. Mullangi). patients remain at substantial risk from recurring cardiovascular events. Epidemiological studies have established that higher levels of high-density lipoprotein cholesterol (HDL-C) are strongly associated with reduced cardiovascular risk, and therefore raising levels of HDL-C may be beneficial in addi- tion to the traditionally well-accepted modality of lowering LDL-C [1]. In this regard the activity of cholesteryl ester transfer protein (CETP) appears to be inversely correlated with HDL-C levels [2]. Therefore, CETP has been projected as an attractive target for intervention to raise levels of HDL-C and potentially reduce residual cardiovascular risk [3,4]. 1570-0232/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2007.07.045