Saxena et al. Int J Pharm Pharm Sci, Vol 6, Issue 10, 565-570 QUANTIFICATION OF URAPIDIL IN HUMAN PLASMA USING ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY–ELECTROSPRAY IONIZATION MASS SPECTROMETRY (UPLC–MS/MS) FOR PHARMACOKINETIC STUDY IN HEALTHY INDIAN VOLUNTEERS Original Article ASHISH SAXENA a,b, *, ARUN KUMAR GUPTA b , PRAVEEN KUMAR V. a,c , M. SUNDARAMOORTHI NAINAR a , MANOJ BOB a , RAVISEKHAR KASIBHATTA a a Bioanalytical Research Department, Lupin Bio-Research Center, Pashan, Pune 411021, Maharastra State, India. * PhD Research scholar, b Faculty of Pharmacy, Pacific University, P.B.-12 Pacific Hills, Airport Road, Pratap Nagar Extension, Debari, Udaipur 313024, Rajasthan State, India. c Faculty of Science, Pacific University, P.B.-12 Pacific Hills, Airport Road, Pratap Nagar Extension, Debari, Udaipur 313024, Rajasthan State, India. *Email: ashishsaxena@lupinpharma.com Received: 03 Aug 2014 Revised and Accepted: 05 Sep 2014 ABSTRACT Objective: A rapid and selective quantitative method was developed and validated in human plasma for urapidil pharmacokinetic study in healthy Indian volunteers. Methods: The ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) method with solid-phase extraction technique utilized Strata X 33µ polymeric reversed phase (30 mg/mL), extraction cartridge. Simple gradient chromatographic conditions and selective reaction monitoring in mass spectrometric detection enabled accurate and precise measurement of urapidil at nanogram levels in 0.1 mL of human plasma. The method used a deuterium labeled internal standard. Results: The method was validated for a linear range of 5–500 ng/mL for urapidil with a correlation coefficient ≥ 0.99 The intra-run and inter-run precision and accuracy were within 10%. The overall recoveries for urapidil and urapidil D4 were more than 90%. The urapidil was found to be stable in plasma matrix and aqueous media. Conclusion: The developed and validated method was specific, sensitive and reproducible in the analysis of clinical samples interspersed with quality control samples under freshly prepared calibration standards. The method was applied for the determination of the pharmacokinetic parameters of urapidil following a single oral administration of urapidil 60 mg capsules in nineteen healthy Indian male volunteers for fasting and fed study. Keywords: Urapidil, UPLC–MS/MS, Human plasma, Pharmacokinetic study, Solid phase extraction INTRODUCTION Urapidil (6-{3-[4-(2-o-methoxyphenyl)-1-piperazinyl]- propylamino}-1, 3-dimethyluracil), is a derivative of pyrimidinedione. It has a melting point range of 156-158 °C and the pKa is 7.01. Its molecular formula and molecular mass is C20H29N5O3 and 387.48 g/mol, respectively [1]. Urapidil is a sympatholytic antihypertensive drug. It blocks peripheral α1- adrenergic receptors and also stimulates central serotonin 1A (5- hydroxytryptamine) (5-HT1A) receptors [2]. It prevents vasoconstrictive action of catecholamines resulting peripheral vasodilation to decrease blood pressure. The absolute bioavailability is approximately 72% and the protein binding is 80%. Urapidil divides rapidly over the tissues. Urapidil is metabolised mainly to the p-hydroxy-urapidil, whose pharmacology activity in humans is not known. In addition, slightly (4%) the O-desmethyl urapidil formed has the same activity as urapidil. 50-70% of the amount of renal urapidil is excreted, about 15% as unchanged drug [3-7]. Reported literature has mentioned, to assess the effect of urapidil on fibrinogen concentration [8]. The pharmacological animal studies [9-13] and in patient studies [14-16] for the pharmacokinetics and pharmacodynamic evaluation of urapidil was done in last decades. Veltkamp AC et al. reported the post-column ion-pair extraction to the on-line radiometric determination of [(14)C]-urapidil and its main metabolises in reversed-phase liquid chromatography (LC) [17]. Large sample volume (1-2 mL) and injection volume (100-50 µL) was used into the automated pre-column system, followed by high-performance liquid chromatography with electrochemical detection by Zech K et al. [18]. A sensitive flow-injection (FI) chemiluminescence (CL) method was developed for the determination of urapidil in pharmaceutical preparation, human urine, and serum by Q. Yue et al. [19]. The urapidil was analyzed in rat plasma by LC-MS/MS and liquid-liquid extraction protocol is followed for a linearity range of 0.1-500 ng/mL by Nirogi R et al. [20]. The aliquots of 0.3 mL plasma was used for Liquid-liquid extraction by ethyl acetate and injected 20 µL to detect urapidil in human plasma in which the linearity range was 2–2503.95 ng/mL and recovery was 74.53%. This method was developed and validated by Ambavaram VBR et al. [21]. As the described methods were liquid- liquid extraction, it was felt necessary to develop a simple, specific, rapid, selective and sensitive analytical method for the quantification of urapidil in human plasma using solid phase extraction with as little as 0.1 mL sample volume. This paper describes development and validation of a LC–MS/MS method for the quantification of urapidil in human plasma having reduced plasma volume and analytical run time with a lower limits of quantification (LOQ) 5.201 ng/mL. Urapidil D4 was used as internal standard. MATERIAL AND METHODS Chemicals and reagents The analytical standards of urapidil and urapidil D4 were obtained from Clearsynth (Mumbai, India). High purity water was prepared in-house using a Milli-Q water purification system obtained from Millipore (Bangalore, India). Gradient grade methanol and acetonitrile were purchased from Merck (Darmstadt, Germany). GR-grade orthophosphoric acid and reagent grade ammonium formate were purchased from Merck (Darmstadt, Germany). Drug-free (blank) buffered human plasma was obtained from Drug Monitoring Research Institute (Mumbai, India) and was stored at –20°C prior to use. International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 6, Issue 10, 2014 Innovare Academic Sciences