Development and validation of a UHPLC-MS/MS bioanalytical method to quantify in plasma the analgesic candidate PT-31 following a preliminary pharmacokinetic study in rats T. C. Bessegato a , M. Niehues b , G. A. Buqui b , N. P. Lopes b , I. R. Pitta c , S. L. Galdino c and T. Dalla Costa a * ABSTRACT: A selective and sensitive UHPLC-MS/MS bioanalytical method to determine PT-31, an analgesic drug candidate, in rat plasma was developed and validated. Analyses were performed using a UHPLC-MS/MS system equipped with an electrospray ionization interface operating in the positive ionization mode using a C 18 reversed-phase column with a mobile phase of wa- ter:acetonitrile (68:31, v/v) containing 0.1% acetic acid eluting in a gradient mode with a flow rate of 0.3 mL/min. Plasma samples were deproteinized with cold acetonitrile containing 0.01% TFA (1:2, v/v) and 50 μL of the supernatant were injected into the sys- tem. PT-31 and phenytoin (internal standard) retention times were roughly 1.0 and 1.5 min, respectively. Linear standard curves were plotted for the 0.01–10 μg/mL concentration range, with a coefficient of determination > 0.99. The method’s precision was over 88%. Maximum intra- and inter-day relative standard deviations were 14.6% and 11.6%, respectively. Interfering substances were not detected in the chromatogram, indicating that the method was specific. PT-31 stability was assessed under different temperature and storage settings. The method was used to characterize PT-31 plasma pharmacokinetics following administra- tion of 5 mg/kg i.v. to Wistar rats. Therefore, the method described is sensitive, linear, precise and specific enough to determine PT-31 in preclinical pharmacokinetic investigations. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: UHPLC-MS/MS PT-31; bioanalytical method validation; PT- 31; analgesic; preclinical pharmacokinetics Introduction Nowadays barbiturates, opioids and benzodiazepins, isolated or in combination, are the drugs of choice for sedation in clinical scenarios (Bagatini et al., 2002; Lemke, 2004). Though these drugs have been proven to produce excellent analgesic and sedative effects, they nevertheless fall short of achieving the required balance between sedation and analgesia, and several of these drugs cause a variety of adverse side-effects, which restricts clinical applications (Bagatini et al., 2002). The current limitations for therapeutic approaches have led to research efforts devoted to discovering new chemical entities for sedation and analgesia for intensive-care patients. The α 2 -adrenergic agonists were first designed and produced in the 1960s, when they were used as nasal decongestants. Later, α 2 - adrenergic agonists were also employed as anti-hypertensive drugs. Studies carried out subsequently showed that this drug class has analgesic, sedative, anxiolytic and sympatholytic action, sparking an interest in these drugs’ anesthetic potential, with a view to reducing the use of regular anesthetics and opioids (Scholz and Tonner, 2000). More specifically in anesthesiology, α 2 -adrener- gic agonists are used mainly in pre-anesthesia, as adjuvants to in- halation, venous and local anesthesia, as well as in analgesia (Alves et al., 2000; Scholz & Tonner, 2000). The clinical benefits of α 2 -adrenergic agonists as painkillers are well known, though few drugs belonging to this class present this activity profile. In this sense, the identification of new compounds for this application is quite relevant in clinical scenarios. With this in mind, 3-(2-chloro-6-fluorobenzyl)-thione-2,4-dione, also called PT- 31 (Fig. 1A), was synthesized by the Research Center for Therapeu- tic Innovation, Federal University of Pernambuco, Brazil. With an analgesic profile that is a result of the activation of α 2 -adrenergic receptors, PT-31 is chemically related to a series of 3-benzyl- imidazolidines. In a previous study, a dose-dependent antinociceptive effect was observed in mice treated with a 1–15 mg/kg PT-31 dose range. The maximum possible analgesic effect (MPE%) induced * Correspondence to: T. Dalla Costa, Universidade Federal do Rio Grande do Sul, Faculdade de Farmácia, Programa de Pós-Graduação em Ciências Farmacêuticas, Av. Ipiranga, 2752, Porto Alegre-RS, 90610–000 - Brazil. Email: dalla.costa@ufrgs.br a Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Fed- eral do Rio Grande do Sul, Porto Alegre, RS, Brazil b Núcleo de Pesquisa de Produtos Naturais e Sintéticos, Universidade de São Paulo, Ribeirão Preto, SP, Brazil c Núcleo de Pesquisa em Inovação Terapêutica, Universidade Federal de Pernambuco, Recife, PE, Brazil Biomed. Chromatogr. 2016; 30: 852–856 Copyright © 2015 John Wiley & Sons, Ltd. Research article Received: 20 March 2015, Revised: 16 July 2015, Accepted: 14 September 2015 Published online in Wiley Online Library: 28 October 2015 (wileyonlinelibrary.com) DOI 10.1002/bmc.3618 852