Research Article Validated HPLC Method for Quantification of Pregabalin in Human Plasma Using 1-Fluoro-2,4-dinitrobenzene as Derivatization Agent Reza Ahmadkhaniha, 1 Siavash Mottaghi, 2 Mohammad Zargarpoor, 2 and Effat Souri 2 1 Department of Human Ecology, School of Public health, Tehran University of Medical Sciences, Tehran, Iran 2 Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran 14155-6451, Iran Correspondence should be addressed to Efat Souri; souri@sina.tums.ac.ir Received 29 May 2014; Accepted 14 July 2014; Published 17 August 2014 Academic Editor: Toyohide Takeuchi Copyright © 2014 Reza Ahmadkhaniha et al. his is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In this study, a sensitive, simple, and reliable HPLC method for quantiication of pregabalin in human plasma was developed and validated. 1-Fluoro-2,4-dinitrobenzene was used as precolumn derivatization agent. For chromatography, an analytical reversed phase (C18) column and a mixture of Na 2 HPO 4 10 mM (pH 8.0)—methanol (35 : 65 v/v) were used as stationary and mobile phase, respectively. Detection was performed using a UV detector tuned at 360 nm. he linearity of the method was tested over the concentration range 1–4500 ng/mL in 500 L of human plasma and satisfactory results were obtained (r 2 > 0.999). he method showed good precision and accuracy in terms of within—between days relative standard deviations and percent deviation from nominated values (in the range of 4.3–12.7% and 2.6–8.0%, resp.). he limit of quantiication of the method was found to be 1 ng/mL which is better than previously reported method and indicates its potential application for sensitive bioanalysis. 1. Introduction Pregabalin (Figure 1), (S)-3-(amino ethyl)-5-methylhexanoic acid, an analogue of gamma amino butyric acid (GABA) with lipophilic properties, is a potent agonist of  2  subunit of voltage dependent calcium channels [1]. Pregabalin reduces release of glutamate, noradrenalin, substance P, serotonin, and dopamine in central nervous system (CNS) [2–4] and could be used for the treatment of pathological conditions such as partial seizure, neuropathic pain, and generalized anxiety disorder [5–9]. Ater being taken orally, pregabalin is absorbed rapidly and reaches maximum plasma concentra- tion ( max ) at about 1.3 h [10]. A range of ( max ) from 3.5 to 4.5 g/mL was reported ater orally administration of 150 mg pregabalin to the volunteers [11]. For determination of pregabalin in biological luids, sophisticated methods such as methods based on LC-MS- MS were employed [11–14]. Although most of LC-MS-MS methods are sensitive and reliable, the instruments are too expensive and unavailable in most of clinical laboratories. Furthermore the carry-over and ion suppression efects are main analytical problems of LC-MS methods which are against the routine use of these methods [15, 16]. Pregabalin is an aliphatic agent without any signif- icant chromophore group, which makes diiculty in its quantiication by general HPLC-UV methods. herefore, derivatizing reagents such as o-phthaldialdehyde (OPA), 3- mercaptopropionic acid, and picrylsulfonic acid were usually used to make better determination [10, 17, 18]. Unfortunately, most of these derivatization methods are complicated and time consuming and sufer from low recovery values. Based on our knowledge, there is only one report about liquid chromatographic analysis of pregabalin in human urine without derivatization [19]. However, the method sensitivity was not adequate for most of bioanalytical application such as pharmacokinetic studies. Pharmacokinetic studies require Hindawi Publishing Corporation Chromatography Research International Volume 2014, Article ID 450461, 6 pages http://dx.doi.org/10.1155/2014/450461