Validated HPLC Method for Quantification of Pregabalin in Human Plasma Using 1-Fluoro-2,4-dinitrobenzene as Derivatization Agent

In this study, a sensitive, simple, and reliable HPLC method for quantification 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 Na2HPO4 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. The linearity of the method was tested over the concentration range 1–4500？ng/mL in 500？μL of human plasma and satisfactory results were obtained (r2 > 0.999). The 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.). The limit of quantification 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 α2δ 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]. After being taken orally, pregabalin is absorbed rapidly and reaches maximum plasma concentration at about 1.3？h [10]. A range of from 3.5 to 4.5？μg/mL was reported after orally administration of 150？mg pregabalin to the volunteers [11]. Figure 1: Chemical structure of pregabalin. For determination of pregabalin in biological fluids, 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 effects 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 significant chromophore group, which makes difficulty in its quantification by general HPLC-UV methods. Therefore, 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