High-Performance Liquid Chromatographic Method for Analysis of Emtricitabine in Rat Plasma: Method Development, Validation and Application to a Pharmacokinetic Study
A new reverse phase liquid chromatographic method for the investigation of emtricitabine in rat plasma was developed after oral administration to Wistar rats. The desired chromatographic separation was achieved on Phenomenex C18 column (250？mm × 4.6？mm I.D., 5？μm) column, under isocratic conditions using UV detection at 280？nm. The optimized mobile phase consisted of a mixture of 10？mM potassium dihydrogen phosphate buffer- (adjusted to pH 6.8) methanol-2% acetic acid in a ratio of (73？:？25？:？2, v/v/v) at a flow rate of 1？mL？min？1. The system was found to produce sharp and well-resolved peaks for emtricitabine with retention time of 5.78？min. The linear regression analysis for the calibration curves showed a good linear correlation over the concentration range 0.050–3.0？μg？mL？1, with determination coefficients, , exceeding 0.9970. The limits of detection (LOD) and quantitation (LOQ) were found to be 0.016？μg？mL？1 and 0.049？μg？mL？1, respectively. The method was successfully applied for the pharmacokinetic in rats. Emtricitabine concentration in plasma reached ( ) was 1.357？μg？mL？1 about 2？h after oral administration of 15？mg/kg/rat. The AUC0-24 was 12.175？μg？mL？1* h and the apparent elimination half-life ( ) was 8.153？h. This method was found to be suitable for examining emtricitabine concentration in rats, after oral administration of emtricitabine in a single dose. 1. Introduction Emtricitabine (5-fluoro-1-(2R, 5S)-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine) (Figure 1(a)) is a potent deoxycytidine nucleoside reverse transcriptase inhibitor for the treatment of human immunodeficiency virus (HIV) infection [1]. In adults, emtricitabine recommended dose is 200？mg once a day (QD) [2]. Both in vitro [3] and in vivo [4] testing demonstrated that emtricitabine presents enough potential to be tested in the prevention of HIV-1, either alone or in combination [5]. Figure 1: Chemical structure of emtricitabine (a) and lamivudine (b). A few HPLC and a brief reference to one UPLC method for simultaneous determination of emtricitabine in combination with other antiretroviral drugs in human plasma and rats have been described in the literature, mainly with the objective of method development for application to a bioequivalence study [6–8]. A simultaneous determination of emtricitabine and tenofovir in human plasma was described [9]. HPLC-UV detection method was developed for simultaneous determination of emtricitabine and tenofovir in tablet dosage form with LOQ of 0.091？μg？mL？1 [10]. A rapid RP-HPLC method for a combination of tenofovir disoproxil fumarate,
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