An analytical method based on reversed phase liquid chromatography (RP-LC) was developed and validated for the determination of acyclovir in human plasma. Acyclovir and guanine (internal standard) were extracted from the plasma by liquid-liquid extraction using acidified acetonitrile as extraction solvent, and separated on a C 18 analytical column (150'mm  4.6 mm I.D.) maintained at 308C. The elution was performed by a fast gradient at a constant flow rate of 1.0 mL/min and the mobile phase A consisted of 1% formic acid, and mobile phase B consisted of acetonitrile. The fluorescence detector was set at 270 nm (excitation) and 380 nm (emission). The chromatographic separation was obtained within 16.0 min and was linear in the concentration range of 20 -3000 ng/mL. The mean extraction recoveries of acyclovir and guanine from plasma were 82.2 and 76.0%, respectively. Method validation investigated parameters such as the specificity, linearity, precision, accuracy, and stability, giving results within the acceptable range. Moreover, the proposed method was successfully applied to a pharmacokinetic study in healthy human volunteers, and results showed that the two acyclovir formulations are bioequivalent in their rate and extent of absorption.
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