Development and Validation of Acyclovir HPLC External Standard Method in Human Plasma: Application to Pharmacokinetic Studies

A simple, rapid, and selective RP-HPLC method was developed for the estimation of acyclovir in human plasma. The method involves a simple protein precipitation technique. Chromatographic separation was carried out on a reverse phase C18 column using mixture of 5？mM ammonium acetate (pH 4.0) and acetonitrile (40？:？60, v/v) at a flow rate of 1.0？mL/min with UV detection at 290？nm. The retention time of acyclovir was 4.12 minutes. The method was validated and found to be linear in the range of 25.0–150.0？ng/mL. Validation studies were achieved by using the fundamental parameters, including accuracy, precision, selectivity, sensitivity, linearity and range, stability studies, limit of detection (LOD), and limit of quantitation (LOQ). It shows recovery at 91.0% which is more precise and accurate compared to the other method. These results indicated that the bioanalytical method was linear, precise, and accurate. The new bioanalytical method was successfully applied to a pharmacokinetic linearity study in human plasma. 1. Introduction Acyclovir, 9-[(2-hydroxyethoxy)-methyl]-guanosine, is an acyclic guanosine derivative which exhibits a selective inhibition of herpesviruses replication with potent clinical antiviral activity against the herpes simplex and varicella-zoster viruses [1, 2]. There are many works published for the determination of acyclovir in biological fluids of different species. For a laboratory, to develop a method is sometimes a compromise between cost, time consumption, and purpose of study. Some of the reported methods about acyclovir quantification in human plasma supposed to be expensive sample extraction method by using liquid-liquid extraction. Several HPLC methods have been reported for determination of acyclovir in human serum using UV [3–13] or fluorescence detection [14–18]. We present herein for the first time, a sensitive and selective HPLC method for the acyclovir assay in human plasma. This paper describes a new sensitive bioanalytical method for acyclovir using RP-HPLC method. By this method, chromatographic conditions have been optimized and validated in accordance with FDA guidelines. This results in a more sensitive, less time consuming, and easier method of quantification compared to the other existing methods and it gives better recovery from the human plasma, which is 91.0%. The present method was found reliable and applicable for the bioequivalence studies. 2. Reagent and Materials Gift sample of acyclovir was obtained from Ranbaxy Pharmaceuticals, Sungai Petani, Malaysia. Acetonitrile (HPLC grade) was obtained from