Abstract

VALIDATION OF SENSITIVE AND RUGGED SPE-LC-MS/MS METHOD FOR DETERMINATION OF ACYCLOVIR IN HUMAN PLASMA: APPLICATION TO FOUR PIVOTAL BIOEQUIVALENCE STUDIES

Nirav P. Patel*, Mallika Sanyal, Naveen Sharma, Pranav S. Shrivastavd, Bhavin N. Patel*, Dinesh S. Patel 

ABSTRACT

A selective, sensitive and rugged liquid chromatography- tandem mass spectrometry (LC-MS/MS) assay for the determination of acyclovir in human plasma is developed using Acyclovir-d4 as an internal standard (IS). The analyte and IS were extracted from 200μL of human plasma via solid phase extraction on Water Oasis HLB cartridges. Chromatographic separation is achieved on a BDS Hypersil C18 (150 mm×4.6 mm, 3μm) column under isocratic conditions. Detection of analyte and internal standard is done by tandem mass spectrometry, operating in positive ion and multiple reaction monitoring (MRM) acquisition mode. The method is fully validated for its selectivity, sensitivity, carryover check, linearity, precision and accuracy, recovery, matrix effect, ion suppression/enhancement, stability and dilution integrity. The limit of detection (LOD) and lower limit of quantitation of the method were 0.2500ng/mL and 5.000ng/mL respectively with a linear dynamic range of 5.000-2500ng/mL for acyclovir. The intra- and inter- batch precision (%CV) and relative recovery across quality control levels is 73.4% respectively. The method is successfully applied to four pivotal bioequivalence studies of 800 mg tablet and 200 mg capsule of acyclovir in 48 and 30 healthy Indian male subjects under fasting and fed condition respectively. The reproducibility of assay method in the measurement of study data is demonstrated by incurred sample reanalysis.

Keywords: Acyclovir; LC-MS/MS; solid phase extraction; human plasma; bioequivalence; incurred sample reanalysis.