Development and validation of a high-performance liquid chromatography–tandem mass spectrometry for the determination of penciclovir in human plasma: Application to a bioequivalence study

Abstract

We developed and validated a simple high-performance liquid chromatography (HPLC) coupled with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection system for determining penciclovir (active metabolite of famciclovir) levels in human plasma using acyclovir as an internal standard (IS). Acquisition was performed in multiple reaction monitoring (MRM) mode by monitoring the transitions: m/ z 254.00 > 152.09 for penciclovir and m/ z 226.00 > 152.09 for IS. The analytes were chromatographed on a Capcellpak MGII C 18 reversed-phase chromatographic column by isocratic elution using 30% methanol and 70% Milli-Q water containing 10 mM ammonium formate (adjusted to pH 3.1 with formic acid). Results were linear over the studied range (0.05–10 μg/ml) with r 2 = 0.9999, and the total analysis time for each run was 2 min. Intra- and inter-assay precisions were 2.3–7.8 and 3.7–7.5%, respectively, and intra- and inter-assay relative errors (RE) were 2.0–8.4 and 1.9–9.1%, respectively. The lower limit of quantification (LLOQ) was 0.05 μg/ml. At this concentration mean intra- and inter-assay precisions were 7.8 and 7.5%, respectively, and mean intra- and inter-assay relative errors were 2.2 and 9.1%, respectively. Penciclovir was found to be stable in plasma samples under short-, long-term storage and processing conditions. The developed assay was successfully applied to a bioequivalence study of penciclovir administered as a single oral dose (500 mg as famciclovir) to healthy volunteers.