Role of RBC Partitioning and Whole Blood to Plasma Ratio in Bioanalysis: A Case Study With Valacyclovir and Acyclovir

Abstract

A LC-MS/MS method was developed for simultaneous estimation of valacyclovir and acyclovir in human plasma. Plasma sample was extracted with solid phase extraction technique and chromatographic condition was set with Inertsil CN-3 (5 μm) column and mobile phase (1 mM ammonium acetate buffer - methanol, 50:50 v/v). Valacyclovir, acyclovir, Valacyclovir D4 and acyclovir D4 were detected in positive polarity in multiple reactions monitoring mode at mass transitions (m/z) 325.2→152.1, 226.2→152.1, 329.3→152.1 and 230.2→152.1, respectively. The validated calibration curve range for valacyclovir is 4.09 to 725.63 ng/mL and for acyclovir is 50.35 to 10017.29 ng/mL. During method development, stability of acyclovir in whole blood could not be established over the period for 2 h as the KWB/P ratio for acyclovir is greater than 1 and although for valacyclovir it is less than 1. Therefore, the drug distribution of acyclovir was investigated in whole blood and plasma. Experimental data showed that an initial drop of acyclovir level in plasma due to the cellular uptake of acyclovir by erythrocytes. Hence, the spiked comparison samples were allowed to reach equilibrium (between RBC and plasma). After reaching the equilibration time (30.0 min), plasma was harvested from the spiked whole blood and processed as per the proposed protocol. From the blood stability data, we concluded that valacyclovir and acyclovir both are stable in blood for 2 h. The developed method was validated as per current regulatory guidelines and applied for valacyclovir and acyclovir bio-equivalence study.