Liquid/liquid extraction using 96-well plate format in conjunction with hydrophilic interaction liquid chromatography–tandem mass spectrometry method for the analysis of fluconazole in human plasma

Abstract

A bioanalytical method using automated sample transferring, automated liquid/liquid extraction (LLE) and hydrophilic interaction liquid chromatography–tandem mass spectrometry was developed for the determination of fluconazole in human plasma. Samples of 0.05 ml were transferred into 96-well plate using automatic liquid handler (Multiprobe™ II). Automated LLE was carried out on a 96-channel programmable liquid handling workstation (Quadra™ 96) using methyl- tetra butyl ether as the extraction solvent. The extract was evaporated to dryness, reconstituted, and injected onto a silica column using an aqueous–organic mobile phase. The chromatographic run time was 2.0 min per injection, with retention times of 1.47 and 1.44 min for fluconazole and internal standard (IS) ritonavir, respectively. The detection was by monitoring fluconazole at m/ z 307→238 and IS at m/ z 721→296, respectively. The standard curve range was 0.5–100 ng ml −1. The inter-day precision and accuracy of the quality control samples were <7.1% relative standard deviation and <2.2% relative error.