High-Performance Liquid Chromatographic Quantification of Busulfan in Human Serum after Fluorescence Derivatization by 2-Naphthalenethiol

Abstract

A simple and highly sensitive fluorometric high-performance liquid chromatographic method was developed for the determination of busulfan in human serum. After busulfan and 1,6-bis(methanesulfonyloxy)hexane as an internal standard were extracted from serum with ethyl acetate, they were derivatized with 2-naphthalenethiol in an alkaline medium. The derivatives were separated by reversed-phase chromatography on a YMC-Pack C₄ column with a mixture of methanol–0.1 M sodium acetate buffer (pH 7.0) (8:2, v/v) as a mobile phase, and were then detected spectrofluorometrically at 370 nm with excitation at 255 nm. Extraction and derivatization efficiencies were 73.9–75.1% and greater than 91.1%, respectively. The detection limit for busulfan added to serum was 2 ng (8 pmol) ml^–1 serum (330 fmol on column) at a signal-to-noise ratio of three with a linear relationship over the 10 ng–3.0 µg ml^–1 (0.04–12 µM) concentration range. The accuracy and precision of this method were within 7.0% and 9.3% even at low concentration (10 ng ml^–1). The within- and between-day variations were lower than 9.3% and 10.9%, respectively.