Analytical Sciences

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

Shuuji HARA, Masahiko TSUCHIE, Riko TSUJIOKA, Masahiko KIMURA, Megumi FUJII,Takeshi KURODA, and Nobufumi ONO

Faculty of Pharmaceutical Sciences, Fukuoka University, Nanakuma, Fukuoka 814-0180, Japan

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 ofmethanol-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 than91.1%, 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 ug ml^-1 (0.04 - 12 uM) 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.

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