Gas-chromatographic analysis of busulfan for therapeutic drug monitoring

The development and validation of a gas chromatographic assay method for determination of total and free busulfan concentrations in human plasma for pharmacokinetic studies is reported. 1,6-Bis(methanesulfonyloxy)hexane, the internal standard, and a potential metabolite, 3-hydroxysulfolane, were synthesized. Plasma and plasma ultrafiltrate samples containing busulfan and internal standard were extracted with ethyl acetate and derivatized with 2,3,5,6-tetrafluorothiophenol prior to gas chromatographic determination. The⁶³Ni electron-capture detector provided a limit of detection of 0.0600 g/ml with a limit of quantitation of 0.100 g/ml busulfan in biological samples. Calibration curves were linear from 0.100 to 3.00 g/ml in plasma (500 l) and 0.100 to 2.00 g/ml in plasma ultrafiltrate (100 l). Extraction and derivatization yields ranged from 78.4% to 89.6% and 56.0% to 71.3%, respectively. Specificity of this assay for busulfan in the presence of its potential metabolites was demonstrated. Also, plasma samples containing co-administered drugs gave no response under these conditions. Clinical samples obtained following administration of a 1 mg/kg oral busulfan dose demonstrate the applicability of this method to analysis of total and free plasma concentrations.This work was supported in part by Burroughs Wellcome Inc. and the British Columbia Cancer Agency.