LC–MS/MS method development for quantification of busulfan in human plasma and its application in pharmacokinetic study

• A simple, cost effective rugged and a high throughput method was developed for estimation of busulfan in human plasma.
• The method consists of a simple sample pretreatment by protein precipitation to give consistent and reproducible recoveries of busulfan.
• In the present work a new LC–MS/MS method for the quantification of busulfan in human plasma was developed for clinical study sample analysis.
• The proposed method is suitable for comparing the pharmacokinetic profiles of formulations.

A simple, rapid, specific and precise liquid chromatography—tandem mass spectrophotometric (LC–MS/MS) method was developed and validated for quantification of busulfan, in human plasma. busulfan d8 was used as internal standard, added to plasma sample prior to extraction using acetonitrile as a precipitating agent. Chromatographic separation was achieved on phenomenex kinetex C18 column (50 mm × 2.1 mm, 2.6 μm) with acteonitrile: 10 mM ammonium formate buffer (80:20 v/v) as an isocratic mobile phase with a flow rate of 0.5 mL min−1. Quantitation was performed by transition of 264.1 → 151.1 (m/z) for busulfan and 272.1 → 159.1 (m/z) for busulfan d8. The lower limit of quantitation was 0.2 ng mL−1 with a 100 μL plasma sample. The concentrations of nine working standards showed linearity between 0.2 and 100 ng mL−1 (r2 ≥ 0.9986). Chromatographic separation was achieved within 2.0 min. The average extraction recoveries of 3quality control concentrations were 92.52% for busulfan and 90.75% for busulfan d8. The coefficient of variation was ≤15% for intra- and inter-batch assays. The developed method was successfully applied for the determination of Busulfan pharmacokinetics after oral administration.

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