Improved assay for determination of busulfan by liquid chromatography using postcolumn photolysis

A highly sensitive and time-reduced HPLC assay for the quantitative analysis of busulfan in plasma and aqueous samples is described. The assay is based on a precolumn derivatization of busulfan to 1,4-diiodobutane and UV-detection of iodide ions generated by a postcolumn photochemical dissociation of the derivative. The extraction and derivatization were carried out in a one-pot reaction without any solid phase extraction and is therefore suitable for high throughput analysis. Quantification was performed by using 1,5-pentanediol-bis-(methanesulfonate), a homologue of busulfan, as an internal standard. Linearity was demonstrated for concentrations from 50 to 10,000 ng/ml. The limit of detection was found at 10 ng/ml. Precision is indicated by an intra-day variety of 2.81% and by an inter-day variety of 6.61% for aqueous samples, 2.93 and 5.76% for plasma samples, respectively. The recovery of busulfan in plasma was more than 95%. No coelution with metabolites of busulfan or other drugs used in cancer therapy was found. The method was generated for measurements of busulfan in aqueous or plasma samples and applied in therapeutic drug monitoring of busulfan.