Determination of the covalent adducts of the novel anti-cancer agent 5,6-dimethylxanthenone-4-acetic acid in biological samples by high-performance liquid chromatography

The reversed-phase HPLC methods were developed to determinate the covalentl y bound protein adducts of the novel anti-cancer drug 5,6-dimethylxanthenon e-4-acetic acid (DMXAA) via its glucuronides after releasing aglycone by al kaline hydrolysis in human plasma and human serum albumin (HSA). An aliquot of 75 mul of the mixture was injected onto a Spherex C-18 column (150x4.6 mm; 5 mum) at a flow-rate of 2.5 ml/min. The mobile phase comprising of ace tonitrile: 10 mM ammonium acetate buffer (24:76, v/v, pH 5.8) was used in a n isocratic condition, and DMXAA was detected by fluorescence. The method w as validated with respect to recovery, selectivity, linearity, precision, a nd accuracy. Calibration curves for DMXAA were constructed in the concentra tion range of 0.5-40 muM in washed blank human plasma or HSA prior to alkal ine hydrolysis. The difference between the theoretical and calculated conce ntration and the relative standard deviation were less than 10% at all qual ity control (QC) concentrations. The limit of detection for the covalent ad duct in human plasma or HSA is 0.20 muM The methods presented good accuracy , precision and sensitivity for use in the preclinical and clinical studies . (C) 2001 Elsevier Science B.V. All rights reserved.