Journal of Chromatography B, 757 (2001) 343–348 www.elsevier.com / locate / chromb 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 a, a a b * Shufeng Zhou , James W. Paxton , Malcolm D. Tingle , Phillip Kestell a Department of Pharmacology and Clinical Pharmacology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand b Auckland Cancer Society Research Centre, University of Auckland, Auckland, New Zealand Received 7 November 2000; received in revised form 27 February 2001; accepted 14 March 2001 Abstract The reversed-phase HPLC methods were developed to determinate the covalently bound protein adducts of the novel anti-cancer drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA) via its glucuronides after releasing aglycone by alkaline hydrolysis in human plasma and human serum albumin (HSA). An aliquot of 75 ml of the mixture was injected onto a Spherex C column (15034.6 mm; 5 mm) at a flow-rate of 2.5 ml/min. The mobile phase comprising of acetonitrile:10 mM 18 ammonium acetate buffer (24:76, v / v, pH 5.8) was used in an isocratic condition, and DMXAA was detected by fluorescence. The method was validated with respect to recovery, selectivity, linearity, precision, and accuracy. Calibration curves for DMXAA were constructed in the concentration range of 0.5–40 mM in washed blank human plasma or HSA prior to alkaline hydrolysis. The difference between the theoretical and calculated concentration and the relative standard deviation were less than 10% at all quality control (QC) concentrations. The limit of detection for the covalent adduct in human plasma or HSA is 0.20 mM. The methods presented good accuracy, precision and sensitivity for use in the preclinical and clinical studies.  2001 Elsevier Science B.V. All rights reserved. Keywords: 5,6-Dimethylxanthenone-4-acetic acid 1. Introduction I/II Clinical Trials Committee. As a biological response modifier, DMXAA’s mode of action is The novel anti-cancer drug 5,6-dimethylxan- different from conventional cytotoxic chemothera- thenone-4-acetic acid (DMXAA, Fig. 1) was de- peutic agents. In murine models, the anti-tumour veloped by the Auckland Cancer Society Research activity of DMXAA appears to be mediated by Centre (ACSRC). It has recently completed Phase I immune modulation and the induction of cytokines, clinical trials in New Zealand and the UK under the such as tumour necrosis factor [1–3], serotonin [4] direction of the Cancer Research Campaign’s Phase and nitric oxide [5,6]. DMXAA is extensively me- tabolised in animals and humans by glucuronidation on its acetic acid side chain, resulting in DMXAA *Corresponding author. Tel.: 164-9-373-7599, ext. 6414; fax: acyl glucuronide (DMXAA-G), which is excreted 164-9-373-7556. E-mail address: shufeng.zhou@auckland.ac.nz (S. Zhou). into bile and urine [7–9]. 0378-4347 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0378-4347(01)00173-6