Objectives: Allopurinol is a uric acid lowering drug used in the treatment of gout and the prevention of tumor lysis syndrome. Allopurinol and its active metabolite oxipurinol inhibit xanthine oxidase, which forms uric acid from xanthine and hypoxanthine. Therapeutic drug monitoring is an important option for evaluation and optimization of allopurinol treatment in case of renal impairment, interaction with uricosuric drugs or to verify patient adherence. In this study we developed and validated a simple quantitative assay using reverse phased high-performance liquid chromatography (HPLC) with UV-detection as a method for quantification of allopurinol and oxipurinol in human serum in the presence of different frequently used drugs.
Methods: The HPLC-UV method uses a mobile phase consisting of sodium acetate (0.02 M; pH 4.5), at a flow rate of 1.0 mL/min. Allopurinol and oxipurinol are detected by UV-absorption at 254 nm with a retention time of 9.9 min for oxipurinol and 12.3 min for allopurinol. Aciclovir is used as internal standard.
Results: Validation showed for allopurinol lower and upper limits of quantification of 0.5 and 10mg/L and for oxipurinol 1 and 40 mg/L, respectively. The assay was linear over the concentration range of 0.5-10mg/L (allopurinol) and 1-40 mg/L (oxipurinol). Intra- and inter-day precision showed coefficients of variation <15% over the complete concentration range; accuracy was within 5% for allopurinol and oxipurinol. Endogenous purine-like compounds were separated from allopurinol, oxipurinol and aciclovir with a resolution factor >1.5. Exogenous purine-like compounds and co-medication frequently used by gout patients did not hinder the analysis due to the dichloromethane washing step or to low UV-absorpion at 253 nm. Serum levels of 66 patients prescribed allopurinol 300 mg/day were determined using this HPLC-UV method. Measured serum allopurinol and oxipurinol concentrations in clinical practice showed large variability with a range of <0.5-4.3 mg/L for allopurinol and <1.0-39.2 mg/L for oxipurinol, respectively.
Conclusion: We developed an easy-to-operate and validated HPLC-UV method for the quantification of allopurinol and oxipurinol in human serum. This method was proven to be valid for samples of gout patients frequently using concomitant medications.