HPLC Determination of 6-Thiouric Acid and 6-Mercaptopurine in Organ Transplant Patient Serum

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Abstract
A sensitive high performance liquid chromatographic method for the simultaneous determination of 6-thiouric acid and 6-mercaptopurine in serum is described. Our intent was to develop a procedure that could be used for pharmacokinetic studies and therapeutic drug monitoring in organ transplant patients taking azathioprine. Serum samples were precipitated with acetonitrile containing 6-n-propyl-2-thiouracil as the internal standard. The chromatographic separation was performed with an octadecylsilane column and gradient solvent system consisting of acetonitrile and 0.01 M sodium dihydrogen phosphate, pH 6.1. An initial acetonitrile concentration of 1% was used to elute 6-thiouric acid but was increased to 16% to recover the 6-mercaptopurine and internal standard. The flow rate was increased from 1.3 ml/min to 1.5 ml/min during the analysis. The column effluent was monitored at 353 nm and 323 nm for detection of 6-thiouric acid and 6-mercaptopurine, respectively. Statistical analysis of standard curve data showed good intra- and inter-day accuracy, precision and reproducibility throughout a concentration range of 10–2500 ng for 6-thiouric acid and 10–500 ng for 6-mercaptopurine/ml of serum. The method has been applied to the quantification of 6-thiouric acid and 6-mercaptopurine in serum from two kidney allograft recipients.