Analysis of Azathioprine and 6-Mercaptopurine in Plasma in Renal Transplant Recipients After Administration with Oral Azathioprine

Studies on the pharmacokinetics of azathioprine (AZA) and its main metabolite, 6-mercaptopurine (6-MP) in patients treated with oral AZA, such as renal transplant recipients, require an analytical method with a high sensitivity. Since the substances differ substantially in their physicochemical properties, it is hardly feasible to develop an extraction procedure and select chromatographic conditions for the simultaneous determination of both substances. Therefore, we have developed two specific chromatographic assays for determination of AZA and 6-MP in plasma. A solid-phase extraction (C8 Isolute) procedure was used for AZA with guaneran as internal standard (IS), while 6-MP was purified on mercurial cellulose and 6-mercaptopurine arabinoside was used as IS. Chromatographic analyses were achieved using C8 and C18 reversed phase columns for AZA and 6-MP, respectively. The methods were reproducible with intra- and inter-assay coefficients of variations below 6 %. The average recoveries of AZA, 6-MP and their respective IS were higher than 79%. The limit of quantitation of AZA and 6-MP in plasma is 0.2 ng.