Reversed-phase high-performance liquid chromatographic assay method for quantitating 6-mercaptopurine and its methylated and non-methylated metabolites in a single sample

Methods of assaying 6-mercaptopurine (6MP) and its methylated and non-methylated metabolites are essential for the therapeutic dose in treating patients with acute lymphoblastic leukemia. However, previous methods are technically complicated and unsuitable for clinical use. Thus, we have now developed a method utilizing reversed-phase high-performance liquid chromatography (HPLC) in order to quantify these compounds in human red blood cells (RBCs) in a single sample to serve as an index of cytotoxic activity. The agents 6MP, 6-thioguanine (6TG) and 6-methylmercaptopurine (6MMP) were well separated by this assay. Linear relationships were observed between the peak areas and the RBC concentrations of 6MP, 6TG and 6MMP over the range of 20-2000, 18-1800 and 18-1800 pmol per 25 mg hemoglobin (Hb), respectively. The limit of quantitation of the assay is 20, 18 and 18 pmol per 25 mg Hb, respectively. This assay system is suitable for routine clinical use.