Quantification of Thiopurine Metabolites in Human Erythrocytes by Liquid Chromatography-Tandem Mass Spectrometry...

The thiopurine drugs, azathioprine, mercaptopurine, and thioguanine, are widely used in the treatment of several malignant and nonmalignant diseases. These inactive prodrugs undergo extensive metabolism to form active cytotoxic metabolites, which act mainly by incorporating into DNA and affecting cell replication. Thiopurine methyltransferase is a highly variable cytosolic enzyme that catalyzes the S-methylation of the thiopurine bases-an inactivating pathway. Patients with low-activity variants of TPMT can be affected by pronounced pharmacologic effects when receiving thiopurine medications. Clinical studies have reported significant interpatient variability in intracellular thiopurine metabolite concentrations in patients receiving thiopurine therapy. In this chapter, we present an LC-MS/MS method to monitor the thiopurine metabolites: 6-thioguanine nucleotides and 6-methylmercaptopurine derivatives in human erythrocytes. This method utilizes acid hydrolysis to release the bases and improves upon previously published procedures by utilizing stable isotope internal standards and a more efficient chromatographic separation.