Development and Method Validation of 6-Thioguanine and 6-Methylmercaptopurine in Erythrocytes Using LC-MS/MS

Aim: Previous studies had shown that the safety and efficacy of 6-mercaptopurine in acute lymphoblastic leukemia (ALL) patients was mainly determined by the concentrations of its metabolites in erythrocytes, which are 6-thioguanine (6-TGN) and 6-methylmercaptopurine (6MeMP). The aim of this research is to develop and validate a method to quantify concentrations of 6-thioguanine and 6-methylmercaptopurine in erythrocytes and apply the validated method in ALL patients undergoing 6-mercaptopurine treatment. Methods: We develop a method to determine 6-TGN and 6-MeMP on LC-MS/MS system, using a 1.8 μm column (2.1 x 50 mm). Mobile phase used for analysis were formic acid in acetonitrile and formic acid in H2O (60:40). Extraction from erythrocytes was done using NH4OH in acetonitrile. Bisoprolol was used as internal standard. The method validation was performed according to the European Medicines Agency (EMEA) guideline. Results: Detection for 6-TGN, 6 MeMP and bisoprolol were done using ion transitions, m/z 168.08>151.02, 167.1>152.06 and 326.12>116.03, respectively The calibration curves were linear over the concentration range of 9.9 – 1979 ng/mL for 6-TGN and 10 – 2000 ng/mL for 6MeMP. The other validation criteria, which consists of selectivity, accuracy, precision, lower limit of quantification, carry over, dilution integrity, matrix effect and stability, were achieved. Concentrations of 6-TGN and 6-MeMP in ALL patients underwent 6-MP treatment were found in the range of 21.05 – 109.56 pmol/8x10 erythrocytes and 13.06 – 80.27 pmol/8x10 erythrocytes, respectively. Conclusion: The present LC-MS/MS method is valid for the quantification of 6-thioguanine and 6-methylmercaptopurine in erythrocytes and can be applied in ALL children treated with 6-MP.