Development and Validation of Simultaneous Quantification of 6TGN and 6MMPN in Whole Blood Using LC-MS/MS: An Application to Therapeutic Drug Monitoring in Hematological Malignancies and in Inflammatory Bowel Diseases.

Thiopurine immunosuppressive drugs are indicated in children with acute lymphoblastic leukemia and in both children and adults with chronic inflammatory diseases. Therapeutic drug monitoring (TDM) of its DNA-incorporated metabolites 6-thioguanine nucleotides (6TGN) and 6-methylmercaptopurine nucleotides (6MMPN) is of high interest to optimize efficacy and prevent toxicity. The objective of this study was to develop and validate a sensitive and simple LC-MS/MS method for the simultaneous quantification of 6TGN and 6MMPN in whole blood for TDM. Following red blood cell count and whole blood extraction with dithiothreitol plus acidic hydrolysis at 100°C, quantification was performed using an LC-MS/MS in positive ionization mode. Calibration curves were linear over the range of 20-1000 ng/mL for 6TGN and 20-20,000 ng/mL for 6MMPN, with correlation coefficients above 0.99 for all analytes. Intra- and inter-day precisions were below 9.45%. Extraction recovery and matrix effects reached 78.9%-83.2% and 121% for 6TGN and 92.2%-95.2% and 112%-118% for 6MMPN, respectively. The method was applied to 749 clinical samples from patients with acute lymphoblastic leukemia or chronic inflammatory diseases. A sensitive and simple LC-MS/MS method was validated for 6TGN and 6MMPN in whole blood and was successfully applied to clinical samples for TDM.