An optimized LC-ESI-MS/MS assay for erythrocyte 6-TG and 6-MMPD: Addressing critical methodological considerations...

Thiopurines, a class of antimetabolites widely used in patients with inflammatory bowel disease (IBD) and acute lymphoblastic leukemia (ALL), are associated with two predominant adverse effects: delayed myelotoxicity and hepatotoxicity. The erythrocyte-based active metabolites 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine nucleotides (6-MMPN) serve as biomarkers reflecting systemic drug exposure and predicting both therapeutic efficacy and adverse reactions. In this study, we successfully developed and validated a rapid, sensitive, and accurate liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analytical method for simultaneous quantification of erythrocyte 6-TG and 6-MMPD concentrations. During method development and validation, we focused on addressing the following five key challenges currently of significant interest in the field: 1) Erythrocytes proved superior to whole blood as biological matrix; 2) Perchloric acid was essential in sample pretreatment; 3) Dual mobile phase additives (HCOOH+NH₄Ac) enhanced chromatographic peak shape and detection sensitivity; 4) Incomplete hydrolysis of 6-TGN and 6-MMPN occurred with heating durations < 60 min; 5) The linear correlation coefficient of the 6-MMPD (6-MMP derivative, 4-amino-5-(methylthio)carbonyl imidazole) standard curve reflected the stable conversion efficiency of 6-MMP. The method exhibited excellent linearity over the concentration ranges of 0.06-50 μmol/L for 6-TG (r = 0.9906) and 0.18-150 μmol/L for 6-MMPD (r = 0.9914). Selectivity, carry-over, intra- and inter-batch accuracy and precision, reproducibility, recovery, matrix effect, and stability all complied with the acceptance criteria outlined in the US Food and Drug Administration (FDA) Bioanalytical Method Validation Guidance (2018). The validated method was successfully employed to quantify 6-TG and 6-MMPD in samples obtained from 20 patients with IBD or ALL.