Validated LC–MS/MS Method for Therapeutic Drug Monitoring of Thiopurine Metabolites in Red Blood Cells

While therapeutic drug monitoring (TDM) of thiopurine metabolites in red blood cells (RBCs) is essential to optimizing dosing regimens and minimizing adverse effects, the analytical methods currently available are lacking the desirable specificity and sensitivity needed for optimum quantification at therapeutic concentrations on a routine basis. This gap has inspired a joint study between the Pandit Jawahar Lal Nehru Government Medical College and Hospital (Chamba, India) and Dayanand Medical College and Hospital (Ludhiana, India) validating and developing a highly repeatable and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the simultaneous quantification of the abalytes 6-mercaptopurine (6-MP), 6-thioguanine (6-TG), and 6-methylmercaptopurine (6-MMP) within human red cells. The researchers hope that their method will be accepted as a reliable analytical tool for monitoring therapeutic drugs and result in the opportunity to provide personalized medicine approaches in therapy optimization. A paper based on their work was published in Cureus (1).

Thiopurines are extensively used for treating inflammatory bowel disease (IBD), acute lymphoblastic leukemia (ALL), and other autoimmune disorders, including systemic lupus erythematosus and rheumatoid arthritis (2). The challenge in employing thiopurines for clinical therapeutics is that their application can become tainted due to interindividual variation between patients in their drug exposure and therapeutic outcome resulting from genetic variation between patients in their major metabolic enzymes, thiopurine S-methyltransferase (TPMT), inosine triphosphate pyrophosphatase (ITPA), and nudix hydrolase 15 (NUDT15) (3). This can result in patients experiencing therapeutic failure because of suboptimal drug levels from insufficient drug exposure, potentially causing serious, life-threatening adverse effects such as myelosuppression and hepatotoxicity (4).

The authors of the study report that the developed method showed excellent linearity for the three analytes, with a coefficient of determination (R²) of ≥0.9997. “Recovery analysis,” they wrote in their study, “showed acceptable accuracy, with the mean recoveries between 88.74% and 117.37% for all the concentration levels” (1). In addition, they state that their method showed high specificity without interference from endogenous matrix components, with a precision study showing a relative standard deviation (RSD) of ≤15% for all the analytes (1).

“The developed LC–MS/MS approach gives a reliable technique for the simultaneous quantification of thiopurine metabolites within erythrocytes,” wrote the authors of the study. “With its reliability and sensitivity, it makes a suitable approach for therapeutic drug monitoring at the clinical level, therefore contributing toward enhanced thiopurine therapy under the banner of personalized medicine” (1).

The researchers admit to some limitations with their study. While they believe that there was sufficient validation presented, they note that it was conducted using spiked samples in a healthy red blood cell matrix rather than patient samples. A full assessment of any interferences that might occur due to pathological matrices, concomitant medications, or metabolite interferences, therefore, could not be carried out. “Future research,” the authors of the study recommended, “should entail clinical validations across patient populations while attempting to correlate concentrations measured with pharmacogenetic and clinical outcomes” (1). In addition, no testing for metabolite stability under different storage conditions was conducted, a factor the authors believe to be necessary when standardizing sample handling in multicentric trials (1).

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References

1. Singh, M.; Kaushal, S.; Gupta, K. et al. Development and Validation of a Sensitive Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Method for the Simultaneous Quantification of Thiopurine Nucleotides in Human Red Blood Cells. Cureus 2025, 17 (12), e98953. DOI: 10.7759/cureus.98953
2. Lennard, L. The Clinical Pharmacology of 6-mercaptopurine. Eur. J. Clin. Pharmacol. 1992, 43, 329–339. DOI: 10.1007/BF02220605
3. Relling, M. V.; Schwab, M.; Whirl-Carrillo, M. et al. Clinical Pharmacogenetics Implementation Consortium Guideline for Thiopurine Dosing Based on TPMT and NUDT15 Genotypes: 2018 Update. Clin.Pharmacol. Ther. 2019, 105, 11095–105. DOI: 10.1002/cpt.1304
4. Dubinsky, M. C.; Lamothe, S.; Yang, H. Y. et al. Pharmacogenomics and Metabolite Measurement for 6-mercaptopurine Therapy in Inflammatory Bowel Disease. Gastroenterology 2000, 118, 705–713. DOI: 10.1016/S0016-5085(00)70140-5