# 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

## TL;DR

This paper presents a new and sensitive method to measure thiopurine metabolites in red blood cells, which can help improve personalized drug therapy.

## Contribution

A novel, validated LC-MS/MS method for simultaneous quantification of three thiopurine metabolites in human red blood cells.

## Key findings

- The method showed excellent linearity with R² values of ≥0.9997 for all three metabolites.
- Recoveries ranged between 88.74% and 117.37%, indicating good accuracy.
- The method demonstrated high specificity and precision with RSD ≤15%.

## Abstract

Background

Therapeutic drug monitoring (TDM) of thiopurine metabolites in red blood cells (RBCs) is essential to optimizing dosing regimens and minimizing adverse effects. Current analytical methods lack the desirable specificity and sensitivity needed for optimum quantification at therapeutic concentrations on a routine basis. The objectives of the current study were to validate and develop a highly repeatable and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of 6-mercaptopurine (6-MP), 6-thioguanine (6-TG), and 6-methylmercaptopurine (6-MMP) within human red cells.

Methods

Hydrophilic interaction liquid chromatography (HILIC) was employed for separation using a gradient elution system. Ammonium formate with formic acid in water (A) and acetonitrile (B) was used as the mobile phases. Multiple reaction monitoring (MRM) detection was carried out in positive mode using an electrospray ionization (ESI) source. The method was validated according to International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines and included testing for linearity, accuracy, recovery, precision, and specificity in the 0.5-1000 ng/mL concentration range.

Results

The developed method showed excellent linearity for the three analytes with a coefficient of determination (R²) of ≥0.9997. Recovery analysis showed acceptable accuracy, with the mean recoveries between 88.74% and 117.37% for all the concentration levels. The method showed high specificity without interference from endogenous matrix components. Precision study showed a relative standard deviation (RSD) of ≤15% for all the analytes.

Conclusions

The developed LC-MS/MS approach gives a reliable technique for the simultaneous quantification of thiopurine metabolites within erythrocytes. 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.