LAUSR

HighlightsNew HILIC‐HPLC‐MS method was developed for determination of TPMT activity.HILIC‐HPLC‐MS combination was approved by excellent performance parameters.HILIC‐HPLC‐MS was compared to traditional RP‐HPLC‐UV as well as advanced RP‐HPLC–MS.Conventional approaches were surpassed in terms of selectivity, LOD, analysis time.HILIC‐HPLC‐MS is favorable for routine assay of 6‐MMP/6‐MP ratio in RBC lysates. Abstract Thiopurine S‐methyltransferase (TPMT) plays an important role in the metabolism of thiopurines used in the therapy of inflammatory bowel diseases (IBD). In this work a new progressive method for the determination of TPMT activity in red blood cells lysates was developed. Analysis was carried out by means of hydrophilic interaction liquid chromatography (HILIC) hyphenated with mass spectrometry (MS). In comparison with reversed‐phase high‐performance liquid chromatography (RP‐HPLC), that has been typically applied in determination of TPMT activity, the HILIC significantly improved the analytical signal provided by MS, shortened analysis time, and improved chromatographic resolution. The HILIC‐HPLC‐MS method was optimized and validated, providing favorable parameters of detection and quantitation limits (5.5 and 16.5 pmol/mL, respectively), linearity (coefficient of determination 0.9999 in the range of 0.01–1.0 nmol/mL), recovery and precision (93.25–100.37% with RSD 1.06‐1.32% in the whole concentration range of QC samples). Moreover, in contrast to the conventional RP‐HPLC‐UV approach, the complex phenotype TPMT profiles can be reliably and without interferences monitored using the HILIC‐HPLC‐MS method. Such advanced monitoring can provide valuable detail information on the thiopurines (e.g. evaluating ratio of methylated and non‐methylated 6‐mercaptopurine) and, by that, TPMT action in biological systems before and during the therapy of IBD.