An Optimized Sample Pretreatment Method Followed by a Validated LC-MS/MS Method for Quantifying 6-TG and 6-MMP...

A rapid and sensitive LC-MS/MS method was developed and validated for quantifying 6-TG and 6-MMP concentrations in erythrocytes. This method is applicable for routine therapeutic drug monitoring in inflammatory bowel disease patients using thiopurines. Red blood cell (RBC) count was performed after washing the blood samples. After storage at -20°C, protein precipitation followed by acidic hydrolysis was performed using 0.062 mol/L DTT and 1.36 mol/L perchloric acid with an incubation time of 60 min at 100°C. Chromatographic separation was performed on an Acquity UPLC HSS T3 column (2.1 × 100 mm, 1.8 μm) using ammonium formate 5 mmol/L in 0.1% aqueous formic acid and methanol in gradient elution at a flow rate of 0.4 mL/min. Detection was performed in multiple reaction monitoring mode using positive electrospray ionization. The method was linear over a calibration range of 25-1500 pmol/100 μL for 6-TG and 250-8000 pmol/100 μL for 6-MMP. The method demonstrated good performance in terms of intraday and interday accuracy (96.2%-110.8%) and precision (CV 0.9%-3.9%). Calibration and QC samples were stable for 3 months at -80°C. The method demonstrated excellent performance in proficiency testing.