Development and validation of a novel HPLC-UV method for simultaneous determination of azathioprine metabolites in human red blood cells

A rapid, specific and accurate high-performance liquid chromatography with tunable ultraviolet detection method was developed to simultaneously determine azathioprine metabolites, 6-thioguanine nucleotides (6-TGN) and 6-methyl mercaptopurine riboside (6-MMPr) in human red blood cells. Erythrocyte lysate sample was precipitated by perchloric acid under the protection of dithiothreitol, with 6-TGN and 6-MMPr being acid hydrolyzed to produce 6-thioguanine (6-TG) and 6-methymercaptopurine (6-MMP). A Waters Cortecs C(18) column (2.1 × 150 mm, 2.7 μm) was used for chromatographic separation with a water (containing 0.01 mol/L ammonium acetate and 0.2% acetic acid)/methanol linear gradient at a flow rate of 0.45 mL/min in a 5.5 min. UV detection wavelengths were 340 nm for 6-TG, 303 nm for 6-MMP and the IS (5-bromouracil). The calibration curves fitted a least squares model (weighed 1/x(2)) from 0.15 to 15 μmol/L for 6-TG (r(2) = 0.9999) and from 1 to 100 μmol/L for 6-MMP (r(2) = 0.9998). This method was validated according to the FDA bioanalytical method validation guidance and ICH M10 bioanalytical method validation and study sample analysis guidance for industry, and successfully utilized in ten IBD patients receiving azathioprine therapy.