Objective: To develop an HPLC method for the determination of 6-methylmercaptopurine concentration and then numerate the thiopurine methyltransferase (TPMT) activity in erythrocyte via formula and analysis of the TPMT activity difference between human and pig.
Methods: The chromatographic apparatus SHIMADZU GC-2010C was used. The stationary phase was a Symmetry C18 reverse-phase column (150 mm x 3.9 mm, 5 microm). The mobile phase was 0.1% acetic acid-methanol and the flow rate 1.0 mL/min. The samples were extracted by ethyl acetate and injected automatically. They were measured at UV 280 nm. 2-amino-6-methyl-mercaptopurine was used as the internal standard.
Results: The retention times for 6-MP, AMMP and 6-MMP were 4.4 min, 7.7 min and 9.3 min respectively. The calibration curves were linear over the range of 6.25-100 nmol/mL. the methodology recovery was 98.08%-100.05%. The extraction recovery of 6-MMP was 88.1%-92.4%. The within-group RSD 1.0%-1.5% and inter-group RSD 1.0%-4.4%. The levels of TPMT activity of human, pig and Banna Minipig Inbred Line (BMI) were (17.45 +/- 3.62) U, (7.65 +/- 1.35) U and (8.73 +/- 1.55) U respectively.
Conclusion: The method is suitable for determination of TPMT activity in erythrocyte of different patients and species in that of clinical medicine and scientific research. TPMT activity of pig and BMI is obviously lower than human's.