Determination of 6-benzylthioinosine in mouse and human plasma by liquid chromatography–tandem mass spectrometry

This paper described the development and validation of a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the quantitative determination of 6-benzylthioinosine (6BT), a novel myeloid leukemia differentiation-inducing agent, in mouse and human plasma. In this method, 2-amino-6-benzylthioinosine (2A6BT) was used as internal standard and ethyl acetate was used as organic solvent for the extraction of 6BT and internal standard from plasma samples. The extracted samples were separated on YMC ODS-AQ® column (2.0 mm × 50 mm), and the eluates from the column were monitored by the positive-electrospray-ionization tandem mass spectrometer (ESI+-MS/MS). Quantification of 6BT by internal calibration with 2A6BT was carried out using multiple-reaction-monitoring (MRM) mode. This method had a linear calibration range of 3.00–1.00 × 103 ng/mL with correlation coefficients of 1.00 and 0.999 in mouse and human plasma. The lower limit of quantification (LLOQ) was 3.00 ng/mL in either mouse or human plasma. The method had recovery of 6BT of 82–87% from mouse plasma and 90–98% from human plasma. Both accuracy (as percent error) and precision (as coefficient of variation) of the method were ≤±15% within the calibration range. This method had been successfully applied to the pharmacokinetic study of 6BT in male C57 mice with intraperitoneal (i.p.) injection at the dose of 1 mg 6BT per kilogram mouse.