Development and validation of an LC-MS/MS method for the determination of a novel thienoquinolin urea transporter inhibitor PU-48 in rat plasma and its application to a pharmacokinetic study.

A specific, sensitive and stable high-performance liquid chromatographic-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantitative determination of methyl 3-amino-6-methoxythieno [2,3-b]quinoline-2-carboxylate (PU-48), a novel diuretic thienoquinolin urea transporter inhibitor in rat plasma. In this method, the chromatographic separation of PU-48 was achieved with a reversed-phase C-18 column (100 x 2.1mm, 3m) at 35 degrees C. The mobile phase consisted of acetonitrile and water with 0.05% formic acid added with a gradient elution at flow rate of 0.3mL/min. Samples were detected with the triple-quadrupole tandem mass spectrometer with multiple reaction monitoring mode via electrospray ionization source in positive mode. The retention time were 6.2min for PU-48 and 7.2min for megestrol acetate (internal standard, IS). The monitored ion transitions were mass-to-charge ratio (m/z) 289.1 229.2 for PU-48 and m/z 385.3 267.1 for the internal standard. The calibration curve for PU-48 was linear over the concentration range of 0.1-1000ng/mL (r(2) > 0.99), and the lower limit of quantitation was 0.1ng/mL. The precision, accuracy and stability of the method were validated adequately. The developed and validated method was successfully applied to the pharmacokinetic study of PU-48 in rats.