Development and validation of a LC-MS/MS method for the quantitative analysis of the adenosine A2a receptor antagonist NIR178 and its monohydroxy metabolite in human plasma: Application to clinical pharmacokinetics.

We report a selective LC-MS/MS method for the simultaneous quantitative determinations of the adenosine A2a receptor antagonist NIR178 (NIR178) and its major metabolite NJI765 in human plasma. Sample preparation steps involved protein precipitation, sample evaporation and reconstitution using a plasma sample volume of 0.1 mL plasma. Separation was achieved in 10 minutes on an Acquity UPLC BEH C18 1.7 μm, 2.1 x 50 mm column heated at 60°C with a gradient elution at 0.6 mL/min mobile phase made of water and acetonitrile both acidified with 0.1% formic acid. The detection was performed in positive ion mode and quantification based on multiple reaction monitoring (MRM). The linear response range was 1.00 to 1000 ng/mL using 1/x2 weighting factor. The intra- and inter-day accuracies (bias %) and intra- and inter day precisions (CV %) obtained for NIR178 and NJI765 were within the acceptance criteria. The normalized NIR178 and NJI765 matrix factor (MF) calculated from 6 lots of normal, lipeamic and haemolyzed plasmas ranged from 0.97 to 1.05. The normalized recoveries of both NIR178 and NJI765 to that of their internal standards were consistent and reproducible with a CV% not greater than 8.0. This method was successfully applied to support pharmacokinetic studies in adult patients with cancer.