Highly sensitive method for the determination of adenosine by LC-MS/MS-ESI: method validation and scope of application to a pharmacokinetic/pharmacodynamic study.

A highly sensitive, rapid assay method has been developed and validated for the estimation of adenosine in rat plasma with liquid chromatography coupled to tandem mass spectrometry with electro-spray ionization in the positive-ion mode. The assay procedure involves extraction of adenosine and phenacetin (internal standard, IS) from rat plasma with a simple protein precipitation extraction process. The method was validated using rat plasma with extinguished adenosine endogenous levels. Chromatographic separation was achieved using a binary gradient using mobile phase A (acetonitrile) and B (0.2% formic acid in water) at a flow rate of 0.50 mL/min on an Atlantis dC(18) column with a total run time of 4.0 min. The MS/MS ion transitions monitored were 268 → 136 for adenosine and 180 → 110 for IS. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.48 ng/mL and the linearity range extended from 0.48 to 1210 ng/mL. The intra- and inter-day precisions were in the ranges 2.32-12.7 and 4.01-9.40%, respectively.