Development of a sensitive bioanalytical method for determination of PNU-83757 in rat, monkey and human plasma: from LC-UV to LC-MS/MS

Abstract

To support pre-clinical pharmacokinetic/toxicokinetic (PK/TK) evaluation, a sensitive bioanalytical method for determination of N-cyano- N′-( tert-pentyl)- N″-(3-pyridinyl) guanidine (PNU-83757), in rat and monkey plasma was required. Although the UV response of PNU-83757 was quite decent and the extracts using solid phase extraction (SPE) were very selective and concentrated, the best limit of quantitation (LOQ) achieved was 0.4 ng ml −1 using 0.5 ml plasma for extraction and 2 ng ml −1 using 0.1 ml plasma for extraction, which was insufficient for PK/TK evaluation at lower doses. When using liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometric detection (LC-APCI-MS/MS, positive ions) and SPE, a LOQ of 0.045 ng ml −1 for PNU-83757 was reached. Quantitation was accomplished using the precursor→product ion combinations of m/ z 232→162 for PNU-83757 and m/ z 236→166 for the internal standard, [ 2H 4]PNU-83757, in the multiple reaction monitoring mode. This method has been successfully utilized for PK/TK evaluation in pre-clinical studies and proved to have sufficient sensitivity to determine plasma concentrations for a dose level as low as 1 μg kg −1 day −1 in the rat and monkey. Further improvement of this method by using electrospray mass spectrometric detection (LC–ESI–MS/MS, positive ions) and automated membrane SPE, gave an LOQ of 0.008 ng ml −1, and allowed analysis of large numbers of samples to support clinical PK studies in μg dose levels.