Quantitative determination of BMS-186716, a thiol compound, in rat plasma by high-performance liquid chromatography-positive ion electrospray mass spectrometry after hydrolysis of the methyl acrylate adduct by the native esterases

Abstract

During method development in support of non-clinical studies in animal models, BMS-186716 was found to be extremely unstable in blood and plasma. Stabilization of the compound was achieved by reacting the compound with methyl acrylate (MA) in blood, from which the plasma was then prepared. While the resulting BMS-186716-MA adduct was found to be stable in dog plasma, and hence it was used as the basis for the method developed for analysis of dog plasma samples, the BMS-186716-MA adduct was found to be unstable in rat plasma as it was readily hydrolyzed to BMS-186716-acrylic acid (AA) by native esterases found in rat plasma. Although the finding of the instability of BMS-186716-MA in rat plasma was not the result of prospective planning, we were able to successfully develop a quantitative bioanalytical method using BMS-186716-AA as the analyte instead of the originally planned BMS-186716-MA analyte. The standard and quality-control (QC) samples were prepared by spiking blank plasma with BMS-186716-MA, and then allowing them to stand at room temperature for 1 h to convert BMS-186716-MA to BMS-186716-AA. After adding the internal standard BMS-188035-AA, each sample was acidified with HCl and then extracted with methyl tert.-butyl ether. The reconstituted extract was injected into a HPLC-electrospray ionization mass spectrometric system for detection by positive ion electrospray ionization. A lower limit of quantitation (LLQ) of 5 ng/ml was achieved, using 0.1 ml plasma and a standard curve range of 5–5000 ng/ml.