cardiovascular-agents and methyl-acrylate

As it is extremely unstable in blood, the thiol compound BMS186716 was stabilized by the addition of methyl acrylate (MA) to blood samples. The blood samples were then kept in ice for 10-15 min for completion of the Michael addition reaction to occur between the thiol group of BMS186716 and MA, after which the plasma was separated by centrifugation under refrigeration. For sample analysis, the standard and quality control samples were prepared by spiking blank plasma with the BMS186716-MA adduct. After addition of the internal standard, BMS 188035-MA, each sample was acidified with HCI and then extracted with methyl tert.-butyl ether. Each reconstituted extract was injected into a high-performance liquid chromatography-positive ion electrospray ionization mass spectrometric system. The electrospray condition was chosen to enhance the [M+NH4]+ signal at the expense of the [M+H]+ signal. Monitoring the [M+NH4]+ signal, a lower limit of quantitation of 2.5 ng/ml was achieved, with 0.5 ml plasma. We have thus shown that a sulfhydryl compound (BMS186716) in blood can successfully be stabilized by reacting it with MA and that the adduct produced is adequately stable in blood and plasma to allow the development of a rugged quantitative bioanalytical method.

Topics: Acrylates; Animals; Antihypertensive Agents; Cardiovascular Agents; Chromatography, High Pressure Liquid; Dogs; Drug Stability; Mass Spectrometry; Metalloendopeptidases; Pyridines; Thiazepines

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.

Topics: Acrylates; Animals; Cardiovascular Agents; Chromatography, High Pressure Liquid; Esterases; Hydrolysis; Mass Spectrometry; Pyridines; Rats; Reference Standards; Sensitivity and Specificity; Thiazepines