Nemani_2018_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_1096_214

Ethyl 3-(2-(4-fluorophenyl)amino)-4-phenylthiazo)-5-yl)-3-oxopropanoate is a novel molecule with potent acetylcholinesterase inhibition property. In this research, we have developed a rapid and selective RP-HPLC bioanalytical method for quantitative measurement of the molecule. The method has been validated following the USFDA bioanalytical method validation guideline. In addition, as a part of drug development, in vitro metabolite identification has also been performed. A Kromasil C18 column was used in eluting the molecule chromatographically. The optimized mobile phase was composed of a mixture of acetonitrile and 10mM ammonium formate buffer (pH4.6) in 70:30 ratio (v/v). The response of the molecule was found to be linear over a calibration range of 0.2mug/mL to 12mug/mL. The inter-day and intra-day accuracy of the method ranged from 89.95% to 101.90% and 99.84% to 104.08%, respectively. On the other hand, the precision (%CV) value for inter-day was in between 3.50% to 6.91% and for intra-day, it was 2.11% to 8.03%. The mean recovery of the molecule at three different quality control levels was more than 85%. The analyte was stable under different stability conditions including 12h autosampler, 8h bench top, 15days long term at -20 degrees C and three freeze-thaw cycles. The method was applied to determine stability of the molecule in human plasma. The molecule was found to be stable with more than 90% remaining even after 120min of incubation in plasma. Two metabolites each in rat liver microsome and human liver microsome has been identified.