Characterization and quantitative analysis of related substances in Coenzyme A by HPLC and LC-MS/MS

Abstract

Ten related substances in coenzyme A (CoA) were detected using a newly developed gradient reverse phase high performance liquid chromatographic (HPLC) method. A highly specific and efficient LC-MS/MS method was developed to characterize process-related substances and the major degradation products, and three unknown related substances were further synthesized and characterized by H-NMR and C-NMR spectroscopy. Synthesized samples of the related substances were used for quantitative analysis by HPLC. The method was validated according to ICH guidelines with respect to specificity, precision, accuracy and linearity. The forced degradation studies included acidic, alkaline, oxidative, photolytic and thermal stress conditions. Furthermore, we depicted and speculated the probable mechanism of formation of related substances and the plausible fragmentation mechanism showed that all the related substances came from the manufacturing process. Characterization, synthesis and quantitative analysis of related substances were discussed in detail, and were critical for quality control, manufacturing process optimization and CoA monitoring, all of which were important to ensure the security of CoA.

Keywords: Characterization; Coenzyme A; LC–MS/MS; Related substances.