Study of relative response factors and mass balance in forced degradation studies with liquid...

A case study was performed using photodiode array detection (PDA) in combination with evaporative light scattering (ELS) detection and mass spectrometry (MS) to assess both mass balance and the relative response factors (RRFs) in the forced degradation studies of the drug substance, glimepiride. The RRF value, which is the ratio of the response factor of the impurity to that of the API, was first determined using calibration curves of standards. This conventional technique was compared to a second, multi-detection technique, which used the PDA and ELS detectors to determine both the ultraviolet (UV) peak area and the concentration (based on measurement by ELS) in a single analysis. The resulting RRF values were then applied to forced degradation studies (acidic hydrolysis and oxidation) of glimepiride drug substance. This analysis was used to assess mass balance as well as impurity quantification. The impact of applying impurity RRF values was evaluated and found to have a significant impact on the percent of impurity quantified and on mass balance calculations at higher degradation levels. In addition, MS was used to quantify a non-chromophoric by-product and assess its impact on mass balance. Analysis of the forced degradation studies by MS detection also provided confirmation of the degradation pathway with new insights. Specifically, MS revealed differences in the stereoselectivity of the degradation processes: the acidic hydrolysis degradation was found to be stereoselective with the formation of the trans by-product while the oxidation degradation produced both the cis/trans isomers in a non-stereoselective reaction.