Analysis of unstable degradation impurities of a benzodiazepine and their quantification without isolation using multiple linear regression

•Molibresib, a benzodiazepine, degrades reversibly in acidic conditions.•Two degradation impurities could not be isolated for relative response factor (RRF) determination by traditional means.•The RRFs of the impurities were determined without isolation using HPLC-UV data and multiple linear regression analysis. This manuscript presents a novel methodology for calculating the relative response factors (RRFs) of unstable degradation impurities of molibresib (1). The degradation impurities were observed by HPLC during stress testing and were accompanied by large mass balance deficits. However, the impurities could not be isolated for traditional RRF determination due to their instability. The RRFs of two degradation impurities were determined without isolation by multiple linear regression analysis of HPLC-UV data. The results permitted accurate quantification of the degradants. The benefits and drawbacks of the approach are discussed, including suggested validation acceptance criteria.