Study on the Impurity Profile and Its Correlation With the Production Process in the Estazolam API and Tablets

Estazolam is a benzodiazepine drug widely used in clinical practice. Currently, estazolam tablets on the Chinese market are generic drugs. To meet the requirements of national standards and uniformity, the impurity analysis methods were developed for active pharmaceutical ingredient (API) and tablets of estazolam.

A high-performance liquid chromatography with ultraviolet detection (HPLC-UV) method was developed to achieve enhanced sensitivity and resolution for the quantitative analysis of related substances. This method was applied to determine impurity levels in generic estazolam tablets from 12 Chinese manufacturers and APIs from 4 manufacturers. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used to determine the impurity profiles. The impurity content and impurity profiles were used as evaluation indicators to trace the correlation between the differences in impurity profiles and the production process.

The content of both eight known and unknown impurities was quantitatively determined by the HPLC-UV method. A principal component external standard method with correction factor was used for calculation, and detailed methodological validation was performed according to ICH guidelines. The structures of impurities in Chinese marketed products and the innovator drugs were qualitatively identified by the LC-MS/MS method, and differences in impurity profiles were compared.

This study identified two USP-listed process impurities, two unknown process impurities, and one non-pharmacopeial degradation product. The unknown impurities were successfully separated and preliminarily characterized.

Three API process impurities were key contributors to preparation impurity profile variations. Impurity levels showed close correlation with API synthesis routes and purification processes. A novel degradation product emerged during formulation, generated under light/heat stress, but minimally impacted tablet impurity variations.

The optimized HPLC method demonstrated enhanced sensitivity and separation efficiency. API manufacturers should prioritize purification process optimization for impurities exceeding 0.1% thresholds to ensure drug safety.