Development of a Suitable HPLC Method for the Analysis of Impurities in Clorprenaline and Bromhexine Capsules and Identification of Unknown Impurities by 2D LC-Q-TOF MS

Abstract

In order to determine the impurities in compound clorprenaline and bromhexine capsules, a new stability-indicating HPLC method was established. A Boston Green ODS column was used, and the UV detection was 225nm. The influences of the gradient elution program, the composition ratio of the mobile phase and the velocity of flow on the separation of impurities in clorprenaline and bromhexine capsules were investigated. The developed methods were validated by assessing linearity, range, accuracy, the limit of detection (LOD), the limit of quantitation (LOQ), and robustness. The experimental results showed that the three components had good linearity, and the correlation coefficients were greater than 0.999. The recoveries of clorprenaline hydrochloride, decloxizine hydrochloride, and bromhexine hydrochloride were in the range of 95.0 to 102.3%. The LOD and LOQ values of clorprenaline hydrochloride, decloxizine hydrochloride, and bromhexine hydrochloride were between 0.20 and 0.82 μg•mL-1. Moreover, two unknown impurities were identified by two-dimensional liquid chromatography-tandem with quadrupole/time-of-flight mass spectrometry in electrospray ionization mode. Impurity 1 was derived from chlorohydroxazine hydrochloride, which has not been reported in the literature. Impurity 2 was derived from bromhexine hydrochloride, and its structure was the same as that of impurity E in EP 10.0 quality standard for bromhexine hydrochloride. The established method was highly specific, sensitive, accurate, and suitable for routine quality control of clorprenaline and bromhexine capsules. The structures of unknown impurities were characterized by the MS/MS data.