Multi-component analysis of bile acids in natural Calculus bovis and its substitutes by ultrasound-assisted solid-liquid extraction and UPLC-ELSD.

An ultrasound-assisted solid-liquid extraction (USLE) coupled to ultra-performance liquid chromatography-evaporative light scattering detection (UPLC-ELSD) method has been developed for the simultaneous extraction and determination of six bile acids (BAs) in natural Calculus bovis and its substitutes, collected from different origins. The USLE conditions, UPLC chromatographic and ELSD conditions for BAs were optimized. Under optimum conditions, the six target analytes were efficiently extracted and baseline separated within 10 min. The limits of detection (LODs) and quantification (LOQs) for six BAs were less than 7 ng and 22 ng, respectively. Average recoveries were within the range of 98.8-100.7% with relative standard deviations (RSDs) <2% for the six analytes. This method, due to its convenience, high selectivity, fast analysis efficiency and good reproducibility can be employed for analyzing the content differences of six BAs in 40 batches of natural C. bovis and its existing substitutes. The differences of the content of each BA in natural C. bovis and its substitutes were significant, and the total contents of six BAs in 13 batches of natural C. bovis were in the range of 7.96-160.17 mg g(-1), in 20 natural C. bovis of 0-245.89 mg g(-1), in 2 artificial cultivated C. bovis of 178.48-194.22 mg g(-1), in 3 cultured C. bovis of 41.01-107.3 mg g(-1), and in 2 counterfeit C. bovis of 144.9-340.25 mg g(-1). The significant differences of multi-component contents reflected the various inherent qualities of these samples, so, the use of these substitutes as the replacers of natural source in clinic should be paid more attention. Some substitutes could not be used as the replacers.