Quantification of Bile Acids in Traditional Animal Medicines and Their Preparations Using Ultra High-Performance Liquid Chromatography-Mass Spectrometry in the Multiple Reaction Monitoring Mode

Abstract

An ultra-high-performance liquid chromatograph-triple quadrupole mass spectrometry has been established and validated for the simultaneous quantification of 15 bile acids in four traditional animal medicines and their preparations. The separations of bile acids were performed on an Agilent ZORBAX Eclipse XDB-C18 column (50 × 2.1 mm; 1.8 μm) with methanol-0.1% formic acid as the mobile phase. Glycyrrhetinic acid was added as internal standard owing to its similar physiochemical properties with the bile acids. Using this condition, detected in the multiple reaction monitoring mode, the 15 bile acids, including three groups of isomers, were well quantified individually. Method validation showed that the linear regression relationship (r(2), 0.9993 - 0.9999), precisions (intra-day RSD, 0.96 - 4.31%; inter-day, 1.73 - 4.43%), and recovery (95.3 - 120.9%) were all satisfactory. The analysis results showed that bear bile and bezoar (Niu Huang) as well as their formulations contained large amounts of most of the 15 bile acids. In addition, this research revealed for the first time the presences of bile acids in animal waste medication used in traditional medicine from two clinics, Hei-Bing-Pian (discharges of wild boar) and Trogopterus Dung. The established method could be used for the quantification of other bile- or animal waste-based crude drugs and their formulated products.

Fecal medicines used in traditional medical system of China: a systematic review of their names, original species, traditional uses, and modern investigations

“…At present, there are some studies involving the quality control of Wu-Ling-Zhi. Yerigui et al [21] quantified five bile acids (i.e., cholic acid, deoxycholic acid, lithocholic acid, ursodeoxycholic acid, and taurocholic acid) in Wu-Ling-Zhi by using ultra high-performance liquid chromatography-mass spectrometry. Jiao et al [22] established a thin layer chromatography (TLC) method for qualitative identification of Wu-Ling-Zhi, and developed a high performance liquid chromatography (HPLC) method to simultaneously quantify three active ingredients (protocatechuic acid, hinokiflavone and amentoflavone) in Wu-Ling-Zhi.…”

“…The elements with high levels are Ca (18,570 μg/g), K (11,625 μg/g), Mg (9975 μg/g), and Fe (7800 μg/g). Furthermore, two bile acids (i.e., cholic acid and taurocholic acid) were detected and quantified in Hei-Bing-Pian by using ultra high-performance liquid chromatography-mass spectrometry method [21]. Besides, Chang et al [39] developed a spectrophotometric method to determine the absorption force of Hei-Bing-Pian on tartrazine, and used this adsorption force as an indicator to control the quality of Hei-Bing-Pian.…”

“…These diet-derived metabolites may be the pharmacologically active ingredients of Wu-Ling-Zhi. Moreover, some bile acids (e.g., deoxycholic acid, lithocholic acid and taurocholic acid) have been found in Wu-Ling-Zhi and Hei-Bing-Pian [21]. These bile acids are the final metabolites of cholesterol under the common metabolism of liver and gut microbiota.…”