[Determination of 30 bile acids in the bile of

Bile acids (BAs), the primary components of bile, play significant roles in sugar, lipid, and cholesterol metabolism. Normal BA metabolism maintains a dynamic equilibrium by regulating gut microbiota to effectively protect the liver and intestines, thereby sustaining overall health. Conversely, abnormal BA metabolism can cause intestinal tissue and liver damage, disruption of enterohepatic circulation homeostasis, dysbiosis of gut microbiota, and gastrointestinal and hepatic diseases. Although largemouth bass (Micropterus salmoides) and grass carp (Ctenopharyngodon idella) are important freshwater fish species in China, their BA profiles have not been fully characterized. Elucidating these profiles could provide valuable data to support the aquaculture industry. In this study, a qualitative and quantitative method for the simultaneous determination of 30 BAs in the gallbladders of M. salmoides and C. idella was developed using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry. The samples were centrifuged and then the supernatant was extracted, vortex-mixed with methanol, and filtered through a 0.22 μm membrane. Various mobile phase systems commonly used in liquid chromatography-mass spectrometry were investigated, including formic acid, acetic acid, ammonium formate, acetic acid-ammonium, and methanol-acetonitrile. Based on the response intensity, chromatographic separation, and peak shape of each substance, the optimal mobile phase was acetic acid and acetonitrile, and the concentration of acetic acid in water was optimized. To improve the ionization efficiency, the most effective ion scanning mode was selected by comparing the response intensities and peak conditions of each BA during mass spectrometry in positive and negative ion modes. Furthermore, the interface voltage for each BA and the ion source interface temperature were investigated to determine the optimal mass spectrometry conditions. Ultimately, separation was performed using a Shim-pack Velox SP-C18 column (100 mm×2.1 mm, 1.8 μm) with gradient elution of 0.01% acetic acid aqueous solution and acetonitrile, and the total analysis time was 14 min. The column temperature was 50 ℃ and the injection volume was 2 μL. Multiple reaction monitoring of 8 positive ions and 22 negative ions was carried out using electrospray ionization. Different series of mixed standard working solutions, prepared based on the response intensity of each BA, were used to construct standard curves. All 30 BAs exhibited good linearities within the investigated concentration ranges, with correlation coefficients (R2) of 0.9975-0.9997, indicating high accuracy. Spiked recoveries were 72.3%-117.2%, and the inter-day precisions were in the range of 0.46%-13.23%. The limits of detection and quantification were 0.01-0.75 ng/mL and 0.02-2.28 ng/mL, respectively, enabling the precise detection of low-concentration BAs. Using the established method, 19 BAs were successfully detected in the bile of M. salmoides, and 16 BAs were detected in the bile of C. idella, enriching the BA profiles of both species. Notably, five BAs present in the gallbladder of M. salmoides were undetected in the gallbladder of C. idella, whereas two BAs found in the gallbladder of C. idella were absent from the gallbladder of M. salmoides, demonstrating differences between the BA compositions of these species. The developed method is characterized by simplicity, speed, high sensitivity, and accuracy, harnessing the high-throughput advantages of ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-MS/MS), and is suitable for the simultaneous detection of the 30 BAs in the gallbladders of M. salmoides and C. idella.