HPLC-refractive index detector(HPLC-RID)method and HPLC-fluorescent detector(HPLC-FLD)method were established for determination of hyocholic acid,hyodeoxycholic acid,chenodeoxycholic acid contents in mixed feed additives,and the results of two HPLC methods,gravimetric method and volumet-ric method were compared and analyzed.In HPLC-RID method,the samples were extracted by methanol-ace-tonitrile-water solution,separated by C18 column and determined by HPLC-RID.Limits of quantification(LOQ)for the three bile acids ranged from 10.0 to 20.0 mg/g,with average recoveries between 82.54％and 108.50％,and relative standard deviations(RSD)spanning 2.90％to 7.61％.In HPLC-FLD method,samples were methanol-extracted,saponified with a potassium hydroxide methanol solution,derivatized using a 1,4,7,10,13,16-hexaoxetane acetonitrile solution and a4-bromomethyl-7-methoxycoumarin acetonitrile solution,fol-lowed by C18 column chromatography and HPLC-FLD quantification.The LOQ for the bile acids were set at 2.0 to 3.0 mg/g,average recoveries at 70.43％to 84.31％,and RSD at 4.82％to 9.80％.The results showed that both HPLC-RID and HPLC-FLD methods could determine the contents of hyocholic acid,hyodeoxycholic acid and chenodeoxycholic acid in mixed feed additives.However,due to the derivatization required in the HPLC-FLD method,results were more susceptible to the sample matrix factors,which leaded to reduce repro-ducibility and stability.The total bile acid content in mixed feed additives was also measured using gravimetric and volumetric methods.Due to the limitation of method characteristics,the results of volumetric method can-not accurately reflect the total bile acid contents in products.The HPLC-RID method,characterized by its high reproducibility,stability,and reliability,is preferable for measuring the contents of hycholic acid,hyodeoxy-cholic acid,and chenodeoxycholic acid in mixed feed additives.The gravimetric method is deemed suitable for total bile acid contents in these additives.[Chinese Journal of Animal Nutrition,2024,36(3):1942-1951]
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