Development of a LC-MS/MS method for simultaneous determination of bile acids and their conjugates in hepatocytes, tissue and fluids in rat

Background: It is still uncertain whether the accumulation of the bile acids (BAs) in hepatocytes beyond the normal stand after taking medicine which can cause hepatic injury or under the pathological state. Because it is lack of valid and simple analytical methods for the quantification of individual BAs and their taurine and glycine conjugates in the hepatocytes. It is necessary to develop methods for BAs quantification in hepatocytes to study their function in detail. Methods: A simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous determination of 6 major BAs, their glycine, and taurine conjugates in rat hepatocytes, liver, bile, urine, and plasma. Results: This method is valid and sensitive with a limit of quantification 5 ng/mL, a large dynamic range (from 4.30 ng/mL to 200000 ng/mL for various analytes). The total run time was 21 min. Detailed BA profiles were obtained from rat hepatocytes, liver, plasma, bile, and urine using this method. The result indicated that total BA concentration in rat hepatocytes was 10892.9 ng/mg. The taurine conjugates BAs constitute 56.2% of the total BAs, unconjugated BAs possess 34.3%, and less than 7.5% are glycine conjugated. Conclusion: A sensitive and a simple LC-MS/MS method was described for the simultaneous quantification of the major BAs, as well as their glycine and taurine conjugates in rat hepatocytes, liver, bile, plasma, and urine.