A new methodology for the analysis of bile acid profile in human serum based on an ultrafiltration clean-up step and LC-MS/MS analysis.

Bile acids (BAs), the major end products of cholesterol catabolism, are useful biomarkers for the diagnosis of many diseases. The pathologies related with bile acids are generally expressed in the first years of life and may lead to serious liver injury. Usually the balance between bile acid synthesis, secretion and reabsorption is tightly regulated: elevated concentrations of bile acids in peripheral circulation are evidence of disorders; similarly, low concentrations may suggest inborn errors of bile acid metabolism [1]. Measurement of total bile acids, as often performed in routine analysis, is only of limited value whereas the analysis of BAs profile in body fluids is an important tool to establish the therapy (pharmacological or surgical) [2]. Here we present a sensitive and rapid method for the analysis of the main 15 bile acids in human serum by liquid chromatography-tandem mass spectrometry. The chromatographic separation is performed using a core-shell column which provides a good separation, particularly useful because of the small structural differences of the analytes. All isomeric BAs of interest were resolved from each other in less than 10 min. Serum sample pretreatment only requires an ultrafiltration step with centrifugal filter devices. This simple procedure on the one hand allows a minimal consumption of serum sample (about 100 μl) and on the other hand is simple, rapid and easily applicable in a routine analysis getting anyhow a satisfying clean up. The calibration curves were linear for all the BAs over a range of 0.005–5 ppm. The extraction recoveries for all the analytes were greater than 80%. Intra-day and inter-day coefficients of variation were all below 15%. The method proposed has been validated according to FDA guidelines for bioanalytical methods and has been applied for the serum analysis of pediatric patients.