Quantitative Analysis of Bile Acids in Human Plasma by Liquid Chromatography-Electrospray Tandem Mass Spectrometry: A Simple and Rapid One-Step Method

Abstract

Bile acids play a pivotal role in the metabolism of cholesterol and lipids. Their blood concentrations are important prognostic and diagnostic indicators of hepatobiliary and intestinal dysfunction. This class of molecules comprises a heterogeneous group of compounds with a common cholesterol scaffold. Recently, the introduction of liquid chromatography coupled to tandem mass spectrometry methods has revealed an innovative path in the quantisation of specific bile acids in biological specimens. A robust and sensitive method has been developed based on high performance liquid chromatography separation coupled to an electrospray triple-quadrupole mass spectrometer. Human plasma samples were analysed on a C18 reverse-phase column. The elution profiles were monitored in multiple reaction-monitoring mode, quantifying and identifying each analyte by its own unique precursor to product patterns. A linear correlation over a broad range of bile acid concentrations (0.1-100 microM) was observed. The average recovery period for all of the analysed bile acids was 98 +/- 3%. Intra-day and inter-day precision averages were 2% and 5.4%, respectively. The determination was achieved within a single chromatographic run for all unconjugated, glycine- and taurine-conjugated isomeric forms of bile acids. As a proof of principle this method has been validated on a small subset of cholestatic patients (n = 7) and compared to appropriate clinical controls (n = 10). Based upon our encouraging experimental results, the described HPLC separation coupled to tandem mass spectrometry method for the analysis of bile acids in biological samples is deemed a robust and accurate procedure. Consequently, we propose this technique as a suitable candidate method for the identification and quantitation of bile acids in routine analysis.

Quantification of the 15 major human bile acids and their precursor 7α-hydroxy-4-cholesten-3-one in serum by liquid chromatography–tandem mass spectrometry

“…Table 4 summarizes and compares extraction recoveries of the here and previously reported methods for the measurement of bile acids and C4. Extraction recoveries for bile acids were quite similar to those reported for other existing methods [16][17][18]20,[22][23][24]. The extraction recovery for C4 was found to be approximately 62%, which was markedly lower than for bile acids and for other C4 methods [12,14,[25][26].…”

“…In contrast to gas chromatography, which requires laborious sample preparation including hydrolysis and derivatisation, liquid chromatography coupled with mass spectrometry (LC-MS(/MS)) nowadays represents the most convenient alternative for this type of analysis, as demonstrated by the number of new quantification methods developed in the past years for bile acids [16][17][18][19][20][21][22][23][24] and C4 [12,14,[25][26].…”

“…Our new methods each required only 100μl of serum sample, which was less than for many of the existing methods [12,14,16,20,[22][23][24] although methods for quantification of C4 in even smaller sample volumes exist [25][26]. A low sample volume represents an important advantage if the method is to be adapted for analysis of mouse samples (muricholic acids), since mice have a very small blood volume.…”

Bile acid profiles over 5 years after gastric bypass and duodenal switch: results from a randomized clinical trial

“…Serum BA profile were analyzed with high performance liquid chromatography (Jasco TM , USA) coupled to tandem mass spectrometry (Applied Biosystems, Cheshire, UK), in the Department of Clinical Biochemistry at the King's College Hospital NHS Foundation Trust in London, using established modifications of a previously described method [25]. The BAs quantified included …”

Intrahepatic Cholestasis of Pregnancy: The Usefulness of Serum Bile Acid Profile for Diagnosis and Treatment

“…In contrast, HPLC coupled to mass (MS) detector, represents a sensitive and selective chromatographic technique to determine BA in biological matrices. Several MS techniques by ionization have been used for BA analysis like HPLC-MS and HPLC-MS/MS, though specially applied to the analysis of BA derivatives (Bootsma et al, 1999;Tagliacozzi et al, 2003), being the electrospray ionization (ESI) the best detection system. In contrast, unconjugated BA are not detectable by MS/MS due to the lack of any specific fragment ions originated from their molecules.…”