HPLC-Fluorescence Determination of Individual Free and Conjugated Bile Acids in Human Serum

Abstract

A method for the quantitative analysis of unconjugated and conjugated bile acids (BA) in serum of patients with primary biliary cirrhosis (PBC) before and after therapy with antibiotic or ursodeoxycholic acid (UDCA) is described. After separation of the free, glycine and taurine conjugated (F, G and T conjugated) fractions by solid‐phase extraction, the isolated T conjugates were hydrolysed enzymatically using cholyglycine hydrolase. The BA fractions were derivatized using 2‐bromoacetyl‐6‐methoxynaphthalene (Br‐AMN) and detected fluorimetrically (λexc=300 nm, λem=460 nm). The derivatization reaction was performed under mild conditions (10 min at 40°C) in an aqueous medium in the presence of tetrakis (decyl) ammonium bromide (TDeABr). The HPLC separation was achieved using an ODS column and with a mobile phase gradient mixture of A–B, where A is water and B is acetonitrile:methanol (60:40 v/v) for elution at a flow‐rate of 1.2 mL/min. The reproducibility, recovery and separation of individual BA under gradient elution conditions were satisfactory, allowing a sensitive detection of each BA in serum samples with a detection limit of about 1–2 pmol. © 1997 by John Wiley & Sons, Ltd.

Evaluation of Serum Bile Acid Profiles as Biomarkers of Liver Injury in Rodents

“…Numerous analytical methods have emerged to determine BA concentrations in plasma and serum (Gatti et al, 1997;Lee et al, 1997;Street et al, 1985;Thompson et al, 1987). The complexity of metabolism, the typically low concentration of BAs in biological fluids, and the existence of multiple isobaric structural isomers make BA separation and quantitation challenging (Janzen et al, 2010;Ostrow, 1993).…”

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

“…1). To improve sensitivity, some derivatization methods have been described by Burkard (Gatti et al, 1997). However, these methods are time consuming.…”

Ultra Performance Liquid Chromatography-Mass Spectrometry Profiling of Bile Acid Metabolites in Biofluids: Application to Experimental Toxicology Studies

“…Hence, sophisticated chromatographic techniques are required in order to obtain comprehensive BA profiles in disordered states. Gas chromatography−mass spectrometry provides a sensitive, specific and reproducible method for BA detection and quantitation, but disadvantages include lengthy derivatization steps and long analysis times . Liquid chromatography (LC) methods can be used with ultraviolet (UV) or fluorescence detection, but disadvantages include (a) unreliability of UV with regard to selectivityBAs lack a strong chromophoreand (b) the need for separation of BAs by conjugation class, as well as hydrolysis and derivatization prior to fluorescence detection.…”