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.

High-performance liquid chromatography–tandem mass spectrometry for the analysis of bile acid profiles in serum of women with intrahepatic cholestasis of pregnancy

“…Its major disadvantage, however, is the time-consuming derivatization prior to analysis [8]. Several HPLC methods have been described that use either ultraviolet (UV) [9] or fluorescence detection [8]. However, the shortcoming of HPLC with UV is unreliability with regard to selectivity.…”

Differentiated quantification of human bile acids in serum by high-performance liquid chromatography–tandem mass spectrometry

“…However, in combination with UV detection, it suffers from limited sensitivity and specificity due to the lack of a strong chromophore in the bile acid molecule. Various derivatization procedures have been described for modifying the bile acid structure to overcome the poor UV absorption [8]. However, such methods are time-consuming.…”

High-performance liquid chromatographic mass spectrometric method for the determination of ursodeoxycholic acid and its glycine and taurine conjugates in human plasma

“…Among the methods described in the literature, there are HPLC methods with ultraviolet, fluorescence or refractive index detection [4][5][6][7][8][9][10], HPLC with mass spectrometric detection [11,12] or gas chromatography with mass spectrometric detection [1,[12][13][14][15]. With most of these methods, free and conjugated bile acids are not analyzed simultaneously.…”