Enzymatic production of bile Acid glucuronides used as analytical standards for liquid chromatography-mass spectrometry analyses.

Abstract

The present study reports a novel method for the production and purification of analytical standards of glucuronide conjugates of bile acids, chenodeoxycholic (CDCA), lithocholic, (LCA) and hyodeoxycholic (HDCA) acids. CDCA-3G (CDCA-3-glucuronide) and -24G, LCA-3G and -24G, and HDCA-6G and -24G were enzymatically formed by using microsomes from human liver, purified by liquid chromatography, digested with recombinant beta-glucuronidase, and quantified by liquid chromatography/electrospray ionization coupled to mass spectrometry (LC-ESI/MS). The position of the glucuronosyl moiety on the bile acids was determined by analyzing the susceptibility to hydrolysis under elevated pH and temperature conditions of the standards. By using the purified analytical standards, a LC-ESI/MS/MS method was developed for the determination of these glucuronide conjugates in in vitro assays. The linearity of the assay ranged from 0.5 to 40 ng/mL for the six glucuronides, and the limit of quantification (LOQ) was 0.5 ng/mL. Intra- and interday precisions and accuracy values were all lower than 10.2%. Furthermore, processed sample stability analyses revealed that the six standards were stable at 4 degrees C for more than 24 h. This method was successfully used for the quantification of CDCA, LCA, and HDCA glucuronides formed by human liver or hepatoma HepG2 cells. In conclusion, such a method allows the purification of high-quality analytical standards of glucuronide derivatives and may easily be used for the quantification of other endo- and xenobiotics that are glucuronidated.

• Authors: Patrick Caron, Jocelyn Trottier, Mélanie Verreault, Julie Bélanger, Jenny Kaeding, Olivier Barbier
• Journal: Molecular pharmaceutics (Mol Pharm) 2006 May-Jun Vol. 3 Issue 3 Pg. 293-302 ISSN: 1543-8384 [Print] United States
• PMID: 16749861 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Validation Study)

Chemical References

• Bile Acids and Salts
• Glucuronides
• Deoxycholic Acid
• Lithocholic Acid
• hyodeoxycholic acid

Topics

• Bile Acids and Salts (chemistry, standards)
• Carcinoma, Hepatocellular (enzymology, metabolism)
• Cell Line, Tumor
• Chromatography, Liquid (methods)
• Deoxycholic Acid (chemistry, standards)
• Glucuronides (chemistry, standards)
• Humans
• Lithocholic Acid (chemistry, standards)
• Microsomes, Liver (chemistry, metabolism)
• Spectrometry, Mass, Electrospray Ionization (methods)
• Statistics as Topic