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Enzymatic production of bile Acid glucuronides used as analytical standards for liquid chromatography-mass spectrometry analyses.
- Source :
-
Molecular pharmaceutics [Mol Pharm] 2006 May-Jun; Vol. 3 (3), pp. 293-302. - Publication Year :
- 2006
-
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.
- Subjects :
- Bile Acids and Salts chemistry
Carcinoma, Hepatocellular enzymology
Carcinoma, Hepatocellular metabolism
Cell Line, Tumor
Deoxycholic Acid chemistry
Deoxycholic Acid standards
Glucuronides chemistry
Humans
Lithocholic Acid chemistry
Lithocholic Acid standards
Microsomes, Liver chemistry
Microsomes, Liver metabolism
Statistics as Topic
Bile Acids and Salts standards
Chromatography, Liquid methods
Glucuronides standards
Spectrometry, Mass, Electrospray Ionization methods
Subjects
Details
- Language :
- English
- ISSN :
- 1543-8384
- Volume :
- 3
- Issue :
- 3
- Database :
- MEDLINE
- Journal :
- Molecular pharmaceutics
- Publication Type :
- Academic Journal
- Accession number :
- 16749861
- Full Text :
- https://doi.org/10.1021/mp060021l