Determination of bile acids in human serum by on-line restricted access material-ultra high-performance liquid chromatography-mass spectrometry

J Chromatogr B Analyt Technol Biomed Life Sci. 2008 Jun 15;869(1-2):1-8. doi: 10.1016/j.jchromb.2008.04.045. Epub 2008 May 13.


This paper describes a new, fully automated on-line method combining restricted access material (RAM) extraction and ultra high-performance liquid chromatography (UHPLC) with mass spectrometric (MS) detection for determining congeners of bile acids (BAs) in human serum. In this method, low-pressure RAM and high-pressure UHPLC-MS are hyphenated by using a 2.5-mL loop-type interface. The compatibility problem between the large volume (1.2mL) of strong solvent (methanol) used for RAM elution and the need for a weak solvent in UHPLC injection has been addressed by using an auxiliary pre-column cross-flow of 0.1% aqueous formic acid. In this way, the complete 2.5mL loop volume can be injected into the UHPLC system, thereby maximizing sensitivity while maintaining good chromatographic performance. The optimised method allows the simultaneous analysis of 13 bile acids in a single run, including glycine- and taurine-conjugated bile acids, cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), ursodeoxycholic acid (UDCA), and litocholic acid. The complete analysis of a 100-microL single serum sample is performed in 30 min, providing detection limits in the pg range (corresponding with clinically relevant concentration levels) for all of the analytes except lithocholic acid, intra-day precision values (%R.S.D.) below 4% (except ursodeoxycholic acid) and inter-day precision lower than 15% (except ursodeoxycholic, glycoursodeoxycholic acid (GUDCA) and lithocholic acid).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bile Acids and Salts / blood*
  • Bile Acids and Salts / chemistry
  • Bile Acids and Salts / isolation & purification
  • Chromatography, High Pressure Liquid / methods*
  • Humans
  • Mass Spectrometry / methods*
  • Online Systems*


  • Bile Acids and Salts