The analysis of bile acids in human plasma is an important diagnostic tool for detection of liver disease and as indicators of potentially harmful side effects of new drugs. There are two main types of bile acids based upon their functional groups: unconjugated bile acids and conjugated bile acids, primarily glycine- or taurine-bound. In this study, 13 bile acids were being analyzed by LC-MS/MS using a Raptor C18 50 x 2.1 mm, 1.8 μm column, but a matrix interference was identified to be coeluting with one of the bile acids standards and causing quantitation issues, so it was necessary to develop a new method on an alternative column chemistry.
Biphenyl, FluoroPhenyl, and ARC-18 stationary phases were tested on a 100 x 2.1 mm, 2.7 μm column dimension using water with 5 mM ammonium acetate as mobile phase A and 50:50 methanol:acetonitrile as mobile phase B. The column temperature was 50°C. Data was collected in ESI- mode.
The biphenyl stationary phase was able to partially resolve the matrix interference and showed selectivity for the taurine and glycine conjugated isomers, but limited selectivity for the unconjugated isomers. The fluorophenyl stationary phase was able to resolve the matrix interference but did not show selectivity for the three isomer sets. The ARC-18 stationary phase was able to resolve the matrix interference and showed selectivity for the three isomer sets. The gradient using the ARC-18 column was optimized to separate all 13 bile acids, including full resolution of the isomer sets and the matrix interference in a 9.5 minute cycle time.
Quantitation of bile acids in matrix is challenging for a number of reasons, such as structural similarities, varying polarity and stereochemistry, the presence of isomers, limited fragmentation of unconjugated bile acids in a mass spectrometer, high endogenous levels, and matrix effects caused by phospholipids or triglycerides. The developed LC-MS/MS method utilizing a Raptor ARC-18 column managed to overcome all of these challenges while also resolving a matrix interference that was preventing proper quantitation.
