Introduction: The quantitative analysis of bile acids in plasma is critical for diagnosing many structural liver diseases. There are two main types of bile acids based upon their functional groups: unconjugated (or free) and conjugated, primarily with glycine- or taurine-based residues. In this study, 17 bile acids were being analyzed by LC-MS/MS using a Raptor C18 column. Through routine validation, a matrix interference was identified to be coeluting with one of the bile acids standards, which resulted in quantitation issues. As the interference could not be resolved on the Raptor C18 column, it was necessary to explore alternative stationary phase chemistries for effectively separating the matrix interference.
Methods: 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.
Preliminary Data: 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 17 bile acids, including full resolution of the isomer sets and the matrix interference in a 9.5-minute cycle time. Acceptable linearity, accuracy, and precision were demonstrated over an analytical measurement range of 0.5-5 µM for all analytes using the developed method.
Novel Aspect: The developed method overcomes challenges associated with bile acids analysis while also resolving a matrix interference that prevented proper quantitation.
