Extraction and quantitative determination of bile acids in feces

With rapid advances in gut microbiome research, fecal bile acids are increasingly being monitored as potential biomarkers of diet related disease susceptibility. As such, rapid, robust and reliable methods for their analysis are of increasing importance. Herein is described a simple extraction method for the analysis of bile acids in feces suitable for subsequent quantification by liquid chromatography and tandem mass spectrometry. A C18 column separated the analytes with excellent peak shape and retention time repeatability maintained across 800 injections. The intra-day and inter-day precision and accuracy was greater than 80%. Recoveries ranged from 83.58 to 122.41%. The limit of detection and limit of quantification were in the range 2.5–15 nM, respectively. The optimized method involved extracting bile acids from wet feces with minimal clean up. A second aliquot of fecal material was dried and weighed to correct for water content. Extracting from dried feces showed reduced recovery that could be corrected for by spiking the feces with deuterated standards prior to drying. Storage of the extracts and standards in a refrigerated autosampler prior to analysis on the LC-MS is necessary. Multiple freeze-thaws of both extracts and standards lead to poor recoveries for some bile acids. The method was successfully applied to 100 human fecal samples. [Display omitted] •Development and validation of a rapid, robust and sensitive LC-MS method for quantification of BA in feces.•Simple and validated extraction procedure for robust and highly sensitive quantification of BA in feces.•Comparison of BA extraction efficiency from wet and dry feces.•Effect of short-term and long-term storage on BA stability in standard solutions and fecal extracts.