Shafaei Armaghan, Rees Joanna, Christophersen Claus T, Devine Amanda, Broadhurst David, Boyce Mary C
Centre for Integrative Metabolomics and Computational Biology, School of Science, Edith Cowan University, Joondalup, WA, 6027, Australia.
School of Medical and Health Sciences, Edith Cowan University, Joondalup, WA, 6027, Australia.
Anal Chim Acta. 2021 Mar 15;1150:338224. doi: 10.1016/j.aca.2021.338224. Epub 2021 Jan 22.
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.
随着肠道微生物组研究的迅速发展，粪便胆汁酸作为饮食相关疾病易感性的潜在生物标志物越来越受到监测。因此，用于其分析的快速、稳健和可靠的方法变得越来越重要。本文描述了一种用于分析粪便中胆汁酸的简单提取方法，该方法适用于随后通过液相色谱和串联质谱进行定量分析。一根C18柱分离分析物，峰形优异，在800次进样中保留时间重复性良好。日内和日间精密度和准确度均大于80%。回收率在83.58%至122.41%之间。检测限和定量限分别在2.5 - 15 nM范围内。优化后的方法包括从湿粪便中提取胆汁酸，且净化步骤最少。取第二份粪便样本干燥并称重以校正水分含量。从干燥粪便中提取显示回收率降低，可通过在干燥前向粪便中加入氘代标准品来校正。在LC-MS分析之前，将提取物和标准品储存在冷藏自动进样器中是必要的。提取物和标准品多次冻融导致某些胆汁酸的回收率不佳。该方法已成功应用于100份人类粪便样本。
