A Validated LC–MS/MS Method for Quantifying Phenolic Acids, Lignans, and Enterolignans from Human Fecal Samples

The human gut microbiome is home to numerous small molecules that impact health. Three prominent classes of molecules in this environment include phenolic acids, lignans, and enterolignans, which have been linked to anti-inflammatory and anti-oxidant effects as well as protection from cancer, cardiovascular disease, and neurodegeneration. The abundance of these molecules in the gut microbiome as well as their biological significance motivated the development of the LC–MS/MS method reported herein, which provides a simple, robust, and high-throughput approach to simultaneously quantify a 17-membered panel of phenolic acids, lignans, and enterolignans in human fecal microbiome samples. This method employs liquid-liquid extraction, which allows for cost-effective sample preparation by using common laboratory materials. Additionally, fecal samples are lyophilized prior to extraction to mitigate confounding effects introduced by variability in water content that exists across individuals’ samples. Inclusion of 0.1% acetic acid in the chromatographic solvents optimized peak shape and signal intensity. Plastic microcentrifuge tubes imparted substantial interferences for some analytes, which was resolved through use of glass vials. Using this method, 60% of human fecal samples from 10 individuals showed that phenolic acids bearing a catechol motif were in significantly greater abundance than were guaiacols. As specific gut bacteria can transform guaiacols into redox-active catechols, determination of guaiacol:catechol ratios in fecal samples may afford a biomarker of gut microbiota anti-oxidant potential.