Development of GC‐MS method for quantification of phenolic acids after in vitro fecal fermentation (1044.4)

Abstract

Unabsorbed polyphenols are subject to metabolism catalyzed by colonic microbiota into a wide range of phenolic acids (PA) with implications for gut health. The aim of this study was to develop a reliable, sensitive method for quantification of PA generated from in vitro anaerobic fecal fermentation. Fecal ferment containing 2% fecal matter (w/v) were purified by solid‐phase extraction (SPE) using a Phenomenex Strata SDB‐L cartridge. PA in the resulting SPE eluate were derivatized sequentially by halogenation using 30% pentafluorobenzyl bromide and 10% diisopropylethylamine and by silylation using 10% trymethylchlorosilane in BSTFA. This 2‐step protocol diminished the impact of ambient humidity on derivatization reproducibility compared to silylation alone. SPE recovery averaged 70% for 25 quantified PA. The derivatized products were separated by a 0.25 mm x 0.25 µm GC column and determined by an Agilent GC‐MS operated in an electron ionization and SIM modes. The internal standard was 4’‐hydroxy‐3’‐methoxyacetophenone. The limit of detection ranged from 1 to 10 ng/mL and the limit of quantification from 10 to 100 ng/mL. The linearity of standard curves constructed from PA spiked into the fecal ferment was r² >0.99. The intra‐day coefficient of variation for authenticated PA standards was <9% and for standards spiked into the fecal ferment was <14%. (Funded by Department of Army, CONACYT, and USDA)Grant Funding Source: Supported by Department of Army, CONACYT, and USDA