B-184 Development and Validation of a High-performance Liquid Chromatography-Mass Spectrometry Method for Analyzing Phenolic Acids Metabolites in Feces
Authors: Odat B , Abdelkader N 
Published: September 2023

Abstract

Background
Phenolic acids are beneficial metabolites produced by Co-metabolism between a human host and the gastrointestinal microbiota. They play a part in signaling pathways involved in intestinal mucosa homeostasis. The synergic relationship between the microbiota and the nutrient in the gut is associated with the content of phenolic compounds found in the gut system. Recent studies have proven that phenolic acids are linked to weight loss, but such an association needs to be better understood. This research aims to develop and validate a Liquid Chromatography tandem Mass spectrometry (HPLC-MS/MS) method for quantifying phenolic acids in human fecal samples according to the food and Drug Administration (FDA) guidelines. Moreover, the research focuses on the synergic relationship between the gut microbiota and the prebiotics, and their ability to enhance the production of phenolic acids, as an increase in the phenolic acid level in the gut may reduce obesity.

Methods
High-pressure liquid chromatography is used to separate the analytes (ferulic acid, gallic acid, and caffeic acid) based on polarity, and AB SCIEX QTRAP triple quadrupole Tendon mass spectrometer is used to conduct mass spectroscopy using electrospray ionization. Solid phase extraction method was used to extract and purify the sample with C18 SPE cartridges before analysis. Quantitative analysis was performed by Analyst software. The developed LC-MS/MS method was applied to fecal samples collected from healthy populations. For testing the influence of prebiotics on the production of phenolic acids. Samples of gut microbiota (lactobacillus acidophilus) mixed with different prebiotics were monitored to measure the outcomes of phenolic acids. Samples were compared with other prebiotics such as fiber, cellulose, and inulin. Target analytes meeting significance of (P &lt; 0.05) will be verified by the development of a targeted LC-MS/MS (MRM) assay. Once compounds of significance have been verified, the method will be evaluated based on the guideline from 2018, Bioanalytical method validation guidance for industry; several parameters must be considered when validating a method. These parameters include selectivity, accuracy, precision & recovery, linearity, sensitivity (LOD and LOQ), stability, and matrix effect. Validation will be applied to the stool samples from an average healthy population.

Results
Phenolic acids were extracted and quantified using a simplified HPLC-MS/MS, with acidified methanol. A total of five phenolic acids with different concentrations were identified in different fecal samples. The method was used to determine the association between gut microbes and nutrient sources and their influence on the production of phenolic acids when mixed. Initial preliminary data indicate that adding prebiotics to a probiotic media increases the peak intensity suggesting an expansion of the quantity of phenolic acid in the mixture. In addition, changing the amount or the type of prebiotics affects the outcomes. Fiber shows the most significant outcomes when compared to other prebiotics.

Conclusion
Based on this research’s findings, certain types of Prebiotics may enhance the generation of phenolic acid quantity through co-metabolism between a human host and Probiotics in the gut. Fiber showed the most significant outcomes of phenolic acid formation compared to inulin and cellulose.