Development and validation of an ultra-high performance liquid chromatography/triple quadrupole mass spectrometry method for analyzing microbial-derived grape polyphenol metabolites

Abstract

Accumulating evidence indicates that the health impact of dietary phenolic compounds, including the principal grape-derived polyphenols, (+)‑catechin and (-)‑epicatechin, is exerted by not only the parent compounds but also their phenolic metabolites generated by the gut microbiota. In this work, a new high-throughput, sensitive and reproducible analytical method was developed employing ultra-high performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS) for the simultaneous analysis of 16 microbial-generated phenolic acid metabolites (PAMs) along with their precursors, catechin and epicatechin. Following optimizing the solvent system, LC conditions and MS parameters, method validation was carried out to evaluate the sensitivity, selectivity, accuracy and precision of the proposed method, and to ensure promising recovery of all analytes extracted from the matrix prior to bioanalysis. Results showed that the optimized analytical method allowed successful confirmation and quantitation of all analytes under dynamic multiple reaction monitoring mode using trans‑cinnamic acid‑d as an internal standard (I.S.). Excellent sensitivity and linearity were obtained for all analytes, with lower limits of detection (LLODs) and lower limits of quantification (LLOQs) in the ranges of 0.225-2.053 ng/mL and 0.698-8.116 ng/mL, respectively. By examining blank matrix spiked with standard mixture at different concentration levels, promising recoveries at two spiking levels (low level, 91.2-115%; high level 90.2-121%), and excellent precision (RSD < 10%) were obtained. This method was then successfully applied to an in vitro study where catechin/epicatechin-enriched broth samples were anaerobically fermented with gut microbes procured from healthy human donors. All sources of bacteria employed showed remarkable activity in metabolizing grape polyphenols and distinct variations in the production of PAMs. The successful application of this method in the in vitro fermentation assays demonstrates its suitability for high-throughput analysis of polyphenol metabolites, particularly catechin/epicatechin-derived PAMs, in biological studies.

Microbial Phenolic Metabolites: Which Molecules Actually Have an Effect on Human Health?

“…In a study by Zhao et al [52], a drink supplemented with catechin and epicatechin was inoculated with gut microbiota from healthy human participants and incubated at 37 °C for 24 h under anaerobic conditions. After this treatment, all catechin and epicatechin was metabolised and significant production of gallic, 3,4-dihydroxybenzoic and homovanillic acids was detected.…”

Chemical, Manufacturing, and Standardization Controls of Grape Polyphenol Dietary Supplements in Support of a Clinical Study: Mass Uniformity, Polyphenol Dosage, and Profiles

“…Due to highly priced commercial phenolic compound standards, we first screened the CGJ using our published UHPLC-QQQ-MS/MS method ( 41 ), and identified 21 phenolic compounds in CGJ DS samples, including 3-hydroxytyrosol isochlorogenic acid, 3,4-dihydroxybenzoic acid, 4-methyl gallic acid, catechin, procyanidin B2, epicatechin, 3-hydroxybenzoic acid, caffeic acid, 4-hydroxybenzoic acid, vanillic acid, dihydromyricetin, syringic acid, resveratrol-3-glycoside, dihydroferulic acid, 3-hydroxycinnamic acid, taxifolin, sinapic acid, ferulic acid, phenylacetic acid, and trans -2-hydroxycinnamic acid. Then, the UHPLC-QQQ-MS/MS method was optimized based on 21 phenolic compounds.…”

Rapid screening of glycoalkaloids in Solanum scabrum and S. nigrum berries using ultra‐high‐performance liquid chromatography with pathway‐specified in‐source fragmentation tandem mass spectrometry

“…The long cycle time could easily be reduced by using a shorter dwell time which, however, could be to the detriment of method sensitivity. Updating the MRM method to the dynamic version (dMRM) may help increase the allotted dwell time for each transition and/or increase collectable data points across chromatographic peaks, 15 but simultaneous determination of the retention time for all glycoalkaloids listed in the transition table in this specific work is very difficult to successfully achieve. Selection of intact glycoalkaloids as the precursor ions further complicates the provision of a clear solution.…”