Screening Antioxidants Using LC-MS: Case Study with Cocoa

Oxidative stress enhances pathological processes contributing to cancer, cardiovascular disease, and neurodegenerative diseases, and dietary antioxidants may counteract these deleterious processes. Because rapid methods to evaluate and compare food products for antioxidant benefits are needed, a new assay based on liquid chromatography-mass spectrometry (LC-MS) was developed for the identification and quantitative analysis of antioxidants in complex natural product samples such as food extracts. This assay is based on the comparison of electrospray LC-MS profiles of sample extracts before and after treatment with reactive oxygen species such as hydrogen peroxide or 2,2-diphenyl-1-picrylhydrazyl radical (DPPH). Using this assay, methanolic extracts of cocoa powder were analyzed, and procyanidins were found to be the most potent antioxidant species. These species were identified using LC-MS, LC-MS/MS, accurate mass measurement, and comparison with reference standards. Furthermore, LC-MS was used to determine the levels of these species in cocoa samples. Catechin and epicatechin were the most abundant antioxidants followed by their dimers and trimers. The most potent antioxidants in cocoa were trimers and dimers of catechin and epicatechin, such as procyanidin B2, followed by catechin and epicatechin. This new LC-MS assay facilitates the rapid identification and then the determination of the relative antioxidant activities of individual antioxidant species in complex natural product samples and food products such as cocoa.