Q. TIAN1, G. D. Stoner2, and S. J. Schwartz1. (1) Dept. of Food Science & Technology, Ohio State Univ., 2015 Fyffe Ct., 110 Parker Food Science Bldg., Columbus, OH 43210-1007, (2) School of Public Health, Ohio State Univ., Div. of Environmental Health Sciences, 300 W. 10th Ave., 1148-B James Cancer Hospital & Research Institute, Columbus, OH 43210
The positive health benefits of anthocyanins reported in numerous epidemiological studies have stimulated interest in the absorption and bioactivity of these phytochemicals from the diet. HPLC and spectrophotometry are commonly used methods for anthocyanin characterization and quantification. However, these techniques are not adequately sensitive for the measurement of anthocyanins in very small sample sizes such as cell extracts and very low concentration of these compounds present in human blood. Our objective was to develop a rapid, sensitive high-performance liquid chromatography electrospray tandem mass spectrometry (LC-ESI/MS-MS) method suitable for anthocyanin characterization and quantification and to demonstrate its application to trace level anthocyanin determination in biological samples. LC-ESI/MS-MS experiments were conducted using a triple quadrupole ion-tunnel mass spectrometer coupled to a Waters 2695 separation module. The identities of anthocyanins were confirmed by mass spectrometry and tandem mass spectrometry using both positive and negative ion analyses. Quantification was conducted using tandem mass spectrometry with selected reaction monitoring (SRM). Four anthocyanins (cyanidin 3-glucoside, cyanidin 3-sambubioside, cyanidin 3-(2G-xylosylrutinoside) and cyanidin 3-rutinoside) were characterized and quantified in black raspberry samples with a detection limit of approximately 1 fmol. Cyanidin 3-glucoside, although presented in black raspberry in lower amounts, was found to be the predominant anthocyanin (40.5 fmol/ml extract) in squamous cells after incubation with black raspberry extracts, indicating that monoglycosidic anthocyanins were more readily absorbed by the cells in comparison to higher molecular weight anthocyanin glycosides. Applying the developed LC-ESI/MS-MS method to the analysis of human plasma after administration of freeze-dried black raspberry (5% in the diet) showed that all four anthocyanins were equally absorbed. In conclusion, LC-ESI/MS-MS represents a powerful tool for the characterization and quantification of anthocyanins in biological samples with limited sample size or when present at trace levels and shows promise for use in bioavailability and absorption studies of anthocyanins from the diet.
Session 33F, Nutraceuticals & Functional Foods: Antioxidants and phytochemical analysis