Tian Q, Failla ML, Bohn T, Schwartz SJ. High-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry determination of cholesterol uptake by Caco-2 cells. Rapid Commun Mass Spectrom 2006.
A simple, sensitive and selective liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometric method (LC/APCI-MS/MS) was developed and applied to quantitative determination of uptake of cholesterol by Caco-2 human intestine cells. Caco-2 cells were cultured in medium containing cholesterol-3,4-13C2 and phytosterols from nutritional supplements after in vitro digestion. Cellular cholesterol (cholesterol-3,4-13C2) and endogenous cholesterol were extracted using methanol/chloroform (1:2, v/v) and directly analyzed using LC/APCI-MS/MS with selected reaction monitoring (SRM), using cholesterol-2,2,3,4,4,6-d6 as an internal standard. Detection and quantification limits were 2.2 and 7.2 pmol, respectively. This method provides an effective tool for rapid determination of cholesterol uptake by cells with increased selectivity and sensitivity in comparison to previously reported LC/APCI-MS analysis using selected ion monitoring (SIM).
Tian Q, Giusti MM, Stoner GD, Schwartz SJ. Characterization of a new anthocyanin in black raspberries (<em>Rubus occidentalis</em>) by liquid chromatography electrospray ionization tandem mass spectrometry. Food Chem 2006.
Purple corn (Zea mays L.) is a rich and economic source of anthocyanin colorants and funtional ingredients. However, high levels of anthocyanin-rich waste are generated during processing, reducing the yields and increasing the costs of the final product. This waste has been associated with anthocyanin complexation with tannins and proteins. Our objective was to evalutate anthocyanin extraction methods to reduce purple corn waste. Different solvents (water, 0.01%-HCl-acified ethanol), temperatures (room temperature, 50, 75, and 100 degrees C), and times of exposure to the solvents were investigated. Acetone (70% acetone in water) extraction was used as control. Anthocyanins, total phenolics, tannins, and proteins in extracts were measured by the pH differential, Folin-Ciocalteu, protein precipitation, and BCA assay methods. Qualitative analyses were done by HPLC coupled to a PDA detector and SDS-PAGE analysis. Water at 50 degrees C achieved the highest yield of anthocyanins (0.94 +/- 0.03g per 100 g dry cornob) with relatively low tannins and proteins, comparable to the anthocyanin yield of anthocyanin yield obtained by 70% acetone (0.98 +/- 0.08 g per 100g dry cornob). Extending the extraction time for 20 to 60 min and using consecutive reextraction procedures reduced anthocyanin purity, increasing the yields of other phenolics. A neutral protease was applied to the extracts and effectively decomposed the major protein that was believed to contribute to the development of anthocyanin complexion and waste generation. Extraction time, consecutive reextraction procedures, and enzyme hydrolysis should be considered for highly yield of anthocyanins and waste reduction.
Tian Q, Rosselot RA, Schwartz SJ. Quantitative determination of intact glucosinolates in broccoli, broccoli sprouts, Brussels sprouts, and cauliflower by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. Anal Biochem 2005.
Many methods have been proposed to analyze glucosinolates, a class of phytochemicals whose breakdown products are thought to be responsible for an improvement in health; however, few are quantitative and many are time consuming. A selective and sensitive quantitative method for direct determination of intact glucosinolates was developed using liquid chromatography coupled to electrospray ionization tandem mass spectrometry with selected reaction monitoring detection. Detection limits for glucoiberin, sinigrin, progoitrin, glucoerucin, and glucotropaeolin were 1.75, 1.38, 1.36, 0.6, and 0.63 pmol, respectively. Intraassay precision of the method was within 10% for each compound. The method was successfully applied to quantify 10 individual glucosinolates in broccoli, broccoli sprouts, Brussels sprouts, and cauliflower. The advantage of the proposed method includes analysis of individual intact glucosinolates rather than the conversion to desulfoglucosinolates, an increased selectivity through the use of mass spectrometry, and a 10-fold improvement in detection sensitivity over conventionally used HPLC techniques.