Development of a new HPLC-MS/MS method for the simultaneous quantification of sixteen polyphenols in pulses.

Pulses represent one of the most important traditionally dietary component worldwide, supplying proteins, dietary fibers, minerals and vitamins. Accumulating evidence supports the cardioprotective, hypolipidemic and hypohomocysteinemic effects of pulses [1] and a recent epidemiological survey on polyphenols-rich food showed that only the consumption of lentils and beans is correlated to a lower incidence of breast cancer [2]. The potential health benefits of legumes is attributed to the presence of a wide range of phytochemicals including polyphenols that are directly associated with antioxidant activity [3]. The aim of this work was to develop a method to simultaneously detect and quantify 16 polyphenols in different kind of pulses by using HPLC-MS/MS Dynamic MRM triple quadrupole. The polyphenols of interest are the following: shikimic acid, gallic acid, delphinidin, catechin, chlorogenic acid, epicatechin, vanillic acid, caffeic acid, siringic acid, cumaric acid, ferulic acid, 3,5 di-CQA, rutin, kaempferol, cyanidin, quercetin. In the current study, we tested different extraction procedures such as acidic and basic hydrolysis, by combining different temperatures and extraction times. After choosing and optimizing the extraction, we proceed with method validation and application to real pulse samples by using HPLC-MS/MS. The separation of the different analytes was performed by using a Kinetex column (100 X 2.1 mm, 2,6 µm) and the mobile phase was composed by water (A) and methanol (B) both containing 0,1% of formic acid. Chromatographic run is fast as polyphenols elute within about 10 minutes. The approach used implied Dynamic MRM that allowed to quantify simultaneously a number of compound assuring excellent linearity and sensitivity. analytes are only monitored while they are eluting from the LC and valuable MS duty cycle is not wasted by monitoring them when they are not expected. The highest polyphenols levels were found in beans such as black beans (458.64 mg/kg) and ruviotto beans (189.02 mg/kg); also lentils displayed consistent level of polyphenols, such as black lentils (137.25 mg/kg) and quality gold lentils (132.27 mg/kg). References [1] J. Anderson, A. W. Major. Pulses and lipaemia, short- and long-term effect: Potential in the prevention of cardiovascular disease. British Journal of Nutrition, 2002, 88 (3), 263-271. [2] C. A. Adebamowo, E. Cho, L. Sampson, M. B. Katan, D. Spiegelman, W. C. Willett, M. C. Holmes. Dietary flavonols and flavonol-rich foods intake and the risk of breast cancer. International Journal of Cancer, 2005, 114, 628-633. [3] Lazze, M.C., Pizzala, R., Savio, M., Stivala, L.A., Prosperi, E., Bianchi, L. (2003). Anthocyanins protect against DNA damage induced by tert-butyl-hydroperoxide in rat smooth muscle and hepatoma cells. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 535, 103-115.