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

Introduction: Pulses are among the most important dietary component worldwide, demonstrating cardioprotective, hypolipidemic [1] and antioxidant activities [2]. These health benefits could be attributed to their content in polyphenols [3]. Thus, this work aims to develop a HPLC-MS/MS method to simultaneously detect and quantify 16 polyphenols in different kind of pulse. Methods: The polyphenols analyzed by HPLC-MS-MS were: 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. Moreover, many extraction procedures such as acidic and basic hydrolysis were compared by combining different temperatures and extraction times. Results: The best extraction recoveries (range 99.7- 107.6%) were obtained in acidic conditions at pH 2 with a temperature of 20o C and an extraction time of 2h. The newly developed analytical method is fast as polyphenols elute within 10 minutes. The method 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. This validated method was applied on thirty one pulse samples including beans, lentils, peas and chickpeas. 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). Conclusions: A new analytical method that uses HPLC-MS/MS was developed for the analysis of sixteen polyphenols in pulses. This study highlights that certain polyphenols are characteristic in specific pulses variety and this aspect may be useful for increasing the health knowledge of legumes and for proposing new functional foods. References 1. J. Anderson, A. W. Major. British Journal of Nutrition, 88 (3), 263-271(2002). 2. C. A. Adebamowo, E. Cho, L. Sampson, M. B. Katan, D. Spiegelman, W. C. Willett, M. C. Holmes. International Journal of Cancer, 114, 628-633 (2005). 3. Lazze, M.C., Pizzala, R., Savio, M., Stivala, L.A., Prosperi, E., Bianchi, L. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 535, 103-115 (2003).