SIMULTANEOUS QUANTIFICATION OF SIXTEEN POLYPHENOLS IN 31 LEGUMES BY USING HPLC-MS/MS DYNAMIC-MRM TRIPLE QUADRUPOLE

Introduction: Pulses represent one of the most important traditionally dietary component worldwide, supplying proteins, dietary fibers, minerals and vitamins. The Food and Agriculture Organization declared 2016 the International Year of Pulses [1]. The potential health benefits of legumes are attributed to the presence of a wide range of phytochemicals including polyphenols that, according to the literature, are directly associated with antioxidant activity [2]. Methods: Different extraction procedures such as acidic, basic and neutral extraction have been tested at different pH, by combining different temperatures and extraction times and the first one was the best. The separation of the different analytes was performed by using a Kinetex PFP column and the mobile phase was composed by water and methanol both containing 0.1% of formic acid. The analysis has been carried out by using HPLC-MS/MS Dynamic MRM triple quadrupole. Results: The polyphenols of interest are: 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. Chromatographic run is very fast as 16 polyphenols elute within about 10 minutes. The approach used implied Dynamic MRM acquisition mode in which 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 sensitivity of the Dynamic MRM method was higher with respect to the MRM one. The LODs achieved with Dynamic MRM method range from 0.0015 mg/l to 0.015 mg/l and LOQs ranged from 0.001 mg/l to 0.05 mg/l. Considering linearity, the obtained coefficients of correlation were in the range 0.9907-0.9998 for the MRM method and 0.9934-1 for the Dynamic MRM method. Conclusions: The application to real samples indicated that black beans (458.64 mg/kg) and ruviotto beans (189.02 mg/kg) displayed the highest amount of total polyphenols. This study highlights that the content of polyphenols is related with the dark color of coat pulses and that certain polyphenols are characteristic in specific pulses variety. These aspects may be useful for increasing the health knowledge of legumes and for proposing new functional foods. Novel Aspect: A new analytical method that uses HPLC-MS/MS and triple quadrupole in Dynamic MRM mode was developed for the analysis of sixteen polyphenols in pulses. References: [1] Food and Agriculture Organization (FAO), 2016. http://www.fao.org/pulses-2016/en/ [2] Adebamowo C.A., Cho E., Sampson L., Katan M.B., Spiegelman D., Willett W.C., Holmes M.C. International Journal of Cancer. 114, 628-633 (2005)