Development of a new analytical 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 (FAO, 1988). They include lentils (Lens culinaris L.), beans (Phaseolus vulgaris L.), peas (Pisum sativum L.), chickpeas (Cicer arietinum L.), and others. Due to the their important nutritional properties and to a low environmental impact that the production of pulses produces worldwide, the Food and Agriculture Organization of the United Nations (FAO) declared 2016 the International Year of Pulses (IYP) (FAO, 2016) [1]. Accumulating evidence supports the cardioprotective, hypolipidemic and hypohomocysteinemic effects of pulses [2] 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 [3]. 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 [4]. Studying these kind of compounds is not certainly easy, because we have to face first of all with a wide difference among their chemical structures and, thus, properties and functions. 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 triple quadrupole. In this way it would be possible to create a specific mixture characterized by particular content of seed legumes to guarantee the right consumption of polyphenols to each population group. 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 very fast as polyphenols elute within about 10 minutes. The approach used implied Dynamic MRM, a new acquisition mode that allowed to quantify simultaneously a number of compounds assuring excellent linearity and sensitivity. The application to real samples indicated that lentils showed a maximum level of total polyphenols of 39.83 mg/kg (laird lentils of Canada), while, on the other side, beans reached a maximum value of total polyphenols of 479.23 mg/kg (Ruviotto beans of Piemonte).