Quantification of Sixteen Polyphenols in Pulses by High-Performance Liquid Chromatography – Dynamic MRM Triple Quadrupole Tandem Mass Spectrometry (HPLC-MS/MS).

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 [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 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 at different pH, 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 PFP 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 16 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 with respect to MRM mode. The sensitivity of the Dynamic MRM method was around ten time higher with respect to the MRM one, for most of compounds. In fact, 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. The application to real samples indicated that red beans and dark beans displayed the highest amount of total polyphenols.