A new extraction method to quantify polyphenols in green coffee using HPLC-MS/MS triple quadrupole

Isoflavones and lignans are considered phytoestrogens, a group of secondary plant metabolites that possess some biological effects due to their similarity to 17β-estradiol [1]. In fact, several studies showed that long term consumption of food rich in polyphenols could reduce the risk of cardiovascular diseases, cancer development, diabetes, osteoporosis and neurodegenerative diseases [2]. The aim of our work was to develop a new analytical method for the extraction of lignans and isoflavones in green coffee using HPLC-MS/MS Triple Quadrupole. The monitored molecules were three lignans, i.e., secoisolariciresinol (SECO), lariciresinol (LARI) and matairesinol (MAT), and six isoflavones, including four aglycons (daidzein, genistein, formononetin and biochanin A) and two glycosylated forms (genistin and daidzin). Several extraction procedures were tested in green coffee samples, including acid and basic hydrolysis and methanolic extraction. Two enzymatic hydrolysis were also performed with different mixtures of enzymes (Clara-Diastase and Taka-Diastase). After a series of attempts, we tried to optimize the procedure combining acid or basic hydrolysis with enzymatic digestion. From our results, alkaline hydrolysis in methanol and alkaline hydrolysis in methanol plus Clara-Diastase seem to be the most promising. The most performing method will be selected and applied to green coffee samples of different origins. The analytical method has been validated using the following criteria: linearity (R2 > 0.995 for all compounds), inter-day and intra-day repeatability (RSD % in the range of 0.6-9.3 for inter-day repeatability and 0.4-4.2 for intra-day repeatability) and sensibility (LODs in the range of 0.1-5 µg L-1, LOQs in the range of 1-16 µg L-1). Numerous researches, concerning the quantification of polyphenols in foodstuffs, have been reported but, to best our knowledge, this is the first time that simultaneous quantification of isoflavones and lignans is applied to green coffee. References [1] Kuhnle GG, Dell’Aquila C, Aspinall SM, Runswick SA, Mulligan AA, Bingham SA. Phytoestrogen content of Beverages, Nuts, Seeds, and Oils. J Agric Food Chem. 2008;56:7311-7315. [2] Rizvi SI, Pandey KB. Plant polyphenols as dietary antioxidants in human health and disease. Oxid Med Cell Longev. 2009;2:270-278.