A new HPLC-MS/MS analytical method for isoflavone and lignan quantification in 25 green coffee samples

A new HPLC-MS/MS analytical method for isoflavone and lignan quantification in 25 green coffee samples Simone Angeloni1,2, Giovanni Caprioli1, Gulzhan Khamitova1,2, Luciano Navarini3, Gianni Sagratini1, Sauro Vittori1 1 School of Pharmacy, University of Camerino, Camerino, Italy; 2 International Hub for Coffee Research and Innovation, Belforte del Chienti (MC), Italy; 3 illycaffè S.p.A., Trieste, Italy Summary: an analytical method for quantification of lignans (lariciresinol, matairesinol and secoisolariciresinol) and isoflavones (biochanin A, daidzein, daidzin, genistein, genistin and formononetin) in green coffee has been developed by using HPLC-MS/MS. Results showed that the best extraction process was a base hydrolysis followed by enzymatic digestion and lignans were more abundant than isoflavones. Keywords: green coffee, phytoestrogen, HPLC-MS/MS 1 Introduction Coffee is one of the most important agricultural products in the international trade and last year 68 million 60 kg bags of green coffee were produced [1]. Green coffee beans, the starting raw material for roasted coffee and coffee beverages, are constituted by carbohydrates (55-65.5%), lipids (10-18%), nitrogen containing compounds (11-15%), purine alkaloids (0.8-4.0%), chlorogenic acids (6.7-9.2%) and minerals (3-5.4%). Other molecules that are found in lower percentages are non-volatile aliphatic acids (citric, malic and quinic acids) and phenols such as phytoestrogens [2]. The most studied and best-known phytoestrogens in foodstuffs are isoflavones and lignans. Both classes have been investigated in coffee powder and beverages but to the best of our knowledge, none have quantified them in green coffee. Hence, we sought to develop a simply and fast method to quantify three lignans (lariciresinol, matairesinol and secoisolariciresinol) and six isoflavones (biochanin A, daidzein, daidzin, genistein, genistin and formononetin) in green coffee beans by using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). For that purpose, firstly, we evaluated different extraction processes and the best one was chosen for lignan and isoflavone extraction. Secondly, we set up an efficient and fast HPLC-MS/MS method for simultaneous quantification of target molecules in green coffee. Finally, after validation the selected method was applied to 25 green coffee samples. 2 Experimental HPLC-MS/MS studies were performed using an Agilent 1290 Infinity series and a Triple Quadrupole 6420 from Agilent Technology (Santa Clara, CA) equipped with an electrospray ionization (ESI) source operating in negative and positive ionization mode. The separation of target compounds was achieved on a Kinetex C18 analytical column (50 mm x 2.10 mm i.d., 2.6 μm) from Phenomenex (Castel Maggiore, Bologna, Italy). The mobile phase for HPLC-MS/MS analyses was a mixture of 85% water (A) and 15% HPLC-grade acetonitrile (B), both with 0.1% formic acid. The separation was obtained by flowing at 0.4 mL/min with gradient elution. Detection was performed in the “multiple reaction monitoring” (MRM) mode. Different extraction processes such as acid and base hydrolysis, enzymatic digestions, organic solvent extraction and a combination of these, were tested and recovery and quantitative data were examined as well. The best one was chosen and applied to 24 Coffea arabica samples having different geographical origins and 1 Coffeea canephora. Chemical differences among the various coffee samples, in terms of lignan and isoflavone contents, were analysed by PCA using the statistical software STATISTICA v.7.1 (Stat Soft Italia S.r.l., Vigonza, Italy). 3 Results A new analytical method for simultaneous quantitation of three lignans and six isoflavones was developed by using HPLC-MS/MS triple quadrupole. For isoflavones in glycosidic form, the precursor ions were protonated molecules [M+H]+ in positive polarity, whereas for the aglyconic form and lignans the precursor ions were deprotonated molecules [M-H]- in negative polarity. The method showed good linearity (R2 for all target compounds were equal to or higher than 0.9952), sensitivity (LODs for isoflavones and lignans ranged from 0.1 to 15 µg L-1) and the separation of studied molecules was obtained within 6 min. The best performing process was a double extraction composed of base hydrolysis in methanol and enzymatic digestion with clara-diastase, since it showed good recovery levels, ranging from 74 to 94%, and the highest total concentration of all the compounds studied (1193.4 μg kg-1). Therefore, this process was chosen and applied to 25 green coffee samples. Results showed that lignans (286.5-8131.8 μg kg-1) were more abundant than isoflavones (3.4-300.0 μg kg-1) and secoisolariciresinol (172.6-5714.1 μg kg-1) and lariciresinol (113.9-2417.7 μg kg-1) were the most abundant compounds followed by genistin (12.6-204.8 μg kg-1). After PCA analysis we found that Ethiopian samples differed significantly from the other origins for the higher content of secoisolariciresinol and lariciresinol suggesting that they are a good source of lignans. 4 Conclusions For the first time, a new analytical method for the simultaneous quantitation in green coffee of three lignans (lariciresinol, matairesinol and secoisolariciresinol) and six isoflavones (biochanin A, daidzein, daidzin, genistein, genistin and formononetin) has been developed, validated and then, applied to 25 green coffee samples. This work provided new knowledge into two important phytonutrients (isoflavones and lignans) in green coffee, one of the most important agricultural products in the international trade. References 1. International Coffee Organization (ICO). Statistics. Trade Statistic Tables. (2018). http://www.ico.org/prices/po-production.pdf. Accessed 25 August 2019. 2. S. I. Mussatto, E. M. Machado, S. Martins, J. A. Teixeira; Food and Bioprocess Technology, 4 (2011), pp 661-672.