Isoflavones in espresso coffee: a new SPE-HPLC-MS/MS method for their determination. (2015)

Isoflavones are a group of phenolic compounds that are structurally and functionally similar to 17-estradiol having a weak estrogen-like effects. Epidemiological data suggest that their consumption is associated with the prevention of several hormone-dependent cancers, cardiovascular diseases, osteoporosis, and hot flushes of menopause [1]. Coffee is a extraordinary combination of numerous bioactive compounds with biological and physiological actions on human health, including isoflavones. Several authors reported the determination of some isoflavones, i.e. daidzein, genistein, formononetin, and biochanin A in coffee matrix, and the found concentrations are quite different by comparing the results. Alves et al. proposed an acidic hydrolysis for extracting isoflavones from espresso coffee and used HPLC-DAD for quantifying genistein (180 g/l), daidzein (523 g/l), formononetin (2833 g/l) [2]. Mazur et al. published an article in which reported the analysis by gas chromatography-mass spectrometry (GC-MS) of three isoflavones in six samples of espresso and instant coffee, by finding daidzein (0.1-0.66 mg/kg), genistein (0.15-0.29 mg/kg) and formononetin (0.72-0.78 mg/kg) [3]. Enzymatic hydrolysis, solid phase extraction (SPE) and GC-MS were combined in the work of Thompson et al. for quantifying formononetin (2 g/l), daidzein (1 g/l) and genistein (1 g/l) in prepared coffee [4]. Kuhnle et al. used liquid chromatography-mass spectrometry (LC-MS) for analyzing daidzein, genistein and biochanin A in coffees by finding total isoflavones content of 9130 g/kg (instant coffee powder), 50 g/kg (instant coffee decaffeinated powder), &lt;10 g/kg (infusion and decaffeinated coffee powder) [5]. Sapozhnikova et al. published an article that uses LC-MS/MS method for analyzing genistein (0.9-1.4 mg/kg), daidzein (3.2-5.2 mg/kg) and formononetin (3-6 g/kg) in ground coffee [6]. Basing on different values of isoflavones content in coffe reported in literature, the aim of this work was to develop a new HPLC-MS/MS method for the simultaneous determination of five isoflavones, i.e. daidzein, genistein, genistin, formononetin and biochanin A in espresso coffee. The certified samples of coffee were purchased by Illy Caffè industry. All samples were 100% Arabica. The espresso coffees were prepared by using an automatic machine Iperespresso X7.1 (Illy, Trieste).&#13;\n1 ml of liquid coffee was extracted with 20 ml of methanol, stored in refrigerator at -20°C for 24 h, then purified by using SPE C18 cartridges (500mg/6ml) and finally injected in HPLC-MS/MS (triple quadrupole as mass analyzer). Separation of the analytes was performed on a Kinetex C18 (50 x 2.10 mm i.d, particle size 2.6 μm, pore diameter 100 Å) by using acetonitrile and formic acid 0.1%/ammonium formiate 5 mM as mobile phase at flow rate of 0.5 ml/min. Calibration curves of six isoflavones in a concentration range 0.001-1 mg/l showed R2 from 0.993 to 0.999. Recovery % calculated at 0.1 mg/l fortification level were in the range 48-106% for all analyzed isoflavones. The validated methodology was applied to the analysis of six samples of certified coffees 100% Arabica. Formononetin was found in a concentration range of 0.40-0.41 g/l, while biochanin A in a range of 0.58-3.26 g/l. Total content of isoflavones was in range of 0.58-3.66 g/l. A HPLC-MS/MS chromatogram of an espresso coffee sample is reported in Fig.1. Samples of coffee 100% Robusta were analyzed to check the content of isoflavones in comparison to 100% Arabica samples.