Quantification of Methylxanthines and Phenolics in Fermented Beans

Defatted samples (250 mg) of fermented and dried beans were subjected to extraction of methylxanthines (theobromine, theophylline and caffeine) and monomeric phenolic compounds (catechin and epicatechin), according to previous studies [14 (link),50 (link),51 (link)]. Aliquots of the extracts (20 µL) obtained were injected into a chromatograph equipped with a diode array detector (HPLC-DAD) at 280 nm (1260 Infinity Agilent Technologies, La Jolla, CA, USA) with Zorbax Eclipse XBD-C18 column (4.6 × 150 mm, 5 µm, Agilent Technologies, La Jolla, CA, USA) at 25 °C, at flow rate of 1.2 mL/min of mobile phases (A) water/acetonitrile (99.8: 0.2, v/v) and (B) methanol, in gradient mode according Chagas Junior et al. [14 (link)].
The tentative identification of the compounds was based on the retention time of each analyzed standard and quantified through the external analytical curves: theobromine (from 3.125to 50 µg/mL, R² ≥ 0.99, LOQ = 0.64 mg/g), theophylline (from 3.125to 100 µg/mL, R² ≥ 0.99, LOQ = 0.02 mg/g), caffeine (from 3.125 to 100 µg/mL, R² ≥ 0.99, LOQ = 0.04 mg/g), catechin (from 3.125 to 50 µg/mL, R² ≥ 0.99, LOQ = 0.31 mg/g) and epicatechin (from 3.125to 100 µg/mL, R² ≥ 0.99, LOQ = 0.11 mg/g) [11 (link)]. All experiments were performed in duplicate.