Co 2060 plus

The hydroethanolic extracts prepared as described in Section 2.4. were filtered through a 0.45 μm nylon syringe filter and analyzed by HPLC-DAD. The chromatographic analysis was performed in a HPLC integrated system from Jasco (Jasco, Tokyo, Japan. This system consisted of an LC-Net II/ADC hardware interface, an automatic sampler (Jasco AS-2057 Plus; Jasco, Tokyo, Japan), a pump (Jasco PU-2089 Plus; Jasco, Tokyo, Japan), a multi-wavelength diode-array detector (DAD, Jasco MD-2018 Plus; Jasco, Tokyo, Japan), and a column oven (Jasco CO-2060 Plus; Jasco, Tokyo, Japan). The gradient elution used was the following: 0 min, 5% B; 40 min, 25% B; 55 min, 45% B; 60 min, 60% B; 65 min, 5% B (solvent A: 0.5% acetic acid; solvent B: 100% methanol), with a flow rate of 1.1 mL/min. The chromatographic column was a Zorbax-SB-C18 (5 μm, 250 mm × 4.6; Agilent Technologies, Santa Clara, CA, USA), at 28 °C. The caffeine, 5-hydroxymethylfurfural, and caffeoylquinic acids were monitored at 274, 280, and 320 nm, respectively. The compound identifications were performed by comparing retention times and co-elution with authentic standards, and by UV absorption spectral analysis. Caffeine, caffeoylquinic acids (3-, 4-, 5-CQA), and 5-hydroxymethylfurfural (HMF) contents were expressed as mg/100 g dw. All determinations were performed in triplicate.

Aliquots (1 mL) of the lyophilized extracts prepared with deionized water were centrifuged (13,000; 10 min) and filtered using syringe filters (0.45 μm) before injection into the HPLC-DAD-FLD system (Jasco, Tokyo, Japan). The system was composed of a LC-NetII/ADC hardware interface, a pump (Jasco PU-2089), an automatic sampler (Jasco AS-2057 Plus) and a multiwavelength diode array detector (Jasco MD-2018 Plus) coupled to a fluorescence detector (Jasco FP-2020 Plus, Jasco Co., Tokyo, Japan) and a column thermostat (Jasco CO-2060 Plus) (Jasco, Tokyo, Japan). Hydroxytyrosol was quantified by fluorescence and monitored (λ excitation = 280 nm; λ emission = 330 nm). The gradient elution program used was: 0 min, 5% B; 30 min, 25% B; 50 min, 75% B; 55 min, 100% B; 60 min, 100% B; 63 min, 5% B, being solvent (A) 1% acetic acid and (B) 100% methanol. A chromatographic column (Zorbax-SB-C18; 250 × 4.6 mm, 5 μm; Agilent Technologies, Amstelveen, The Netherlands) was used at 20 °C with a flow rate of 1 mL/min and an injection volume of 20 µL. The hydroxytyrosol content was expressed as g/100 g (dry weight).

Residual sugar was determined by high-performance liquid chromatography, using a Shimadzu chromatograph with a Nucleogel Ion 300 OA column, a LC-9A pump, a CTO-10A oven at 30°C, and a RID-6A refractive index detector. H₂SO₄ 0.008 Ν was used as mobile phase with a flow rate of 0.8 mL/min, and propanol-1 was used as an internal standard. A volume of 0.25 mL of the sample and 0.625 mL of a 1% (v/v) solution of propanol-1 were diluted to 25 mL. Then, 60 μL of the final solution was injected directly to the column. Residual sugar and concentrations were calculated using standard curves.
Volatile fatty acids were determined by high-performance liquid chromatography, using a Jasco chromatograph LC-2000 Plus (Jasco Analytical Instruments, Tokyo, Japan) with a Bio-rad Aminex HPX-87H column (7.8 mm ID × 300 mm, 9 μm particle size), a PU-2089 plus quaternary gradient pump, a Jasco CO-2060 Plus oven at 50°C, a MD-2018 plus photodiode array detector operated at 210 nm, and an autosampler AS 2050 plus. H₂SO₄ 0.008 Ν was used as mobile phase with a flow rate of 0.6 mL/min. The samples were filtered by a membrane filter of 0.45 nm, and all the data were processed with the ChromNav program.