Lc solution

The quantification of lactic acid in the fermentative liquid was performed using a Shimadzu high-performance liquid chromatograph, equipped with a quaternary pump r coupled to a degassing system (DGU-20A5r). The system holds an oven to control the column temperature (set at 28 °C) and an automatic injector (20 µL injection) with a diode array detector (SPD-M20A; range 190–800 nm). An ion exchange column (300 mm × 7.8 mm × 9 µm; Aminex^® HPX-87H, Bio-Rad, Hercules, CA, USA) was used. The elution was conducted isocratically with a mobile phase composed of 5 mM H₂SO₄ and with a flow of 0.6 mL/min. The software used was LC-Solutions, manufactured by Shimadzu Corporation (Kyoto, Japan) [26 (link)]. For each test, the concentration of lactic acid in an unfermented juice with the same pulp concentration (unfermented controls) was also detected. The production of lactic acid (g/L) was determined by the difference between the concentration of lactic acid in each fermented liquid and its respective unfermented control. The fermentative conditions with the best results were selected for further assays.

The quantification of lactic acid was performed as described by Farias, Soares and Gouveia [26 (link)], using a Shimadzu high-performance liquid chromatograph system equipped with a quaternary pump coupled to a degassing system (DGU-20A5r). The apparatus contains an oven to control the column temperature (set at 28 °C) and an automatic injector (20 µL injection) with a diode array detector (SPD-M20A; range 190–800 nm). An ion exchange column (300 mm × 7.8 mm × 9 µm; Aminex^® HPX-87H, Bio-Rad, Hercules, CA, USA) was used. The elution was carried out isocratically with a mobile phase composed of 5 mM H₂SO₄ and with a flow of 0.6 mL/min. The software used was LC-Solutions manufactured by Shimadzu Corporation (Kyoto, Japan).
For each test, the concentration of lactic acid in an unfermented juice containing the same concentration of pulp (unfermented controls) was also detected. The production of lactic acid (g/L) was determined by the difference between the concentration of lactic acid in each fermented liquid and its respective unfermented control.

The receptor samples were analysed by a validated HPLC method using a Shimadzu Prominence Ultra flow liquid chromatography (UFLC) system with a SIL20-AHT autosampler and an SPD20A UV detector. The samples were prepared by mixing 50 µL of the sample aliquotes with 50 µL each of 25% ethanol in water and propylparaben 25 µg/mL in ethanol (as internal standard). The standards were prepared by mixing 50 µL of standard with 50 µL each of propylparaben 25 µg/mL of ethanol and 6% (w/v) Brij™ O20 in PBS. The column used was Luna C18-2, 150 × 4.6 mm, 5 µm. The mobile phase was 52:48 Acetonitrile: 0.1% phosphoric acid in water at a flow rate of 1 mL/min, and the injection volume was 50 µL [27 (link)]. Salicylic acid, methyl salicylate, and propylparaben were detected at 235 nm and had retention times of 3.5, 5.5, and 7.5 min, respectively. The method provided good precision and linearity in the required concentration range (methyl salicylate; 1.56–500 μg/mL, R² = 0.999: salicylic acid; 0.78–500 μg/mL, R² = 0.999). The lower limit of quantification (LLOQ) for methyl salicylate and salicylic acid was 0.2 μg/mL. The chromatography software used was LC solutions^® (Shimadzu, Kyoto, Japan).