Le. Determination of Total Tannin Content (TTC) The TTC was estimated by a modified version in the process created by Hong et al. [29]. Briefly, 25 of sample was mixed with 150 of vanillin methanolic answer (four w/v) within a 96-well plate and 25 32 H2 SO4 in methanol was added. The mixture was incubated for 15 min at 25 C and also the absorbance was measured at 500 nm within a microplate reader. The results have been obtained using a normal calibration curve of epicatechin solution in methanol at concentrations of 120, 220, 350 500, 650, 800, 950, 1000 /mL. Outcomes are expressed as g of epicatechin (EE) equivalents in dry weight (DW) of each sample. two.3.three. Identification and Quantification of Polyphenolic Compounds by LC-MS/MS Analysis Analytical Options and Sample Preparation Stock options of each and every analyte have been prepared in methanol for concentrations ranging from 90 to 2400 /mL. The stock solutions had been maintained at -20 C and utilised for the preparation of an intermediate methanolic stock resolution containing all analytes for 20 /mL concentration. Before each evaluation, the respective stock options had been diluted in concentrations ranging from 50 to 1500 ng/mL. The latter had been utilized for the building of calibration curves immediately before sample analyses. The DP web samples in the extracts had been prepared by diluting 1 g of extract in 1 mL of methanol just prior to the analysis. All standards solutions and all of the samples have been analyzed in triplicate. LC-MS/MS Analysis LC-MS/MS was selected because the analytical technique for assessment of phenolic compound presence due to its selectivity and sensitivity [30]. The identification of phenolic compounds was performed utilizing an Accela Ultra-High-Performance Liquid Chromatography program coupled with a TSQ Quantum Access triple quadrupole mass spectrometer equipped with an autosampler (HSP70 Molecular Weight Thermo Fischer Scientific, Waltham, MA, USA). The stationary phase of your chromatographic evaluation was a C18 column (Fortis Technologies Ltd. Neston, UK; C18, 150 2.1 mm, 3 ) having a guard column (10 2 mm, three ) with the identical material and organization. The mobile phase consisted of two solutions, both containing formic acid (0.1 ) and water (A) or acetonitrile (B). The mobile phase gradient system was: 0.0.0 min: ten B, 2.06.7 min from ten B to 100 , 16.78.7 min one hundred B, and 18.82.0 min 10 B to re-equilibrate the column. The flow rate was 0.two mL/min. The injection volume was 10 and also the temperature from the tray along with the column was set at 25 and 35 C, respectively. Mass spectrometer was operated on electrospray ionization (ESI) approach in adverse and optimistic polarities plus the chosen reaction monitoring (SRM) mode for enhanced sensitivity. Before every analysis, all target analytes’ molecular ion transitions and their collision energies have been obtained by direct infusion in complete scan (mass range: 100500). The ion supply and vacuum parameters were optimized to become applicable for all analytes. A nitrogen generator (Peak Scientific) was used to create nitrogen as sheath and auxiliary gas. The respective gas pressures have been set at 25 and 10 Arb, respectively. The spray voltage was set at 3.5 kV inside the damaging polarity and three.0 kV within the optimistic polarity, capillary temperature was regulated at 300 C, and collision pressure was adjusted at 1.5 mTorr. The signals in the selected ion transitions on the deprotonated molecules of m/z utilized had been: gallic acid (169.939 126.089 (17 eV), 169.939 125.047 (17 eV)), caftaric acid (312.1.
