Gold 126 solvent module

The HPLC system consisted of gold 126 solvent modules (Beckman Coulter), a RID-10A differential refractometric detector (Shimadzu, Kyoto, Japan), an analytical C₁₈ column (Zorbax 300SB-C₁₈ 250 mm × 4.6 mm I.D., 5 μm particle size; Agilent Technologies), and a one-channel recorder (Klipp and Zonen BD 40, Rotterdam, The Netherlands). The mobile phase, a mixture of acetonitrile, isopropanol, 0.02 M phosphate buffer, pH = 4.6 (340:150:510, v/v) was eluted at 1.0 ml/min.

Total phenolic compounds of the cocoa extract supplement (Cocoavia; Mars Inc., Hackettstown, NJ, USA) made by patented process (Cocoapro; Mars Inc., Hackettstown, NJ, USA) were extracted with three volumes of ice-cold methanol (w/v)). Samples were mixed, sonicated (15 min, 4 °C), centrifuged (16,000× g rpm, 10 min, 4 °C), and filtered through 0.2 µm pore filters. The supernatant samples were diluted 1:100 and 1:10 (v/v) with LC-MS grade methanol for HPLC-ESI-MS. The samples were further diluted 1:2 with LC-MS grade water and passed through Minisart 0.2 µm filters following protocol [19 (link)]. Each sample was analyzed in two technical and three biological replicates, with 20 µL injection volumes. The unknown metabolites of samples were analyzed using HPLC-ESI-MS (Esquire 6000, Bruker Daltonics, Billerica, USA), followed by ESI (Electrospray Ionization)-base peaks, MS/MS, and MS3 fragmentation at its retention time and mass to charge ratio (m/z). For this purpose, an “in house” library of commercial standard spectra, scientific literature, and online databases such as Mass bank (www.massbank.jp) were used. The quantification of the metabolites in the methanolic extracts was carried out through HPLC-DAD (Beckman Coulter Gold 126 Solvent Module coupled with a Gold 168 Diode Array Detector), relying on the calibration curves of authentic standard compounds.