Esquire 6000

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.

Samples collected in 2011 were extracted and analyzed by HPLC-ESI-MS as previously described^{26 (link)}. In brief, the extracts were analyzed by HPLC using a Beckman Coulter Gold 127 Solvent Module coupled to a Bruker Esquire 6000 ion trap mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) equipped with an electrospray ionization source. Negative ion spectra were recorded in the range 50–2000 m/z (full scan mode, 13,000 m/z per second). For metabolite identification, MS/MS and MS3 spectra were recorded in negative mode. Metabolites were putatively identified by comparing the m/z values, fragmentation patterns (MS/MS and MS3) and retention times of each signal with those of an in-house library of authentic standards. When commercial standards were not available, m/z and fragmentation patterns were compared with those published in the literature or on-line databases such as MassBank (www.massbank.jp/en/database.html) and Human Metabolome Database (http://www.hmdb.ca/).

Chromatographic analyses were performed with an Agilent Series 1100 HPLC system with a G1315B diode array detector (Agilent Technologies) and an ion trap mass spectrometer (Esquire 6000, Bruker Daltonics) with an electrospray interface operating in negative ion mode. Separation was performed in a Luna Omega Polar C₁₈ analytical column (150 × 3.0 mm; 5 µm particle size) with a Polar C₁₈ Security Guard cartridge (4 × 3.0 mm), both purchased from Phenomenex. Detailed chromatographic conditions are available in [39 (link)].
The most abundant compounds (flavonoids) were quantified by UV signal at 350 nm and the following analytical standards: vicenin-2, kaempferol, luteolin, and quercetin. Calibration graphs were constructed in the 0.5–100 mg L⁻¹ range. Peak areas at 350 nm were plotted against analyte concentration. Each analytical standard was used to quantify the corresponding compound or compounds of the same chemical family. Detection limits (3σ criterion) were 0.1–0.2 mg L⁻¹. Repeatability (n = 10) and intermediate precision (n = 9, three consecutive days) were lower than 4 and 8%, respectively. The robustness of the chromatographic method was evaluated by recording analyte signals at ±2 nm of the optimum wavelength and by slightly varying the percentage of the mobile phase (2% changes), observing variations lower than 5% for all the analytes concerning the optimum conditions.

The pepsin-digested cyt c was manually injected and trapped by the Trap column (Optimize, 3 mm cartridge). The deuterated peptides were subsequently separated using a high-performance liquid chromatography C18 column (Biobasic 5 μm 50 mm × 1 mm, Thermo Scientific) and then sequentially analyzed through ESI–MS or ESI–MS/MS (Esquire 6000, Bruker, Billerica, MA). The gradient of the mobile phase was varied from 0% A solution (0.1% TFA) and 100% B solution (0.01% TFA, 80% ACN) to 100% A in 40 min at a flow rate of 0.1 mL/min. The buffer, column, and tubing were immersed in ice water as previously described^{29 (link)}. Back exchange levels were calculated based on 24-h fully deuterated samples as previously reported^{62 (link)}.

The phenolic fraction of the extracts was characterized by resorting to an HPLC Agilent 1100 Series with a G1315B diode array detector. A Luna Omega Polar C₁₈ analytical column of 150 × 3.0 mm and 5 µm particle size (Phenomenex), with a Polar C₁₈ Security Guard cartridge (Phenomenex) of 4 × 3.0 mm, was used. The HPLC system was connected to an ion trap mass spectrometer (Esquire 6000, Bruker Daltonics) equipped with an electrospray interface operating in negative mode. Detailed conditions are reported elsewhere [46 (link)]. Compounds identification was carried out based on analytical standards and mass spectra, whereasultra violet (UV) spectra were used for quantification purposes.

SSi6-trans (Figure S1A) and SSi6-cis (Figure S1B) were isomers synthesized and purified by classic liquid chromatography, as described early, with some modifications [23 (link)]. Gingerol (400 mg) and 2,4-dinitrophenylhydrazine (280 mg) were reacted in a flask at 0 °C, using anhydrous methanol as solvent, molecular sieve 3A, and hydrochloric acid (HCl) as the catalyst, under stirring for 60 min. Products extraction was performed with dichloromethane after the addition of cold water. Compounds isolation was performed by liquid chromatography on silicon oxide (SiO₂) column (15.0 × 3.4 cm i.d.) using n-hexane/ethyl acetate, 60:40 (V/V). Twenty fractions (30 mL) were collected and monitored by thin-layer chromatography (TLC). The SSi6-trans and SSi6-cis were identified in the fractions 10–16 and 19–20, respectively. The compounds structures were elucidated by 1H NMR (Figure S2A,B) using a Bruker DRX400 instrument (Bruker, Billerica, MA, USA), operating at 400 MHz for 1H spectra, with TMS as internal standard and by electrospray ionization-ion trap mass spectrometry (ESI-MS/MS, Bruker model Esquire 6000) in positive mode (Figure S3A,B).