Schroeter et al. 10.1073/pnas.0510168103. |
Fig. 4. Dietary flavanols increase urinary nitrite+nitrate levels. Higher urinary excretion of nitrite+nitrate (open columns) and epicatechin equivalents (filled columns) in indigenous island dwelling Kuna Indians compared with urbanized mainland inhabitants. Columns depict mean values (n = 6) ± SEM. *, P < 0.05 vs. island dwellers.
Fig. 5. HPLC chromatogram of authentic standards for epicatechin, catechin, and their metabolites. Standards were separated by HPLC using the method detailed above and detected by either FL or UV detection. The peaks represent epicatechin-5-O-b-D-glucuronide (A), IS [internal standard (catechol)], epicatechin-7-O-b-D-glucuronide (B), 3′-O-methyl-epicatechin-5/7-O-b-D-glucuronide (C), catechin (D), 4′-O-methyl-epicatechin-7-O-b-D-glucuronide (E), epicatechin (F), 3′-O-methyl-epicatechin (G), 4′-O-methyl-epicatechin (H), 3′-O-methyl-catechin (J), 4′-O-methyl-catechin (K), 3′-O-ethyl-epicatechin (L). Chromatogram represents 125 pmol on column for each compound.
The internal standard (IS) amount on column was 75 pmol. The UV trace was recorded as absorbance at 280 nm. FLD conditions were 276 nm for excitation and 316 nm for emission. Individual UV spectra (220–400 nm) of compounds E, K, and L are depicted as Insets.
Fig. 6. MS/MS spectrum of authentic standard 1, 4′-O-methyl-epicatechin-7-O-b-D-glucuronide. Product ion scan of m/z 479 in negative ion mode: m/z 303 represents a methylated epicatechin fragment after neutral the loss of glucuronic acid, m/z 175 indicates a glucuronic acid fragment after the loss of methylated epicatechin, and m/z 137 represents a classic epicatechin-related A-ring fragment that arises from a Retro-Diels-Alder reaction.
Fig. 7. MS/MS spectrum of authentic standard 5, epicatechin-7-O-b-D-glucuronide. Product ion scan of m/z 465 in negative ion mode: m/z 289 represents an epicatechin fragment after neutral the loss of glucuronic acid, m/z 175 indicates a glucuronic acid fragment after the loss of epicatechin, and m/z 137 represents a classic epicatechin-related A-ring fragment that arises from a Retro–Diels–Alder reaction.
Supporting Materials and Methods
Authentic Standards.
Standards for flavanols/metabolites [4′-O-methyl-epicatechin-7-O-b-D-glucuronide (1), 4′-O-methyl-epicatechin (2), 3′-O-methyl-epicatechin-5/7-O-b-D-glucuronide (3), 3′-O-methyl-epicatechin (4), epicatechin-7-O-b-D-glucuronide (5), epicatechin (6), and catechin (7), 4′-O-methyl-catechin (8), 3′-O-methyl-catechin (9)] were provided by Mars Incorporated. 3′-O-ethyl-epicatechin (3′EthylEC), a not naturally occurring epicatechin derivative (provided by Mars Incorporated), was used as an internal standard. The structural identity of the standards was verified by using mass spectroscopy (Figs. 6 and 7) and NMR spectroscopy (Tables 1 and 2). Figs. 6 and 7 exemplify our mass spectroscopic analyses by providing LC-MS data for compounds 1 and 5, two of the main epicatechin derivatives investigated during this study. In addition, the results of NMR-based structural analyses for compounds 1–6 are provided as the 1H-NMR (Table 1) and 13C-NMR (Table 2) chemicals shifts, respectively. The structural identity with regard to the site of glucuronidation of compounds 1, 3, and 5 was further confirmed by hetero-multiple bond correlation (HMBC) experiments (data not shown). The HMBC spectrum for compound 1 revealed a cross peak corresponding to anomeric proton, H-1′′ (d 4.93) of the glucuronide moiety and C-7 (d157.4), thereby confirming glucuronidation in position 7 of the flavanol ring structure. The HMBC spectrum for compound 5 confirmed the structure as epicatechin-7-O-glucuronide by demonstrating the presence of a cross-peak corresponding to anomeric proton, H-1′′ of the glucuronide moiety with the C-7 (d157.2) of the epicatechin moiety. HMBC experiments of compound 3 were inconclusive with regard to precisely distinguishing between positions 5 or 7 as sites of glucuronidation, thus the compound was designated as 3′-O-methyl-epicatechin-5/7-O-b-D-glucuronide.Sample Preparation.
Sample preparation procedures were optimized/validated for all compounds quantified by using human plasma spike-recovery experiments. The average recovery for all compounds investigated was 85 ± 9%. Plasma samples were drawn into EDTA-containing vials, supplemented with ascorbate (1 mg/ml), snap-frozen in liquid nitrogen, and stored at –80°C. For HPLC analysis, samples were defrosted on ice and prepared by using a three-step procedure. Step 1: Samples were mixed with twice their volume of acidified methanol (100% vol/vol, –20°C, internal standard A = 3′EthylEC) and centrifuged at 17,000 ´ g for 15 min at 4°C. Step 2: The supernatant was collected and the pellet was resuspended in methanol as detailed for step 1. This resuspension was centrifuged (as above), the supernatant was collected, and the pellet was washed (step 3) in methanol (50% vol/vol, internal standard A). After centrifugation, the supernatant was collected and combined with the supernates from steps 1 and 2. This mixture was centrifuged (as above) and the solvents were removed by using a rotary evaporation system at 4°C. Residues were resuspended in 200 ml of methanol (25% vol/vol, internal standard B = catechol), which represents a 5-fold concentration as compared with plasma.HPLC.
Fifty to 100 ml of the extracted sample was injected onto a HPLC system and separated on a reversed-phase column [Phenomenex Luna C18(2), 150 × 4.6 mm, 3 mm] with guard column (Phenomenex C18 4 × 3.0 mm). Separation of analytes was accomplished by using an acetonitrile gradient in 50 mM methanolic (4% vol/vol) sodium acetate (pH 4.4) with a flow rate of 0.8 ml/min. Acetonitrile concentrations were linearly increased from 0% to 10% between minutes 5 and 20. Thereafter, acetonitrile concentrations were further increased in linear segments (20–28 min, 10–12%; 28–34 min, 12–20%; 34–41 min, 20–30%; 41–45 min, 30–71%) and held at 71% for 10 min. We routinely used fluorescence (FL) detection for sample analysis, and in addition we used DAD, CoulArray, and LC-MS detection for confirmation and verification purposes. A representative HPLC chromatogram obtained by using FL detection and demonstrating the separation of 11 flavanol derivatives is shown in Fig. 5.Peak Identification.
Peaks were identified by (i) comparing their retention time with the retention time of the authentic standards (Fig. 5), (ii) on-top spiking of the samples with authentic standards, (iii) isolating the unknown peaks and analyzing the isolated compound by using LC-MS detection, and (iv) treatments with glucuronidase/sulfatase as detailed previously were used to confirm glucuronidation/sulfation when necessary.Data Analysis.
Throughout these investigations, the analytical chemists were blinded with regard to sample information. Peak/area ratios of individual authentic standards and internal standards were plotted against concentrations to achieve a multilevel external calibration curve using linear regressions. Peak/area ratios (analyte/internal standards) were compared with standard curves to quantify the analytes.Mass Spectroscopic Analysis.
LC-MS analyses were performed by using an Agilent 1100 HPLC coupled to an Agilent 1100 MSD/LC Trap equipped with an API-ES chamber. Samples were either directly infused by using a continuous-flow infusion system or subjected to on-line HPLC prior to infusion. Before analyses, ionization parameters were optimized by direct infusion of authentic standards. Conditions for the mass spectral analysis in negative ion mode included a capillary voltage of 5,000 V, a nebulizing pressure of 40 psi, a drying gas flow of 10 liters/min, and a temperature of 350°C.NMR Analysis.
NMR spectra were obtained by using a Bruker 500-MHz instrument. 1HNMR and 13CNMR spectra were recorded in deuterated methanol (d4-MeOH), deuterated acetone (d6-acetone), deuterated acetonitrile (d3-ACN), or dideuterium oxide (D2O). Additional HMBC experiments were conducted to determine the position of glucuronide moiety. Chemical shifts were expressed in parts per million relative to trimethylsilylpropionic acid as a reference standard.