Synthesis and quantitative analysis of plasma-targeted metabolites of catechin and epicatechin

Article abstract of DOI:10.1021/jf505922b

Grape seed polyphenolic extract (GSPE) rich in the flavan-3-ols (+)-catechin and (-)-epicatechin beneficially modulates Alzheimers Disease phenotypes in animal models. The parent molecules in the extract are converted to a series of methylated and glucuronidated derivatives. To fully characterize these metabolites and establish a robust quantitative assay of their levels in biological fluids, we have implemented a partial synthetic approach utilizing chemical methylation followed by enzymatic glucuronidation. Liquid chromatography/time-of-flight mass spectrometry (LC-TOF-MS) and nuclear magnetic resonance (NMR) spectroscopy were used to assign unequivocal structures to the compounds. An analytical method using solid-phase extraction and LC-MS/MS in selective reaction monitoring mode (SRM) was validated for their quantitation in plasma. These studies provide a basis for improvements in future work on the bioavailability, metabolism, and mechanism of action of metabolites derived from dietary flavan-3-ols in a range of interventions.

Full text of DOI:10.1021/jf505922b

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Article
Synthesis and quantitative analysis of plasma-
targeted metabolites of catechin and epicatechin
Jack W Blount, Benjamin W Redan, Mario Ferruzzi, Bradley L Reuhs, Bruce R. Cooper,
John S. Harwood, Vladimir Shulaev, Giulio M. Pasinetti, and Richard Arthur Dixon
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf505922b • Publication Date (Web): 11 Feb 2015
Downloaded from http://pubs.acs.org on February 18, 2015
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1 Synthesis and quantitative analysis of plasma-targeted metabolites
2 of catechin and epicatechin
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Jack W. Blount1, Benjamin W. Redan2, Mario G. Ferruzzi2,3, Bradley L. Reuhs2,
Bruce R. Cooper5, John S. Harwood6,7,Vladimir Shulaev1, Giulio Pasinetti4 and
Richard A. Dixon1*
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1University of North Texas, Department of Biological Sciences, 1155 Union Circle #305220
Denton, TX 76203-5017
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2Department of Nutrition Science, Purdue University, 700 W. State St., West Lafayette, IN
47907
3Department of Food Science, Purdue University, 745 Agriculture Mall Dr, West Lafayette, IN
47907, USA
4Department of Psychiatry, The Mount Sinai School of Medicine, One Gustave L. Levy Place,
Box 1230, New York, NY 10029, USA
5Bindley Bioscience Center Metabolite Profiling Facility, Purdue University, 1203 W. State
Street, West Lafayette, IN 47907
6Purdue Interdepartmental NMR Facility, Purdue University, 560 Oval Drive, West Lafayette,
IN 47907, USA
7Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907, USA
* To whom all correspondence should be addressed:
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(phone +1-940-565-2308; fax +1-580-224-6692; E-mail Richard.Dixon@unt.edu)
ABSTRACT: Grape seed polyphenolic extract (GSPE) rich in the flavan-3-ols (+)-catechin and
(-) epicatechin beneficially modulates Alzheimer’s Disease phenotypes in animal models. The
parent molecules in the extract are converted to a series of methylated and glucuronidated
derivatives. To fully characterize these metabolites, and establish a robust quantitative assay of
their levels in biological fluids, we have implemented a partial synthetic approach utilizing
chemical methylation followed by enzymatic glucuronidation. Liquid chromatography/time-of-
flight mass spectrometry (LC-TOF-MS) and nuclear magnetic resonance (NMR) spectroscopy
were used to assign unequivocal structures to the compounds. An analytical method using solid-
phase extraction and LC-MS/MS in selective reaction monitoring mode (SRM) was validated for
their quantitation in plasma. These studies provide a basis for improvements in future work on
the bioavailability, metabolism and mechanism of action of metabolites derived from dietary
flavan-3-ols in a range of interventions.
KEYWORDS: catechin, glucuronosylation, O-methylation, phase II metabolites, semi-
synthesis, structure determination, two-site validation
■INTRODUCTION
Plant derived polyphenolic compounds possess diverse biological activities, and some have been
reported to show anti-tumor, anti-oxidant, anti-inflammatory, or anti-microbial activities (1-3).
Previous work from our laboratories has shown that polyphenolic compounds from multiple
sources, including a specific grape seed polyphenolic extract (GSPE), are capable of improving
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cognitive functions and reducing brain neuropathology in animal models of Alzheimer’s Disease
through multiple mechanisms (4, 5). At least a part of this effect may be mediated through
interference with aggregation of β-amyloid peptides into neurotoxic, soluble high-molecular
weight species (5).
GSPE is a complex mixture of proanthocyanidins (PACs, both oligomeric and polymeric)
and their monomeric units consisting of the flavan-3-ols (+)-catechin (C) and (-)-epicatechin
(EC) (6). The flavan-3-ol units are assembled into the various types of oligomers and polymers
through C4→C8 or C4→C6 interflavan bonds, and the individual units in the polymer can also
be esterified with gallic acid. The monomeric units, rather than the oligomers or polymers, are
bioavailable and therefore candidates for being the active components in the GSPE as regards
amelioration of Alzheimer’s Disease symptoms (4). These components, in the form of
glucuronidated and/or methylated phase II metabolites, reach the brain at a concentration of 300-
400 nM after 10 days of repeated dosing (4).
While knowledge of all metabolites found in plasma may be of broader significance for
understanding the therapeutic effects of GSPE on a range of disease phenotypes, mechanistic
studies require knowledge of the exact structures of metabolites that accumulate in brain or other
tissue. To initiate such studies, we have previously implemented a semi-synthetic approach to
generate sufficient quantities of flavan-3-ol metabolites for unequivocal identification. We
demonstrated that recombinant human glucuronosyltransferases of the UGT1A and UGT2B
families can efficiently glucuronidate EC or 3ʹ-O-methyl-EC in vitro, suggesting a method for
the semi-synthesis of brain-targeted flavan-3-ol metabolites (7). Of twelve enzymes tested,
UGT1A9 was the most efficient, and this enabled us to generate 3ʹ-O-methyl-EC-5-O-
glucuronide to 50% overall yield (7).
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In the present study, we have extended the above approach, in combination with chemical
methylation of C and EC with iodomethane, to generate authentic standards of the C and EC
metabolites found in the plasma and brains of rats treated with GSPE. The structures of the
metabolites were rigorously identified, and we present a robust, validated protocol for the
measurement of the levels of these compounds in animal fluids.
■MATERIALS AND METHODS
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Materials. UDP‐glucuronosyl transferase enzymes (recombinant or as mouse
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microsomes), buffers and UDP‐glucuronic acid were purchased from BD Biosciences (San
Diego, CA). (-)-Epicatechin, (+)-catechin, DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) and
iodomethane were from Sigma Aldrich, St Louis, MO. 3ʹ- and 4ʹ -O-methyl-epicatechin were
purchased from Nacalai USA, Inc. (San Diego, CA).
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Chemical Methylation of C/EC. (Epi)catechin (1 g) was dissolved in 240 mL
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dimethylformamide in a 1 L bottle, and the bottle saturated with nitrogen gas for 5 min. DBU
(14 mL) and iodomethane (300 mL) were then added, mixed well, and the solution re-saturated
with nitrogen gas for 30 s. The bottle was capped and placed at 40 oC in the dark for 3 h. The
reaction was then quenched by the addition of 400 mL methanol with mixing. The solution was
dried under nitrogen and re-suspended in HPLC grade methanol for HPLC analysis and
purification.
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Large Scale Purification of Methylated C/EC. Methylated (epi)catechins were
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purified on a large scale using a Biotage Isolera flash chromatography system equipped with a 10
g SNAP C18 cartridge at a flow rate of 18 mL/min, monitoring at 280 nm with a UV/Vis
detector. The sample in methanol was added to the column via a preloading concentrator disc.
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Solvent A was milli-Q water, and solvent B was HPLC grade acetonitrile. The column was
equilibrated at 5% B, and the gradient for elution was as follows: isocratic at 5% B for 12
column volumes (CV) or 180 mL; from 5% B to 13% B in 48 CVs or 720 mL; from 13% B to
100% B in 4 CVs or 60 mL; then washed in 100% B for 20 CVs or 300 mL. Fractions were
collected in 16 CV or 240 mL fractions.
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Glucuronidation of EC and Methyl-C/EC and Purification of the Products.
The glucuronidation reaction was performed at room temperature, and the mixture consisted of
210 ꢀL purified water, 40 ꢀL UGT Reaction Mix Solution A (containing UDP-glucuronic acid,
BD Biosciences),100 ꢀL UGT Reaction Mix Solution B (containing MgCl2, Tris-HCl buffer and
alamethicin, BD Biosciences), 10 ꢀL of 20 mM (epi)catechin derivative, and 120 ꢀL human
UGT1A9 (for EC and methyl-EC) or pooled male mouse liver microsomes (5 mg protein per
mL) (for C and methyl-C). After 6 h, an additional 40 ꢀL of UGT Reaction Mix Solution A, 40
ꢀL of Reaction Mix Solution B, 120 ꢀL UGT1A9 or mouse microsomes, and 10 ꢀL of 20 mM
(epi)catechin derivative were added. Then, at 12 h, a further 40 ꢀL UGT Reaction Mix Solution
A and 120 ꢀL UGT1A9 or mouse microsomes were added. The reactions were stopped after 24
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h by the addition of 400 ꢀL of methanol containing 3% of 85% phosphoric acid. The solvent was
removed under nitrogen gas, and the aqueous fractions were desalted using a Waters Sep‐Pak
Plus C18 cartridge. The cartridge was first washed with 10 mL methanol, followed by 20 mL
water. The sample (5 mL) was loaded onto the cartridge, then washed twice with 5 mL water,
and finally eluted twice with 5 mL methanol. The sample was dried under nitrogen and then re-
suspended in a small volume of methanol for HPLC purification. Product peaks were partially
purified on the analytical HPLC and the solvent removed under nitrogen gas. The aqueous
fractions were then acidified to 1% phosphoric acid final concentration, and desalted using a
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Waters Sep‐Pak Plus C18 cartridge. The methanol was dried under nitrogen and the fractions
analyzed for comparison to products found in the plasma of mice/rats fed the monomer enriched
GSPE fraction. All samples were dissolved in 100 ꢀL acidified water and acetonitrile (4:1, v/v),
and further analyzed by HPLC coupled to LC-TOF-MS (see below).
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HPLC for Analysis and Purification of C/EC Metabolites. HPLC analysis and
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purification of (epi)catechin derivatives were performed on an Agilent HP1200 HPLC,
monitoring at 280 nm. The HPLC with ChemStation software version B.02.01.SRI was equipped
with a G1322A degasser, a G1311A quaternary pump, a G1367B autosampler, a G1316A
thermostatic column compartment, and a G1315C diode array detector. A Varian Metasil 5 Basic
C18 250 x 4.6 mm column was used for analytical and micro‐preparative HPLC on the Agilent
HPLC system. Analytical and preparative HPLC were performed using the following gradient:
isocratic at 5% B for 5 min; 5% B to 10% B in 5 min; 10% B to 17% B in 15 min; 17% B to 23%
B in 5 min; then 23% B to 50% B in 35 min. Alternatively, a shorter method was used which
retained the same gradient except that the last step gradient was from 23% B to 28% B in 5 min,
reducing the run time by 30 min. Solvent A was 1% phosphoric acid in MilliQ water, and solvent
B was HPLC grade acetonitrile. Flow rate was 1 mL/min.
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Solid Phase Extraction (SPE) of C and EC Metabolites from Rodent
132 Plasma. Rodent plasma samples were obtained from a previous pharmacokinetic study using
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GSPE (4) that received approval from the Animal Care and Use Committee at Purdue
University. Plasma aliquots from control animals (no GSPE) and treated animals (with GSPE)
that were collected at sacrifice were thawed from -80 oC storage and quickly pooled separately at
Purdue University. These control and GSPE plasma samples were then re-aliquoted into
individual vials and re-frozen at -80 °C until analysis. For target assessment purposes, typical
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plasma was diluted fivefold with blank plasma to produce low dose plasma. Blank, low, and
normal plasma samples were then used for analytical validation at Purdue University and a
parallel set of samples was sent to the University of North Texas for cross-lab validation.
Samples of frozen plasma were thawed at room temperature, and 300 µL of acidified saline
(9g NaCl in 1 L 0.1% v/v formic acid/H2O) were added to 200 µL of plasma with vortex mixing.
A SPE cartridge (30 mg Oasis HLB column, Waters Co, Milford, MA), was activated by
addition of 1 mL MeOH followed by 1 mL DI H2O, after which the plasma/saline mixture was
loaded. The loaded SPE column was first washed with 1 mL 1.5 M formic acid, followed by 1
mL 95:5 v/v water: MeOH, and the eluate discarded. Metabolites were then eluted with 2 mL
0.1% v/v formic acid/MeOH.
Extraction recovery was assessed in a pilot experiment by spiking 10 µL of ethyl gallate
stock solution (10 µM) into freshly thawed plasma samples prior to extraction and analysis.
Extraction recovery was found to be 96.0 3.1%. Following validation of recovery, ethyl gallate
was used as a daily internal standard (10 ꢀL of stock 10 ꢀM solution) added to the eluates from
solid phase extraction prior to samples being dried under vacuum (<50 mm Hg) at 37 °C. The
residues were re-suspended in 160 µL 0.1% v/v formic acid/H2O + 40 µL 0.1% v/v formic
acid/acetonitrile, sonicated for 10s, vortex mixed for 10s, then transferred to centrifuge tubes
which were centrifuged at 18,000 g for 5 min to remove any particulates. The clear supernatants
were carefully transferred to 300 µL HPLC vials for analysis.
The ability of this extraction method to account for potential matrix effects was assessed by
comparing the response of individual metabolite standards in the presence or absence of blank
plasma extracted matrix. Briefly, standards of C and its major metabolites in GSPE rodent
plasma at 50, 100, and 500 nM were resuspended either in pure solvent (160 µL 0.1% v/v formic
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acid/H2O + 40 µL 0.1% v/v formic acid/acetonitrile -Standard) or in resolubilized blank plasma
extracted by SPE (Standard+Matrix). By comparison of response between standards injected
with or without plasma matrix an estimate of matrix effects was established by using the
following equation: % matrix effect (% ME) = (peak area of standard with matrix – peak area of
pure standard)/(peak area of pure standard) x 100 (8).
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NMR Analysis
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NMR spectroscopy was performed using a Bruker Avance DRX 500 MHz spectrometer
equipped with a cryoprobe. The metabolites were dissolved in CD3OD (99.96% atom D; Sigma,
St. Louis, MO) and then transferred to a 5 mm Shigemi tube (Wilmad Glass, Vineland, NJ).
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HPLC/MS Analysis of C/EC Metabolites from Rodent Plasma.
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1. LC-TOF-MS for unknown metabolite characterization. Separation was achieved on an
Agilent 1100 HPLC system using a Waters XBridge BEH shield RP-C18 XP (2.1 x 100 mm 2.5
µm) column following a 2 µm frit filter. The column and autosampler were maintained at 40 °C
and 10 °C, respectively. Mobile phase A was 0.1% v/v formic acid/water and B was 0.1% v/v
formic acid/acetonitrile. A linear gradient for elution was used at a constant flow rate of 0.26
mL/min. Initial conditions 5% B; 0-12 min: 5-15% B; 12-18 min: hold at 15% B; 18-24 min: 15-
40% B; 24-30 min: 40-50% B; 30-31 min: 50-5% B; 31-36 min: hold at 5% B to reset gradient.
Injection volume was set to 10 µL. Mass spectra of flavan-3-ol metabolites were obtained using
an Agilent 6200 time-of-flight (TOF) mass spectrometer, with negative mode electrospay
ionization, using a mass range of 100-1000 m/z and acquisition rate of 1.4 spectra/s. Source
parameters were as follows: gas temperature = 350 °C, drying gas = 9 L/min, nebulizer pressure
= 35 psi, capillary potential = 3500 V, fragmentor voltage = 145 V, skimmer voltage = 60 V, and
OCT 1 RF Vpp = 250 V.
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2. LC-MS/MS for metabolite quantification. Separation was achieved on an Agilent 1200
HPLC system as described in the previous section, except for the gradient conditions as follows:
initial conditions 10% B; 0-9 min: 10-35% B; 9-10 min: 35-65% B; 10-12 min: 65-10% B; 12-16
min: hold at 10% B to reset gradient. Analytes were quantified using tandem mass spectrometry
(MS/MS) with an Agilent 6460 triple quadrupole with electrospray ionization (ESI) in negative
mode using selected reaction monitoring (SRM) mass transitions. Collision energy was 17 eV.
Acquisition MRMs were: C/E, 289 → 245 m/z; C/E glucuronide, 465 → 289 m/z; methyl C/E
glucuronide, 479 → 303 m/z; ethyl gallate, 197 → 169 m/z. All mass transitions used a dwell
time of 200 ms and fragmentor voltage of 135 V. Source parameters were as follows: gas
temperature = 350 °C, gas flow = 11 L/min, nebulizer pressure = 30 psi, sheath gas temperature
= 350 °C, sheath gas flow = 11 L/min, capillary potential = 3500 V, and nozzle voltage = 1000
V. Intraday repeatability was assessed by extracting and analyzing pooled plasma
n = 5 times.
Interday reproducibility was determined across n = 5 days using aliquot pooled plasma.
Repeatability and reproducibility were determined as relative coefficient of variation (% CV) as
calculated by standard deviation/mean x 100. Limit of detection (LOD) was defined as S/N = 3
and limit of quantification (LOQ) as S/N = 5. Both values were obtained by producing serial
dilutions of parent non-metabolized compounds and metabolites. Data are presented as mean
standard deviation. Signal to noise (S/N) ratio was calculated using MassHunter software using
the peak-to-peak method.
3. Second site validation. Samples were analyzed using a Waters Xevo-TQ mass
spectrometer interfaced with a Waters Acquity UPLC system. Sample preparation and LC
parameters were according to the original protocol above. Mass spectrometry source parameters
were as follows: ESI, negative mode; capillary voltage, 2 kV; source temperature, 150 oC; sheath
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gas flow, 800 L/h; desolvation temperature, 400 oC; cone voltage, 28 V. Acqusition MRMs were:
C/EC glucuronide, 456 → 289 m/z, collision energy 22; C/EC methyl glucuronide, 479 → 303
m/z, collision energy 22; ethyl gallate, 197 → 169 m/z, collision energy 16. Dwell time was 200
ms.
■RESULTS AND DISCUSSION
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Metabolites of Flavan-3-ols in Rat Plasma and Strategy for Generation of
214 Standards. Multiple metabolites derived from the flavan-3-ols C (2,3-trans) and EC (2,3-cis)
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are found in mouse or rat plasma following the feeding of monomer enriched GSPE. LC-MS
analysis has indicated that these compounds are primarily glucuronides of C/EC or methyl-C/EC
(4) (Figure 1A,B). One of the most abundant metabolites in plasma was previously identified by
micro NMR as 3ʹ-O-methyl-EC-5-O-β-D-glucuronide (Figure 1C) (7), and we have developed a
semi-synthetic route to this compound (7). Because of the low levels of the metabolites, and the
need to develop a robust method for their quantitation, we decided to develop semi-synthetic
routes to the remaining compounds.
Our strategy for generation of standards of the other C/EC metabolites found in rat plasma
was to: 1) synthesize methyl derivatives of C and EC; 2) subject the (methylated) C and EC to
enzymatic glucuronidation; 3) compare LC-MS mobility and fragmentation pattern of the
synthetic compound to those of the metabolites in plasma; 4) scale up the synthesis of
compounds that match those in plasma and confirm their structures by NMR.
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Methylation of C and EC. Although 3′-and 4′-O-methyl-EC are commercially
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available, they are expensive (synthetic 4′-O-methyl-EC currently costs over 1,000 Euros per
mg; tebu-bio, Le Perray-en-Yvelines, France). We have previously explored the methylation of
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EC using catechol-O-methyltransferase from porcine liver (7); this leads predominantly to the 3′-
O-methyl derivatives. Although this method was effective (and is a useful approach if it is
desired to incorporate radiolabel in the methyl group through incubation with labeled methyl
donor S-adenosyl-L-methionine), it is also relatively expensive, and we therefore investigated
the cheaper alternative of chemical methylation of the core flavan-3-ols with iodomethane, using
previously established procedures (9). This approach leads to generation of multiple methylated
products from both C and EC (Figure 2), with the more complex multi-methylated products
constituting a larger proportion of the reaction products with longer incubation times. However,
because the different products and isomers can easily be separated by preparative HPLC, and
because of the low cost of the starting materials, this method proved effective for generation of
3ʹ- and 4ʹ-O-mono-methyl derivatives of both C and EC, suitable for subsequent glucuronidation.
The methylated EC derivatives were authenticated by comparison to the commercial standards.
Standards are not available for the methyl C derivatives; identity was first inferred based on the
retention times of the corresponding EC derivatives, and subsequently confirmed (for the 3 ʹ-O-
methyl derivative) by NMR of the resulting glucuronidated products (see below). Optimization
of reaction conditions, particularly reaction time, resulted in maximum yields of 3ʹ-O-methyl-
C/EC of around 20%. The methylated compounds were purified by chromatography using a
Biotage Isolera system.
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Glucuronidation of (Methyl)C/EC, and Matching of Synthetic Compounds
249 to Metabolites in Plasma. Human recombinant glucuronosyltransferase UGT1A9, in the
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presence of UDP-glucuronic acid, converts EC to a number of products, the major one being the
3′-O-glucuronide (a compound not found in the plasma of rats or mice fed GSPE), with minor
amounts of the 5-O-glucuronide (a minor metabolite in the plasma) (7). 3′-O-Methyl-EC is
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efficiently converted by UGT1A9 to 3′-O-methyl-EC-5-O-glucuronide (a major plasma
metabolite). The other compounds in plasma appear to be derivatives of C. We therefore
investigated conditions (reaction times, enzyme and substrate concentrations and
supplementation of reaction mixtures with additional enzyme/substrate) for the optimal
glucuronidation of methylated and non-methylated C by mammalian glucuronosyltransferases
and UDP-glucuronic acid, as previously described for EC (7). In contrast to EC, we found that
pooled male mouse microsomes were more efficient at glucuronidation of C and its derivatives
than was recombinant UGT1A9, resulting in two major products initially named CG14 and
CG18 based on their approximate retention times by HPLC (Supplemental Figure 1A). CG14
and CG18 were further purified by preparative HPLC (Supplemental Figure 1B). Both
compounds possessed a molecular ion of 465, and matched peaks in the plasma of rats fed
GSPE: CG14 was a minor component (Supplemental Figure 1C), whereas CG18 corresponded to
the second most abundant metabolite (Supplemental Figure 1D).
In a similar manner, glucuronidation of (presumed) 3′-O-methyl-C by mouse microsomes in
the presence of UDP-glucuronic acid led to the formation of multiple products of which two, of
retention time 19 and 24 min and molecular ions of 479 (MCG19 and MCG24) matched
metabolites in the rat plasma. Glucuronidation products of (presumed) 4ʹ-O-methyl-C did not
match any of the compounds found in plasma. Figure 3 summarizes the matches between the
synthetic (methyl)-Cs and plasma-derived metabolites, and indicates the diagnostic major
fragmentation products of the molecular ions.
273 Structural characterization of C and EC metabolites. We have previously reported
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the structural characterization of 3ʹ-O-methyl-EC-5-O-glucuronide (7). To elucidate the
structures of CG14, CG18, MCG19 and MCG24, 2D NMR experiments using HSQC
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(heteronuclear single quantum coherence spectroscopy), HMBC (heteronuclear multiple bond
correlation spectroscopy), and NOESY (nuclear Overhauser effect spectroscopy) were
performed. The spectral data (chemical shifts) for the four compounds are shown in Table 1, and
1H, HSQC and HMBC, and NOESY data are shown in Supplemental Figures 2 to 8. The
glucuronic acid moiety was assigned to the 7 position on (+)-C for CG-14 and MCG-19 based on
the presence of a HMBC cross peak between the anomeric proton (H-1ʹʹ) and C-7 (Supplemental
Figure 7). The assignment on CG-18 and MCG-24 was to the 5 position due to a HMBC cross
peak between H-1ʹʹ and C-5 (Supplemental Figure 4). The methyl group was assigned to the 3ʹ
position on MCG-19 and MCG-24 due to a NOESY cross peak between H-2ʹ and the methoxy
group, confirming the identity of the precursor as 3ʹ-O-methyl-C (Supplemental Figure 8). Figure
4 summarizes the structures of the four C metabolites, and its caption provides further
description of how the positions of methylation and glucuronidation were assigned.
Several previous studies have reported the presence of PAC metabolites in blood plasma
after feeding animals with PA preparations or foods rich in PACs (10-13). The metabolites were
partially identified as sulfonated, glucuronidated and/or methylated derivatives of EC and C (11,
14-16), with most studies focusing on the EC derivatives. Definitive structures, particularly with
respect to the position of glucuronidation, were not systematically assigned for many of the
biologically relevant metabolites. The present systematic approach to the synthesis and
identification of predominant plasma-derived metabolites of flavan-3-ols present in plasma of
rodents dosed with GSPE provides a blueprint for the development of improved methods for the
evaluation of bioavailability of plant polyphenols, as well as a source of standard target
molecules for evaluating modes of action.
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A Cross-Validated Analytical Method for Determining Plasma Levels of
299 C and EC Metabolites. LC-MS using an Agilent 6460 Triple Quadrupole system was
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selected as the analytical method to determine the levels of C and EC metabolites in samples of
plasma from rats fed GSPE, due in part to the sensitive and selective nature of this method as
well as the broad application of this method in the literature (17-19). Initially, the linear dynamic
range for each molecule (C, EC, two C-glucuronides, two methyl-C-glucuronides and one
methyl-EC -glucuronide) was determined for concentrations ranging over 4 orders of magnitude
(pM-ꢀM), with lowest concentrations in the 250-650 pM range. The linear dynamic range was
observed to be excellent (Table 2) and provides the ability to quantify these compounds across a
range of concentrations typical in biological fluids and tissues. Metabolites were extracted from
plasma by solid phase extraction as described in Materials and Methods. Measurement of effects
of matrix and plasma concentration (Supplemental Table 1), and intraday and interday variability
(Supplemental Tables 2 and 3) revealed the method to be highly robust and efficient at limiting
ion suppression or other matrix effects commonly reported, but rarely accounted for, in analysis
of biological samples (20-22). As expected, there was a greater matrix effect (ME) at lower
concentrations. The % ME when measuring C ranged from -7.84% at 500 nM to -20.7% at 50
nM. The ME for MCG24 ranged from -13.5% at 500 nM to -23.0% at 50 nM. Finally, the ME
for CG18 ranged from -13.1% at 500 nM to -15.7% at 50 nM. Since metabolites appear in rodent
plasma at approximately 300-400 nM after repeated dosing of GSPE, using the SPE procedure
results in underestimating metabolites by about 13%.
The Horwitz ratio (HorRat), a well-recognized normalized performance parameter that
provides a measure of laboratory precision, was calculated for individual metabolites based on
the equations described by Horwitz and Albert (23). HorRat values ranging from 0.13 to 1.29
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indicated the high reproducibility of this method. While lower values of the HorRat (0.13-0.21)
do fall outside of the generally accepted range, it is important to note that these were for minor
metabolites (C-7-O-glucuronide, 3ʹ-O-Me-C-7-O-glucuronide and 3 ʹ-O-Me-EC-7-O-
glucuronide). Comparatively, values for major metabolites identified in this method, (C-5-O-
glucuronide, 3 ʹ-O-Me-C-5-O-glucuronide) were 1.29 and 0.51 respectively. Overall these data
are consistent with that of a highly reproducible analytical method.
Calculated LOD (defined as 3:1 signal:noise) ranged from 1.03-3.41 fmol on column for
metabolites of C and EC compared to 55.5 and 48.4 pmol on column for the parent compounds C
and EC, respectively. This suggest the potential for more efficient ionization of metabolites
relative to parent compounds. These values translated to LOQs of 2.1-10.6 fmol on-column or
~11-53 fmol per mL of plasma for metabolites.
Finally, the analyses were repeated at a second site, using the same column and separation
parameters, but a different mass spectrometer (see Materials and Methods). The C and EC
metabolites in the plasma were resolved by HPLC with very similar profiles to those recorded
previously (compare Supplemental Figure 9 and Figure 3). Calibration with authentic standards
resulted in absolute values and standard deviations that closely matched the results from the first
site analyses, with the exception of MCG24 (3ʹ-O-methyl-C-5-O-glucuronide) which was
determined based on calibration with the corresponding 7-O-glucuronide (Supplemental Table
4). It was observed that the C and EC metabolites were unstable in thawed plasma samples, as
recently reported elsewhere (24), such that reproducible results were only obtained if the samples
were injected onto the HPLC within a few hours. Samples left at room temperature were almost
totally degraded after 3 days.
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Advantages and Disadvantages of the Present Approach. Alternative
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approaches for identifying metabolites of flavan-3-ols in biological fluids have either generated
standard compounds by chemical synthesis (9), or attempted to identify the compounds directly
from the fluids (14-16, 25); most of these previous approaches have only unequivocally
identified a sub-set of the metabolites present. A recent study has reported the chemical synthesis
and full characterization of a complete range of EC glucuronides and sulfates (24). Such
compounds will be of great value for future studies on bioavailability, although their chemical
synthesis is complex, requiring, as starting point, the availability of a complete series of
orthogonally protected EC derivatives.
The present study has combined a semi-synthetic/biosynthetic scheme with matching of
compounds to authentic metabolites. Although the glucuronosyl transferase enzymes are not
completely regiospecific, their mammalian origin means that they can generate physiologically
relevant metabolites, and the matching of synthetic compounds to plasma metabolites prior to
final purification means that only those metabolites that are physiologically relevant are pursued.
Furthermore, an enzymatic strategy has advantages for the facile introduction of radiolabel, for
example in the methyl (from S-adenosyl-L-methionine) or glucuronide (from UDP-glucuronic
acid) moieties, should labeled compounds be required for mechanism of action studies or
analytical internal standards. The limiting steps from a cost perspective are the price of the
enzymes, which cannot be recycled, and the cost of UDP-glucuronic acid. Now that standards of
all glucuronidated metabolites are available for subsequent identification of products, this
problem could be circumvented by a total biological synthesis using recombinant enzymes
expressed in E. coli, as we have previously shown for synthesis of flavonol and isoflavone
glucosides (26). It should be noted that the present work does not address the sulfated
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derivatives that also arise from mammalian metabolism of flavan-3-ols, particularly in humans
(27).
■ASSOCIATED CONTENT
*S Supporting Information
Supplemental Figure 1. Glucuronidation of catechin by mouse microsomes in the presence of
UDP-glucuronic acid, and comparison of major products to metabolites of catechin in plasma
from rats fed GSPE.
Supplemental Figure 2. 1H-NMR spectrum of catechin glucuronide CG-18.
Supplemental Figure 3. 1H-NMR spectrum of methyl catechin glucuronide MCG-24.
Supplemental Figure 4. HSQC and HMBC spectra of CG-18 and MCG-24.
Supplemental Figure 5. 1H-NMR spectrum of methyl catechin glucuronide MCG-19.
Supplemental Figure 6. 1H-NMR spectrum of catechin glucuronide CG-14.
Supplemental Figure 7. HSQC and HMBC spectra of CG-14 and MCG-19.
Supplemental Figure 8. NOESY spectra of methylcatechin glucuronide MCG-19.
Supplemental Figure 9. Chromatographic separation and identification of C/EC metabolites for
each mass transition in the second site validation experiment.
Supplemental Table 1. Target assessment (plasma dilutions) and matrix effects for quantitation
of catechin and epicatechin metabolites in rat plasma by LC-MS.
Supplemental Table 2. Intraday variability for quantitation of catechin and epicatechin
metabolites in rat plasma by LC-MS, n = 5.
Supplemental Table 3. Interday variability for quantitation of catechin and epicatechin
metabolites in rat plasma by LC-MS. Experiments were performed over 5 days, n = 17.
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Supplemental Table 4. Quantitation of catechin and epicatechin metabolites in rat plasma (n
mol/l) by LC-MS in the second site validation experiment, n = 5.
This material is available free of charge via the Internet at http://pubs.acs.org.
■AUTHOR INFORMATION
395 Corresponding Author
396
397
398
*(R.A.D.) Mail: University of North Texas, Department of Biological Sciences, 1155 Union
Circle #305220, Denton, TX 76203-5017. E-mail: Richard.Dixon@unt.edu. Phone +1-940-565-
2308. Fax +1-580-224-6692.
399
400 Funding
401
402
403
404
405
406
407
408
409
410
411
This work was supported by National Institutes of Health grant 5 PO1 AT004511-05
Administrative Supplement to GMP, and by the University of North Texas and Purdue
University. BWR is supported by National Science Foundation grant DGE-1333468.
■ABBREVIATIONS USED
GSPE, grape seed polyphenol extract; PAC, proanthocyanidin; C, (+)-catechin; EC, (-)-
epicatechin; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; UGT, uridine diphosphate
glycosyltransferase; CV, column volume; SPE, solid phase extraction; DI, deionized; LC-TOF-
MS, liquid chromatography- time of flight mass spectrometry; NMR, nuclear magnetic
resonance; SRM, selective reaction monitoring; HSQC, heteronuclear single quantum coherence
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spectroscopy; HMBC, heteronuclear multiple bond correlation spectroscopy; NOESY, nuclear
Overhauser effect spectroscopy; LOD, limit of detection; LOQ, limit of quantitation.
■
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with respect to precision. J. AOAC Int. 2006, 89, 1095-109.
(24) Zhang, M.; Jagdmann, G. E., Jr.; Van Zandt, M.; Sheeler, R.; Beckett, P.; Schroeter, H.,
Chemical synthesis and characterization of epicatechin glucuronides and sulfates: bioanalytical
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(25) Natsume, M.; Osakabe, N.; Oyama, M.; Sasaki, M.; Baba, S.; Nakamura, Y.; Osawa, T.;
Terao, J., Structures of (−)-epicatechin glucuronide identified from plasma and urine after oral
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(27) Ottaviani, J.I.; Momma, T.Y.; Kuhnle, G.K.; Keen, C.L.; Schroeter, H. Structurally related
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■FIGURE CAPTIONS
Figure 1. Flavan-3-ol derivatives in rodent plasma. A, B. Representative mass spectra of
unknown metabolites in rodent plasma obtained on a LC/TOF system with a recording range of
100-1000 m/z. The mass spectrum of A is consistent with the mass of methyl-C/EC glucuronide,
while that of B is consistent with C/EC glucuronide. C. Structure of 2,3-cis-flavan-3-ols.
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Epicatechin: R1 = R2 = R3 = OH; 3ʹ-O-methyl-epicatechin-5-O-β-D-glucuronide: R1 =
glucuronic acid; R2 = OMe; R3 = OH.
Figure 2. O-Methylation of epicatechin (A) and catechin (B) by reaction with iodomethane.
HPLC traces show the multiple products formed, with the 3ʹ- and 4ʹ-O-methyl derivatives
identified by comparison to authentic standards.
Figure 3. Chromatographic separation and identification of C/EC metabolites for each mass
transition. Peak identification: (a) CG14, C-glucuronide; (b) CG18, C-glucuronide; (c) MCG24,
3ʹ-O-methyl-C-glucuronide; (d) MCG19, 3ʹ-O-methyl-C-glucuronide; (e) 3ʹ-O-methyl-EC-5-O-
glucuronide.
Figure 4. Structures assigned to CG14, CG18, MCG19 and MCG24. The glucuronic acid moiety
was able to be assigned to the 7 position on (+)-catechin for CG-14 and MCG-19 based on the
presence of a HMBC cross peak between the anomeric proton (H-1ʹʹ) and C-7. The chemical
shift of C-7 was determined by a HMBC cross peak between H-8 and C-7. Further support of
this connectivity was provided by the absence of a cross peak between H-1ʹʹ and C-5 (chemical
shift determined by a cross peak between H-4 and C-5). The assignment on CG-18 and MCG-24
was to the 5 position due to a HMBC cross peak between H-1ʹʹ and C-5. The methyl group was
assigned to the 3ʹ position on MCG-19 and MCG-24 due to a NOESY cross peak between H-2ʹ
and the methoxy group. HMBC:
NOESY:
TABLES
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Table. 1. NMR spectral data (chemical shifts) for CG14, CG18, MCG19 and MCG24.
Table 2. Linear dynamic ranges for the quantitation of (methyl)catechin and
(methyl)epicacatechin glucuronides by LC-MS/MS.
530
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CG-14 Chem. Shift (ppm)
13C
MCG-19 Chem. Shift (ppm)
13C
MCG-24 Chem. Shift (ppm)
13C
CG-18 Chem. Shift (ppm)
1H
Position
1H
Position
1H
Position
1H
Position
13C
2
4.59
82.55
2
4.63
82.54
2
4.62
83.04
2
4.59
82.53
3
3.97
2.87
2.53
6.25
6.15
6.83
6.71
6.76
4.83
3.45
3.44
3.53
3.77
68.17
28.04
---
3
4a
4b
6
4.01
2.90
2.54
6.22
6.14
6.97
3.84
6.79
6.83
4.84
3.48
3.43
3.56
3.87
68.29
30.88
---
3
4a
4b
6
4.13
2.61
2.17
6.34
6.00
6.96
3.83
6.77
6.85
4.82
3.50
3.49
3.55
3.69
66.49
27.08
---
3
4a
4b
6
4.05
3.00
2.60
6.27
6.01
6.81
6.70
6.75
4.84
3.48
3.48
3.55
3.81
66.31
28.35
---
4a
4b
6
96.94
96.81
114.86
120.27
115.62
102.06
74.23
77.27
72.98
76.16
96.94
96.59
111.46
55.83
115.42
121.44
102.03
74.06
74.42
72.65
76.13
97.49
98.08
111.61
56.40
115.97
121.21
102.81
74.60
77.94
73.71
76.59
96.76
97.97
114.83
115.75
115.50
102.23
74.43
77.43
73.12
76.57
8
8
8
8
2′
2′
2′
2′
OMe
5′
OMe
5′
5′
5′
6′
6′
1′′
2′′
3′′
4′′
5′′
6′
6′
1′′
2′′
3′′
4′′
5′′
1′′
2′′
3′′
4′′
5′′
1′′
2′′
3′′
4′′
5′′
Table 1
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LOD
(S/N=3)
MOC
Linearity
LOQ (S/N=5)
MOC
Compound
Range
Regression line
(R2)
C
5nM-50µM
y = 7E+09x+ 528.31
y = 7E+09x+ 689.79
y=5E+11x– 3699.8
y = 4E+11x+ 403.99
y=6E+11x– 16601
y=2E+11x+ 12580
y=4E+11x– 31920
0.9997
0.9997
0.9998
0.9999
0.9998
0.9990
0.9994
138.9 pmol
106.4 pmol
2.05 fmol
10.6 fmol
3.68 fmol
5.80 fmol
4.70 fmol
55.5 pmol
48.4 pmol
1.07 fmol
3.41 fmol
1.2 fmol
EC
5nM-50µM
3’OMe-EC-5-O-glucr
CG18
250pM-2.5µM
500pM-10µM
500pM-10µM
650pM-10µM
500pM-20µM
CG14
MCG19
2.80 fmol
1.03 fmol
MCG24
Table 2
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Figure 1
479.121
465.104
A
C
3’
2’
4’
5’
B
8
9
7
2
6’
A
C
B
6
3
10
5
4
600
m/z
800
1000
200
400
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Figure 2
A
B
DAD1C,Sig=280,4Ref=500,100(JWB12DECD2\UGTID0000012.D)
3ʹ-O-Methyl
4ʹ-O-Methyl
mAU
4ʹ-O-methyl
Standards
400
60
50
Methylation reaction
300
200
100
0
3ʹ-O-methyl
40
3ʹ-O-Methyl
Methylation reaction
Catechin
30
20
10
15
20
25
30
35
40
45
50
55
min
15
20
25
30
35
40
45
50
55
15
20
25
30
35
40
45
50
55
Retention time (min)
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Figure 3
e
External standards
c
A
d
479 → 303 m/z
a
2
b
465 → 289 m/z
14
16
4
6
8
min
10
12
e
GSPE plasma extract
d
B
b
c
479 → 303 m/z
465 → 289 m/z
a
8
min
14
16
2
4
6
10
12
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