Vol. 30, Issue 12, 1357-1363, December 2002
Application of Directly Coupled High Performance Liquid
Chromatography-NMR-Mass Spectometry and 1H NMR
Spectroscopic Studies to the Investigation of 2,3-Benzofuran Metabolism
in Sprague-Dawley Rats
John C.
Connelly,1
Susan C.
Connor,2
Soria
Monte,3
Nigel J.C.
Bailey,
Nathan
Borgeaud,
Elaine
Holmes,
Jeff
Troke,
Jeremy K.
Nicholson, and
Claire L.
Gavaghan
Department of Biological Chemistry, Division of Biological
Sciences, Faculty of Medicine, Imperial College of Science, Technology,
and Medicine, London, United Kingdom
 |
Abstract |
The urinary excretion of metabolites of 2,3-benzofuran was studied
in Sprague-Dawley rats (n = 5) given a single dose
of 150 mg/kg i.p. Urine samples were collected at defined intervals up to 7 days postdose and analyzed using 1H NMR and directly
coupled high performance liquid chromatography (HPLC)-NMR,
HPLC-(mass spectrometry) MS and HPLC-MS-NMR methods. The principal
metabolites were determined to be 2-hydroxyphenylacetic acid and
2-(2-hydroxyethyl)phenyl hydrogen sulfate, representing 24.3 ± 6.0% and 19.6 ± 6.4% of the dose, respectively. This indicates that metabolism of benzofuran to the polar species excreted in urine
involves cleavage of the furan ring.
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Introduction |
The
general population is potentially exposed to 2,3-benzofuran
(BF4) through its release into the environment as
a result of waste incineration (Junk and Ford, 1980
) and from exhaust
gases from gasoline and diesel engines (Seizinger and Dimitriades,
1972; Hampton et al., 1982). The use of BF in the manufacture of
coumarone-indene resins is an industrial hazard and may result in
environmental contamination with BF via various waste streams
(Budavari, 1989). Additionally, BF has been detected in drinking (Svec
et al., 1974) and ground water (Rostad et al., 1985), coffee aroma
(Silwar and Tressl, 1989), and human milk (Pellizzari et al., 1982),
and it is a component of coal tar (McNeil, 1983). The
probability of human exposure to BF has led to limited work on hazard
identification being carried out. BF induces lesions in the liver and
kidney in both mouse and rat (Connelly, 1983; National Toxicology
Program, 1989) and is associated with intrahepatic cholestasis in the
rat after short periods of daily i.p. administration (Connelly, 1983).
It was concluded by IARC (1995) that BF presents a risk of
carcinogenicity in humans as studies have shown BF to be genotoxic in
the mouse and carcinogenic in both the mouse and female rat. However,
the role of metabolism in the clearance of toxicity of BF has not been
fully characterized. The current work was initiated to identify the
urinary metabolites of BF, as part of a wider study to determine the
significance of metabolism of BF and the validity of the rat as a model
for human risk assessment.
Directly coupled HPLC-NMR has proved useful for the metabolic profiling
of xenobiotics via structural characterization of their metabolites
(Albert, 1995; Lindon et al., 1996). However, NMR spectroscopic methods
are not always sufficient for metabolite structure elucidation, for
example in the determination of sulfate conjugates, because the
introduced moiety does not contain NMR-detectable nuclei. In such
cases, directly coupled HPLC-NMR with parallel MS has the potential to
allow full structural characterization of xenobiotic metabolites in a
complex biological matrix in a single chromatographic run (Shockcor et
al., 1996; Abel et al., 1999; Bailey et al., 2000). By combining the
NMR and MS data acquisition in a directly coupled system, the problems
of synchronizing LC-MS and HPLC-NMR data are overcome. Here, we apply
this approach to identify the metabolites of BF in the urine of rats
given BF intraperitoneally.
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Materials and Methods |
Materials.
The following chemicals were obtained from the Sigma-Aldrich
(Gillingham, Dorset, UK): ammonium formate, BF, deuterium oxide, formic
acid, 2-hydroxyphenylacetic acid, 2-(2-hydroxyethyl)phenol, and methanol.
Animal Dosing.
Appropriate dose levels of benzofuran for an acute toxicity study were
selected via a dose range finder study based on literature data
(Connelly, 1983). The lowest dose of BF producing detectable histopathological changes in the liver (increased incidence and degree
of focal inflammatory cell infiltration with associated single cell
necrosis) was determined as 150 mg/kg and hence was selected as the
dose level for the current study.
Fifteen male Sprague-Dawley (SD), aged 9 to 10 weeks, were placed in
grid-based plastic cages (North Kent Plastics, Rochester, Kent,
UK). Animals Animals were given access to water and food (pelleted irradiated Rat and Mouse Diet 1; Special Diet Services, Witham, Essex) ad libitum and kept at a temperature of 21 ± 2°C. Groups of five rats were given either a single i.p. dose of BF (150 mg/kg) or saline (controls). Urine was collected over the following time periods: predose (
24-0 h), and at 0 to 8 h, 8 to
24 h, 24 to 32 h, 32 to 48 h, 48 to 72 h, 72 to
96 h, 96 to 120 h, 120 to 144 h and 144 to 168 h
postdose. All urine samples were collected into labeled tubes over
solid CO2 and stored at 20°C until analysis.
All animals were killed by cervical dislocation after collection of the
final urine sample. A separate group of rats (n = 5)
was dosed with BF and killed after 48 h to assess tissue
pathology. Major organs, including the liver and kidney, were fixed in
10% buffered formol saline, processed through paraffin wax, sectioned,
and stained with hematoxylin and eosin for examination by light microscopy.
Offline Solid Phase Extraction Chromatography with NMR Detection
(Offline SPEC-NMR) and 1H NMR Analysis of Whole Urine.
BF metabolites were isolated by offline SPEC-NMR, a procedure that
allows the concentration and purification of analytes from complex
biological matrices such as urine or plasma (Wilson and Nicholson,
1987). A urine sample collected at 0 to 8 h postdose was purified
using C8 and C18 SPEC
columns (Bond Elut; Jones Chromatography, Hengoed, Wales, UK). Using
the VacElut system, each 2-ml SPEC column was conditioned by washing
under vacuum (10 bar) with methanol (2 ml) and then acidified water (2 ml, acidified with formic acid to pH 2). The urine sample (1 ml) was
applied to the conditioned column and pulled through slowly under
vacuum and the eluate (sample) collected. The column was then washed
with water (2 × 1 ml, pH 2) to elute all nonretained molecules
from the dead volume followed by a step gradient of methanol/water (1 ml) at 20, 40, 60, 80, and 100%. Methanol was removed before all SPEC
fractions were lyophilized and reconstituted in
D2O (400 µl) for 1H NMR
spectroscopic analysis.
1H NMR spectroscopic data of SPEC extracts and
whole rat urine were acquired at ambient temperature using a 1D version
of the Nuclear Overhauser Enhancement SpectroscopY pulse
sequence (Jeener et al., 1979).
Secondary irradiation of the water signal was achieved during the
mixing time tm (150 ms). NMR data of
the SPEC extracts were acquired on a Bruker Avance spectrometer (Bruker
BioSpin GmbH, Rheinstetten, Germany) operating at 600.13 MHz.
For each spectrum 64 free induction decays (FIDs) were collected into
64 K data points using a spectral width of 12019.23 Hz, an acquisition
time of 2.73 s, and a total pulse recycle delay of 4.73 s.
NMR data of whole rat urine were acquired on a Bruker DRX 500 operating at 1H frequency 500.13 MHz. For each spectrum,
400 FIDs were collected into 64 K data points using a spectral width of
8992.81 Hz, an acquisition time of 3.64 s and a total pulse recycle
time of 6.64 s. The FIDs acquired at both frequencies were
multiplied by an exponential weighting function corresponding to a line
broadening of 0.3 Hz prior to Fourier transformation.
2D 1H-1H Long-Range Correlation
Spectroscopy (Long-Range COSY).
A 1H NMR two-dimensional spectrum of a
C8 SPEC fraction containing both BF metabolites
(water wash, pH 2) was acquired on a Bruker Avance spectrometer
operating at 600.13 MHz. 1H NMR spectra were
acquired at ambient temperature using a modified version of the
long-range correlation spectroscopy (COSY) pulse sequence (Bax and
Freeman, 1981) using a 1.5 s relaxation delay and a 300 ms delay
to allow modulation of the long-range couplings. Two hundred and
fifty-six increments with thirty-two FIDs were collected into 2 K data
points with a spectral width of 8503.40 Hz.
Analytical Chromatography for HPLC-MS, HPLC-NMR, and HPLC-NMR-MS.
The HPLC method was developed on a Hypersil ODS2 5-µm column
(250 × 4.6 mm, i.d; Thermo Hypersil-Keystone Ltd., Runcorn,
Cheshire, UK) using a step gradient elution with 0.01 M ammonium
formate buffer, pH 6.8 (100%), for 10 min; followed by
acetonitrile/0.01 M ammonium formate buffer, pH 6.8 (0:100 to 30:70
v/v), from 10 to 35 min; acetonitrile/0.01 M ammonium formate buffer,
pH 6.8 (30:70 to 50:50 v/v), from 35 to 45 min; and acetonitrile/0.01 M
ammonium formate buffer, pH 6.8 (50:50 v/v), from 45 to 55 min with a
flow rate of 1 ml/min. For the HPLC-NMR and HPLC-NMR-MS analyses,
ammonium formate buffer was prepared using D2O
(Philip Harris Scientific, London, UK). For analysis by HPLC-MS, the
ammonium formate buffer was prepared using H2O.
All methods used acetonitrile (Pestanal grade; Riedel de Haen, Seelze,
Germany) in the mobile phase.
HPLC-NMR and Mass Spectroscopic Analysis of SPEC Fractions.
The HPLC system consisted of a Bruker LC22 pump using a Bischoff 1000 Lambda variable wavelength detector (Bischoff Chromatography, Stuttgart, Germany) operated at 254 nm. Separation was effected at ambient temperature using the HPLC method as described previously. Chromatography was controlled using the Bruker HyStar software operating in the stop-flow mode. For stop-flow 1H
NMR spectra were acquired on a Bruker Avance spectrometer operating at
600.13 MHz using a presaturation pulse sequence, with double presaturation for suppression of the water and acetonitrile signals. FIDs were collected into 64 K computer data points with a
spectral width of 12019.40 Hz, an acquisition time of 2.73 s, and
a total pulse recycle time of 4.27 s with 128 summed FIDs. For
stop-flow HPLC-NMR-MS studies, FIDs were collected into 32 K computer
data points with a spectral width of 12019.40 Hz, an acquisition time of 2.73 s, and a total pulse recycle time of 4.27 s with 2000 summed FIDs. The FIDs from both studies were multiplied by an exponential weighting function corresponding to a line broadening of
0.3 Hz prior to Fourier Transform. 1H NMR
chemical shifts were referenced to the acetonitrile signal at 
2.0.
Mass spectrometric data were acquired on a Bruker Esquire ion-trap mass
spectrometer with electrospray ionization operating in either positive
or negative ion modes. Mass spectra were acquired between
m/z 100 and 1000. MS-MS experiments were effected
by isolation and fragmentation of the peak of interest using helium
gas. For HPLC-NMR-MS using mass-directed stop-flow NMR detection, a
reconstructed ion chromatogram was used to search in negative ion mode
for the molecular ion fragment of metabolite B at
m/z 218. A 20:1 splitter was positioned
immediately after the HPLC column, with 5% of the flow to the mass
spectrometer and approximately 95% of the flow to the NMR flow cell
via the UV detection cell, using polyether ether ketone tubing. The
system was arranged such that the eluent reached the mass spectrometer
6 s before it reached the UV cell. This allowed detection of the
peak of interest by MS, before the same peak was observed by UV and
subsequently parked in the NMR flow cell for NMR analysis.
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Results |
1H NMR Spectroscopy of Urine and SPEC Fractions of
Urine.
The 600 MHz 1H NMR spectra of SD rat urine
samples collected over a 4-day period prior to and after administration
of BF are shown in Fig. 1. Comparison of
the pre- and postdose 1H NMR spectra (Fig. 1) and
a spectrum of BF (99.5% purity, spectrum not shown) showed that no
parent compound was detectable but identified new resonances relating
to BF metabolites present in both the 0 to 8 h and 8 to 24 h
p.d. urine collections. Signals and consideration of and
J patterns indicated these peaks were from two BF
metabolites, termed provisionally metabolite A and metabolite B (Fig.
1). Visual comparison of the peak intensities and signal-to-noise
(s/n) ratio of the BF metabolites present in the
1H NMR spectra obtained for the 0 to 8 h and
8 to 24 h p.d. urine collections indicated that the 0 to 8 h
sample contained higher concentrations of BF metabolites. Thus, for
metabolite identification, a urine sample collected over the period 0 to 8 h after administration of BF was subjected to SPEC, and the
extracts, analyzed by 1H NMR spectroscopy,
confirmed the existence of two BF metabolites. Metabolite A coeluted
with metabolite B in the 40% methanol C18 extract, but the 80% methanol C18 extract
contained only A (Fig. 2).
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Fig. 1.
600 MHz 1H NMR spectra of urine
over a 4-day period (predose and postdose) from Sprague-Dawley rats
treated with BF.
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Fig. 2.
Expanded 600 MHz 1H NMR spectra
of metabolites A and B.
Metabolites A and B were separated by SPEC of SD urine, using a
C18 column and step-wise methanol gradient. These
metabolites were present predominantly in (a) the 80% and (b) the 40%
methanol washes, respectively, although metabolite A coeluted with B in
the 40% methanol fraction.
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Identification of BF Metabolite A.
The 80% methanol C18 extract, containing
metabolite A, was re-analyzed using directly coupled HPLC-NMR. The
metabolite was isolated in the HPLC-NMR probe, the
1H NMR spectrum acquired (Fig.
3a) and the eluate containing the peak
collected for analysis by ion-trap mass spectrometry (Fig. 3b). The
aromatic splitting pattern of two pairs of doublet (7.12 and 6.85)
and triplet (7.15 and 6.88), together with an integral value
corresponding to a total of four aromatic protons, relative to the
aliphatic singlet (3.46) corresponding to two protons, is
characteristic of an ortho substituted phenyl moiety. A 2D long-range COSY experiment (Fig. 4) of
urine containing BF metabolites A and B established a connectivity
between the singlet and the aromatic resonances corresponding to
metabolite A. Ion-trap LC-MS of the eluate, operating in positive ion
mode, identified the molecular ion with m/z of
153 (Fig. 3b) and a loss of a CO2 fragment, yielding a base peak at m/z 109. The MS and NMR
data were consistent with the structure of 2-hydroxyphenylacetic acid.
Authentic 2-hydroxyphenylacetic acid standard spiked into the original
SPEC fraction gave exactly coincident resonances, confirming the
assignment of metabolite A (data not shown). The urinary excretion of
2-hydroxyphenylacetic acid was observed in the 0 to 8 h and 8 to
24 h post-dose collection period (Fig. 1).
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Fig. 3.
Identification of metabolite A by stop-flow
HPLC-NMR using UV-directed detection and MS.
a, 600 MHz 1H NMR spectrum acquired after stop-flow
HPLC-NMR with UV-directed detection of metabolite A from a SPEC
fraction (80% methanol wash); and b, mass spectrum of the eluate from
HPLC-NMR analysis obtained by ion-trap mass spectroscopy, operated in
positive ion mode. Molecular ion m/z
153.
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Fig. 4.
2D 1H-1H long-range
COSY NMR spectrum of a C8 SPEC fraction (water wash, pH 2)
containing BF metabolites.
The off-diagonal cross-peaks show the connectivities for the side chain
proton resonances and phenyl ring proton resonances for metabolites A
and B. The corresponding peaks are also labeled in the 1D
1H NMR spectrum.
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Identification of BF Metabolite B.
Integration of the 1H NMR resonances relating to
metabolite B (Fig. 2b) established four aromatic protons (two doublets
at 7.43 and 7.38 and two triplets at 7.34 and 7.28) and a
pair of methylene protons (triplets at 2.98 and 3.85). A 2D
1H-1H long-range COSY
experiment of a C8 SPEC fraction (water wash, pH
2) containing BF metabolites A and B (Fig. 4) established
connectivities between the pair of triplets at 2.98 and 3.85, the
aromatic resonances between 7.28 and 7.43, and the triplet at
2.98 with the aromatic resonances corresponding to metabolite B. The
ortho substitution aromatic splitting pattern and the
connectivity between the aliphatic and aromatic resonances indicated
that metabolite B could be 2-(2-hydroxyethyl)phenol (2-HEP). However,
authentic 2-(2-hydroxyethyl)phenol spiked into a 40% methanol
C18 SPEC extract containing metabolite B showed
that the 1H NMR resonances did not coincide (data
not shown).
The 40% methanol C18 SPEC extract containing
metabolite B was analyzed by ion-trap mass spectrometry. Negative-ion
mass spectroscopic analysis of the SPEC extract dissolved in
H2O resulted in a molecular ion with
m/z 217, and MS-MS analysis of this molecular ion
yielded a fragment with m/z of 137, consistent
with the identification of a sulfate conjugate of
2-(2-hydroxyethyl)phenol. When this sample was dissolved in
D2O, the negative ion mass spectrum (data not
shown) resulted in a molecular ion with m/z of
218, and MS-MS analysis of this ion yielded a fragment with
m/z of 138, consistent with the loss of one
sulfate group (80 mass units). In a D2O matrix, all exchangeable protons in the metabolite would exchange with deuterium, thus the molecular ion becomes m/z of
218 (instead of m/z 217). To confirm that the
mass spectrum corresponded to metabolite B, the sample was analyzed by
directly coupled HPLC-NMR-MS using mass-directed detection in negative
ion mode, observing selected ion chromatograms at
m/z of 218. The resultant mass spectrum (Fig.
5a) observed a molecular ion peak with
m/z of 218 and the presence of a peak with
m/z of 220 at approximately 4% intensity of the
m/z 218 molecular ion, consistent with containing
the 34S isotope. The mass spectrum (Fig. 5a) also
showed the presence of fragment ions with two peaks associated with
each fragment separated by one mass unit. Since
D2O was used in the HPLC mobile phase, it would
be expected that all exchangeable protons would exchange for deuterium.
However, since the eluent was mixed with additional acetonitrile that
contains residual H2O in addition to the presence
of protons from the SPEC process, partial re-exchange of the deuteriums
for protons had occurred, complicating the fragmentation pattern
observed.
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Fig. 5.
Identification of metabolite B by directly
coupled on-flow HPLC-NMR-MS with mass-directed detection.
Analysis by HPLC-NMR-MS and MS was performed on a 40% methanol SPEC
fraction. Mass-directed detection of molecular ion at
m/z 218 identified the LC peak for
1H NMR acquisition. Panel a, extracted mass spectrum of
metabolite B, acquired in negative ion mode. Molecular ion
m/z = 218. Panel b, subsequently
acquired 600 MHz 1H NMR spectrum of metabolite B, after
mass-directed detection during the on-flow HPLC-NMR-MS analysis.
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The corresponding HPLC peak was "parked" in the NMR flow cell and
the 1H NMR spectrum acquired (Fig. 5b). The
1H NMR and mass spectra indicate the structure to
be 2-(2-hydroxyethyl)phenol conjugated with one sulfate group but do
not identify whether conjugation occurs at the phenolic or the
aliphatic hydroxyl position. Sulfate groups are known to cause a down
field shift in the 1H NMR resonances of directly
adjacent proton (Kemp, 1991). The 1H NMR
resonances of metabolite B were compared (Table
1) with those resulting from authentic
2-(2-hydroxyethyl)phenol to establish which signals had shifted down
field (to a higher chemical shift value). A down field shift was
observed in all of the resonances for metabolite B relative to those of
2-(2-hydroxyethyl)phenol, but the largest shifts were observed in the
protons attached directly to the phenyl ring, with a smaller down field
shift for the aliphatic protons. Additionally, connectivities from the
long-range COSY spectrum (Fig. 4) identified the triplet at 2.98 as
arising from the aliphatic protons directly adjacent to the aromatic
ring (Ar-CH2), and these protons show
a greater down field shift than the aliphatic protons furthest from the
aromatic ring
(Ar-CH2-CH2-OH).
This suggests that sulfate conjugation probably occurred at the
phenolic OH. 2-(2-hydroxyethyl)phenyl hydrogen sulfate was observed
in all urine samples obtained between 0 and 8 h and between 8 and 24 h postdose.
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TABLE 1
Chemical shift values of metabolite B and authentic
2-(2-hydroxyethyl)phenol (2-HEP)
1H NMR spectra acquired at 600 MHz, chemical shift values
referenced to TSP (0.00) and sample pH 7.4.
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Quantification of BF Metabolites.
The mean percentages of the dose recovered in urine as
2-hydroxyphenylacetic acid (24.3 ± 6.0%) and
2-(2-hydroxyethyl)phenyl hydrogen sulfate (19.6 ± 6.4%) over the
first 24 h after dosing BF, as determined by
1H NMR spectroscopy, are shown in Table
2. Total recovery of material identified
as derived from BF over the 24 h period was 44.0 ± 11.5% of
the dose given.
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TABLE 2
Percentage of the dose recovered in urinea as the metabolites
2-hydroxyphenylacetic acid and 2-(2-hydroxyethyl)phenyl hydrogen
sulfate during the first 24 h after administration of 150 mg BF/kg i.p.
to SD rats
The urinary BF metabolites, 2-hydroxyphenylacetic acid and
2-(2-hydroxyethyl)phenyl hydrogen sulfate, were quantitated using
1H NMR spectroscopic data. Shown is the total per cent of dose
represented for both of these metabolites excreted in the urine ± the standard deviation.
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Discussion |
The urinary excretion profile and identity of the metabolites of
BF following a single i.p. dose at 150 mg/kg in SD rats were determined
using a combination of 1H NMR, HPLC-NMR, and
HPLC-NMR-MS. A total of 44.0 ± 11.5% of the dose was eliminated
as two urinary metabolites, detectable by 1H NMR
spectroscopy up to 24 h p.d. These metabolites were identified as
2-hydroxyphenylacetic acid (24.3 ± 6.0% of dose) and
2-(2-hydroxyethyl)phenyl hydrogen sulfate (19.6 ± 6.4% of dose).
The proposed mechanism for the metabolism of BF to metabolite A and
metabolite B involves formation of the intermediate 2-(2-hydroxyphenyl) ethanal (II, Fig. 6). The furan ring is
cleaved, probably via cytochrome P450-catalyzed oxidation, to form II.
This intermediate is likely to be a substrate either for aldehyde
oxidase, yielding metabolite A (III) or for alcohol dehydrogenase
yielding the reduced product, 2-(2-hydroxyethyl)phenol sulfate (IV,
Fig. 6). Sulfation of IV will yield metabolite B (V).
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Fig. 6.
Proposed metabolism of 2,3-benzofuran in the
rat.
Key, I = 2,3-benzofuran; II = 2-(2-hydroxyphenyl) ethanal;
III = 2-(2-hydroxyphenyl) acetic acid; IV = 2-(2-hydroxyethyl)phenol; V = 2-(2-hydroxyethyl)phenyl hydrogen
sulfate.
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As a methodology for structural characterization of xenobiotic
metabolites, directly coupled HPLC-NMR-MS offers the potential for full
structural characterization in a single chromatographic run with
minimal sample preparation. In this study we have used HPLC-NMR-MS with
mass-directed NMR detection and demonstrated how this approach
facilitates the structural characterization of metabolites containing
"NMR-silent" moieties, as exemplified by the identification of the
sulfate group in 2-(2-hydroxyethyl)phenyl hydrogen sulfate.
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Footnotes |
Received May 17, 2002; accepted August 26, 2002.
1
Present address: Metabometrix Limited, RSM Prince
Consort Road, London, SW7 2BP, UK
2
Present address: Department of Safety Assessment,
GlaxoSmithKline Pharmaceuticals, Ware, Herts, SG12 0DP, UK.
3
Present address: Department of Analytical Sciences, New
Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK.
This study was supported by GlaxoSmithKline Pharmaceuticals.
Address correspondence to: Claire L. Gavaghan,
Biological Chemistry, Biomedical Sciences Division, Imperial College of
Science, Technology and Medicine, University of London, Sir Alexander
Fleming Building, South Kensington, London, SW7 2AZ, UK. E-mail:
c.gavaghan{at}ic.ac.uk
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Abbreviations |
Abbreviations used are:
BF, 2,3-benzofuran;
HPLC, high performance liquid chromatography;
MS, mass spectrometry;
LC, liquid chromatography;
SD, Sprague-Dawley;
SPEC, solid phase
extraction chromatography;
xD, 1 or 2 dimension(s);
FID, free induction
decays;
COSY, correlation spectroscopy;
MS-MS, tandem mass
spectrometry;
metabolite A, 2-(2-hydroxyphenyl) acetic acid;
metabolite B, 2-(2-hydroxyethyl)phenyl hydrogen sulfate;
2-HEP, 2-(2-hydroxyethyl)phenol;
p.d., postdose.
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Abstract
Introduction
