A comprehensive approach to the profiling of the cooked meat carcinogens 2-amino-3,8-dimethylimidazo[4,5- f ]quinoxaline, 2-Amino-1-methyl-6- phenylimidazo[4,5- b ]pyridine, and their metabolites in human urine

Article abstract of DOI:10.1021/tx900436m

A targeted liquid chromatography/tandem mass spectrometry-based metabolomics type approach, employing a triple stage quadrupole mass spectrometer in the product ion scan and selected reaction monitoring modes, was established to profile 2-amino-3,8-dimeth

Full text of DOI:10.1021/tx900436m

788
Chem. Res. Toxicol. 2010, 23, 788–801
A Comprehensive Approach to the Profiling of the Cooked Meat
Carcinogens 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline,
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, and Their
Metabolites in Human Urine
Dan Gu,† Lynn McNaughton,‡ David LeMaster,‡ Brian G. Lake,§ Nigel J. Gooderham,|
Fred F. Kadlubar,⊥ and Robert J. Turesky*,†
DiVision of EnVironmental Health Sciences and DiVision of Translational Medicine, Wadsworth Center, New
York State Department of Health, Albany, New York 12201, Centre for Toxicology, Health and Medical
Sciences, UniVersity of Surrey, GU2 7XH, U.K. and LFR Molecular Sciences, Randalls Road, Leatherhead,
Surrey KT22 7RY, United Kingdom, Biomolecular Medicine, Faculty of Medicine, Imperial College London, Sir
Alexander Fleming Building, London SW7 2AZ, United Kingdom, and Winthrop P. Rockefeller Cancer Institute,
UniVersity of Arkansas for Medical Sciences, Little Rock, Arkansas 72205
ReceiVed December 7, 2009
A targeted liquid chromatography/tandem mass spectrometry-based metabolomics type approach,
employing a triple stage quadrupole mass spectrometer in the product ion scan and selected reaction
monitoring modes, was established to profile 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx),
2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and their principal metabolites in the urine
of omnivores. A mixed-mode reverse phase cation exchange resin enrichment procedure was employed
to isolate MeIQx and its oxidized metabolites, 2-amino-8-(hydroxymethyl)-3-methylimidazo[4,5-
f]quinoxaline (8-CH2OH-IQx) and 2-amino-3-methylimidazo[4,5-f]quinoxaline-8-carboxylic acid (IQx-
8-COOH), which are produced by cytochrome P450 1A2 (P450 1A2). The phase II conjugates N2-
(ꢀ-1-glucosiduronyl)-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and N2-(3,8-dimethylimidazo[4,5-
f]quinoxalin-2-yl)-sulfamic acid were measured indirectly, following acid hydrolysis to form MeIQx.
The enrichment procedure permitted the simultaneous analysis of PhIP, N2-(ꢀ-1-glucosidurony1)-
2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, N3-(ꢀ-1-glucosidurony1)-2-amino-1-methyl-6-
phenylimidazo[4,5-b]pyridine, 2-amino-1-methyl-6-(4′-hydroxy)-phenylimidazo[4,5-b]pyridine (4′-
HO-PhIP), and the isomeric N2- and N3-glucuronide conjugates of the carcinogenic metabolite,
2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (HONH-PhIP), which is formed by P450
1A2. The limit of quantification (LOQ) for MeIQx, PhIP, and 4′-HO-PhIP was ∼5 pg/mL; the LOQ
values for 8-CH2OH-IQx and IQx-8-COOH were, respectively, <15 and <25 pg/mL, and the LOQ
values for the glucuronide conjugates of PhIP and HONH-PhIP were 50 pg/mL. The metabolism
was extensive; less than 9% of the dose was eliminated in urine as unaltered MeIQx, and <1% was
eliminated as unaltered PhIP. Phase II conjugates of the parent amines accounted for up to 12% of
the dose of MeIQx and up to 2% of the dose of PhIP. 8-CH2OH-IQx and IQx-8-COOH accounted
for up to 76% of the dose of MeIQx, and the isomeric glucuronide conjugates of HONH-PhIP
accounted for up to 33% of the dose of PhIP that were eliminated in urine within 10 h of meat
consumption. P450 1A2 significantly contributes to the metabolism of both HAAs but with marked
differences in substrate specificity. P450 1A2 primarily catalyzes the detoxification of MeIQx by
oxidation of the 8-methyl group, whereas it catalyzes the bioactivation of PhIP by oxidation of the
exocyclic amine group.
of the relative extent of bioactivation, as opposed to detoxifi-
cation, undergone by the chemicals in vivo (1). These measure-
ments can also reveal interindividual differences in metabolism
due to genetic polymorphisms that code for enzymes involved
in xenobiotic metabolism; such differences can affect the
genotoxic potency of many procarcinogens. A number of
biomarkers of carcinogens present in tobacco smoke (1), as well
as biomarkers of the hepatocarcinogen aflatoxin B1 (2, 3), have
been measured in human urine.
Introduction
Urine is a useful biological matrix for the assessment of recent
exposures to carcinogens, since large quantities can be obtained
noninvasively. Moreover, the characterization of the urinary
metabolic profiles of the genotoxicants can provide an estimate
* To whom correspondence should be addressed. Tel: 518-474-4151.
Fax: 518-473-2095. E-mail: Rturesky@wadsworth.org.
† Division of Environmental Health Sciences, New York State Depart-
ment of Health.
The measurement of urinary biomarkers of carcinogens can
also be used to assess the efficacy of chemoprotective agents
in modulating the activities of phase I and II enzymes involved
in carcinogen metabolism (4, 5). This is an area of research
that we are currently investigating and involves heterocyclic
‡ Division of Translational Medicine, New York State Department of
Health.
§ University of Surrey.
| Imperial College London.
⊥ University of Arkansas for Medical Sciences.
10.1021/tx900436m  2010 American Chemical Society
Published on Web 03/01/2010

Biomonitoring of MeIQx, PhIP, and Their Metabolites in Urine
Chem. Res. Toxicol., Vol. 23, No. 4, 2010 789
Scheme 1. Major Pathways of Metabolism of MeIQx in Humans
aromatic amines (HAAs),1 a class of carcinogens formed in
high-temperature-cooked meats (6).
experimental laboratory animals (18, 20–22) and humans
(5, 23–28). The major pathways of metabolism of MeIQx and
PhIP are depicted in Schemes 1 and 2. Metabolic activation
occurs by cytochrome P450-mediated N-oxidation of the
exocyclic amine group, to produce N-hydroxy-2-amino-3,8-
dimethylimidazo[4,5-f]quinoxaline (HONH-MeIQx) and N-hy-
droxy-2-amino-1-methyl-6-phenylmidazo[4,5-b]pyridine (HONH-
PhIP). This oxidation step is catalyzed primarily by P450 1A2
in liver and by P450s 1A1 and 1B1 in extrahepatic tissues
(16, 17). The HONH-HAAs undergo further metabolism by
N-acetyltransferases (NATs) or by sulfotransferases (SULTs)
to produce highly reactive esters that bind to DNA (29).
2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and
2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are
two of the most mass-abundant carcinogenic HAAs formed in
cooked meats: Concentrations can range from less than 1 part
per billion (ppb) to greater than 15 ppb in meats prepared under
common household cooking conditions (7). Both compounds
induce tumors in multiple organs of rodents during long-term
feeding studies (6). Putative DNA adducts of MeIQx (8) and
PhIP (9–12) have been detected in human tissues. Thus, the
chronic consumption of foods containing these HAAs constitutes
a potential human health hazard: The Report on Carcinogens,
11th edition, of the National Toxicology Program, concluded
that several prevalent HAAs, including MeIQx and PhIP, are
“reasonably anticipated” to be human carcinogens (13).
The metabolism of MeIQx and PhIP has been extensively
studied in vitro with tissue fractions, purified and recombinant
enzymes (14–17), and hepatocytes (18, 19) and in vivo in
Competing pathways of metabolism serve as mechanisms of
detoxification for both HAAs. In the case of MeIQx, P450 1A2
also catalyzes oxidation of the 8-CH3 group to form 2-amino-
8-(hydroxymethyl)-3-methylimidazo[4,5-f]quinoxaline (8-CH2OH-
IQx), which undergoes further oxidation by P450 1A2, to form
2-amino-3-methylimidazo[4,5-f]quinoxaline-8-carboxylic acid
(IQx-8-COOH) (30). This latter metabolite is the major detoxi-
fication product of MeIQx in human hepatocytes (31) and in
humans (23). It is noteworthy that IQx-8-COOH is not formed
in rodents or nonhuman primates (21, 31). Human P450s
catalyze oxidation at the 4′-position of the phenyl ring of PhIP
to form 2-amino-4′-hydroxy-1-methyl-6-phenylimdazo[4,5-
b]pyridine (4′-HO-PhIP); however, the rates of formation of 4′-
HO-PhIP are significantly lower than the rates of HONH-PhIP
formation (16, 22, 32). Although 4′-HO-PhIP is a minor
metabolic product of human P450 1A2, it is a principal
detoxification product of PhIP in rodents and nonhuman
primates (18, 21).
1 Abbreviations: MeIQx, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline;
MeIgQx, 2-amino-1,7-dimethylimidazo[4,5-g]quinoxaline; HONH-MeIQx,
N-hydroxy-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline; 8-CH2OH-IQx,
2-amino-8-(hydroxymethyl)-3-methylimidazo[4,5-f]quinoxaline; IQx-8-
COOH, 2-amino-3-methylimidazo[4,5-f]quinoxaline-8-carboxylic acid; MeIQx-
N2-Gl, N2-(ꢀ-1-glucosiduronyl)-2-amino-3,8-dimethylimidazo[4,5-f]quinox-
aline; HON-MeIQx-N2-Gl, N2-(ꢀ-1-glucosiduronyl)-2-(hydroxyamino)-3,8-
dimethylimidazo[4,5-f]quinoxaline;MeIQx-N2-SO3H,N2-(3,8-dimethylimidazo[4,5-
f]quinoxalin-2-yl-sulfamic acid; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-
b]pyridine; HONH-PhIP, N-hydroxy-2-amino-1-methyl-6-phenylmidazo[4,5-
b]pyridine; 4′-HO-PhIP, 2-amino-4′-hydroxy-1-methyl-6-phenylimdazo[4,5-
b]pyridine; 5-HO-PhIP, 2-amino-1-methyl-6-(5-hydroxy)phenylimidazo[4,5-
b]pyridine; HON-PhIP-N2-Gl, N2-(ꢀ-1-glucosiduronyl-2-(hydroxyamino)-
1-methyl-6-phenylimidazo[4,5-b]pyridine; HON-PhIP-N3-Gl, N3-(ꢀ-1-
glucosiduronyl-2-(hydroxyimino)-1-methyl-6-phenylimidazo[4,5-
b]pyridine; PhIP-N2-Gl, N2-(ꢀ-1-glucosiduronyl-2-amino-1-methyl-6-
phenylimidazo[4,5-b]pyridine; PhIP-N3-Gl, N3-(ꢀ-1-glucosiduronyl-2-
amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; AMS, accelerator mass
spectrometry; fwhm, full width at half-maximum; GC-NICI-MS, gas
chromatography with negative ion chemical ionization-mass spectrometry;
HAAs, heterocyclic aromatic amines; LC/MS, liquid chromatography/mass
spectrometry; LC-ESI/MS/MS; liquid chromatography-electrospray ioniza-
tion/mass spectrometry/tandem mass spectrometry; LOQ, limit of quanti-
fication; MR, metabolic ratio; NATs, N-acetyltransferases; ppb, parts per
billion; SPE, solid-phase extraction; SRM, selected reaction monitoring;
SULTs, sulfotransferases; UGTs, uridine diphosphate glucuronosyltrans-
ferases.
MeIQx and PhIP also undergo detoxification by phase II
conjugation reactions. MeIQx undergoes sulfamation to form
N2-(3,8-dimethylimidazo[4,5-f]quinoxalin-2-yl-sulfamicacid(MeIQx-
N2-SO3H); the process is catalyzed by sulfotransferase 1A1
(SULT1A1) (33). A second phase II metabolite of MeIQx is
the glucuronide conjugate, N2-(ꢀ-1-glucosiduronyl)-2-amino-
3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx-N2-Gl), which
is formed by uridine diphosphate glucuronosyltransferases
(UGTs), apparently UGT1A isoforms (34–36). An N2-glucu-
ronide conjugate of HONH-MeIQx has been characterized as
N2-(ꢀ-1-glucosiduronyl)-2-(hydroxyamino)-3,8-dimethylimida-

790 Chem. Res. Toxicol., Vol. 23, No. 4, 2010
Scheme 2. Major Pathways of Metabolism of PhIP in Humans
Gu et al.
zo[4,5-f]quinoxaline (HON-MeIQx-N2-Gl) (37). Both PhIP and
HNOH-PhIP undergo conjugation by UGT1A isoforms to
produce N2- and N3-glucuronide conjugates (34–36). The
glucuronide conjugates of HNOH-MeIQx and HONH-PhIP are
viewed as detoxification products (27).
for one another, in assessment of exposures in humans consum-
ing unrestricted diets (46). Moreover, assessment of the effica-
cies of chemoprotective agents requires a multipurpose analytical
method that can be employed to quantitate both HAAs and their
metabolites in urine. We recently reported a facile SPE method
to isolate PhIP and several of its major metabolites in human
urine; its use is followed by quantitative measurements by liquid
chromatography/mass spectrometry (LC/MS) (47). In this article,
we describe a refinement of the SPE procedure to create a rapid,
one-step method to isolate MeIQx and PhIP and several of their
P450 1A2-derived metabolites in urine, followed by quantitative
LC-ESI/MS/MS analysis. With this validated method, we have
examined the interrelationship between the oxidative metabolism
of MeIQx and that of PhIP in urine samples from 10 volunteers.
The analysis of unaltered MeIQx and PhIP and their
metabolites in human urine is an analytical challenge, because
usually only ∼1 to several micrograms of each compound is
ingested per day in individuals eating well-done meat (38). Thus,
the concentrations of these HAAs and their metabolites are often
well below the ppb level in urine. The polar and ionic nature
of the metabolites also presents difficulties for their isolation,
along with their parent HAAs, from thousands of other
components in the urine matrix. Various analytical approaches
have been devised to isolate MeIQx or PhIP from human urine:
Such techniques have included solvent extraction (39, 40), solid-
phase extraction (SPE) (41), the use of molecularly imprinted
polymers (28), and immunoaffinity methods (24), followed by
quantification by gas chromatography with negative ion chemi-
cal ionization-mass spectrometry (GC-NICI-MS) (39, 40, 42)
or liquid chromatography-electrospray ionization/mass spec-
trometry/tandem mass spectrometry (LC-ESI/MS/MS) (28, 41)
or alternatively followed by fluorescence detection (25). [14C]-
MeIQx, [14C]-PhIP, and [14C]-radiolabeled metabolites have
been identified in human urine by accelerator mass spectrometry
(AMS) (23, 27, 43). Urinary metabolites have also been detected
by liquid chromatography-electrospray ionization/mass spec-
trometry/tandem mass spectrometry (LC-ESI/MS/MS) (5, 26),
or indirectly, after chemical reduction or acid hydrolysis of
HONH-PhIP conjugates, with detection LC-ESI/MS/MS or GC-
NICI-MS (44, 45).
Experimental Procedures
Caution: MeIQx, PhIP, and seVeral of their deriVatiVes are
potential human carcinogens and should be handled with caution
in a well-Ventilated fume hood with the appropriate protectiVe
clothing.
Materials and Methods. MeIQx, PhIP, and 2-amino-3-tri-
deutromethyl-8-methylimidazo[4,5-f]quinoxaline ([2H3C]-MeIQx)
and2-amino-1-trideutromethyl-6-phenylimidazo[4,5-b]pyridine([2H3C]-
PhIP) (both at 99% isotopic purity) were purchased from Toronto
Research Chemicals (Toronto, ON, Canada). NADPH, NADH,
glucose-6-phosphate, uridine-5′-diphosphoglucuronic acid (UD-
PGA), glucose-6-phosphate dehydrogenase, alamethicin, ꢀ-glucu-
ronidase type IX-A from Escherichia coli, and sulfatase from
abalone entrails type VIII were all purchased from Sigma (St. Louis,
MO). Pooled male rabbit liver microsomes (New Zealand White)
were purchased from BD Biosciences (Woburn, MA). Male SD
rat liver microsomes of animals pretreated with polychlorinated
biphenyls (PCBs, Aroclor-1254) were obtained from Moltox
(Boone, NC). Human liver microsomal samples were from Ten-
nessee Donor Services (Nashville, TN) and were a gift kindly
provided by Dr. F. P. Guengerich, Vanderbilt University. The P450
1A2 protein expression and metabolic activity were previously
characterized (16). All solvents used were high-purity B & J Brand
To our knowledge, no report in the literature has described
the simultaneous analysis of MeIQx and PhIP and their principal
metabolites in human urine. The concurrent analysis of these
biomarkers is important since the urinary excretion levels of
either MeIQx or PhIP can only serve as an approximate measure

Biomonitoring of MeIQx, PhIP, and Their Metabolites in Urine
Chem. Res. Toxicol., Vol. 23, No. 4, 2010 791
from Honeywell Burdick and Jackson (Muskegon, MI). ACS
reagent grade HCO2H (88%) was purchased from J. T. Baker
(Phillipsburg, NJ), Retain CX resins (30 mg) were purchased from
ThermoFisher Scientific (Palm Beach, FL), and Baker C18 SPE
resins (500 mg) were purchased through Krackeler Scientific Inc.
(Albany, NY). All other chemical reagents were ACS grade and
were purchased from Sigma Aldrich.
male volunteers who participated in a previous investigation, and
full details were reported previously (5, 51). In brief, each subject
consumed 275 g of cooked minced beef patties that had been fried
without added oil or fat for 6 min on each side, using a hot metal
griddle at 300 °C, until the meat was well-browned. The average
amount of MeIQx ingested was 920 ng, and the average amount
of PhIP ingested was 4950 ng (51). A urine collection was then
commenced over 10 h, and urine samples were stored at -80 °C.
A subset of samples were sent blind coded on dry ice to the
Wadsworth Center for further analyses. This study was approved
by the Institutional Review Board at the Wadsworth Center.
General Methods. Mass spectra of synthetic and biosynthetic
derivatives were obtained on a Finnigan Quantum Ultra triple stage
quadrupole mass spectrometer (Thermo Electron, San Jose, CA).
Typical instrument tuning parameters were as follows: capillary
temperature, 275 °C; source spray voltage, 3.5 kV; sheath gas
setting, 35; tube lens offset, 95; capillary offset, 35; and source
fragmentation, 15 V. Argon, set at 1.5 mTorr, was used as the
collision gas. Analyses were conducted in positive ionization mode.
NMR resonance assignment experiments were carried out at 25
and 45 °C for the rabbit and human liver PhIP-N-glucuronides,
respectively, on a Bruker Avance DRX 600 MHz spectrometer
equipped with a triple resonance cryoprobe (Bruker BioSpin Corp.,
SPE of MeIQx and PhIP and Their Metabolites from
Urine. The enrichment procedure was previously reported (47). In
this present workup, the organic precipitation and vacuum concen-
tration steps were omitted prior to SPE. The urine samples (1.0
mL) were acidified with HCO2H (88% v/v, 20 µL) and centrifuged
at 15000g for 2 min to remove particulates. The supernatants were
applied to ThermoFisher HyperSep Retain CX (30 mg of resin)
cartridges that had been prewashed with CH3OH containing 5%
NH4OH (1 mL), followed by 2% HCO2H in H2O (1 mL). The resins
were attached to a vacuum manifold, under slight positive pressure
(∼5 in. of Hg), to achieve a flow rate of the eluent of approximately
1 mL/min. After application of the samples, the cartridges were
washed with 2% HCO2H in H2O (1 mL), followed by 2% HCO2H
in CH3OH (1 mL), H2O (1 mL) and 5% NH4OH (1 mL). The resin
was allowed to run to dryness. Next, the biomarkers were eluted
from the resin with CH3OH containing 1% NH4OH (1.5 mL). This
solvent was allowed to absorb into the resin for 3 min, prior to
gentle manual elution of the solvent with a disposable 1 mL syringe.
The extract was collected into Eppendorf tubes (2.0 mL) and placed
in a ventilated hood for 15 min to allow the NH3 to evaporate. The
extracts were concentrated to approximately 0.1 mL by vacuum
centrifugation. Then, the samples were transferred into silylated
glass conical vials (0.35 mL volume) from MicroLiter Analytical
Supplies, Inc. (Suwanee, GA) and evaporated to dryness by vacuum
centriguation. The samples were resuspended in 1:1 H2O:DMSO
(20 µL).
LC-ESI/MS/MS Analyses. Chromatography was performed with
an Agilent 1100 series capillary LC system (Agilent Technologies)
equipped with an Agilent Zorbax-SB-C18 column (0.3 mm × 250
mm; 5 µm particle size). Analytes were separated by a gradient.
The A solvent contained 0.01% HCO2H in H2O, and the B solvent
contained 0.01% HCO2H and 5% H2O in CH3CN. The flow rate
was set at 6 µL/min, starting at 100% A and holding for 1 min,
followed by a linear gradient to 60% B at 35 min, and then to
100% B at 36 min, and holding for 4 min. The gradient was reversed
to the starting conditions over 1 min, and a postrun time of 14 min
was required for re-equilibration. The mass spectral data were
acquired on a Finnigan Quantum Ultra Triple Stage Quadrupole
MS; data manipulations were carried out with Xcalibur version 2.07
software. Analyses were conducted in the positive ionization mode
and employed an Advance nanospray source from Michrom
Bioresources Inc. (Auburn, CA). The spray voltage was set at 2100
V, the in-source fragmentation was -10 V, and the capillary
temperature was 220 °C. No sheath or auxiliary gas was used. The
peak widths (in Q1 and Q3) were set at 0.7 Da, and the scan width
was 0.002 Da.
1
Billerica, MA). The H chemical shifts were referenced directly
from the DMSO-d6 multiplet at 2.50 ppm. A standard DQF-COSY
experiment was employed to collect 1024 t1 increments over a 7507
Hz spectral window. Selected NMR spectra are provided in the
Supporting Information. HPLC separations of biomarkers were done
with an Agilent (Palo Alto, CA) model 1100 HPLC system
equipped with a photodiode array detector, equipped with a
Rheodyne 7725i (Rhonert Park, CA) manual injector.
Syntheses. 2-Amino-3-methylimidazo[4,5-f]quinoxaline-8-car-
baldehyde, 8-CH2OH-IQx, IQx-8-COOH, and MeIQx-N2-SO3H.
MeIQx (5 mg, 2.3 µmol) in dioxane (20 mL) was oxidized with
selenium dioxide (5 mg, 45 µmol) by heating the solution at reflux
for 5 h, as previously described (31). The two major products, the
8-carbaldehyde derivative of MeIQx and IQx-8-COOH, were
formed in approximately 30 and 60% yields, respectively. Treatment
of the reaction mixture with NaCNBH3 (5 mg, 78 µmol, 1 h at 37
°C) resulted in complete reduction of the aldehyde to 8-CH2OH-
IQx (31). MeIQx-N2-SO3H was prepared by treatment of MeIQx
with a 1.2 mol excess of chlorosulfonic acid in anhydrous pyridine,
as previously described (48).
Biosynthesis of NOH-MeIQx-N2-Gl and MeIQx-N2-Gl. These
metabolites were prepared by incubating MeIQx or HONH-MeIQx
(0.5 mM) (or [2H3C]-MeIQx and [2H3C]-HONH-MeIQx) with 20
mg of liver microsomal protein from rats pretreated with PCBs in
10 mL of 100 mM Tris-HCl buffer (pH 7.5), containing 10 mM
MgC12 and UDPGA (5 mM) for 6 h at 37 °C (49). The microsomal
mixture was preincubated with alamethicin (50 µg/mg protein) on
ice for 15 min, prior to the addition of the MeIQx compounds.
The glucuronide metabolites were isolated as previously described
(47). HONH-MeIQx was synthesized as previously described (50).
Biosynthesis of 4′-HO-PhIP, N2-(ꢀ-1-Glucosidurony1)-2-
amino-1-methy1-6-phenylimidazo[4,5-b]pyridine (PhIP-N2-Gl),
N3-(ꢀ-1-Glucosiduronyl-2-amino-1-methyl-6-phenylimidazo[4,5-
b]pyridine (PhIP-N3-Gl), N2-(ꢀ-1-Glucosidurony1)-N-hydroxy-
2-amino-1-methy1-6-phenylimidazo[4,5-b]pyridine (HON-PhIP-
N2-Gl), and N3-(ꢀ-1-Glucosiduronyl-2-(hydroxyimino)-1-methyl-
6-phenylimidazo[4,5-b]pyridine (HON-PhIP-N3-Gl) Conjugates.
The biosynthesis of N2-(ꢀ-1-glucosiduronyl-2-amino-1-methyl-6-
phenylimidazo[4,5-b]pyridine (PhIP-N2-Gl) was carried out with
rabbit liver microsomal protein (1 mg/mL), and the biosynthesis
of PhIP-N3-Gl was done with human liver microsomal protein (2
mg/mL) in 100 mM Tris-HCl buffer (pH 7.5), containing 10 mM
MgC12, PhIP (0.5 mM), and UDPGA (5 mM). The mixture was
preincubated with alamethicin (50 µg/mg protein) on ice for 15
min, followed by incubation at 37 °C for 6 h. The products were
isolated as previously described (47). The isomeric HON-PhIP-
N2-Gl and HON-PhIP-N3-Gl metabolites were prepared from human
or rat liver microsomes as previously described (47). 4′-HO-PhIP
was produced from the oxidation of PhIP with rat liver microsomes
pretreated with PCBs (47).
The following transitions and collision energies were used for
the quantification of MeIQx, PhIP, and their metabolites: MeIQx
and [2H3C]-MeIQx: 214.1 f 199.1 and 217.1 f 199.1 at 30 eV;
8-CH2OH-IQx and [2H3C]-8-CH2OH-IQx: 230.1 f 212.1, 197.1,
and 233.1 f 215.1, 197.1 at 20 or 30 eV; 8-COOH-IQx and [2H3C]-
8-COOH-IQx: 244.1 f 198.1, 183.1, and 247.1 f 201.1, 183.1 at
20 or 30 eV; PhIP and [2H3C]-PhIP: 225.1 f 210.1 and 228.1 f
210.1 at 33 eV; 4′-HO-PhIP: 241.1 f 226.1 at 35 eV; isomeric
HON-PhIP-N-Gl and [2H3C]-HON-PhIP-N-Gl: 417.1 f 225.1,
224.1, and 223.1 and 420.1 f 228.1, 227.1 and 225.1 @ 34 eV.
The dwell time for each transition was 10 ms. Argon was used as
the collision gas and was set at 1.5 mTorr. Product ion spectra
were acquired on the protonated molecules [M + H]+, scanning
Human Subjects and Meat Consumption. The analyses of
MeIQx and PhIP metabolites were conducted on urine samples from

792 Chem. Res. Toxicol., Vol. 23, No. 4, 2010
Gu et al.
from m/z 50 to 500 at a scan speed of 250 amu/s using the same
acquisition parameters as above.
ESI/MS/MS. The transition employed to monitor MeIQx ([M
+ H]+ f [M + H - 15]+• at 34 eV) produces a radical cation.
The product ion arises by homolytic cleavage of the 3-N-CH3
bond (53). The transition used to monitor 8-CH2OH-IQx ([M
+ H]+ f [M + H - 18]+ at 20 eV) is due to the loss of H2O
from the alcohol group (30). The transition employed to monitor
IQx-8-COOH ([M + H]+ f [M + H - 46]+ at 20 eV) is
attributed to loss of H2O and CO (30). These collision energy
values provided the maximum sensitivity in the selective
reaction monitoring (SRM) mode. MeIQx was not detected in
the pre-exposure urine sample, but it and its linear, tricyclic
ring isomer, 2-amino-1,7-dimethylimidazo[4,5-g]quinoxaline
(MeIgQx) (54), were observed in urine collected after meat
consumption by the subject (Figure 1). The internal standard
[2H3C]-8-CH2OH-IQx was discerned in both pre- and postex-
posure urine extracts; however, the background signal of the
transition employed to monitor 8-CH2OH-IQx was elevated in
the pre-exposure urine, and numerous isobaric interfering peaks
were observed in the urine extract after meat consumption. The
transitions employed to measure IQx-8-COOH and [2H3C]-IQx-
8-COOH were even less selective. The internal standard was
barely resolved from interfering peaks in the urine extract
analyzed after meat consumption.
Calibration Curves. Calibration curves were generated in
triplicate by the addition of a fixed amount of [2H3C]-PhIP and
[2H3C]-MeIQx (100 pg) and [2H3C]-8-CH2OH-IQx (300 pg) and
0, 6, 12, 16, 20, 40, 60, or 100 pg of the unlabeled standards per
1.0 mL urine from a volunteer who had not consumed cooked meat
for at least 48 h. [2H3C]-IQx-8-COOH was added at a level of 300
pg/mL of urine, and the unlabeled standard was added at 0, 30, 60,
80, 100, 200, 300, or 500 pg per mL of urine. The calibration curves
of PhIP metabolites were also constructed in triplicate with [2H3C]-
PhIP-N3-Gl, [2H3C]-HON-PhIP-N2-Gl, and [2H3C]-HON-PhIP-N3-
Gl added at a fixed concentration of 1000 pg per mL of urine, and
the unlabeled analytes were each added at concentrations of 0, 60,
120, 160, 200, 400, 600, or 1000 pg/mL of urine. The calibration
data were fitted to a straight line using the ordinary least-squares
method with equal weightings. Each urine sample was subjected
to the SPE processing conditions described above. The within-day
and between-day precisions for MeIQx and PhIP and their
metabolites were calculated in triplicate or quadruplicate as
described (52), with use of urine samples from three different
subjects collected during 10 h, after consumption of cooked meat
(5). The measurements were done on three different days over a
time period of 1 month. The response of the signal of 4′-HO-PhIP
was assumed to be comparable to PhIP, and [2H3C]-PhIP was
employed as an internal standard to estimate the levels of 4′-HO-
PhIP.
Enzyme and Acid Hydrolysis Assays. Urine samples from
omnivores (0.5 mL) were spiked with the isotopically labeled
internal standards of MeIQx and PhIP or their metabolites and then
diluted with 0.5 mL of 100 mM sodium acetate buffer (pH 5.5) or
0.5 mL of 100 mM sodium phosphate buffer (pH 6.5). Enzyme
hydrolysis was conducted with ꢀ-glucuronidase (14 U/mL) and
sulfatase (2 U/mL) at 37 °C for 4 h. The samples were then diluted
with glacial acetic acid (1 mL) and processed by SPE. Urine
samples (0.5 mL) were also subjected to acid hydrolysis in 50%
(v/v) glacial acetic acid or in 1 N HCl, by heating at 80 °C for 8 h.
After they were cooled, the samples were processed by SPE. Under
these acid hydrolysis conditions, MeIQx-N2-SO3H, MeIQx-N2-Gl,
and PhIP-N2-Gl were quantitatively hydrolyzed to the parent amines,
whereas the PhIP-N3-Gl was stable and <5% of the conjugate
underwent hydrolysis.
The Finnigan Quantum Ultra triple stage quadrupole MS has
the capability of scanning at enhanced resolution, thereby
increasing the mass resolving power from ∼500 [m/∆m at full
width at half-maximum (fwhm) peak height], when it is scanning
at 0.7 Da in either Q1 or Q3 (55), to a resolution of ∼2000 for
MeIQx and its oxidized metabolites, when the scan width is
set at 0.1 Da at fwhm resolution. However, the use of the
enhanced resolution scan mode did not significantly improve
the signal-to-noise for the transitions of 8-CH2OH-IQx, 8-IQx-
8-COOH, or their internal standards (data not shown).
The collision energy used to fragment these oxidized me-
tabolites of MeIQx was then increased from 20 to 30 eV. Under
the higher collision energy conditions, both metabolites undergo
a second fragmentation at the 3-N-CH3 bond and lose a CH3
radical (30). Although the response of the signals was decreased
at 30 eV, there was a dramatic diminution in the background
signals at all transitions ([M + H]+ f ([M + H - 33]+ and
[M + H]+ f ([M + H - 36]+ for 8-CH2OH-IQx and [2H3C]-
8-CH2OH-IQx, respectively, and [M + H]+ f ([M + H - 61]+
and [M + H]+ f ([M + H - 64]+ for IQx-8-COOH and
[2H3C]-IQx-8-COOH, respectively): At 30 eV, the target
compounds and internal standards of both metabolites appear
as distinct peaks (Figure 2). 8-CH2OH-IQx or 8-IQx-8-COOH
was not detected in the predose urine extract, but both
metabolites were readily discernible in the urine sample after
meat consumption. Remarkably, the high sensitivity provided
by the Quantum Ultra triple quadrupole MS with the Michrome
Advance nanospray source has enabled us to acquire good
quality product ion spectra of the urinary 8-CH2OH-IQx and
IQx-8-COOH metabolites. The spectra are in good agreement
with the spectra of the reference compounds (Figure 3) (30).
The product ion spectra of MeIQx and MeIgQx were also
successfully acquired on urine samples that underwent acid
hydrolysis, resulting in an increase in the concentrations of these
HAAs by up to 5-fold (vide infra) (Figure 4).
Statistical Analysis. Spearman’s rank correlation coefficient (rs)
determinations for the urinary metabolites and unmetabolized HAAs
were done with GraphPad Prism Version 4 software (San Diego,
CA).
Results
Rapid SPE and LC-ESI/MS/MS Analysis of MeIQx,
8-CH2OH-IQx, IQx-8-COOH, PhIP, PhIP-N2-Gl, 4′-HO-
PhIP, HON-PhIP-N2-Gl, and HON-PhIP-N3-Gl in Urine
of Omnivores. A facile SPE procedure previously developed
to purify PhIP and its metabolites from urine (47) was employed
for the concurrent isolation of MeIQx, 8-CH2OH-IQx, and IQx-
8-COOH. We determined that the treatment of urine with an
organic solvent, to precipitate salt and protein, and the ensuing
vacuum concentration step, to remove the organic solvent, are
not required prior to SPE. The recovery of the analytes, the
purity of the extracts, and the limit of quantification (LOQ) of
MeIQx, PhIP, and their metabolites were not affected by the
omission of these pre-SPE processing steps. The exclusion of
these procedures has resulted in a rapid, one-step, high-
throughput method for isolation of these HAA urinary biomarkers.
The analyses of MeIQx, 8-CH2OH-IQx, and IQx-8-COOH
in urine from an omnivore who had refrained from eating cooked
meat for 48 h (pre-exposure) and in urine collected over a 10 h
time point, after consumption of grilled meat (postexposure),
is shown in Figure 1. The compounds were monitored by LC-
The analyses of PhIP, 4′-HO-PhIP, PhIP-N2-Gl, PhIP-N3-
Gl, HON-PhIP-N2-Gl, and HON-PhIP-N3-Gl in urine of the
subject, pre- and postmeat consumption, are shown in Figure
5. None of these biomarkers were present in urine at detectable
levels, when the subject had refrained from eating meat.
However, PhIP and all of its metabolites, except for PhIP-N3-

Biomonitoring of MeIQx, PhIP, and Their Metabolites in Urine
Chem. Res. Toxicol., Vol. 23, No. 4, 2010 793
Figure 1. SRM traces for MeIQx, 8-CH2OH-IQx, and IQx-8-COOH in a urine sample collected before and after meat consumption. The transition
employed to monitor 8-CH2OH-IQx and IQx-8-COOH and their internal standards was 20 eV. The retention time (tR), area, and ion intensity are
reported. The large peak eluting at tR ) 23.0 min and monitored with the transition 214.1 > 199.1 is 7-MeIgQx, an isomer of MeIQx, which elutes
at tR ) 23.8 min.
Gl, were detected in the urine of the subject after consumption
of meat. The product ion spectra of HON-PhIP-N2-Gl and HON-
PhIP-N3-Gl are in excellent agreement with the spectra of the
reference compounds previously published (data not shown)
(47). Product ion spectra of PhIP and 4′-HO-PhIP were
successfully acquired on urine samples that underwent acid
hydrolysis: Acid treatment increased the concentrations of these
compounds by up to several-fold (vide infra) (Figure 6).
is the major human liver microsomal metabolite, and PhIP-N2-
Gl is the principal glucuronide conjugate produced by rabbit
liver microsomes. Despite the strong preference, by human liver
UGTs, to glucuronidate PhIP at the N3 imidazole atom, only
the PhIP-N2-Gl isomer is eliminated in urine at levels above
the limit of detection. However, the amounts of PhIP-N2-Gl
eliminated in urine are low, and the direct glucuronidation of
PhIP appears to be a minor route of detoxication of PhIP in
these 10 subjects of our pilot study.
Performance of the Analytical Method. The recoveries of
each internal standard [2H3C]-MeIQx and [2H3C]-PhIP (100 pg/
mL), [2H3C]-8-CH2OH-IQx and [2H3C]-IQx-8-COOH (each at
300 pg/mL), and PhIP-N2-Gl, HON-PhIP-N2-Gl, and HON-
PhIP-N3-Gl (each at 1000 pg/mL), added to urine prior to
sample processing, were consistently between 40 and 80%,
based on the response of the signals to those of pure standards
measured by LC-ESI/MS/MS. The response of the signals of
the processed internal standards is a function of the recoveries
of the compounds and the potential ion suppression effects of
the urine matrix (41). The calibration curves for MeIQx,
8-CH2OH-IQx, and IQx-8-COOH (generated from three inde-
pendent replicates per calibrant level), constructed from urine
samples from a subject who had refrained from eating cooked
meat for 48 h, displayed good linearity (R2 ) >0.997; Supporting
The relative amounts of isomeric glucuronide conjugates of
PhIP formed by human liver microsomes in vitro, and their
formation in vivo, based on urinary elimination, appear at
variance. Human liver microsomes fortified with UDPGA
produced 10-fold greater amounts of PhIP-N3-Gl than PhIP-
N2-Gl (47, 56); yet, PhIP-N2-Gl is the main isomer known to
be present in urine of meat eaters (Figure 5, tR ) 27.1 min)
(27, 43). To confirm the sites of conjugation and structural
assignments of these isomeric glucuronide conjugates, we re-
examined, by 1H NMR spectroscopy, the human and rabbit liver
microsomal PhIP-N-Gl metabolites: The glucuronide conjugate
formed by rabbit liver was previously characterized as PhIP-
N2-Gl, whereas the conjugate produced by human liver was
1
assigned as the PhIP-N3-Gl (56). Our H NMR spectral data
(Supporting Information, Figures S-1A-C and Table S-1) are
consistent with the structures previously assigned: PhIP-N3-Gl

794 Chem. Res. Toxicol., Vol. 23, No. 4, 2010
Gu et al.
Figure 2. SRM traces for MeIQx, 8-CH2OH-IQx, and IQx-8-COOH in the same urine samples, as in Figure 1, collected before and after meat
consumption. The transition employed to monitor 8-CH2OH-IQx and IQx-8-COOH and their internal standards was 30 eV.
Information, Figure S-2). The calibration curves for PhIP, PhIP-
N3-Gl, HON-PhIP-N2-Gl, and HON-PhIP-N3-Gl have previ-
ously been reported (47): The new sets of calibration curves of
these biomarkers obtained in the current study are presented in
the Supporting Information (Figure S-3). The LOQ values were
derived based on a threshold of 10σ SD units above the
background signal levels (57) in the urine samples from three
volunteers, during the pre-exposure phase of the study. The LOQ
values were ∼5 pg/mL for MeIQx, PhIP, and 4′-HO-PhIP,
whereas the LOQ for 8-CH2OH-IQx was 10 pg/mL, and the
LOQ for IQx-8-COOH was 35 pg/mL. The LOQ values for
the glucuronide conjugates of PhIP and HONH-PhIP were
estimated at 50 pg/mL.
The precision CV (%) of the estimates in the calibration curve
at the lowest calibrant levels of MeIQx and 8-CH2OH-IQx (6
pg/mL) was e11%, and the CV (%) for IQx-8-COOH at the
lowest calibrant levels (30 pg/mL) was e10%; the precision
values improved at the higher calibrant levels. The performance
of the method was assessed by the within-day and between-
day estimates and precision of measurements of MeIQx,
8-CH2OH-IQx, IQx-8-COOH, PhIP-N2-Gl, HON-PhIP-N2-Gl,
and HON-PhIP-N3-Gl, in urine samples from three randomly
selected volunteers determined over 3 separate days (n ) 3 or
4 independent measurements per day), within a time period of
1 month. The results are summarized in Table 1. The within-
day and between-day CV (%) in estimates of MeIQx, 8-CH2OH-
IQx, and IQx-8-COOH were well below 10.0%. The perfor-
mance of the method was previously reported for PhIP and its
N2- and N3-glucuronides of HONH-PhIP (47); a similar degree
of precision is observed in these newly assayed urine samples
(Table 1) and in the within-day and between-day CV (%) values,
which are also below 10% (Supporting Information, Table S-2).
Thus, the analytical method is precise, and intraday and interday
estimates for the quantification of MeIQx and PhIP and their
metabolites are highly reproducible.
Estimation of MeIQx and PhIP Urinary Metabolites
and Correlations among Urinary Metabolic Ratios
(MRs). The estimates of MeIQx and PhIP and their metabolites
in urine samples from 10 subjects are summarized in Table 2.
Both HAAs underwent extensive metabolism: The proportion
of unaltered MeIQx ranged from 2.2 to 8.7% of the ingested
dose, whereas the proportion of unmetabolized PhIP ranged from
0.2 to 0.8% of the ingested dose. The major pathway of
metabolism of MeIQx occurred through oxidation of the C8-
methyl group to form IQx-8-COOH, but the major pathway of
PhIP metabolism occurred through oxidation of the exocyclic
amine group to form HONH-PhIP. The latter metabolite
underwent glucuronidation to form the isomeric N-glucuronide
conjugates. 4′-HO-PhIP was a minor component, but it was
detected in the urine of all subjects, after meat consumption.
4′-HO-PhIP can form at the low ppb concentration in beef
cooked well-done (58, 59). Hence, the 4′-HO-PhIP present in

Biomonitoring of MeIQx, PhIP, and Their Metabolites in Urine
Chem. Res. Toxicol., Vol. 23, No. 4, 2010 795
Figure 3. Product ion spectra for the urinary 8-CH2OH-IQx and 8-IQx-8-COOH metabolites and synthetic compounds (background spectra have
been subtracted).
the dose eliminated in urine as unmetabolized MeIQx and PhIP
was rs ) 0.86. The correlation coefficient relating the MR of
the two major oxidative metabolites of MeIQx and PhIP, IQx-
8-COOH/MeIQx and HON-PhIP-N2-Gl/ PhIP, was rs ) 0.92;
the correlation for IQx-8-COOH/MeIQx and HON-PhIP-N3-
Gl/PhIP was rs ) 0.78; the correlation for IQx-8-COOH/MeIQx
and 8-CH2OH-IQx/MeIQx was rs ) 0.64; and the correlation
for HON-PhIP-N3-Gl/PhIP and HON-PhIP-N2-Gl/PhIP was rs
) 0.90. These correlations were all significant (p value two-
tailed R < 0.05), except for the correlation of the MR relating
IQx-8-COOH/MeIQx and 8-CH2OH-IQx/MeIQx (rs ) 0.60, P
value R ) 0.07) (Figure 7). The inter-relationship between these
latter two metabolites could be obscured because P450 1A2
catalyzes the further oxidation of 8-CH2OH-IQx to form IQx-
8-COOH (30).
Indirect Measurement of Phase II Detoxification
Products of MeIQx and PhIP. MeIQx-N2-SO3H, MeIQx-N2-
Gl, and HON-MeIQx-N2-Gl did not bind to the mixed-mode
reversed phase cation exchange SPE resin. The poor binding
of the sulfamate was not surprising, given the compound’s strong
polarity and negative charge. MeIQx-N2-Gl and HON-MeIQx-
N2-Gl did bind to the SPE resin, when applied as pure standards
in 1.8% HCO2H. However, the binding of the metabolites to
the SPE resin was poor, when applied in acidified urine,
Figure 4. Product ion spectra for MeIQx and MeIgQx in urine following
regardless of preprocessing of the urine samples by organic
precipitation to remove salt and protein. The poor binding of
these two MeIQx glucuronide conjugates was unexpected, given
that the pKa values for MeIQx (pKa 5.94) and PhIP (pKa 5.65)
are similar (61) and given that the binding of the urinary
glucuronide metabolites of PhIP to the SPE resin was satisfactory.
Acid hydrolysis of MeIQx-N2-SO3H and MeIQx-N2-Gl
quantitatively transforms these conjugates to MeIQx (62). The
increase in the amount of MeIQx, after acid hydrolysis of urine,
was used as a means to estimate the contribution of N2-
sulfamation and N2-glucuronidation to the metabolism of MeIQx
(24, 62). We concurrently measured, following acid treatment,
the amounts of PhIP and the 4′-HO-PhIP; this latter metabolite
can undergo further metabolism to form 4′-sulfate or 4′-
acid hydrolysis (background spectra have been subtracted).
urine either can be a minor metabolite of PhIP or may be derived
from unaltered 4′-HO-PhIP ingested in the cooked beef. The
amounts of 4′-HO-PhIP excreted in urine at the 10 h time point
of these subjects are minor, and the levels ranged from an
equivalent of 0.2-1.0% of the ingested dose of PhIP.
The urinary MR is often used as an indirect method of
assessing drug-metabolizing enzyme activity in vivo (60). We
observed that the extent of MeIQx and PhIP metabolism and
the MR (% dose of urinary metabolite/% dose of unmetabolized
urinary HAA) for several oxidative urinary metabolites of
MeIQx and PhIP were correlated for a given subject. The
Spearman’s rank correlation coefficient for the percentage of

796 Chem. Res. Toxicol., Vol. 23, No. 4, 2010
Gu et al.
Figure 5. SRM traces for PhIP and its glucuronide metabolites in a human urine sample collected before and after the consumption of cooked beef.
The retention time (tR), area, and ion intensity are reported.
glucuronide conjugates (18, 27). The LC-ESI/MS/MS traces of
MeIQx, PhIP, and 4′-HO-PhIP in urine from a subject who ate
meat, before and after acid hydrolysis of urine, are shown in
the Supporting Information (Figure S-4). The SPE procedure
is highly effective in purifying these HAAs from acidified urine:
The acid hydrolysis treatment increased the amounts of all three
biomarkers. The contribution of phase II conjugation to the
metabolism of MeIQx and PhIP in urine samples from four
subjects is summarized in Figure 8. The amounts of MeIQx,
PhIP, and 4′-HO-PhIP increased by 3-5-fold, following acid
hydrolysis. Approximately 8-13% of the ingested dose of
MeIQx was present as phase II conjugates, but <3% of the
ingested dose was recovered as PhIP or 4′-HO-PhIP, following
acid hydrolysis.
The pretreatment of urine with a mixture of ꢀ-glucuronidase
and arylsulfatase did not increase the urinary concentration of
MeIQ because both MeIQx-N2-SO3H and MeIQx-N2-Gl are
resistant to these hydrolytic enzymes (37). The isomeric PhIP-
N-Gl conjugates are poor substrates of ꢀ-glucuronidase, whereas
ꢀ-glucuronidase treatment of urine resulted in complete hy-
drolysis of HON-PhIP-N3-Gl, but HON-PhIP-N2-Gl remained
intact (data not shown). The relative susceptibilities of these
glucuronide conjugates of PhIP to ꢀ-glucuronidase are consistent
with a previous observation (63). We did observe a modest
∼2.5-fold increase in the urinary concentrations of PhIP and
4′-HO-PhIP, following enyzme hydrolysis with a combination
of arylsulfatase and ꢀ-glucuronidase (data not shown). The
amount of the isomeric PhIP-N2-Gl conjugate present in urine
samples was just at the LOQ (∼50 pg/mL), whereas the level
of PhIP-N3-Gl was below the LOQ (Figure 5). These findings
demonstrate that direct phase II conjugation is more important
for the detoxification of MeIQx than for the detoxification of
PhIP, in agreement with the findings of a previous study (24).
Discussion
A facile, one-step SPE enrichment procedure was devised to
isolate MeIQx and PhIP and several of their principal metabo-
lites in the urine of omnivores. With this cleanup procedure,
20 urine samples can be processed in 1 day. Both HAAs undergo
extensive metabolism. In the case of MeIQx, the major urinary
metabolite is IQx-8-COOH, a detoxification product (31). The
primary metabolite of PhIP is HON-PhIP-N2-Gl, a conjugate
of HONH-PhIP, which is a genotoxic metabolite that covalently
adducts to DNA (64). The formation of both IQx-8-COOH
(30, 31) and HONH-PhIP (14, 16) is catalyzed by P450 1A2.
Thus, P450 1A2 serves a dual role in the metabolism of HAAs:
It catalyzes both the bioactivation and the detoxification of
MeIQx, but it only catalyzes the bioactivation of PhIP.
Comprehensive analyses of urinary metabolites of MeIQx and
PhIP in humans are limited to studies that have employed 14C-
labeled compounds (23, 27, 43). In a pilot study, five volunteers

Biomonitoring of MeIQx, PhIP, and Their Metabolites in Urine
Chem. Res. Toxicol., Vol. 23, No. 4, 2010 797
as 30-50% of the ingested dose of PhIP is excreted in urine
between 8 and 48 h postmeat consumption (5, 27, 43). In human
metabolic studies conducted with 14C-PhIP, the isomeric HON-
PhIP-N-glucuronide conjugates were reported to account for
approximately 50% of the dose eliminated in urine within 24 h
and, combined with 4′-HO-PhIP conjugates, PhIP-N-Gl, and
unaltered PhIP, accounted for 60-82% of the dose of PhIP
eliminated in urine within 24 h (27). We have also detected, by
LC/MS, significant quantities of HON-PhIP-N-glucuronide
conjugates in urine of our subjects at the 10-24 h time point
(unpublished observations) (5). Thus, our LC/MS method covers
a large proportion of the PhIP dose that is eliminated in urine.
A method was recently developed to measure 2-amino-1-
methyl-6-(5-hydroxy)phenylimidazo[4,5-b]pyridine (5-HO-
PhIP) in urine of omnivores (28). 5-HO-PhIP is a solvolysis
product of N-acetoxy-PhIP, a penultimate metabolite that reacts
with DNA (Scheme 2) (28, 40). The analysis of 5-HO-PhIP
required heat treatment of urine with hydrazine, under acidic
pH conditions, to hydrolyze potential glucuronide conjugates
of 5-HO-PhIP, prior to LC/MS (28). The authors estimated high
levels of 5-HO-PhIP in human urine, following consumption
of meat (28). In a previous study, we did not detect 5-HO-PhIP
in urine of omnivores (47), but we did identify several urinary
analytes at m/z 417.1, an m/z that is consistent with the molecular
weight of protonated glucuronide conjugates of hydroxylated-
PhIP (47). The full scan product ion spectra of these analytes
were acquired under elevated collision-induced dissociation
conditions to fragment the aglycone ions [M + H - 176]+;
however, the product ion spectra of these analytes differed from
the spectra of either 4′-HO-PhIP or 5-HO-PhIP (47). These
urinary components appeared to be isobaric interferents. The
major pathways of biotransformation of 14C-PhIP have been
characterized in human urine (27, 43) and in human hepatocytes
(19): Neither 5-HO-PhIP nor its glucuronide or sulfate conju-
gates were identified as prominent metabolites. Quantitative LC/
MS methods with a stable, isotopically labeled internal standard
will be required to determine the amounts of 5-HO-PhIP in urine
of omnivores (28).
Figure 6. Product ion spectra for PhIP and 4′-HO-PhIP in urine
following acid hydrolysis (background spectra have been subtracted).
who were about to undergo colorectal cancer surgery were given
the dietary equivalent of 14C-labeled MeIQx in a capsule (23).
Between 20 and 59% of the ingested dose of MeIQx was
excreted in urine within 26 h. Unmetabolized MeIQx and the
five principal urinary metabolites were estimated by HPLC with
liquid scintillation counting. The estimates of MeIQx and the
formed metabolites were as follows (noted as the range in
percentages of the ingested dose): MeIQx (0.7-2.8%), MeIQx-
N2-SO3H (0.6-3.1%), 8-CH2OH-IQx (1.0-4.4%), MeIQx-N2-
Gl (1.6-6.3%), HON-MeIQx-N2-Gl (1.4-10.0%), and IQx-8-
COOH (8-28%).
In this current study, the rates of metabolism and elimination
of MeIQx in healthy subjects ingesting MeIQx in well-done
cooked beef were greater than the rates determined in the
subjects who underwent colorectal surgery; this discrepancy may
be reflective of the poorer health status of those subjects (23).
We estimated, by LC-ESI/MS/MS, that from 60 to 85% of the
ingested dose of MeIQx was eliminated in the urine of our
healthy subjects, as a combination of unaltered MeIQx, P450
1A2 derived metabolites, and phase II conjugates, within 10 h
of consumption of cooked beef (Table 2). IQx-8-COOH and
8-CH2OH-IQx (30, 31) combined accounted for 34.8-76.2%
of the ingested dose (Table 2), underscoring the strong contribu-
tion of P450 1A2 to the metabolism of MeIQx.
The prominent role of P450 1A2 in the metabolism of MeIQx
and PhIP in humans was previously inferred from a pharma-
cokinetics study on subjects given furafylline (39), a selective
and mechanism-based inhibitor of P450 1A2 (65), prior to
consumption of cooked beef. In that study, P450 1A2 was
estimated to account for 90% of the elimination of MeIQx and
70% of the elimination of PhIP (39). Consistent with that
finding, we previously showed that the pretreatment of human
liver microsomes with various amounts of furafylline led to a
concentration-dependent inhibition of 8-CH2OH-IQx, IQx-8-
COOH, HONH-MeIQx, and HONH-PhIP formation by up to
95% (16, 30). The formation of 8-CH2OH-IQx and IQx-8-
COOH and the glucuronide conjugates of HONH-MeIQx and
HONH-PhIP was also inhibited to a similar degree in human
hepatocytes pretreated with furafylline (19, 31).
The estimates of unaltered PhIP (0.2-0.8% of the dose), as
well as the contribution of HON-PhIP-N2-Gl (14.1-29.7% of
the dose) and HON-PhIP-N3-Gl (1.0-3.5% of the dose) to the
metabolism of PhIP, are in good agreement to values previously
estimated in the 10 h urine samples of these subjects (5), and
the estimates of these PhIP metabolites formed are comparable
to the data obtained by liquid scintillation counting of radio-
activity (27) or by LC-ESI/MS/MS in other feeding studies (26).
The smaller percentage of the dose of PhIP that is eliminated
in urine as compared to MeIQx, at the 10 h time point of our
study, is due to the slower rate of elimination of PhIP: As much
We examined the extent of MeIQx and PhIP metabolism and
compared the urinary MR values for several of their P450 1A2-
catalyzed oxidation products. The use of MR values and their
correlations must be interpreted with caution. A high urine flow
rate can limit the utility of MR in assessment of enzyme
metabolizing activity in vivo, if the elimination of the parent
compound or the metabolite is dependent upon the flow rate of
urine (60). In a previous study, the renal clearance of MeIQx
and PhIP was reported not to be urine flow-dependent (39). In
the present pilot study of 10 subjects, we observed that the renal
clearances of MeIQx, 8-CH2OH-IQx, IQx-8-COOH, HON-

798 Chem. Res. Toxicol., Vol. 23, No. 4, 2010
Table 1. Intraday and Interday Measurements of MeIQx, 8-CH2OH-IQx, and IQx-8-COOHa
Gu et al.
amount (pg/mL)
subject
9
metabolite
day 1
day 2
day 3
overall mean
245
CV (%) within-day
5.8
CV (%) between-day
5.4
IQx-8-COOH
mean
SD
RSD (%)
mean
SD
RSD (%)
mean
SD
RSD (%)
mean
SD
RSD (%)
mean
SD
RSD (%)
mean
SD
RSD (%)
mean
SD
RSD (%)
mean
SD
237
8.1
3.4
34.7
2.1
6.1
22.0
0.8
3.6
179
3.6
2.0
23.0
0.5
2.2
11.1
0.6
5.4
395
16.4
4.2
49.0
1.7
3.5
29.9
0.4
249
19.1
7.7
34.6
1.5
4.3
22.8
1.1
4.8
184
3.2
1.7
22.9
1.2
248
12
4.8
34.3
1.4
4.1
21.5
1.1
5.1
177
4.0
2.3
22.6
1.1
4.9
9.9
0.5
8-CH2OH-IQx
MelQx
34.5
5.1
4.5
2.0
4.4
5.0
4.0
4.2
4.2
4.2
4.8
2.8
3.7
7.2
4.6
7.3
4.2
22.1
14
IQx-8-COOH
8-CH2OH-IQx
MelQx
180
22.8
10.4
5.2
10.2
0.5
4.9
5.1
20
IQx-8-COOH
8-CH2OH-IQx
MelQx
418
10.3
2.5
45.9
1.0
2.2
29.3
1.3
394
20.4
5.2
43.1
2.7
6.3
28.5
0.8
402
46.0
29.2
RSD (%)
mean
SD
RSD (%)
1.3
4.4
2.8
a Mean ( SD; n ) 3 or 4 replicated per day.
Table 2. Measurements of MeIQx and PhIP and Their Metabolites in Urine Collected for 10 h after the Consumption of
Cooked Beefa
MelQx
8-CH2OH-IQX
IQx-8-COOH
PhlP
HONH-PhlP-N2-GI HONH-PhlP-N3-GI
4′-HO-PhlP
%
dose
%
dose
%
dose
%
dose
%
dose
%
dose
%
dose
subject
ng
ng
ng
ng
ng
ng
ng
S-001 80.3 ( 2.7 8.7
134 ( 10.4 13.5 447 ( 22.9 42.5 37.1 ( 3.3 0.8 2201 ( 37.3 24.3
238 ( 7.4
189 ( 10.6
176 ( 4.2
222 ( 10.0
191 ( 5.5
319 ( 6.0
179 ( 7.0
2.6 20.1 ( 9.9 0.4
2.1 35.0 ( 3.4 0.7
1.9 41.7 ( 0.9 0.8
2.5 27.0 ( 3.8 0.5
2.1 25.2 ( 2.8 0.5
3.5 50.1 ( 2.8 1.0
2.0 37.2 ( 5.2 0.7
1.0 18.4 ( 1.2 0.4
2.4 18.3 ( 2.9 0.3
2.8 38.3 ( 2.5 0.7
S-003 26.7 ( 1.6 2.9 49.2 ( 2.6
S-005 32.4 ( 1.5 3.5 70.1 ( 5.9
S-008 33.8 ( 1.3 3.7 57.4 ( 1.2
S-009 45.7 ( 1.4 5.0 72.0 ( 3.8
5.0 630 ( 11.6 59.8
7.1 649 ( 17.8 61.6 11.4 ( 0.9 0.2 2112 ( 88.5 23.3
5.8 565 ( 25.9 53.6 9.5 ( 0.6 0.2 2198 ( 88.2 24.3
6.5 ( 1.1 0.1 2692 ( 121 29.7
7.3 493 ( 14.7 46.8 18.5 ( 1.6 0.4 2009 ( 31.0 22.2
11.8 678 ( 16.8 64.4 17.0 ( 0.7 0.3 2621 ( 86.0 29.0
8.6 596 ( 19.7 56.6 14.1 ( 1.2 0.3 1848 ( 25.6 20.4
S-012 48.3 ( 1.9 5.2
117 ( 9.0
S-013 60.9 ( 3.2 6.6 85.7 ( 4.2
S-014 20.6 ( 0.9 2.2 42.8 ( 0.8
S-015 39.2 ( 2.0 4.2 89.0 ( 6.9
S-020 51.5 ( 2.2 5.6 84.4 ( 2.6
4.3 332 ( 5.6
9.0 662 ( 20.5 62.9
8.5 680 ( 24.3 64.6 18.9 ( 1.6 0.4 2223 ( 15.6 24.6
31.5
8.9 ( 0.6 0.2 1274 ( 29.0 14.1 89.5 ( 3.3
8.2 ( 1.0 0.2 2067 ( 103 22.8
216 ( 12.0
253 ( 4.9
a Means ( SDs of three independent measurements. The estimates of 4′-HO-PhlP were based on the use of [2H3C]-PhIP as an internal standard, and
comparable ionization efficiencies were assumed. We assumed that 4′-HO-PhIP was a metabolite of PhIP and that 4′-HO-PhIP was not formed in
cooked beef.
PhIP-N2-Gl, HON-PhIP-N2-Gl, and 4′-HO-PhIP were indepen-
dent of urine flow rate (Spearman rs values <0.52, two-tailed
R, p > 0.14, urine volume vs percent dose of biomarkers
collected in urine over 10 h), but the elimination of PhIP did
appear to be correlated with urine flow output (rs ) 0.74, two-
tailed R, p ) 0.02). The urinary MR value relating the two major
oxidation metabolites of MeIQx and PhIP, IQx-8-COOH/
MeIQx, and HON-PhIP-N2-Gl/PhIP were strongly correlated
among the 10 subjects of the study (Figure 7). These correlations
support the notion that P450 1A2 is an important enzyme in
the metabolism of both procarcinogens in vivo. A study on a
larger number of subjects will be required before we can firmly
establish the MR values and the inter-relationship between P450
1A2-mediated metabolism of MeIQx and that of PhIP.
efficiency of P450 1A2 in the first oxidation step of the 8-methyl
group of MeIQx to form 8-CH2OH-IQx (kcat/Km ) 0.03, Vmax
) 0.1 nmol/min/mg protein, Km ) 3.1 µM) is ∼10-fold lower
than the catalytic efficiency of P450 1A2 in the production of
HONH-MeIQx (kcat/Km ) 0.26, Vmax ) 3.9 nmol/min/mg of
protein, Km ) 15 µM) (30). Thus, we postulate that a portion
of the HONH-MeIQx metabolite formed in vivo undergoes
enzymatic reduction back to MeIQx (66), which ultimately
undergoes oxidation at the 8-methyl group to form 8-CH2OH-
IQx and IQx-8-COOH (Scheme 1).
HON-MeIQx-N2-Gl was not measured, due to its poor binding
to the SPE resin. The acid treatment of HON-MeIQx-N2-Gl
produces the deaminated derivative 2-hydroxy-3,8-dimethylimi-
dazo[4,5-f]quinoxaline with high yield. This biomarker was
employed as an indirect measure of urinary HON-MeIQx-N2-
Gl by GC/MS (67). HON-MeIQx-N2-Gl was deduced to account
for 9.4 ( 3.0% (mean ( SD) of the dose and varied in range
from 2.2 to 17.1% (mean ( SD) of the dose in 66 subjects. We
observed that 2-hydroxy-3,8-dimethylimidazo[4,5-f]quinoxaline
The large contribution of IQx-8-COOH to the metabolism
of MeIQx is unexpected, when the steady-state kinetic param-
eters of P450 1A2 for the formation of 8-CH2OH-IQx, the
precursor to IQx-8-COOH, are considered. On the basis of
kinetic studies with human liver microsomes, the catalytic

Biomonitoring of MeIQx, PhIP, and Their Metabolites in Urine
Chem. Res. Toxicol., Vol. 23, No. 4, 2010 799
In summary, we have established a robust SPE method to
measure MeIQx and PhIP and several of their major urinary
metabolites that are produced by P450 1A2. With this validated
analytical method, we plan to explore the influence of genetic
polymorphisms that encode enzymes involved in xenobiotic
metabolism and assess the efficacy of chemoprotective dietary
constituents to modulate the metabolism of these two potential
human carcinogens.
Acknowledgment. This work was funded by R01CA122320
(D.G. and R.J.T.) from the National Cancer Institute, by Grant
2007/58 funded by the World Cancer Research Fund Interna-
tional (D.G., R.J.T., and F.F.K.), and by T01009 funded by the
Food Standards Agency, United Kingdom (N.J.G. and B.G.L.).
We acknowledge the assistance of the NMR Structural Biology
Facility at the Wadsworth Center.
Supporting Information Available: 1H NMR chemical shift
data (ppm) for PhIP-N-glucuronides (Table S-1); performance
of the analytical method for PhIP and its metabolites (Table
1
S-2); 2D H-1H COSY NMR spectra of PhIP-N2-Gl (Figure
S-1A) and PhIP-N3-Gl (Figure S-1B,C); calibration curves of
MeIQx and its metabolites (Figure S-2A-C), PhIP and its
metabolites (Figure S-3A-D); and LC-ESI/MS/MS traces of
MeIQx, PhIP, and 4′-HO-PhIP in urine of an omnivore, before
and after acid hydrolysis of urine (Figure S-4). This material is
available free of charge via the Internet at http://pubs.acs.org.
Figure 7. Scatter plots relating the percentage of the unmetabolized
dose of MeIQx and PhIP and relating the MR of oxidized MeIQx and
PhIP metabolites (% of dose of metabolite/% of dose of unmetabolized
MeIQx or PhIP) eliminated in urine collected for 10 h after meat
consumption.
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