Quantitation of 13 heterocyclic aromatic amines in cooked beef, pork, and chicken by liquid chromatography-electrospray ionization/tandem mass spectrometry

Article abstract of DOI:10.1021/jf072461a

The concentrations of heterocyclic aromatic amines (HAAs) were determined, by liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-ESI-MS/MS), in 26 samples of beef, pork, and chicken cooked to various levels of doneness. The HAAs id

Full text of DOI:10.1021/jf072461a

68 J. Agric. Food Chem. 2008, 56, 68–78
Quantitation of 13 Heterocyclic Aromatic Amines in
Cooked Beef, Pork, and Chicken by Liquid
Chromatography-Electrospray Ionization/Tandem
Mass Spectrometry
WEIJUAN NI,† LYNN MCNAUGHTON,‡ DAVID M. LEMASTER,‡
RASHMI SINHA,§ AND ROBERT J. TURESKY*,†
Division of Environmental Disease Prevention and Division of Molecular Medicine, Wadsworth
Center, New York State Department of Health, Albany, New York 12201-0509, and Nutritional
Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute,
6120 Executive Boulevard, EPS 3046, Rockville, Maryland 20892-7273
The concentrations of heterocyclic aromatic amines (HAAs) were determined, by liquid chromatography-
electrospray ionization/tandem mass spectrometry (LC-ESI-MS/MS), in 26 samples of beef, pork,
and chicken cooked to various levels of doneness. The HAAs identified were 2-amino-3-methylimi-
dazo[4,5-f]quinoline, 2-amino-1-methylimidazo[4,5-b]quinoline, 2-amino-1-methylimidazo[4,5-g]qui-
noxaline (IgQx), 2-amino-3-methylimidazo[4,5-f]quinoxaline, 2-amino-1,7-dimethylimidazo[4,5-
g]quinoxaline (7-MeIgQx), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, 2-amino-1,6-dimethyl-
furo[3,2-e]imidazo[4,5-b]pyridine, 2-amino-1,6,7-trimethylimidazo[4,5-g]quinoxaline, 2-amino-3,4,8-
trimethylimidazo[4,5-f]quinoxaline, 2-amino-1,7,9-trimethylimidazo[4,5-g]quinoxaline, 2-amino-1-methyl-
6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-9H-pyrido[2,3-b]indole, and 2-amino-3-methyl-9H-
pyrido[2,3-b]indole. The concentrations of these compounds ranged from <0.03 to 305 parts per
billion (micrograms per kilogram). PhIP was the most abundant HAA formed in very well done
barbecued chicken (up to 305 µg/kg), broiled bacon (16 µg/kg), and pan-fried bacon (4.9 µg/kg).
7-MeIgQx was the most abundant HAA formed in very well done pan-fried beef and steak, and in
beef gravy, at concentrations up to 30 µg/kg. Several other linear tricyclic ring HAAs containing the
IgQx skeleton are formed at concentrations in cooked meats that are relatively high in comparison to
the concentrations of their angular tricyclic ring isomers, the latter of which are known experimental
animal carcinogens and potential human carcinogens. The toxicological properties of these recently
discovered IgQx derivatives warrant further investigation and assessment.
KEYWORDS: Heterocyclic aromatic amines; food mutagens; food carcinogens; LC-ESI-MS/MS
INTRODUCTION
multiple sites of rodents during long-term feeding studies:
included are tumors of the liver, colorectum, prostate, and female
mammary gland (4). A number of these tissues in humans are
capable of bioactivation of HAAs to genotoxic species (7).
Moreover, several epidemiological studies have reported that
frequent consumption of well-done cooked beef containing
HAAs leads to an increased risk of development of cancers in
these tissues (8, 9). These observations have led to the
hypothesis that HAAs play a causal role in colorectal and
possibly other common forms of human cancers (10–12).
Heterocyclic aromatic amines (HAAs) are potent bacterial
mutagens, and carcinogens in experimental animals, that occur
in well-done grilled meats (1, 2). Since the discovery of HAAs
three decades ago (3), more than 20 HAAs have been identified
in cooked meats (4). Several HAAs, including 2-amino-9H-
pyrido[2,3-b]indole (ARC) and 2-amino-3-methyl-9H-pyrido[2,3-
b]indole (MeARC), also arise in tobacco smoke condensate and
diesel exhaust (5, 6). HAAs in high doses induce tumors at
The concentrations of HAAs in cooked meats can vary by
more than 100-fold range. The amounts of HAAs formed are
dependent upon the type of meat, method of cooking, and the
temperature and duration of cooking (1, 2). Quantitative
measurements of HAAs in foods are essential, if we are to
accurately estimate exposures and human cancer risks (11).
Many studies reported on the estimates of HAAs in foods
* To whom correspondence should be addressed. Phone: 518-474-
4151. Fax: 518-486-1505. E-mail: rturesky@wadsworth.org.
† Division of Environmental Disease Prevention, New York State
Department of Health.
‡ Division of Molecular Medicine, New York State Department of
Health.
§ Division of Cancer Epidemiology and Genetics, National Cancer
Institute.
10.1021/jf072461a CCC: $40.75
2008 American Chemical Society
Published on Web 12/11/2007

LC/MS Analysis of HAAs in Cooked Meats
J. Agric. Food Chem., Vol. 56, No. 1, 2008 69
b]pyridine (IFP) was kindly provided by Dr. M. Knize, Lawrence
Livermore National Laboratory, Livermore, CA. 2-Amino-1,7,9-
trimethylimidazo[4,5-g]quinoxaline (7,9-DiMeIgQx) was kindly pro-
vided by Dr. K. Wakabayashi, National Cancer Center Research
Institute, Tokyo, Japan. CNBr, pyruvaldehyde (40%), hydrazine hydrate
(50% v/v), 1-[2H3C]creatinine, [2H3C]I, [2H3C]-NH2-HCl (all >99%
isotopic purity), and palladium/carbon (10%) were purchased from
Sigma-Aldrich (Milwaukee, WI). 4-Fluoro-5-nitrobenzene-1,2-diamine
was purchased from Maybridge through Ryan Scientific Inc. (Isle of
Palms, SC). Oasis MCX LP extraction (500 mg) and borosilicate glass
total recovery capLC vials were purchased from Waters (Milford, MA).
Extrelut-20 resin was obtained from EMD Chemicals (Gibbs Town,
NJ). A Baker C18 cartridge (1 g) and Whatman glass-backed silica
preparative TLC plates (1000 µm) were purchased through Krackeler
Scientific, Inc. (Albany, NY).
General Methods. Mass spectra of synthetic derivatives were
obtained on a Finnigan TSQ Quantum Ultra triple quadrupole mass
spectrometer (Thermo Electron, San Jose, CA). Typical instrument
tuning parameters used were as follows: source spray voltage 3.0 kV,
N2 sheath gas setting 10, auxiliary gas setting 0, capillary temperature
350 °C, and tube lens offset 80; the in-source collision-induced
dissociation (CID) offset was 10 V. The collision energy was set at 28
V for all compounds in the MS/MS scan mode. Argon, set at 1.5 mTorr,
was used as the collision gas. Full-scan or product ion spectra (100-300
Da) were acquired with these MS parameters at a scan rate of 200
Da/s. Analyses were conducted in the positive ionization mode. 1D
NMR spectra were collected on a Bruker 500 MHz DRX spectrometer
(Bruker BioSpin Corp., Billerica, MA) using solutions in DMSO-d6.
2D spectra were collected on a Bruker 600 MHz DRX Spectrometer,
equipped with a cryoprobe. HPLC separations were done with an
Agilent 1100 HPLC system equipped with a photodiode array detector
(Palo Alto, CA) equipped with a Rheodyne 7725i (Rhonert Park, CA)
manual injector. The accurate mass measurement data of the products
of syntheses were obtained at the Mass Spectrometry Center in the
School of Chemical Sciences, University of Illinois, Urbana–Cham-
paign. The MS instrument was a Q-tof Ultima API (Waters Corp.,
Milford, MA).
employed HPLC coupled with UV photodiode array or fluo-
rescence detection for measurement (13). This approach has
been successfully used to estimate the concentrations of known
HAAs in a variety of cooked meats. However, the known HAAs
have been reported to account for only about 30% of the total
mutagenicity attributed to this class of genotoxicants in well-
done cooked beef (1, 14). We recently employed liquid
chromatography-electrospray ionization/tandem mass spec-
trometry (LC-ESI-MS/MS) to characterize HAAs in urine of
meat eaters; in that study, we discovered 2-amino-1,7-dimeth-
ylimidazo[4,5-g]quinoxaline (7-MeIgQx) (15, 16), an isomer
of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx),
which is a potent bacterial mutagen and rodent carcinogen (4).
Thereafter, we identified 7-MeIgQx in fried ground beef patties
and also characterized, by LC-ESI-MS/MS, other putative HAAs
that contain an imidazoquinoxaline skeleton (15, 17). Thus, a
number of previously uncharacterized HAAs of undetermined
genotoxic potential are shown to be present in cooked meats.
A food frequency questionnaire (FFQ) has been devised to
assess exposure to HAAs and other genotoxicants in cooked
meats and these toxicants’ potential causal role in dietary-linked
cancers (11). A meat-cooking practices module and a database
were established to provide estimates on the amounts of
8-MeIQx, 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-
DiMeIQx), and PhIP, three of the principal HAAs formed in
various types of meat cooked by a number of methods and to
varying degrees of doneness. Photographs depicting these meat
samples were incorporated into the FFQ (18, 19) to serve as a
means to gauge an individual’s preference of meat “doneness”
and to estimate the HAA intake. Our recent discovery of
7-MeIgQx and other previously unreported HAAs (15–17)
prompted us to reexamine several of the commonly eaten meat
staples listed in this FFQ to determine the concentrations of
7-MeIgQx and other presumed HAAs containing the 2-amino-
1-methylimidazo[4,5-g]quinoxaline (IgQx) structure, relative to
the amounts of known carcinogenic HAAs formed in these
foods. Our findings demonstrate that 7-MeIgQx is one of the
most abundant HAAs formed in cooked meat sample types of
this database. Several other HAAs containing the IgQx
skeleton (17, 20) also occur in cooked meats and contribute to
the daily burden of HAA exposure. Studies on the toxicological
properties of 7-MeIgQx and other linear tricyclic ring HAAs
are warranted.
Syntheses. 2-Amino-1-methylimidazo[4,5-g]quinoxaline (IgQx) and
2-Amino-1,6,7-trimethylimidazo[4,5-g]quinoxaline (6,7-DiMeIgQx). The
syntheses of IgQx, 6,7-DiMeIgQx, and their trideuterated internal
standards were done by modification of a method described for the
synthesis of 7-MeIgQx (Scheme 1) (15).
5-Fluoro-6-nitro-1H-benzimidazole-2-amine (2). CNBr treatment of
the commercially available 4-fluoro-5-nitrobenzene-1,2-diamine (1)
gave a 94% yield of 5-fluoro-6-nitro-1H-benzimidazole-2-amine (2),
as previously reported (15).
5-Fluoro-1-methyl-6-nitro-1H-benzo[d]imidazole-2-amine (3a) and
6-Fluoro-1-methyl-5-nitro-1H-benzo[d]imidazole-2-amine (3b). Com-
pound 2 (75 mg, 0.38 mmol) in anhydrous DMSO (4 mL), containing
anhydrous K2CO3 (330 mg, 3.06 mmol), was methylated by the addition
of CH3I (27 µL, 0.42 mmol) over 10 min with stirring. The reaction
was continued overnight at room temperature with stirring. Then, water
(50 mL) was added, and the mixture was applied to a Baker C18 SPE
cartridge (1 g). The cartridge was washed with water, and the desired
product was eluted with CH3OH. The eluent was concentrated, and
the residue was purified by flash chromatograpy with CH3OH-CH2Cl2
(starting from 1:100 to 1:30 with 0.5% NH4OH) to give a light yellow
solid mixture of 3a and 3b (38 mg, 0.18 mmol, 48%). Major species
(tentatively 3a): 1H NMR (DMSO-d6) δ 7.99 (d, JHF ) 6.8 Hz, 1H,
H-7), 7.41 (br, 2H, 2-NH2), 7.13 (d, JHF ) 13.1 Hz, 1H, H-4), 3.60 (s,
EXPERIMENTAL PROCEDURES
Caution. Heterocyclic aromatic amines are carcinogenic. They
should be handled in a well-ventilated hood with extreme care and use
of appropriate protective clothing and equipment.
Chemicals. The following chemicals were purchased from Toronto
Research Chemicals (Downsview, Ontario, Canada): 2-amino-3,4-
dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3-methylimidazo[4,5-
f]quinoline (IQ), and trideuterated 3-[2H3C]-IQ (isotopic purity >99%);
2-amino-1-methylimidazo[4,5-b]quinoline (IQ[4,5-b]); 2-amino-3-me-
thylimidazo[4,5-f]quinoxaline (IQx); 8-MeIQx, 3-[2H3C]-8-MeIQx
(isotopic purity ∼96%); PhIP and trideuterated 1-[2H3C]-PhIP (isotopic
purity >99%); ARC; MeARC; and 5-chloro-4-nitrobenzo[1,2,5]-
selenadiazole. [4b,5,6,7,8,8a-13C6]-2-ARC (isotopic purity >99%) was
a kind gift from Dr. D. Doerge, NCTR, Jefferson, AR. 2-Amino-3,4,8-
trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), trideuterated 3-[2H3C]-
4,8-DiMeIQx (isotopic purity >99%), and 3-[2H3C]-IQx (isotopic purity
>99%) were synthesized as previously described (17, 21). Trideuterated
1-[2H3C]-IQ[4,5-b] was synthesized from 1-[2H3C]creatinine (>99%
isotopic purity) and 2-aminobenzaldehyde (22, 23). 7-MeIgQx and
trideuterated 1-[2H3C]-7-MeIgQx (>99% isotopic purity) were syn-
thesized as previously described with [2H3C]I used for the methylation
step, instead of CH3I (15). 2-Amino-1,6-dimethylfuro[3,2-e]imidazo[4,5-
1
3H, N1-CH3). Minor species (tentatively 3b): H NMR (DMSO-d6) δ
7.81 (d, J ) 6.7 Hz, 1H, H-4), 7.44 (d, JHF ) 12.1 Hz, 1H, H-7), 7.05
(br, 2H, 2-NH2), 3.60 (s, 3H, N1-CH3). HRMS ESI-MS m/z for [M +
H]+: C8H8FN4O2, calculated, 211.0631; found, 211.0628.
1-Methyl-6-nitro-1H-benzo[d]imidazole-2,5-diamine (4a) and 1-Methyl-
5-nitro-1H-benzo[d]imidazole-2,6-diamine (4b). The substituted amines
4a and 4b were obtained by treatment of a mixture of the isomers 3a
and 3b (8 mg, 0.038 mmol) in DMSO (1.2 mL) with NH4OH (26%
v/v) (0.4 mL) for 5 days at 130 °C. After the mixture was cooled,
water (100 mL) was added, and the solution was applied to a Baker
C18 SPE cartridge (1 g). The product was collected as described above.

70 J. Agric. Food Chem., Vol. 56, No. 1, 2008
Ni et al.
Scheme 1. Chemical Synthesis of IgQx and 6,7-DiMeIgQx
The eluent was concentrated and purified by preparative TLC to give
an isomeric mixture of 4a and 4b (5.7 mg, 0.028 mmol, 74%). In order
to distinguish the two isomers, homonuclear ROESY experiments were
conducted to identify through-space dipolar interactions between protons
in spatial proximity. ROE cross-peaks were observed between the N1-
CH3 signal and the 2-NH2 and H-7 resonances for both 4a and 4b.
The identification of the phenyl NH2 signal was independently verified
by an ROE cross-peak to H-7 in compound 4a and to H-4 in compound
4b. 1H NMR (DMSO-d6): 4a, δ 7.589 (s, 1H, H-7), 7.22 (br, 2H,
2-NH2), 7.06 (br, 2H, 5-NH2), 6.49 (s, 1H, H-4), 3.41 (s, 3H, N1-CH3);
4b, δ 7.593 (s, 1H, H-4), 7.13, (br, 2H, 6-NH2), 6.749, (br, 2H, 2-NH2),
6.494 (s, 1H, H-7), 3.38 (s, 3H, N1-CH3). HRMS ESI-MS m/z for [M
+ H]+: C8H10N5O2, calculated, 208.0834; found, 208.0826.
parts (flesh and skin) of the cooked chicken were finely minced to form
a composite sample for the specific chicken type, method of cooking,
and degree of doneness. The cooking levels for skinless, boneless
chicken breast samples that were grilled/barbecued were just done, well
done, and very well done. The internal temperature of the chicken at
80 °C was designated as just done; the internal temperature was at
least 5 °C higher for well done and at least 10 °C higher for very well
done. The pan-fried chicken breasts (without skin and bones) were
cooked in a Teflon-coated frying pan with 2 tablespoons of cooking
oil. Oven-broiled chicken breasts (with or without skin and bones) were
cooked in a commercial gas range broiler, with the meat placed 12.5
cm away from the heat source. Grilled/barbecued chicken breasts (with
or without skin and bones) were prepared on a home propane gas
barbecue unit with ceramic briquettes (Sunbeam Model 44M39, 271/2
in. × 15 in., 44000 BTU).
2-Amino-1-methylimidazo[4,5-g]quinoxaline (6) and 2-Amino-1,6,7-
trimethylimidazo[4,5-g]quinoxaline (7). A mixture of 4a and 4b isomers
(2 × 20 mg, 0.097 mmol) was added to two tubes of ethanol (6 mL)
and reduced with H2 at atmospheric pressure, using 10% Pd/C (10 mg)
as a catalyst. The reaction mixtures were stirred for 1.5 h to give the
unstable triamine, 1-methyl-1H-benzo[d]imidazole-2,5,6-triamine (5)
(100% yield, on the basis of LC-ESI-MS infusion [M + H]+ at m/z
178.1). After filtration of the reaction mixtures through a Celite pad,
the filtrates were concentrated in Vacuo to ≈2 mL, followed by the
addition of glyoxal (40%, 12.5 µL, 0.11 mmol) or 2,3-butanedione
(97%, 9.6 µL, 0.11 mmol). The solutions were heated to 50 °C for 1 h.
After concentration, the residues were purified by a preparative silica
TLC plate using 15% CH3OH in CH2Cl2 in the presence of 0.5%
NH4OH to give compound 6 (16 mg, 0.080, 82%) and compound 7(21
mg, 0.092 mmol, 95%), respectively. Compound 6: 1H NMR (DMSO-
d6) δ 8.70 (d, J ) 1.9 Hz, 1H, H-6), 8.68 (d, J ) 1.9 Hz, 1H, H-7),
7.70 (s, 1H, H-9), 7.62 (s, 1H, H-4), 7.35 (br, 2H, 2-NH2), 3.68 (s, 3H,
N1-CH3). An ROE cross-peak was observed between the N1-CH3 signal
and the H-9. ROE cross-peaks were also observed between H-6 and
H-4 and between H-7 and H-9. HRMS ESI-MS m/z calculated for [M
+ H]+: C10H10N5, calculated, 200.0936; found, 200.0934. Compound
Beef was purchased from a supermarket: hamburger patties of freshly
ground lean beef (15% fat, 1.5-2.0 cm thick × 10 cm diameter), steaks
(top loin, New York strip, USDA choice steaks, 2.6-3.3 cm thick),
and roasts (eye round roast, 4-6 lbs). Hamburger patties and steaks
were pan-fried, oven-broiled, or grilled/barbecued, while roasts were
cooked in an oven. The hamburger patties and steaks were cooked to
four levels of doneness: rare, medium, well done, and very well done.
The degree of doneness for the varying types of beef products was
primarily defined by internal temperature: the internal temperature of
60 °C as rare, 70 °C as medium, 80 °C as well done, and 90 °C as
very well done. Hamburger patties and steaks were pan-fried in a
Teflon-coated frying pan without added oil. Oven-broiled hamburger
patties and steaks were cooked in a commercial gas range broiler with
the meat placed 12 cm away from the heat source. Grilled/barbecued
patties and steaks were prepared on the Sunbeam gas barbecue unit
with ceramic briquettes. Roast beef was cooked, in a gas oven heated
to 160 °C, to three levels of doneness: rare, medium, and well done.
Drippings were collected from two roasts and combined. Gravy was
made from the crusty drippings after skimming off all the fat except
one-quarter cup (56 g). In a saucepan, 1/4 cup of flour, 13/4 cups of
water, and 1/4 cup of fat with all the drippings were mixed. This mixture
was cooked until thick and bubbly (18).
Tandem SolVent-Solid-Phase Extraction (SPE) of HAAs from
Cooked Meat. The extraction procedure followed the method that was
developed by Gross (24) and then modified by Turesky (17). The
cooked meat samples (2 g) were homogenized with 1 N NaOH (4 mL/g
of meat, spiked with isotopically labeled internal standards at 2.5 ng/
g of meat), and mixed thoroughly with an Extrelut-20 resin (3 g of
resin/g of meat). The mixture was placed into an empty polypropylene
cartridge (50 mL) containing a 200 µm pore size frit and connected to
a Waters Oasis MCX LP extraction cartridge (150 mg). The MCX resin
was prewashed with 5% NH4OH in CH3OH (2 mL), followed by 2%
CH3CO2H in CH3OH (2 mL) prior to connection to the Extrelut-20
resin. The HAAs were eluted from the Extrelut-20 resin with 5% toluene
in CH2Cl2 (50 mL) and adsorbed onto the MCX cartridge. The Extrelut-
20 resin was discarded, and the MCX cartridge was then sequentially
1
7: H NMR (DMSO-d6) δ 7.53 (s, 1H, H-9), 7.47 (s, 1H, H-4), 7.18
(br, 2H, 2-NH2), 3.60 (s, 3H, N1-CH3), 2.60 (s, 3H, 7-CH3), 2.52 (s,
3H, 6-CH3). An ROE cross-peak was observed between the N1-CH3
signal and the H-9, and ROE cross-peaks were observed between 6-CH3
and H-4, as well as between 7-CH3 and H-9. HRMS ESI-MS m/z for
[M + H]+: C12H14N5, calculated, 228.1249; found, 228.1253.
Cooking of Meat Samples. The meat types (beef, pork, and chicken)
and cooking methods were previously reported in detail (2, 18, 19).
The cooked samples, which were stored at -80 °C after cooking, were
blind coded, and identities and methods of cooking were not revealed
until all of the quantitative analyses of HAAs in meat samples had
been completed. We assayed selected samples from the FFQ represent-
ing the meats most frequently consumed in the United States and the
cooking methods most commonly used: pan frying, oven broiling,
grilling/barbecueing, or roasting.
Two types of chicken were assayed: skinless, boneless chicken
breasts and breasts with skin and bones. The bones (from whole chicken
and breasts with skin and bones) were removed manually, and edible
washed with toluene-CH2Cl2 (5:95,
2 mL), 0.01 N HCl in

LC/MS Analysis of HAAs in Cooked Meats
J. Agric. Food Chem., Vol. 56, No. 1, 2008 71
H2O-CH3OH (40:60, 2 mL), and CH3OH (2 mL), followed by H2O
(2 mL) and NH4OH-CH3OH-H2O (2:15:85, 2 mL). Then, the HAAs
were eluted from the resin with 2 mL of 5% NH4OH in CH3OH and
collected into Eppendorf tubes. The eluent was concentrated to dryness
by vacuum centrifugation (Savant ISS110 Speedvac concentrator). The
residue was resuspended in 40 µL of DMSO-0.1% HCO2H (1:1) with
a final concentration of internal standards at 125 pg/µL (assuming 100%
recovery). Two microliters of the extract was quantified for the HAA
content.
Characterization and Quantification of HAAs in Meat Samples by
LC-ESI-MS/MS. Chromatography of HAAs was performed with an
Agilent Technologies 1100 series capillary LC system (Palo Alto, CA)
equipped with an Aquasil C18 reversed-phase column (3 µm particle
size, 1 × 200 mm) from Thermo Electron Corp. (Bellefonte, PA) with
a Javelin precolumn (1 × 10 mm) containing the C18 reversed-phase
resin. The A solvent was 1 mM CH3CO2NH4-0.1% HCO2H-10%
CH3CN-89.9% H2O. The B solvent contained 0.1% HCO2H-4.9%
H2O-95% CH3CN. The flow rate was set to 40 µL/min. The solvent
composition was isocratic at 4% B for 10.5 min, followed by a linear
gradient to 15% B at 15 min; it was then increased to 100% B at 25
min and held at 100% B for 7 min, followed by reequilibration to
starting conditions.
The MS analyses were performed on a Finnigan TSQ Quantum Ultra
triple quadrupole (TSQ) mass spectrometer employing the MS param-
eters described in General Methods. Quantitative analyses were
conducted in the selective reaction monitoring (SRM) scan mode. The
transitions monitored (0.02 s dwell time per transition) and approximate
chromatographic retention times (tR) were as follows: [M + H]+
>
•
[M + H - 15]+• (loss of CH3 ) for IQ (199 > 184, tR ) 9.4 min),
IgQx (200 > 185, tR ) 12.0 min), IQx (200 > 185, tR ) 12.9 min),
7-MeIgQx (214 > 199, tR ) 19.8 min), 8-MeIQx (214 > 199, tR
)
20.7 min), IFP (203 > 188, tR ) 21.60 min), IQ[4,5-b] (199 > 184, tR
) 21.6 min), 6,7-DiMeIgQx (228 > 213, tR ) 22.5 min), 4,8-DiMeIQx
(228 > 213, tR ) 23.2), 7,9-DiMeIgQx (228 > 213, tR ) 23.8 min),
and PhIP (225 > 210, tR ) 24.6 min) and [M + H]+ > [M + H -
18]+• (loss of CD3 •) for the respective trideuterated internal standards.
The transition [M + H]+ > [M + H - 17]+ (loss of NH3) was used
for ARC (184 > 167, tR ) 25.5 min), MeARC (198 > 167, tR ) 27.0
min), and [13C6]ARC (190 > 173).
The quantification of HAAs was performed with an external
calibration curve using 125 pg/µL of internal standards containing
unlabeled HAAs at nine calibrant levels ranging from 0 to 1250 pg
injected on the column; these levels were equated to 0, 0.025, 0.05,
0.3, 0.5, 1.0, 2.5, 5.0, 10.0, and 25 µg/kg of HAAs in cooked meat.
Due to the high concentrations of 7-MeIgQx and PhIP found in some
meat samples, the concentration of the calibrants in the upper range
was increased to 100 µg/kg for 7-MeIgQx and 300 µg/kg for PhIP.
Since isotopically labeled internal standards were not available for 7,9-
DiMeIgQx, MeARC, and IFP, [2H3C]-6,7-DiMeIgQx, [13C6]-ARC, and
[2H3C]-IQ[4,5-b] were used as the respective internal standards. The
coefficient of determination (r2) of all HAA calibration curves exceeded
0.995.
Figure 1. Chemical structures of HAAs investigated in this study.
formed in fried ground beef patties. Two novel HAAs, which
appeared to contain the IgQx skeleton, on the basis of their ESI
product ion spectra (15, 17), were identified. These two HAAs
were thought to be IgQx (tR ) 12.0 min) and 6,7-DiMeIgQx
(tR ) 22.5 min).
Syntheses of 6-MeIgQx and 6,7-DiMeIgQx. We undertook
the chemical syntheses of IgQx and 6,7-DiMeIgQx to determine
whether either compound was formed in cooked beef. The
synthesis is depicted in Scheme 1. The key intermediates were
the isomeric compounds 1-methyl-6-nitro-1H-benzo[d]imida-
zole-2,5-diamine (4a) and 1-methyl-5-nitro-1H-benzo[d]imida-
zole-2,6-diamine (4b), both of which underwent reduction to
form 1-methyl-1H-benzo[d]imidazole-2,5,6-triamine (5). The
triamine readily condensed with glyoxal or 1,3-butadione to form
the desired IgQx (6) and 6,7-DiMeIgQx (7) homologues. The
incorporation of [2H3C]I in the reaction scheme was done to
prepare the stable, isotopically labeled compounds for internal
standards.
Quantification of HAAs in Cooked Meats. The tandem SPE
method employed to isolate HAAs from cooked meats (25) was
originally developed for the analysis of HAAs by HPLC with UV
and fluorescence detection (24). We adapted the method by
replacing the individual propylsulfonic acid (PRS) and C18 resins
with an Oasis MCX LP extraction cartridge, which contains a
mixture of hydrophobic and cation-exchange resins (17). The use
of the MCX resin permitted the recovery of all of the HAAs in
one eluent. We previously validated this method by LC-ESI-MS/
MS measurements of six prevalent HAAs spiked to pan-fried beef
and barbecued chicken cooked well done at a level of 1 µg/kg.
The accuracy of the method was on average within 15% of the
target value with coefficients of variation e15%, based upon
isotopic dilution with internal standards. The lower limit of
quantification (LOQ) approached 0.030 µg/kg (17).
RESULTS
Identification of Known HAAs and Detection of Novel
HAAs in Cooked Meat by LC-ESI-MS/MS. The chemical
structures of 14 HAAs formed in cooked meat and investigated
in this study are depicted in Figure 1. Thirteen out of the 14
HAAs were identified in our cooked beef, pork, and poultry
samples. MeIQ was the only HAA not detected in these cooked
meat samples, consistent with findings of previous studies (18, 19).
A representative LC-ESI-MS/MS analysis of HAAs formed in
ground beef patties pan-fried to the well-done level at 191 °C
is presented in Figure 2. Eleven of the HAAs under study were
detected in this fried meat sample. The carboline derivatives
ARC and MeARC were below the limit of detection (LOD) in
all cooked beef samples, consistent with their occurrence only
in fried beef cooked at elevated temperatures (>250 °C) (1, 17).
7-MeIgQx (Figure 2, tR ) 19.8 min) was the principal HAA
Many HAAs were readily detected in all cooked meat
products of the FFQ database, except for those samples cooked

72 J. Agric. Food Chem., Vol. 56, No. 1, 2008
Ni et al.
Figure 2. LC-ESI-MS/MS analysis of HAAs formed in pan-fried beef cooked well done. The approximate tR values are IQ (9.4 min), IgQx (12.0 min),
IQx (12.9 min), 7-MeIgQx (19.8 min), 8-MeIQx (20.70 min), IFP (21.6 min), IQ[4,5-b] (21.6 min), 6,7-DiMeIgQx (22.5 min), 4,8-DiMeIQx (23.2 min),
7,9-DiMeIgQx (23.8 min), PhIP (24.6 min), ARC (25.5 min), and MeARC (27.0 min). Peaks of HAAs and isotopically labeled internal standards are
shaded, and approximate tR values and peak area are reported.
at low temperature (100 °C); for the latter, the amounts of all
HAAs were below 1 µg/kg (data not shown). The estimates of
HAAs in cooked pork, chicken, beef, and beef gravy are reported
in Tables 1-4. The relative levels of HAAs formed in several
of these cooked meat samples are presented in Figures 3
and 4.
µg/kg (Figure 3B and Table 2), consistent with the estimates
of PhIP obtained by HPLC with UV/fluorescence detection (2).
Other prominent HAAs formed in barbecued chicken cooked
very well done were ARC (40 µg/kg), IFP (26 µg/kg), 7-MeIgQx
(11.3 µg/kg), 8-MeIQx (7.7 µg/kg), and MeARC (6.1 µg/kg).
HAA Content in Cooked Beef. 7-MeIgQx was the major
HAA formed in hamburgers and steak pan-fried at a surface
temperature of 191 °C for all levels of doneness (Figure 4 and
Tables 3 and 4). Increasing the time of cooking and degree of
well doneness increased the HAA content. Other HAAs identi-
fied at concentrations above 1 µg/kg in hamburgers or steak
were IgQx, 6,7-MeIgQx, 7,9-DiMeIgQx, 8-MeIQx, 4,8-
DiMeIQx, IFP, and PhIP. As previously reported, oven broiling
of hamburgers produced much lower concentrations of HAAs
than did pan frying (18).
HAA Content in Roast Beef Gravy. Roast beef was reported
to contain low amounts of 8-MeIQx, 4,8-DiMeIQx, and PhIP
(<1 µg/kg) at all degrees of doneness, but the gravy prepared
from well-done drippings of roast beef contained 8-MeIQx (7.1
µg/kg) and PhIP (4.1 µg/kg) (18). Our estimates of both HAAs
in gravy, by quantitative LC-ESI-MS/MS (Table 4), agree with
those published values (18); markedly, the amount of 7-MeIgQx
formed in gravy exceeded the amounts of 8-MeIQx and PhIP,
by 3.6- and 8.7-fold, respectively, in the present study.
Confirmation of Previously Characterized and Recently
Discovered HAAs by Product Ion Spectra Scan Mode. The
identities of the 13 HAAs were confirmed by acquiring full-
scan product ion spectra of the analytes; these spectra proved
HAA Content in Pork Meats. Sausage patties pan-fried very
well done contained 8-MeIQx at a concentration of 5.1 µg/kg.
Low amounts of PhIP (0.2 µg/kg) were also detected. The
estimates of these HAAs are comparable to the values previously
determined by HPLC with UV/fluorescence detection (19).
However, the recently discovered 7-MeIgQx was the most
abundant HAA, at an estimated concentration of 8.4 µg/kg
(Figure 3A). Three other linear tricyclic ring HAAs, IgQx, 6,7-
DiMeIgQx, and 7,9-DiMeIgQx, were detected at concentrations
that ranged between 0.2 and 1.8 µg/kg. Pan-fried and oven-
broiled bacon strips contained high amounts of PhIP, consistent
with the findings of previous reports (19, 26). 7-MeIgQx was
the second most abundant HAA formed in cooked bacon,
followed by 8-MeIQx. Pork chops pan-fried very well done
contained only trace quantities of HAAs; 7-MeIgQx was the
most abundant, at 0.4 µg/kg. The remaining HAAs were formed
at concentrations below 1.0 µg/kg in all cooked pork meats
(Table 1).
HAA Content in Cooked Chicken. PhIP was the principal
HAA formed in all types of chicken samples prepared by
barbecuing, oven broiling or pan frying. PhIP was formed in
very well done barbecued chicken at a concentration of 305

LC/MS Analysis of HAAs in Cooked Meats
J. Agric. Food Chem., Vol. 56, No. 1, 2008 73

74 J. Agric. Food Chem., Vol. 56, No. 1, 2008
Ni et al.

LC/MS Analysis of HAAs in Cooked Meats
J. Agric. Food Chem., Vol. 56, No. 1, 2008 75
Figure 3. HAA formation in (A) cooked bacon, sausage patties, and pork chops and (B) pan-fried and barbecued chicken prepared at a range of
temperatures and doneness.
Figure 4. HAA formation in (A) pan-fried ground beef patties and (B) pan-fried beef steak prepared at a range of doneness.
to be in excellent agreement with the spectra of the synthetic
compounds. Many of the ESI product ion mass spectra have
been published previously (16, 17). The product ion spectra of
two newly discovered HAAs, IgQx and 6,7-DiMeIgQx, and the

76 J. Agric. Food Chem., Vol. 56, No. 1, 2008
Ni et al.
Figure 5. LC-ESI-MS product ion spectra of (A) IgQx, (B) 7-MeIgQx, (C) IFP, and (D) 6,7-DiMeIgQx formed in pan-fried meat cooked very well done
(upper panels) and the respective synthetic compounds (lower panels).
spectrum of 7-MeIgQx, and IFP, the only HAA investigated in
this study that contains an oxygen atom in the 5-membered
heterocyclic ring (27), and the synthetic reference compounds,
are presented in Figure 5. The product ion spectra corroborate
the identities of these analytes in the cooked meat samples.
Other Putative HAAs in Cooked Meats. One peak (Figure
2, tR ) 23.6 min), which displayed the same SRM transition
(199 > 184) as did IQ and IQ[4,5-b], was found in 17 out of
26 meat samples. Moreover, the product ion spectra displayed
prominent fragment ions at m/z 184, 157, and 131, as do the
product ion spectra of several isomers of IQ (23). There are
several plausible structures for this presumed isomer of IQ;
additional studies are required to unambiguously determine its
identity. If we assume an ionization efficiency comparable to
that of IQ, this compound occurs in cooked meat at concentra-
tions ranging from 0.2 to 1.0 µg/kg. Two putative isomers of
DiMeIQx [M + H]+ at m/z 228 occurred in many pan-fried
beef samples cooked well done or very well done: one isomer
eluted just prior to 4,8-DiMeIQx (tR ) 23.2 min), and the second
compound eluted just before 7,9-DiMeIgQx (tR ) 23.8 min)
(Figure 2). The product ion spectra of these analytes are
suggestive of structures that contain the amino-N-methylimi-
dazo[4,5-f]quinoxaline or amino-N-methylimidazo[4,5-g]qui-
noxaline ring structure (17). The identities of these compounds
remain to be elucidated.
methods, to varying degrees of doneness, has been determined
by quantitative LC-ESI-MS/MS using the stable, isotope dilution
method. Ten known HAAs and three recently characterized
HAAs were identified; these latter compounds were postulated
to be linear tricyclic ring isomers of IQx, 8-MeIQx, and 4,8-
DiMeIQx (17). The syntheses of 7-MeIgQx, IgQx, and 6,7-
DiMeIgQx were undertaken to determine whether these com-
pounds were formed in cooked beef (Scheme 1) (15). The tR
values of the reference compounds, the trideuterated internal
standards, and the analytes in cooked meats were identical.
Moreover, the product ion spectra of the analytes and of the
respective reference compounds were in excellent agreement
(Figure 5) (15). Thus, these three HAAs, along with 7,9-
DiMeIgQx, which was previously identified in beef extract (20),
represent a new class of linear HAAs, which can arise at
appreciable concentrations in cooked meats.
The most important finding of our study is the detection of
7-MeIgQx in cooked beef at high levels relative to the
concentrations of other HAAs. 7-MeIgQx arises in cooked beef
at higher levels than do 8-MeIQx, 4,8-DiMeIQx, and PhIP,
which have been recognized as three of the most mass-abundant
HAAs formed in cooked beef (1, 28). The sum of the masses
of 7-MeIgQx and the three other IgQx-ring-structured HAAs
exceeds 50% of the total HAA content of hamburgers and steak
cooked well done and very well done and the content of gravy
obtained from very well done roast beef. Moreover, these IgQx-
structured HAAs represent a significant proportion of the total
daily intake of HAAs in bacon.
DISCUSSION
The HAA content of hamburger patties, beef steak, roast beef
gravy, pork, and chicken cooked by common household

LC/MS Analysis of HAAs in Cooked Meats
J. Agric. Food Chem., Vol. 56, No. 1, 2008 77
In general, the HAA content of all of the meat samples
increased with degree of doneness, but the individual HAAs
measured were not produced to the same extent by each cooking
method and doneness level. Barbecuing or pan frying produced
larger amounts of HAAs than did oven broiling, a result in
agreement with earlier findings on HAA formation as a function
oftemperatureandmethodanddurationofcooking(1,2,17–19,29).
The amounts of HAAs determined, by LC-ESI-MS/MS, in
various cooked meat samples of the FFQ are in good agreement
to those estimates previously obtained by HPLC with UV/
fluorescence detection (2, 18, 19). The average concentrations
of 8-MelQx, PhlP, and 4,8-DiMelQx in a high-temperature
quality control (high QC) cooked ground beef sample were
[mean (coefficient of variation)] 7.2 (0.36), 10.9 (0.24), and 1.66
(0.40) ng/g, respectively, determined by HPLC with UV/
fluorescence detection. The average concentrations of 8-MelQx,
PhlP, and 4,8-DiMelQx from five batches of this high QC meat
sample, estimated by LC-ESI-MS/MS, were 6.0 (0.04), 7.8
(0.08), and 1.8 (0.12) ng/g, respectively. The amount of
7-MeIgQx estimated in this high QC meat sample was 31.0
(0.16) ng/g. The precision of the LC-ESI-MS/MS assay is
superior to the precision of the HPLC with UV/fluorescence
detection because the former’s use of stable, istopically labeled
internal standards accurately compensates for recovery of the
analytes from complex food matrices (17).
and 2-napthylamine (2-NA) in the Ames Salmonella typhimurium
tester strains TA98 and YG1024 (frameshift-specific) or YG1029
(primarily point mutation-specific) (35, 36). Both 4-ABP and 2-NA
are bladder carcinogens in the experimental dog model, and both
compounds are recognized human urinary bladder carcinogens
(37). Given that the concentrations of 7-MeIgQx and its
homologues in cooked meats are high in comparison to the
concentrations of angular tricyclic ring isomers, some of which
are strong experimental animal carcinogens (4), studies on the
toxicological properties of 7-MeIgQx and other linear tricyclic
HAAs are warranted.
ABBREVIATIONS USED
IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; IQ[4,5-b], 2-amino-
1-methylimidazo[4,5-b]quinoline; IgQx, 2-amino-1-methylimida-
zo[4,5-g]quinoxaline; IQx, 2-amino-3-methylimidazo[4,5-f]qui-
noxaline;
7-MeIgQx,
2-amino-1,7-dimethylimidazo[4,5-
g]quinoxaline; 8-MeIQx, 2-amino-3,8-dimethylimidazo[4,5-
f]quinoxaline; IFP, 2-amino-1,6-dimethylfuro[3,2-e]imidazo[4,5-
b]pyridine; 6,7-DiMeIgQx, 2-amino-1,6,7-trimethylimidazo[4,5-
g]quinoxaline; 4,8-DiMeIQx, 2-amino-3,4,8-trimethylimidazo[4,5-
f]quinoxaline; 7,9-DiMeIgQx, 2-amino-1,7,9-trimethylimidazo[4,5-
g]quinoxaline; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-
b]pyridine;ARC,2-amino-9H-pyrido[2,3-b]indole;MeARC,2-amino-
3-methyl-9H-pyrido[2,3-b]indole; CID, collision-induced dissociation;
FFQ, food frequency questionnaire; LC-ESI-MS/MS, liquid
chromatography-electrospray ionization/tandem mass spectrom-
etry; MS, mass spectrometry; SPE, solid-phase extraction; SRM,
selected reaction monitoring.
The estimates of PhIP, 8-MeIQx, and 4,8-DiMeIQx in cooked
meat samples, measured by HPLC with UV/fluorescence, have
been incorporated into a meat-cooking module within the FFQ
to assess dietary intake of these HAAs (11). Epidemiological
studies investigating the risk of development of colorectal and
prostate cancers have examined meat consumption, and the
investigators have proposed that some of these HAAs are
potential causal agents of cancer (11, 12). Our examination of
these same meat samples, by LC-ESI-MS/MS, has shown that
10 additional HAAs are present at variable concentrations in
many of these meat staples; clearly, some of these additional
compounds should be taken into account when the causal role
of HAAs in the risk of developing cancer is being assessed.
ACKNOWLEDGMENT
We acknowledge the assistance of the NMR Structural
Biology Facility at the Wadsworth Center.
LITERATURE CITED
(1) Knize, M. G.; Dolbeare, F. A.; Carroll, K. L.; Moore, D. H.;
Felton, J. S. Effect of cooking time and temperature on the
heterocyclic amine content of fried beef patties. Food Chem.
Toxicol. 1994, 32, 595–603.
(2) Sinha, R.; Rothman, N.; Brown, E. D.; Salmon, C. P.; Knize,
M. G.; Swanson, C. S.; Rossi, S. C.; Mark, S. D.; Levander, O. A.;
Felton, J. S. High concentrations of the carcinogen 2-amino-1-
methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) occur in chicken
but are dependent on the cooking method. Cancer Res. 1995, 55,
4516–4519.
(3) Sugimura, T.; Nagao, N.; Kawachi, T.; Honda, M.; Yahagi, T.;
Seino, Y.; Stao, S.; Matsukura, N.; Matsushima, T.; Shirai, A.;
Sawamura, M.; Matsumoto, H. Mutagen-carcinogens in food, with
special reference to highly mutagenic pyrolytic products in broiled
foods. In Origins of Human Cancer, Book C; Hiatt, H. H., Watson,
J. D., Winstein, J. A., Eds.; Cold Spring Harbor Laboratory: Cold
Spring Harbor, NY, 1977; pp 1561–1577.
(4) Sugimura, T.; Wakabayashi, K.; Nakagama, H.; Nagao, M.
Heterocyclic amines: Mutagens/carcinogens produced during
cooking of meat and fish. Cancer Sci. 2004, 95, 290–299.
(5) Manabe, S.; Wada, O.; Kanai, Y. Simultaneous determination of
amino-alpha-carbolines and amino-gamma-carbolines in cigarette
smoke condensate by high-performance liquid chromatography.
J. Chromatogr. 1990, 529, 125–133.
Short-term bacterial mutagenesis assays, such as the Ames
assay, have been an effective screening tool for the identification
of many mutagenic HAAs in complex food matrices. However,
the mutagenic potencies of HAAs vary over a >1000-fold range
in bacterial assays (4), and only HAAs either possessing high
mutagenic activity or present at great abundance have been
successfully isolated and characterized from cooked meats when
bacterial mutagenicity assays are employed for screening (28, 30).
HAAs containing the IgQx ring structure, such as 7-MeIgQx
and 7,9-DiMeIgQx, are ≈1000-fold weaker bacterial mutagens
than their respective angular tricyclic ring isomers (15).
Consequently, the IgQx molecules present in cooked meats are
not easily detected in the Ames assay. Screening for HAAs by
LC-ESI-MS/MS-based methods instead of biological assays has
enabled us to identify novel IgQx-structured HAAs and other
putative compounds in cooked beef (17).
While bacterial mutation assays have served as a valuable
tool to detect HAAs of high genotoxic potency in cooked meat,
the mutation assays do not reliably predict carcinogenic potency
in mammals (31). The large differences in the ranges of potency
of HAAs observed in bacterial mutation assays (4) are not
observed in mammalian cell assays (32–34). Moreover, the total
doses of HAAs required to induce tumors in 50% of the animals
tested in carcinogen bioassays fall generally within a factor of
10 (4). The genotoxic potency of 7-MeIgQx (15, 20) is comparable
to the potencies of the aromatic amines 4-aminobiphenyl (4-ABP)
(6) Manabe, S.; Izumikawa, S.; Asakuno, K.; Wada, O.; Kanai, Y.
Detection of carcinogenic amino-alpha-carbolines and amino-
gamma-carbolines in diesel-exhaust particles. EnViron. Pollut.
1991, 70, 255–265.
(7) Turesky, R. J. Interspecies metabolism of heterocyclic aromatic
amines and the uncertainties in extrapolation of animal toxicity

78 J. Agric. Food Chem., Vol. 56, No. 1, 2008
Ni et al.
data for human risk assessment. Mol. Nutr. Food Res. 2005, 49,
101–117.
(8) Cross, A. J.; Sinha, R. Meat-related mutagens/carcinogens in the
etiology of colorectal cancer. EnViron. Mol. Mutagen. 2004, 44,
44–55.
(9) Knize, M. G.; Felton, J. S. Formation and human risk of
carcinogenic heterocyclic amines formed from natural precursors
in meat. Nutr. ReV. 2005, 63, 158–165.
(10) Sinha, R.; Gustafson, D. R.; Kulldorff, M.; Wen, W. Q.; Cerhan,
J. R.; Zheng, W. 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyri-
dine, a carcinogen in high-temperature-cooked meat, and breast
cancer risk. J. Natl. Cancer Inst. 2000, 92, 1352–1354.
(11) Sinha, R. An epidemiologic approach to studying heterocyclic
amines. Mutat. Res. 2002, 506–507, 197–204.
(12) Cross, A. J.; Peters, U.; Kirsh, V. A.; Andriole, G. L.; Reding,
D.; Hayes, R. B.; Sinha, R. A prospective study of meat and meat
mutagens and prostate cancer risk. Cancer Res. 2005, 65, 11779–
11784.
(13) Pais, P.; Knize, M. G. Chromatographic and related techniques
for the determination of aromatic heterocyclic amines in foods.
J. Chromatogr. B 2000, 747, 139–169.
(14) Felton, J. S.; Knize, M. G.; Dolbeare, F. A.; Wu, R. Mutagenic
activity of heterocyclic amines in cooked foods. EnViron. Health
Perspect. 1994, 102 (Suppl. 6), 201–204.
(15) Turesky, R. J.; Goodenough, A. K.; Ni, W.; McNaughton, L.;
LeMaster, D. M.; Holland, R. D.; Wu, R. W.; Felton, J. S.
Identification of 2-amino-1,7-dimethylimidazo[4,5-g]quinoxaline:
an abundant mutagenic heterocyclic aromatic amine formed in
cooked beef. Chem. Res. Toxicol. 2007, 20, 520–530.
(16) Holland, R. D.; Taylor, J.; Schoenbachler, L.; Jones, R. C.;
Freeman, J. P.; Miller, D. W.; Lake, B. G.; Gooderham, N. J.;
Turesky, R. J. Rapid biomonitoring of heterocyclic aromatic
amines in human urine by tandem solvent solid phase extraction
liquid chromatography electrospray ionization mass spectrometry.
Chem. Res. Toxicol. 2004, 17, 1121–1136.
(24) Gross, G. A.; Gruter, A. Quantitation of mutagenic/carcinogenic
heterocyclic aromatic amines in food products. J. Chromatogr.
1992, 592, 271–278.
(25) Gross, G. A. Simple methods for quantifying mutagenic hetero-
cyclic aromatic amines in food products. Carcinogenesis 1990,
11, 1597–1603.
(26) Gross, G. A.; Turesky, R. J.; Fay, L. B.; Stillwell, W. G.; Skipper,
P. L.; Tannenbaum, S. R. Heterocyclic aromatic amine formation
in grilled bacon, beef and fish and in grill scrapings. Carcino-
genesis 1993, 14, 2313–2318.
(27) Pais, P.; Tanga, M. J.; Salmon, C. P.; Knize, M. G. Formation of
the mutagen IFP in model systems and detection in restaurant
meats. J. Agric. Food Chem. 2000, 48, 1721–1726.
(28) Felton, J. S.; Knize, M. G.; Shen, N. H.; Andresen, B. D.;
Bjeldanes, L. F.; Hatch, F. T. Identification of the mutagens in
cooked beef. EnViron. Health Perspect. 1986, 67, 17–24.
(29) Holder, C. L.; Preece, S. W.; Conway, S. C.; Pu, Y. M.; Doerge,
D. R. Quantification of heterocyclic amine carcinogens in cooked
meats using isotope dilution liquid chromatography/atmospheric
pressure chemical ionization tandem mass spectrometry. Rapid
Commun. Mass Spectrom. 1997, 11, 1667–1672.
(30) Wakabayashi, K.; Kim, I. S.; Kurosaka, R.; Yamaizumi, Z.;
Ushiyama, H.; Takahashi, M.; Koyota, S.; Tada, A.; Nukaya, H.;
Goto, S. Identification of new mutagenic heterocyclic amines and
quantification of known heterocyclic amines. In Heterocyclic
aromatic amines: Possible Human Carcinogens. Proceedings of
the 23rd International Princess Takamatsu Symposium, 23 ed.;
Adamson, R. H., Gustafson, D. R., Ito, N., Nagao, M., Sugimura,
T., Wakabayashi, K., Yamazoe, Y., Eds.; Princeton Scientific
Publishing: Princeton, NJ, 1995; pp 39–49.
(31) Sugimura, T. Successful use of short-term tests for academic
purposes: Their use in identification of new environmental
carcinogens with possible risk for humans. Mutat. Res. 1988, 205,
33–39.
(32) Thompson, L. H.; Tucker, J. D.; Stewart, S. A.; Christensen, M. L.;
Salazar, E. P.; Carrano, A. V.; Felton, J. S. Genotoxicity of
compounds from cooked beef in repair-deficient CHO cells versus
Salmonella mutagenicity. Mutagenesis 1987, 2, 483–487.
(33) Wu, R. W.; Tucker, J. D.; Sorensen, K. J.; Thompson, L. H.;
Felton, J. S. Differential effect of acetyltransferase expression on
the genotoxicity of heterocyclic amines in CHO cells. Mutat. Res.
1997, 390, 93–103.
(34) Wu, R. W.; Panteleakos, F. N.; Kadkhodayan, S.; Bolton-Grob,
R.; McManus, M. E.; Felton, J. S. Genetically modified Chinese
hamster ovary cells for investigating sulfotransferase-mediated
cytotoxicity and mutation by 2-amino-1-methyl-6-phenylimi-
dazo[4,5-b]pyridine. EnViron. Mol. Mutagen. 2000, 35, 57–65.
(35) Scribner, J. D.; Fisk, S. R.; Scribner, N. K. Mechanisms of action
of carcinogenic aromatic amines: an investigation using mutagen-
esis in bacteria. Chem.-Biol. Interact. 1979, 26, 11–25.
(36) Thompson, P. A.; DeMarini, D. M.; Kadlubar, F. F.; McClure,
G. Y.; Brooks, L. R.; Green, B. L.; Fares, M. Y.; Stone, A.;
Josephy, P. D.; Ambrosone, C. B. Evidence for the presence of
mutagenic arylamines in human breast milk and DNA adducts in
exfoliated breast ductal epithelial cells. EnViron. Mol. Mutagen.
2002, 39, 134–142.
(17) Turesky, R. J.; Taylor, J.; Schnackenberg, L.; Freeman, J. P.;
Holland, R. D. Quantitation of carcinogenic heterocyclic aromatic
amines and detection of novel heterocyclic aromatic amines in
cooked meats and grill scrapings by HPLC/ESI-MS. J. Agric.
Food Chem. 2005, 53, 3248–3258.
(18) Sinha, R.; Rothman, N.; Salmon, C. P.; Knize, M. G.; Brown,
E. D.; Swanson, C. A.; Rhodes, D.; Rossi, S.; Felton, J. S.;
Levander, O. A. Heterocyclic amine content in beef cooked by
different methods to varying degrees of doneness and gravy made
from meat drippings. Food Chem. Toxicol. 1998, 36, 279–287.
(19) Sinha, R.; Knize, M. G.; Salmon, C. P.; Brown, E. D.; Rhodes,
D.; Felton, J. S.; Levander, O. A.; Rothman, N. Heterocyclic amine
content of pork products cooked by different methods and to
varying degrees of doneness. Food Chem. Toxicol. 1998, 36, 289–
297.
(20) Nukaya, H.; Koyota, S.; Jinno, F.; Ishida, H.; Wakabayashi, K.;
Kurosaka, R.; Kim, I. S.; Yamaizumi, Z.; Ushiyama, H.; Sugimura,
T. Structural determination of a new mutagenic heterocyclic
amine, 2-amino-1,7,9-trimethylimidazo[4,5-g]quinoxaline (7,9-
DiMeIgQx), present in beef extract. Carcinogenesis 1994, 15,
1151–1154.
(21) Turesky, R. J.; Bur, H.; Huynh-Ba, T.; Aeschbacher, H. U.; Milon,
H. Analysis of mutagenic heterocyclic amines in cooked beef
products by high-performance liquid chromatography in combina-
tion with mass spectrometry. Food Chem. Toxicol. 1988, 26, 501–
509.
(37) Beland, F. A.; Kadlubar, F. F. Formation and persistence of
arylamine DNA adducts in vivo. EnViron. Health Perspect. 1985,
62, 19–30.
(22) Ronne, E.; Olsson, K.; Grivas, S. One-step synthesis of 2-amino-
1-methylimidazo[4,5-b]quinoline. Synth. Commun. 1994, 24,
1363–1366.
(23) Holland, R. D.; Gehring, T.; Taylor, J.; Lake, B. G.; Gooderham,
N. J.; Turesky, R. J. Formation of a mutagenic heterocyclic
aromatic amine from creatinine in urine of meat eaters and
vegetarians. Chem. Res. Toxicol. 2005, 18, 579–590.
Received for review August 15, 2007. Revised manuscript received
November 8, 2007. Accepted November 10, 2007. This research is
supported by Grant 05B025 from the American Institute for Cancer
Research and funded in part by the Wadsworth Center, New York
State Department of Health.
JF072461A