Identification of urinary metabolites of orally administered N,N-dimethyl-p-toluidine in male F344 rats

Article abstract of DOI:10.1080/15287390701206176

The metabolism of orally administered N,N-dimethyl-p-toluidine (DMPT) in male F344 rats was investigated. The rat urinary metabolite profile was determined by analytical reverse-phase high performance liquid chromatography (HPLC). Four radiolabeled peaks were observed, isolated, and purified by solid-phase extraction (SPE) and preparative HPLC methods. The 4 peaks were identified as p-(N-acetylhydroxyamino)hippuric acid (M1), DMPT N-oxide (M2), N-methyl-p-toluidine (M3), and parent DMPT. Metabolites M1 and M2 were identified by spectrometric and spectroscopic methods, including mass fragmentation pattern identification from both liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry, and from chemical analysis of nuclear magnetic resonance spectra. Structural confirmation of metabolite M2 was accomplished by comparison with a synthetic standard. Peaks M3 and the peak suspected to be DMPT were identified by comparison of their HPLC retention times and mass fragmentation patterns with authentic standards of N-methyl-p-toluidine and DMPT, respectively. DMPT metabolism is similar to that reported for N,N-dimethylaniline. Copyright Taylor & Francis Group, LLC.

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Identification of Urinary Metabolites of Orally
Administered N,N-Dimethyl-p-Toluidine in Male F344
Rats
Nam-Cheol Kim a , Katayoon Ghanbari a , Dean A. Kracko a , Waylon M. Weber a , Jacob D.
McDonald a & Kelly J. Dix a
a Lovelace Respiratory Research Institute , Albuquerque, NM, USA
Published online: 17 Apr 2007.
To cite this article: Nam-Cheol Kim , Katayoon Ghanbari , Dean A. Kracko , Waylon M. Weber , Jacob D. McDonald & Kelly J.
Dix (2007) Identification of Urinary Metabolites of Orally Administered N,N-Dimethyl-p-Toluidine in Male F344 Rats , Journal of
Toxicology and Environmental Health, Part A: Current Issues, 70:10, 781-788, DOI: 10.1080/15287390701206176
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Journal of Toxicology and Environmental Health, Part A, 70: 781–788, 2007
Copyright © Taylor & Francis Group, LLC
ISSN: 1528-7394 print / 1087-2620 online
DOI: 10.1080/15287390701206176
UTEH
Identification of Urinary Metabolites of Orally Administered
N,N-Dimethyl-p-Toluidine in Male F344 Rats
Metabolism of orally administered DMPT in rats
Nam-Cheol Kim1, Katayoon Ghanbari, Dean A. Kracko, Waylon M. Weber,
Jacob D. McDonald, and Kelly J. Dix
Lovelace Respiratory Research Institute, Albuquerque, NM, USA
preparations, and is an intermediate in the synthesis of dyes
and pesticides. There is the potential for human exposure to
DMPT in prosthesis users, individuals with dental plates, and
occupational settings (Potter et al., 1988; Taningher, et al.,
1993; Haddad et al., 1996). Human exposure to DMPT has
resulted in methemoglobinemia and allergic responses (Pegam
and Medhurst, 1971; Bunn and Forget, 1986; Potter et al.,
1988; Tosti et al., 1990; Dutrée-Meulenberg et al., 1992;
Haddad et al., 1995; Kao et al., 1997). Accidental ingestion of
artificial fingernail solutions containing DMPT by a 16-month-
old girl resulted in an acute cyanotic episode due to methemo-
globinemia and a 5-month-old boy suffered from a less severe
case after ingestion of an artificial fingernail solution contain-
ing DMPT (Potter et al., 1988; Kao et al., 1997).
The induction of methemoglobinemia produced by DMPT
ingestion is suspected to be due to the formation of a toxic
metabolite, p-methylphenylhydroxylamine. This metabolite
is analogous to phenylhydroxylamine, which is likely respon-
sible for methemoglobinemia observed after exposure to
aniline (Potter et al., 1988; Bunn and Forget, 1986). Aniline,
N,N-dimethylaniline (DMA), and toluidines (ortho-, meta-,
and para-) are structurally similar to DMPT, and the known
metabolism of these compounds may provide useful informa-
tion on DMPT metabolism. It was reported that the primary
metabolites of ortho-, meta-, and para-toluidine resulted from
ring hydroxylation and subsequent conjugation (Cheever et al.,
1980). Pathways of DMA metabolism include N-demethylation
to form N-methylaniline and aniline; N-oxidation to form
N,N-dimethylaniline N-oxide; and N-glucuronidation of N-
methylaniline to form N-methylaniline N-glucuronide. In
The metabolism of orally administered N,N-dimethyl-p-
toluidine (DMPT) in male F344 rats was investigated. The rat
urinary metabolite profile was determined by analytical
reverse-phase high performance liquid chromatography
(HPLC). Four radiolabeled peaks were observed, isolated, and
purified by solid-phase extraction (SPE) and preparative HPLC
methods. The 4 peaks were identified as p-(N-acetylhy-
droxyamino)hippuric acid (M1), DMPT N-oxide (M2), N-
methyl-p-toluidine (M3), and parent DMPT. Metabolites M1
and M2 were identified by spectrometric and spectroscopic
methods, including mass fragmentation pattern identification
from both liquid chromatography/mass spectrometry and gas
chromatography/mass spectrometry, and from chemical analy-
sis of nuclear magnetic resonance spectra. Structural confirma-
tion of metabolite M2 was accomplished by comparison with a
synthetic standard. Peaks M3 and the peak suspected to be
DMPT were identified by comparison of their HPLC retention
times and mass fragmentation patterns with authentic stan-
dards of N-methyl-p-toluidine and DMPT, respectively. DMPT
metabolism is similar to that reported for N,N-dimethylaniline.
Keywords N,N-dimethyl-p-toluidine, metabolism, glycine conju-
gate, N-oxidation, N-demethylation
INTRODUCTION
N,N-dimethyl-p-toluidine (DMPT) is a high-production-
volume chemical used as a polymerization accelerator in the
manufacture of bone cements and dental materials. DMPT
is also found in industrial glues and artificial fingernail
Received 12 April 2006; accepted 25 July 2006.
This manuscript has not been published elsewhere and has not addition, ring hydroxylation in the para-position is another
been submitted simultaneously for publication elsewhere.
pathway involved in DMA metabolism and results in the for-
mation of 4-aminophenol, N-methyl-4-aminophenol, and
N,N-dimethyl-4-aminophenol (Gorrod and Gooderham,
1981; Gooderham and Gorrod, 1981; Sherrat and Damani,
1Current Address: Global Technology & Quality, Kraft Foods, 801
Waukegan Road, Glenview, IL 60025.
Address correspondence to Kelly J. Dix, PhD, Lovelace
Respiratory Research Institute, 2425 Ridgecrest Dr. SE, Albuquerque,
NM 87108, USA. Tel: 348-505-9450, Fax: 505-348-4980. E-mail: 1989). This study reports the identification of urinary metab-
kdix@LRRI.org
olites of orally administered DMPT in F344 rats.
781

782
N.-C. KIM ET AL.
MATERIALS AND METHODS
and data acquisition. Signals were monitored at 254 nm and
the column was maintained at 40°C. Preparative HPLC was
carried out on the same HPLC system with the exception of
a Phenomenex Luna semi-preparative column (5 μm, 250 ×
10 mm i.d.). The HPLC mobile phase was 5% acetonitrile in
H2O for 5 min, then linearly increasing acetonitrile content to
95% over 30 min. The flow rate was 1 mL/min for the analyti-
cal HPLC and 5 mL/min for semi-preparative HPLC. Four
radiolabeled peaks were present as shown in Figure 1.
Chemicals
DMPT and N-methyl-p-toluidine standards were pur-
chased from Sigma-Aldrich Chemical Co. (St. Louis, MO).
Radiolabeled DMPT (25.3 mCi/mmol of 14C, uniformly ring
labeled; PerkinElmer, Boston, MA) was provided by the
National Institute of Environmental Health Sciences (NIEHS).
High-performance liquid chromatography (HPLC)-grade sol-
vents were purchased from Fisher Scientific Co. (Denver,
CO) for column chromatography and HPLC. Deuterated
nuclear magnetic resonance (NMR) solvents (CDCl3 and
CD3OD) and tetramethylsilane (TMS), acetyl chloride, Diaz-
ald, and (trimethylsilyl)diazomethane were purchased from
Sigma-Aldrich Chemical Co.
Isolation and Purification of Radioactive Peaks
Prior to HPLC analysis, urine samples were cleaned using
an Eppendorf 5417C microcentrifuge for 10 min at 10,000
RCF (relative centrifugal force) to pellet any particulate
O
Animals
H
N
O
A
Adult male F344 rats weighing 180-227 g (10-11 weeks of
age) on dose day were purchased from Charles River Laborato-
ries (Raleigh, NC) and housed two to a cage during a 2-week
quarantine. For 1 day prior to and following dosing, animals
were housed individually in all-glass metabolism chambers.
Animal studies were conducted in facilities accredited by the
Association for Assessment and Accreditation of Laboratory
Animal Care International and in compliance with the Guide
for the Care and Use of Laboratory Animals (National
Research Council, 1996). Animals received a single oral dose
of [14C]DMPT (2.5 – 250 mg/kg) in 10% aqueous Alkamuls
(Rhodia, Cranbury, NJ).
OH
CH3
N
CH3
HO
CH3
O
CH3
M1
N
H3C
H
N
H3C
CH3
O
M3
N
H3C
25
CH3
M2
DMPT
5
10
15
20
30
35
min
Sample Collection
B
C
Urine was collected separately from each animal into
receivers cooled over dry ice. Samples were stored in tightly
capped containers in the dark at −22 °C until analysis. The col-
lection-interval composites of rat urine from the 250-mg/kg
dose group were prepared by combining 10% by weight of the
total urinary output from 4 rats to yield composites in 5 differ-
ent hourly collections (6, 12, 24, 48, and 72 hr). Samples were
analyzed for total radioactivity using a Packard Model 2500
TR Liquid Scintillation Analyzer (Packard Instrument Co. Inc.,
Meridien, CT). Samples were assayed for 14C by directly dis-
solving them in Ultima Gold scintillation cocktail (Packard
Instrument Co. Inc.).
0
5
10
15
20
25
30
35
min
HPLC Analysis of Urine Samples
Analytical HPLC was performed using an Agilent Model
1100 HPLC system (Agilent Technologies, Palo Alto, CA)
coupled with a β-RAM-Model 3 radioactivity detector (IN/
US Systems, Tampa, FL) attached to a Phenomenex
(Torrance, CA) Luna C-18 column (5 μm, 150 × 4.6 mm
internal diameter [i.d.]). ChemStation (version A.09.01 or
A.09.03, Agilent Technologies) was used for system control
0
5
10
15
20
25
30
35
min
FIG. 1. Radiochromatogram of radiolabeled components (M1, M2, M3, and
parent DMPT) isolated from urine of [14C]DMPT-treated male F344 rats (A),
nonradiolabeled standards of N-methyl-p-toluidine (B) and N,N-dimethyl-p-
toluidine (C). The spectra were obtained on the HPLC system;
radiochromatogram signals commonly appear approximately 0.5 min after the
UV signals.

METABOLISM OF ORALLY ADMINISTERED DMPT IN RATS
783
material. Prior to semi-preparative HPLC, solid-phase extrac- Chemical Synthesis
tion (SPE) was performed for crude purification of metabolites
using Sep-Pak reversed-phase (C18) cartridges (3 mL, 500 mg
sorbent; Waters, Milford, MA). The C18 SPE cartridge was
preconditioned with 3 mL of H2O prior to sample loading.
Urine (3 mL) from rats was loaded onto the conditioned car-
tridge. Urine was washed through the cartridge with no elution
solvent and collected as fraction 1. The cartridge was then
washed with 3 mL of purified water (fraction 2) followed by 3
mL of acetonitrile (fraction 3). Fraction 1 contained M1 and
M2 with M1 as the major peak. Fraction 2 also contained M1
and M2 but with M2 as the major peak. Fraction 3 contained
M3 and what appeared to be parent DMPT based on chromato-
graphic retention time. Although there were only 4 signals in
the radiochromatograms, multiple peaks were detected in the
ultraviolet (UV) chromatogram indicating the fractions con-
tained contaminants. The three SPE fractions were purified by
semi-preparative HPLC to yield peaks M2, M3, and DMPT as
pure samples. M1 still contained multiple UV peaks and addi-
tional purification steps were carried out by SPE using Sep-Pak
reversed phase (C18, 3 mL, 500 mg sorbent) and Strata ™ X-C
(Phenomenex) cation exchange (3 mL, 200 mg sorbent) car-
tridges. The Strata X-C cartridge was conditioned with 2 mL
methanol before equilibration with 2 mL H2O. Partially puri-
fied M1 in a 1-mL H2O solution was acidified with 20 μL of
H3PO4, loaded onto the cartridge, and eluted with 2 mL 0.1%
aqueous H3PO4. The cartridge was dried for 2 min, eluted with
2 mL of 100% methanol and again with 2 mL of 5% aqueous
NH4OH. The methanol fraction containing M1 was further
purified by reversed-phase SPE (Sep-Pak C18). The sample
was dissolved in 2 mL aqueous 1% NH4OH. The Sep-Pak was
conditioned with 2 mL methanol then 2 mL H2O. The sample
solution was loaded and eluted with 2 mL H2O then 2 mL of
methanol to yield pure M1 in both the radioactive and UV
chromatograms.
DMPT N-oxide was synthesized according to a method
described by Seto and Guengerich (1993) and purified by silica
gel column chromatography. The product was analyzed by
HPLC, LC/MS, and NMR.
p-Acetaminohippuric acid was synthesized by reaction of p-
aminohippuric acid with acetyl chloride. p-Aminohippuric acid
was stirred in a solution of sodium hydroxide (1.3 mL, 2N in
water) in methanol (7.7 mL) at 0°C. Acetyl chloride (1.0 mL) was
added and the mixture stirred for 10 min. Hydrochloric acid (25
mL, 8% in water) was added and the mixture stirred for an addi-
tional 30 min. The resulting precipitate was filtered and washed
with hydrochloric acid (25 mL, 8% in water) and water (100 mL).
The product was analyzed by HPLC, LC/MS, and NMR.
M1 and p-aminohippuric acid were derivatized using a solu-
tion of diazomethane in diethyl ether. The diazomethane was
generated from Diazald using a Diazald kit (Sigma-Aldrich) to
give an approximate concentration of 2N in ethyl ether. Diaz-
omethane (3 mL, 2N) was added to a solution of either M1 or
p-aminohippuric acid (39.1 mg) in ethanol (1 mL) and the
solutions stirred for 1 hr at room temperature. The solvents
were removed under a stream of nitrogen and the products
were analyzed by HPLC, LC/MS, and NMR.
M1 and p-aminohippuric acid were also derivatized with tri-
methylsilyldiazomethane in methanol. Trimethylsilyldiaz-
omethane (100 μL, 2N in diethyl ether) was added to a solution
of either M1 or p-aminohippuric acid (38 mg) in methanol
(1 mL) and the solutions stirred for 30 min at room tempera-
ture. The solvents were removed under a stream of nitrogen
and the products were analyzed by HPLC, LC/MS, and NMR.
RESULTS AND DISCUSSION
Four radiolabeled peaks were observed by analytical HPLC in
urine from [14C]DMPT-treated rats (Figure 1). Off-column recov-
ery of injected radioactivity was >95%. Urine samples were stored
frozen and analyzed periodically over a period of 2 – 3 years. The
same chromatographic profile was reproduced over the 2- to 3-yr
storage period, indicating that the metabolites are stable in the
urine matrix under these conditions. These peaks were separated
and purified by SPE and semi-preparative HPLC. The purified
peaks were analyzed by analytical HPLC, LC/MS, GC/MS, and
NMR and compared to authentic standards to identify their struc-
tures. HPLC retention times and NMR data are shown in Table 1.
M3 and a peak suspected to be DMPT were tentatively
identified as N-methyl-p-toluidine and parent DMPT by com-
paring analytical HPLC retention times of M3 (20.9 min) and
the suspected DMPT (25.2 min) to authentic standards (Figure
1). The mass fragmentation patterns of M3 and DMPT
obtained from GC/MS confirmed M3 as N-methyl-p-toluidine
(Figure 2) and the fourth peak as DMPT (Figure 3). This is
consistent with P450 2B1-mediated N-dealkylation of N,N-
dialkylarylamines reported by Seto and Guengerich (1993).
NMR and MS Analysis
NMR spectra (1D and 2D) were recorded in CDCl3 or
CD3OD using a Bruker AVANCE™ DRX 500 spectrophotome-
ter (500 MHz; Bruker BioSpin Corp., Billerica, MA) with
TMS as an internal standard. Liquid chromatography/mass
spectrometry (LC/MS) was performed on an Agilent 1100
Series LC system with photodiode array (PDA) detection coupled
with an Applied Biosystems MDS Sciex API 4000 G-TRAP
Triple Quad MS/MS (Foster City, CA). LC/MS data were
acquired with Analyst software (version 1.4.1, Agilent Tech-
nologies). The gas chromatography/mass spectrometry (GC/
MS) system consisted of an Agilent 6890N Network GC system,
a 5973 inert mass selective detector with electron ionization (at
70 eV) or chemical ionization using methane, and a 7683 series
injector. Data were acquired with MDS ChemStation software
(version D.01.02.16, Agilent Technologies).

784
N.-C. KIM ET AL.
TABLE 1
HPLC retention times and corresponding 1H NMR and MS data
MS Fragmentation
Analyte
TR (min)
2.5
1H NMR (δ)
Pattern (m/z)
M1 (p-(N-acetylhydroxyamino)
hippuric acid)
2.50 (s, 3H), 4.09 (s, 2H), 7.67 (d, 2H, J=8.5 Hz), 77, 94, 120, 134, 162, 180, 254
7.96 (d, 2H, J=8.9 Hz)
p-Acetaminohippuric acid
1.7
2.15 (s, 3H), 4.09 (s, 2H), 7.67 (d, 2H, J=8.5 Hz), 58, 65, 92, 120, 162, 192, 237
7.82 (d, 2H, J=8.5 Hz)
M1-methyl ester
16.1
2.17 (s, 3H), 3.61 (s, 3H), 4.13 (s, 2H),
7.51 (m, 2H), 7.93 (m, 2H)
65, 77, 93, 108, 120, 162,
194, 251, 268
(p-(N-acetylhydroxyamino)
hippuric acid methyl ester)
p-Acetaminohippuric acid
methyl ester
M2 (N,N-dimethyl-p-toluidine
N-oxide)
12.1
10.4
10.4
2.16 (s, 3H), 3.75 (s, 3H), 4.12 (s, 2H),
7.67 (d, 2H, J=8.5 Hz), 7.83 (d, 2H, J=9.0 Hz)
2.40 (s, 3H), 3.68 (s, 6H), 7.26 (m, 2H),
7.80 (m, 2H)
2.32 (s, 3H), 3.52 (s, 6H), 7.19 (d, 2H, J=9.0 Hz), 120, 135, 152
7.73 (d, 2H, J=8.5 Hz)
65, 92, 120, 162, 192, 219, 251
120, 135, 152
N,N-dimethyl-p-toluidine
N-oxide
M3 (N-methyl-p-toluidine)
N-Methyl-p-toluidine
DMPT-parent
(N,N-dimethyl-p-toluidine)
N,N-Dimethyl-p-toluidine
20.9
20.9
25.2
N/A
N/A
N/A
51, 59, 65, 77, 91, 106, 120, 121
51, 59, 65, 77, 91, 106, 120, 121
51, 65, 77, 91, 105, 119,
134, 135
25.2
2.14 (s, 3H), 2.67 (s, 6H), 6.87 (d, 2H, J=8.5 Hz), 51, 65, 77, 91, 105, 119,
7.08 (d, 2H, J=8.3 Hz) 134, 135
In the mass spectra of M2, obtained by LC/MS, the molecu- consistent. The proton NMR spectra of the synthesized DMPT
lar ion peak at m/z 152 [M+H] is 16 amu larger than the parent N-oxide and M2 confirmed the identification of M2 as DMPT
peak of DMPT (m/z 135), indicating the metabolite may be N-oxide. The mass fragmentation pattern was also consistent
oxygenated. DMA, a DMPT analog, is metabolized to DMA with the findings of Seto and Guengerich (1993).
N-oxide. Therefore, the hypothesized structure of M2 was
Due to the difficulty of separating peaks with early HPLC
DMPT N-oxide. Formation of DMPT N-oxide was previously retention times, M1 was chemically modified to its methyl
reported by Seto and Guengerich (1993) and is likely mediated ester using trimethylsilyldiazomethane. Mass fragmentation
by the flavin-containing monooxygenaeses as reported for patterns obtained by LC/MS indicated the possible presence of
N,N-dimethylaniline and other N,N-dialkylarylamines (Sherratt a glycine conjugate. The proton NMR of derivatized M1
and Damani, 1989; Agosin and Ankley, 1987; Gorrod and showed two major signals (δ 7.51 and 7.93 ppm) in the aro-
Gooderham, 1981; Gold and Ziegler, 1973; Hamill and Cooper, matic region, which is consistent with DMPT. These signals
1984; and Grothusen et al., 1996).
integrated for two protons in each of two different environ-
The proton NMR spectra of M2 show signals for the 4 sets ments on the aromatic ring. This indicated that the ring was not
of protons in M2 and DMPT, indicating that the core structure more substituted than DMPT. The signal at δ 4.13 ppm also
of DMPT is present in M2. However, the chemical shifts for integrated for two protons which correlated to a methylene
the 4 signals in M2 are shifted downfield by δ 0.26 – 1.01 ppm moiety, possibly from glycine. Signals at δ 2.17 and 3.61 ppm
from DMPT depending on the distance from the proposed integrated for three protons each, one signal for the methyl pro-
N-oxide. For instance, DMPT displays a signal at δ 2.67 ppm tons of the methyl ester of the hippuric acid (derivatized M1)
for the methyl protons associated with the amine, and M2 dis- and the other signal for methyl protons of a possible N-acetyl
plays a signal at δ 3.68 ppm also for the methyl protons associ- group. Heteronuclear multiple bond correlation (HMBC) NMR
ated with the amine bearing the proposed oxygen. These studies indicated a three-bond correlation between a set of
results suggested that DMPT N-oxidation occurred to yield methylene protons and a carbonyl carbon, which is additional
M2. To confirm this observation, DMPT N-oxide was synthe- evidence that M1 is a glycine conjugate. HMBC two-bond cor-
sized. The analytical HPLC retention time of the synthesized relation studies indicated a set of methyl protons adjacent to a
DMPT N-oxide (10.2 min) was compared with the retention carbonyl carbon indicating the presence of an N-acetyl group,
time of metabolite M2 (10.4 min) and the retention times were which was confirmed by the LC/MS mass fragmentation

METABOLISM OF ORALLY ADMINISTERED DMPT IN RATS
785
FIG. 2. Mass spectra of M3 (A) and N-methyl-p-toluidine (B). The molecular ions are identified with arrows.
pattern of the derivatized M1 (m/z 120 and m/z 162) as shown showed a similar proton with NMR as metabolite M1. How-
in Figure 4. With this data at hand, the derivatized M1 was ten- ever, M1 displayed a mass fragment at m/z 180 (Figure 4),
tatively identified as p-acetaminohippuric acid methyl ester, which was absent in p-acetaminohippuric acid (Figure 4). The
which suggested that M1 is p-acetaminohippuric acid. To con- derivatized M1 and p-acetaminohippuric acid methyl ester
firm this, p-acetaminohippuric acid methyl ester was synthe- showed common fragmentation patterns by LC/MS (m/z 120,
sized. However, when the retention time of p-acetaminohippuric 162) but derivatized M1 displayed an additional mass fragment
acid methyl ester (12.1 min) was compared with the retention at m/z 194 (Figure 4), suggesting the possible presence of an
time of derivatized M1 (16.1 min) by analytical HPLC, the additional hydroxyl group. The mass fragmentation pattern
retention times were significantly different, indicating that M1 along with the proton NMR spectrum allowed localization of
was not p-acetaminohippuric acid. The synthesized compound the hydroxyl group as attached to the N-acetyl nitrogen. The

786
N.-C. KIM ET AL.
FIG. 3. Mass spectra of DMPT-parent (A) and N,N-dimethyl-p-toluidine (B). The molecular ions are identified with arrows.
N-oxidation in M1 was confirmed by synthesis. Derivatiza- methylate an aromatic hydroxyl group. When these two
tion of M1 and p-acetaminohippuric acid to their respective reagents were employed, the M1 derivatization products dis-
methyl esters using both diazomethane and (trimethylsi- played the same HPLC chromatogram retention times and
lyl)diazomethane would distinguish if the hydroxyl group was LC/MS fragmentation patterns, indicating that no aromatic
located on the aromatic ring or on the N-acetyl nitrogen. Diaz- hydroxyl group was present in M1. Therefore, the data indi-
omethane is a potent methylating agent and methylates an cate that the structure of M1 is p-(N-acetylhydroxyamino)hip-
aromatic hydroxyl group, whereas (trimethylsilyl)diaz- puric acid. A proposed metabolism scheme for DMPT is
omethane is a weaker methylating agent and may not readily shown in Figure 5.

METABOLISM OF ORALLY ADMINISTERED DMPT IN RATS
787
FIG. 4. Mass spectra of M1: (A) metabolite M1, (B) p-acetaminohippuric acid, (C) metabolite M1 methyl ester, and (D) p-acetaminohippuric acid methyl
ester. The molecular ions are identified with arrows.
In summary, 4 major radiolabeled peaks in [14C]DMPT- Spectral Data
treated rats were isolated and identified as DMPT-parent, N-
methyl-p-toluidine, DMPT N-oxide, and p-(N-acetylhydroxy-
lamino)hippuric acid. N-demethylation and N-oxidation are
known cytochrome P450-mediated metabolic pathways for
DMPT (Seto and Guengerich, 1993; MacDonald et al., 1989).
Based on the metabolism of DMA to phenylhydroxlamine,
which produces methemoglobinemia, the metabolite putatively
responsible for DMPT-induced methemoglobinemia is p-meth-
ylphenylhydroxylamine. p-Methylphenylhydroxylamine was
not identified in urine. However, p-(N-acetylhydroxyamino)
hippuric acid, which is the glycine conjugate of N-acetylated p-
methylphenylhydroxylamine was present. Therefore, it is
reasonable to speculate that p-methylphenylhydroxylamine is
formed from DMPT in vivo. Overall DMPT metabolism is
consistent with the metabolism of DMA.
NMR spectra were recorded in CDCl3 or CD3OD on a
Bruker Avance DRX 500 spectrophotometer (500 MHz) with
TMS as an internal standard. Chemical shifts are reported in
parts per million (Table 1). Retention times (TR) were
obtained by analytical HPLC on an Agilent Model 1100
HPLC system coupled with a β-RAM-Model 3 radioactivity
detector attached to a Phenomenex Luna C-18 column.
Retention times are recorded in minutes. Mass spectra for M1
and M2 were obtained by LC/MS on an Agilent 1100 Series
LC system with photodiode assay detection coupled with an
Applied Biosystems MDS Sciex API 4000 G-TRAP Triple
Quad MS/MS. Mass Spectra for M3 and DMPT were
obtained by GC/MS on an Agilent 6890N Network GC sys-
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ACKNOWLEDGMENTS
This work was supported by Contract Number N01-ES-
25483 from the NIEHS. We thank Dr. L.T. Burka at NIEHS
for his critical review of the manuscript. We thank Dr. Karen
Ann Smith in the Department of Chemistry, University of New
Mexico, for her assistance with the NMR experiments. We also
thank Mark Gurule, Briana Hedtke-Weber, and Krystal
Pacheco of Lovelace Respiratory Research Institute for techni-
cal assistance and Ms. Vicki Fisher for assistance with manu-
script preparation.
Seto, Y., and Guengerich, F. P. 1993. Partitioning between N-dealkylation and
N-oxidation in the oxidation of N,N-dialkylamines catalyzed by cyto-
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by isolated rat hepatocytes: identification of a novel N-conjugate. Xenobi-
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N,N-dimethylaniline and N,N-dimethyl-p-toluidine. Environ. Mol.
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