Chemical Reduction of Zoalene to ANOT for Use in Zoalene Residue Studies

Article abstract of DOI:10.1021/jf9802068

A novel chemical synthesis and purification scheme to produce ANOT (3-amino-5-nitro-o-toluamide) was developed to yield an analytical standard grade material for zoalene residue investigations. An original conversion of zoalene (3,5-dinitro-o-toluamide) t

Full text of DOI:10.1021/jf9802068

J. Agric. Food Chem. 1998, 46, 3623−3629
3623
Ch em ica l Red u ction of Zoa len e to ANOT for Use in Zoa len e Resid u e
Stu d ies
Gregory K. Webster*
Chemsyn Laboratories, 1701 Vine Street, Harrisonville, Missouri 64701
Richard J . Pastore and Kenneth A. Hawkins
Animal Health Division, Alpharma, Inc., 400 State Street, Chicago Heights, Illinois 60411
Arnold L. Hirsch
NSC Technologies, 601 East Kennsington, Mount Prospect, Illinois 60056-1300
A novel chemical synthesis and purification scheme to produce ANOT (3-amino-5-nitro-o-toluamide)
was developed to yield an analytical standard grade material for zoalene residue investigations.
An original conversion of zoalene (3,5-dinitro-o-toluamide) to ANOT was developed. Several
literature references were available for the partial reduction of dinitro aryl compounds, but none
detailed the conversion of zoalene to ANOT. A chemical synthesis was chosen over a biological
synthesis due to the latter’s complex reaction matrix, difficult recovery, and low batch yields. Using
general partial reduction of dinitro aryl schemes, it was found that 10% palladium on carbon coupled
to a cyclohexene reducing agent provided an optimum ANOT yield. Chromatographic techniques
were utilized to isolate ANOT from the product mixture and to purify it to a standard grade material
(>99% pure). The chemically synthesized ANOT product was characterized using colorimetric, TLC,
FTIR, NMR, and LC/MS techniques.
Keyw or d s: ANOT; zoalene; zoaline residue; chemical reduction
INTRODUCTION
zoalene under a nitrogen blanket using cyclohexene (a
reducing agent) in 3A ethanol with a reaction temper-
ature of 51 °C and a catalyst of 10% palladium on carbon
(Figure 1). Zoalene was dissolved in the reaction
mixture while cyclohexene was slowly added via a
peristaltic pump. The reaction progress was monitored
by the LC analysis of small aliquots taken over regular
intervals. When zoalene could no longer be detected in
the LC chromatogram of the reaction mixture, the
reaction was stopped. The product mixture was stable
at room temperature for several days as long as the
headspace was kept purged with nitrogen.
3-Amino-5-nitro-o-toluamide (ANOT) is the primary
metabolite of zoalene (3,5-dinitro-o-toluamide), a poultry
feed additive used as a coccidiostat (Hymas et al., 1960;
Runnels et al., 1958). A sufficient quantity of ANOT
was desired for use as an analytical standard. Because
a current source of ANOT could not be obtained, an
attempt was made to synthesize ANOT.
A biological synthesis of ANOT from zoalene in
chicken liver tissue (Smith et al., 1963) resulted prima-
rily in the formation of ANOT. The other isomer formed
was 5-amino-3-nitro-o-toluamide (5-ANOT). The bio-
logical synthesis of Smith et al. was a selective reduction
of zoalene to ANOT; however, only 30 mg of ANOT could
be produced per batch, and it would be difficult to scale
up for a higher yield. Chemical means of zoalene
reduction were not found to be selective for the synthesis
of ANOT; rather, the diamino and 5-amino isomers of
ANOT are more prevalent. Aromatic dinitro reduction
synthesis schemes were available from the literature,
but this search did not provide specific details as to the
synthesis of ANOT (Entwistle et al., 1977; J ohnstone
et al., 1985; Ayyangar et al., 1981, 1983).
The purification of the reaction product mixture to
ANOT was accomplished using multiple liquid chro-
matographic schemes: (1) the side reaction products
were removed using dry column chromatography; (2) the
3-amino and 5-amino isomers of the reaction product
were separated on a low-pressure LC system utilizing
an alumina column; and (3) the isolated 3-ANOT
product was purified using ion exchange chromatogra-
phy.
The presence of ANOT and the 5-amino isomer in the
reaction product was verified by chromatographic (TLC,
LC) and colorimetric investigations. The successful
synthesis of ANOT in the reaction product was con-
firmed using mass spectrometry, nuclear magnetic
resonance spectroscopy, and infrared spectroscopy. The
melting point range of the final purified product was
also taken and compared to the reference range.
The combination of the optimum reduction scheme
coupled to chromatographic techniques allowed for an
effective recovery of a standard grade of ANOT (Hart-
man and Silloway, 1955; Loev, 1970; Thiegs et al., 1961).
The synthesis scheme of choice consisted of reducing
* Author to whom correspondence should be addressed.
S0021-8561(98)00206-4 CCC: $15.00 © 1998 American Chemical Society
Published on Web 08/11/1998

3624 J. Agric. Food Chem., Vol. 46, No. 9, 1998
Webster et al.
filtered through a 0.2 µm PTFE membrane (Gelman Acrodisc,
25 mm diameter) and applied to the top of the column. When
the Celite became saturated, the mobile phase (chloroform/
ethyl acetate/methanol 5:5:1) was pumped onto the column at
8 mL/min and gradually slowed to 2 mL/min over a 4 h period.
The dry column tube was then laid on a glass plate and
examined under a 254 nm light.
The yellow band was removed from the column and ex-
tracted with 700 mL of methanol. The extract was filtered
through a 12.5 cm Bu¨chner funnel using Whatman No. 40
filter paper. Evaporation of the filtrate was performed with
a tared 2 L round-bottom flask at 50 °C. The dry residue was
diluted with methanol to produce a 3% (w/v) solution.
Separation of 3-Amino and 5-Amino ANOT Isomers Using
Low-Pressure Chromatography. Isolation of crude ANOT was
carried out with a 48 mm i.d. × 30 cm glass column (Kontes
Chromaflex) filled with 11 cm of dry alumina (Acros, neutral,
activated, 50-200 µm). The column was part of a low-pressure
liquid chromatography system that consisted of a Waters
M6000-A isocratic pump, a Gilson model 115 UV detector, and
a Gilson FC 203B fraction collector.
The 3% solution of crude ANOT in methanol was filtered
prior to application to the column. A Rheodyne type 50 valve
equipped with a 1.0 mL Teflon sample loop was used. The
column was washed with ∼850 mL of the dry column mobile
phase prior to use. The ANOT component eluted between 45
and 85 min. After the elution of ANOT, the mobile phase was
changed to methanol to elute the 5-ANOT isomer. This isomer
eluted between 100 and 150 min. The multiple ANOT frac-
tions collected were evaporated to a dry residue under a stream
of compressed air while standing on a heating pad under a
hot lamp. The collected residues at this point typically
exhibited a relative ANOT peak area of >98% and were
combined with other residues of equal or greater purity for
further processing.
Final Purification of ANOT Using Ion Exchange Chroma-
tography. On the basis of a purification scheme found in the
literature (Thiegs et al., 1961), a 113.5 mg sample of ANOT
isolated from the low-pressure chromatography procedure was
dissolved in 4.00 mL of 80% aqueous ethanol (v/v) and
quantitatively transferred to a 10 mm i.d. × 30.5 cm length
chromatographic column containing a 6 cm length of wet
Dowex 50W-X8 H+ resin. The Dowex resin bed was covered
at the top by a 1 cm layer of purified sand. Slight air pressure
was needed to elute the components at this desired flow rate
(1 drop/5 s). The remainder of the 30.5 cm column was
carefully filled with 80% ethanol without disturbing the top
of the resin bed.
Fraction I (11 mL) was very light yellow in color and did
not contain ANOT. Fraction II (49 mL) was a considerably
deeper yellow than either fraction I or the very light yellow
fraction III (141 mL). Fractions II and III contained ANOT
and were combined. LC analysis of samples from continued
elution with 80% ethanol confirmed the absence of ANOT. The
recovered ANOT was found to be 99% pure, based on peak
area.
F igu r e 1. Reduction of zoalene to ANOT.
EXPERIMENTAL PROCEDURES
Syn th esis of ANOT Mixtu r e. ANOT was synthesized
from the chemical reduction of zoalene (Alpharma, Chicago
Heights, IL). The starting material (1.41 g, 6.25 mM) was
weighed into a 500 mL four-neck flask equipped with an
efficient overhead stirrer (furnished with a water-cooled stirrer
gland), thermometer, and Hopkins condenser. After the
apparatus had been thoroughly purged with nitrogen, 190 mL
of anhydrous 3A ethanol was added to the flask. The mixture
was stirred at room temperature until total dissolution oc-
curred. A small aliquot was removed to monitor the progress
of the reaction. After 3.33 g of 10% Pd on carbon (Aldrich)
was added to the flask, the apparatus was again purged with
nitrogen.
The reaction was sampled using a 50 mL pear-shaped
separatory funnel that was connected to a peristaltic pump
by means of C-Flex tubing. Initially, 50.0 mL of a 3.88% (v/v)
cyclohexene solution in 3A ethanol was pipetted into the
separatory funnel. The temperature of this apparatus was
maintained at 51 ( 1 °C.
The reduction of zoalene started with the addition of 50.0
mL of the cyclohexene solution at a nominal rate of 0.05 mL/
min. This initial cyclohexene addition is time zero for the
reaction. The progress of the reaction was monitored at
designated time intervals by removing six drops of sample.
This aliquot was diluted with 500 µL of LC mobile phase and
filtered prior to injection with a 0.2 µL Teflon syringe filter.
The reaction appeared to follow first-order kinetics; samples
after 13 h show the absence of zoalene. To determine the
actual rate of cyclohexene addition, the addition of the cyclo-
hexene solution was continued until all of the 50.0 mL of
solution was added. This addition was completed in 13.5 h.
Therefore, the actual rate of cyclohexene solution addition was
0.062 mL/min (equivalent to the addition of 2.4 µL of cyclo-
hexene/min).
At the point of completion, the reaction mixture was filtered
through a fine glass fritted funnel and the 10% Pd on carbon
was washed thoroughly with anhydrous 3A ethanol. The
reaction mixture was evaporated to a moist residue using a
Rotovapor under reduced pressure and a temperature setting
of 50 °C. The reaction mixture was quantitatively transferred
to a recovery flask using methanol. The residue was further
evaporated to dryness with the aid of a Rotovapor under the
final reduced pressure of 3 mmHg and at 50 °C to give a crude
residue weighing 1.37g.
The ANOT-containing fractions were evaporated under the
house vacuum (19 in Hg gauge pressure) using a temperature
of 50 °C. The resulting residue was recrystallized from 1.9
mL of 190 proof ethanol. The wet crystals were washed with
cold 190 proof ethanol. The recovered ANOT crystals were
dried using an Abderholden drying pistol at the boiling point
of methyl ethyl ketone and at a reduced pressure of 3.5 mmHg.
The crystals were dried for 1 h at 3.5 mmHg. Using this
procedure, 37.8 mg of chromatographically pure ANOT was
recovered.
LC An a lysis of ANOT. The progress of the reaction was
monitored using an LC method for the separation of ANOT,
5-ANOT, and zoalene (Morawski and Kyle, 1984; Parks, 1985).
The system consisted of a Waters model 6000A isocratic pump
and model 490E UV detector, a Wescan model 728 autoinjector
configured with a 20 µL fixed volume injection loop, and a
Hewlett-Packard Chromjet integrator. Using a Kromasil C8,
5 µm column (Phenomenex, Torrance, CA), the separation was
optimized using a 15% (v/v) acetonitrile in water mobile phase
Isola tion of ANOT. Isolation of ANOT from the crude
synthesis product was a three-step process: (1) removal of side
reaction products; (2) isolation of crude ANOT; and (3) final
purification of crude ANOT.
Removal of Side Reaction Products Using Dry Column
Chromatography. Dry column tubing (32 mm diameter,
Kontes Chromaflex) was sealed at one end by stapling and
tightly packing with dry untreated silica gel (J . T. Baker 40
µm flash chromatography packing) to a height of ∼25 in. To
the top of the column was applied 1 in. of diatomaceous earth
(Fisher Scientific Celite 545) and 1 in. of chromatography sand.
A 1.8 g mass of crude dry product in 10 mL of methanol was

Chemical Reduction of Zoalene to ANOT
J. Agric. Food Chem., Vol. 46, No. 9, 1998 3625
dure used to monitor the reaction did not use a buffer in the
mobile phase, no adjustment was necessary for the LC/MS
system. The system used was a Waters liquid chromatography
system consisting of a model 745 data module, a model 440
UV detector, a model 510 LC pump gradient controller, and a
Vestec thermospray interface coupled to a Finnagan TSQ 70
mass spectrometer. The mass spectrometer was tuned to the
positive ion (EI) and negative ion (CI) modes with FC43.
Thermospray performance was checked using the buffer ions
and a 1 mg/mL injection (10 µL) of a caffeine standard. A 1.18
mg sample from the initial reaction product residue was placed
into a 10 mL volumetric flask. Acetonitrile (1.5 mL) was
added, and the solution was brought to volume with 18 MΩ
water. Dissolution was effected by heating on a steam table.
Again, a Kromasil C8, 5 µm, 4.6 mm × 15 cm column
(Phenomenex) was used for the separation. The injection
volume was 100 µL, and the flow rate was 1.0 mL/min. The
full scale of the absorbance was set to 0.5, and a 254 nm
wavelength was used. The mobile phase was mixed using the
gradient controller in an isocratic mode.
Color im etr ic An a lysis of P r ep a r a tive LC F r a ction s.
Colormetric analysis of preparative chromatography ANOT
fractions was performed using a Beckman DU-64 spectropho-
tometer according to the method of Smith et al. (1963). The
analysis solution was prepared by adding 50 µL of a 9 µg/µL
ANOT fraction solution in methanol to 2.5 mL of 0.25 N HCl
and 125 µL of 0.1% NaNO2 solution. Upon standing for 5 min,
125 µL of a 0.5% ammonium sulfamate solution was added.
After an additional 3 min, 125 µL of a 0.1% N-1-(naphthyl)-
ethylenediamine dihydrochloride solution was added. A col-
ored solution developed immediately, which was scanned from
450 to 650 nm.
In fr a r ed Sp ectr oscop ic An a lysis. FTIR scans of the dry
ANOT preparative chromatography fractions were performed
using a Matteson Galaxy Series FTIR 5000 spectrometer. A
diluted mixture of each ANOT dry fraction in anhydrous KBr
was prepared and ground to a fine powder in a small marble
mortar (∼1% w/w). Each dilute mixture was scanned using
the technique of diffuse reflectance. The FTIR was used in
transmittance mode with a resolution of 4 cm-1 and a scan
range of 400 to 4000 cm-1. The Fourier transform was set to
operate on 64 scans of each ANOT fraction.
Nu clea r Ma gn etic Reson a n ce Sp ectr oscop ic An a lysis.
For NMR analysis, each sample (30.6 mg of ANOT, 30.5 mg
of 5-ANOT) was dissolved in 750 µL of DMSO-d6 (Cambridge
Labs, DLM-10, CAS Registry No. 2206-27-11). After dissolu-
tion, 2.3 µL of TMS (Cambridge Labs, CAS Registry No. 75-
76-3) was added to each solution. NMR spectra of the final
solutions in the 13C and proton NMR mode were recorded on
a GE QE Plus 300 NMR. (CAS Registry No. were supplied
by the author.)
F igu r e 2. LC profile of reaction mixture: (A) ANOT; (B)
5-ANOT; (C) zoalene.
flowing at 1 mL/min. The column dimensions were 4.6 × 150
mm. A wavelength of 254 nm was used for detection. ANOT
was found to elute with a 5 min retention time, whereas the
5-ANOT isomer had a retention time of 7 min. Zoalene eluted
in 19 min. The peaks were symmetrical and base-line re-
solved. (Figure 2).
P r ep a r a tive Sca le LC An a lysis of ANOT. The LC
separation was scaled up to collect the separation peaks for
identity confirmation. The preparative scale system consisted
of two Shimadzu LC-8A pumps configured for binary gradient
controlled by a Shimadzu SCL-6B system controller and a 9A
autoinjector. A Shimadzu 6A UV detector was configured with
a preparative flow cell and used at 254 nm. The profile of
Figure 2 retained its integrity using a 25 × 250 mm Kromasil
C8, 5 µm column (Phenomenex) flowing at 18 mL/min using
the same 15% acetonitrile mobile phase used with the smaller
sized column. Using a 1 mL injection volume, the profile peaks
were collected using a Gilson model 201 fraction collector. The
ANOT fractions collected were evaporated off using a vacuum
rotavap. This system allowed the collection of sufficient
quantities of ANOT and 5-ANOT for TLC and colorimetric
investigations. The system proved to be too inefficient for large
scale fraction collections; thus, a low-pressure LC scheme was
developed.
Meltin g P oin t Deter m in a tion . Melting ranges of the
preparative LC ANOT fractions were obtained using an
Electrothermal model 9200 melting point apparatus.
TLC An a lysis of P r ep a r a tive Ch r om a togr a p h y ANOT
F r a ction s. TLC analyses were performed using the procedure
from Owens (1985) on fractions obtained from the preparative
LC system. Each fraction was evaporated to a dry residue
and dissolved in sufficient methanol to make a 0.3 mg/mL
solution. The fraction solutions were applied to Whatman
linear HPTLC 10 × 10 cm silica gel (5 µm) plates using 10 µL
microcaps (Drummond). The plate was developed in a 20 ×
20 cm chromatographic developing chamber using a developing
solvent of chloroform/ethyl acetate/methanol in the proportions
5:5:1 (v/v), respectively. The fully developed plate was pho-
tographed under ultraviolet light using a wavelength of 366
nm. The plate was exposed for 5 s to nitrous acid vapors
generated by the addition of sodium nitrite to 8% phosphoric
acid. Finally, the plate was sprayed with a Bratton-Marshall
solution (0.4% naphthylethylenediamine dihydrochloride in
methanol) for spot detection and photographed.
RESULTS AND DISCUSSION
Syn th esis a n d Isola tion of ANOT. The Zinnin
reaction (a reduction using sodium sulfide), Raney
nickel, and palladium on carbon catalysts have all been
successfully used in the partial reduction of dinitro
aromatic compounds. However, no references were
found with specific details for the synthesis of ANOT.
Other potential routes of synthesis of ANOT were
considered in our laboratory. The high-pressure homo-
geneous catalyzed reduction of zoalene with tris(tri-
phenylphosphene)ruthenium chloride [RuCl2(PPh3)3]
looked promising due to its successes with other aro-
matic compounds (Knifton, 1975a,b) but was not tried
in our laboratory due to instrumental constraints.
Nitration of 3-amino-o-toluamide was investigated, but
preliminary experiments yielded nitration in all posi-
tions and this scheme was abandoned. Dow Chemical,
Liqu id Ch r om a togr a p h y/Ma ss Sp ectr om etr ic An a ly-
sis. For molecular weight confirmation, the reaction product
mixture was analyzed using LC/MS. Because the LC proce-

3626 J. Agric. Food Chem., Vol. 46, No. 9, 1998
Webster et al.
Ta ble 1. Rever se P h a se LC Reten tion Tim es of ANOT
a n d Rela ted Com p on en ts
Ta ble 2. TLC Da ta of ANOT F r a ction s fr om P r ep a r a tive
LC
Parks and Doerr
reten,a min
rel reten
this work
preparative
LC fraction
Parks and Doerr:
this work:
Rf value
Rf value
component
reten,b min
rel reten
ANOT
0.56
0.45
0.74
0.53
0.44
0.73
ANOT
5-ANOT
zoalene
11.9
16.1
13.2
0.74
1.0
n/a
4.9
6.8
18.4
0.72
1.0
NA
5-ANOT
sulfadimethoxidea
a
Sulfadimethoxide used as a control.
a
ANOT and 5-ANOT separated using acetonitrile/water (10 +
b
90). Zoalene separated using methenol/water (30% + 70%). All
three components separated using acetonitrile/water (15% + 85%).
Dry column chromatography was applied to this
isolation strategy after it was discovered that fluores-
cent impurities were still in samples purified solely
using the low-pressure chromatographic system. This
technique was very simple and a highly effective way
to remove side reaction products from the ANOT
isomers. The chromatography was performed in a
flexible nylon tube using silica gel as the stationary
phase packing. Elution continued until the mobile
phase had reached the bottom of the 25 in. column. Side
reaction products were found to adhere to the top of the
column, with the ANOT compounds eluting further
down. At least two different side reaction products were
retained at the top of the column: a dark brown band
(visible in white light) at the apex and a strongly
fluorescent green band immediately following. The
column was cut at a region between the fluorescent
green band and a further eluting yellow band which
contained the ANOT isomers. This fluorescence was
used to determine the region of separation between the
side reaction products and the ANOT isomers. Whereas
there was no appreciable separation between the ANOT
isomers, there was a satisfactory separation between
the side reaction products and the ANOT isomers. The
portion containing ANOT isomers was extracted with
methanol, filtered on a Bu¨chner funnel, and washed
with additional methanol. The filtrate (containing
isolated ANOT isomers) was transferred to a round-
bottom flask and evaporated to a dry residue. This
residue was diluted with methanol to obtain a 3%
solution.
previously the sole origin of ANOT compounds, used a
biological synthesis. As ANOT is no longer available
from this source and was needed as an analytical
standard for metabolism studies, its in-house synthesis
was required. The synthesis of ANOT from zoalene is
a hydrogenation reaction, and it seemed logical that the
use of the Zinnin reaction using either of the two
mentioned catalysts could be satisfactorily used for the
production of ANOT.
The Zinnin reaction was first attempted for the
production of ANOT. This reaction, when applied to the
zoalene, was found to be vigorous and resulted primarily
in the formation of the 5-ANOT isomer (∼90% yield).
The formation of ANOT with the Zinnin reaction ap-
peared to be sterically hindered by the methyl group
adjacent to the amide.
Raney nickel catalytic hydrogenation was then evalu-
ated for the synthesis of ANOT. Because of the signifi-
cant number of side reaction products that resulted from
the use of Raney nickel, the use of this scheme was
discontinued. The yield of ANOT was found to be
unacceptably low. Finally, a synthesis scheme found
in the literature to reduce aromatic amines utilizing
10% palladium on carbon with cyclohexene as a reduc-
ing agent was investigated. When this reaction scheme
was used, the number of side reaction products was
greatly reduced. In addition, the yield of ANOT had
increased to ∼44% ANOT on the basis of isomer LC
peak areas. This reaction scheme still produced un-
characterized side products. A method of isolating
ANOT from the product mixture was needed to use the
10% palladium on carbon reaction procedure.
The procedure developed using 10% palladium on
carbon catalysis was optimized to the conditions re-
ported. Optimization experiments showed that use of
cyclohexene in the reaction mixture at the start did not
produce a significant amount of ANOT. The principal
product produced was 3,5-diamino-o-toluamide. A slow
addition of cyclohexene produced ANOT and 5-ANOT,
but in an unfavorable ratio for our purposes. The
synthesis of ANOT was much more favorable using a
cyclohexene solution in 3A ethanol. For this scheme, a
3.88% (v/v) solution of cyclohexene was used. Another
important factor for increasing the ratio of ANOT to
5-ANOT may have been the activity of the catalyst.
Different batches of 10% Pd on carbon can produce
varying isomer ratios based on the activity of the
catalyst.
Low-pressure preparative chromatography allowed
for the separation and collection of ANOT and 5-ANOT.
Though the 3-amino and 5-amino isomers of ANOT
could be resolved and collected on the preparative scale
LC system, the system proved to be too inefficient for
collecting large amounts of ANOT. Transferring the
TLC procedure that had already proved to be effective
in resolving the ANOT isomers to a low-pressure column
chromatography system allowed the use of more reac-
tion product on the column while maintaining the
effective resolution of isomers.
ANOT was the first major band to elute as the
5-ANOT isomer was strongly retained by the stationary
phase. A change of mobile phase was required to
effectively elute the 5-ANOT isomer. Each injection of
a 3% solution prepared at the end of the dry column
step onto a 480 mm × 11 cm alumina column yielded
∼12 mg of pure product (∼98% pure).
Final purification of the synthesized ANOT was
performed using ion exchange chromatography. This
scheme was incorporated because it was used not only
to purify the original ANOT standards (Thiegs et al.,
1961) but also to up the baseline of the LC chromato-
gram of the recovered ANOT fractions. This final step
yielded an analytical grade material suitable for me-
tabolism and residue studies.
The LC method made it possible to monitor the
progress of the ANOT synthesis and to verify the
relative amount of ANOT in the product mixture.
Retention times of the ANOT fractions obtained from
the LC analysis of the ANOT reaction mixture were
found to be consistent with the literature values (Table
1).
Ch a r a cter iza tion of th e Ch em ica lly Syn th esized
ANOT. The Rf values from the TLC analysis of frac-

Chemical Reduction of Zoalene to ANOT
J. Agric. Food Chem., Vol. 46, No. 9, 1998 3627
F igu r e 3. Colorimetric analysis of ANOT fractions.
tions collected from the preparative scale LC system
were calculated and compared to the Dow Chemical
reference values (Table 2). The relative Rf values were
found to match the literature values. The identity of
the fractions as ANOT and 5-ANOT was supported by
this analysis.
ing to Smith et al. (1963). Slight shifting of the
absorbance maxima from the literature values was
noted (Table 3) and attributed to normal instrumental
error. The colorimetric spectra correlated for the posi-
tive identification of the preparative LC fractions as
ANOT and 5-ANOT.
Colorimetric analysis of preparative LC fractions of
the prepared ANOT fraction solutions showed maxi-
mum absorbencies that were consistent with ANOT
absorbance values from the literature (Figure 3) accord-
Liquid chromatography/mass spectrometry (LC/MS)
analysis of the synthesis reaction product yielded results
that are consistent with the molecular weight of ANOT.
Positive ion thermospray mass spectra for the ANOT

3628 J. Agric. Food Chem., Vol. 46, No. 9, 1998
Webster et al.
F igu r e 4. Mass spectrum of ANOT from the LC/MS analysis of the reaction product.
Ta ble 3. Color im etr ic Absor ba n ce Ma xim a of ANOT
F r a ction s fr om P r ep a r a tive LC
Ta ble 4. NMR Resp on se for ANOT a n d 5-ANOT
ANOT
5-ANOT
Smith et al:
absorbance max, nm
this work:
absorbance max, nm
position/
proton
calcd
(ppm)
found
(ppm)
position/
proton
calcd
(ppm)
found
(ppm)
fraction
ANOT
5-ANOT
525
540
520
535
13C NMR
C3
C5
148.4
146.3
148.5
146.0
C3
C5
150.0
169.7
150.8
170.0
1H NMR
-CH3
-NH2
-CONH2
C4
2.2
6.6
7.6
7.1
7.9
2.2
5.8
7.6, 7.9
7.3
7.5
2.6
5.9
7.6
7.0
7.4
2.2
5.9
7.6. 7.9
6.8
7.0
C6
The 13C NMR spectra for the C3 and C5 carbon agree
fairly well with the calculated values for the 3- and
5-ANOT isomers found in Table 4. The proton NMR
spectra for the two isomers are consistent with their
respective structures. Chemical shifts for the protons
(Table 4) other than those on the aromatic ring are very
similar and agree closely with the calculated spectra for
the two compounds. The two amido hydrogens show
different chemical shifts (0.35 ppm shift for ANOT and
0.33 ppm shift for 5-ANOT). Integration of the proton
signals for both compounds is consistent with their
structures. The signals from protons C4 and C6 on the
aromatic ring are shifted downfield (7.32 and 7.51 ppm,
respectively) in the ANOT because of their proximity
to the 5-nitro group compared to that in the 5-ANOT.
This is expected due to the larger contribution of
shielding from the nitro group at this position. In the
5-ANOT structure, the C4 and C6 protons resonate
downfield at 6.81 and 7.05 ppm, respectively, because
the shielding effect is not as large. As a final confirma-
tion of the identity and purity of the synthesized ANOT,
the melting point of the recovered product was deter-
mined. The light yellow crystals melted quickly at 198
°C, well within a 1 °C range. The melting point reported
in the literature is within the range of 198.5-199.5 °C
(Thiegs, 1994; The Merck Index, 1996).
F igu r e 5. FTIR analysis of isolated ANOT.
peak was consistent with the structure of ANOT (Fig-
ure 4). The analysis produced the characteristic M H+
+
(196 amu) and M NH4 (213 amu) ions expected for
ANOT. The negative ion thermospray analysis pro-
duced a single ion representing M CH3COO- (254 amu),
which is also consistent with the ANOT structure.
The FTIR spectra of the preparative LC fraction
produced the characteristic IR stretches associated with
the functional groups of ANOT (Figure 5) according to
Smith et al. (1963). The figure illustrates the important
response of the ANOT functional groups (Shriner et al.,
1980; Silverstein et al., 1991): the NsH stretch of the
primary amine coupled doublet (asymmetric 3373 cm-1
;
symmetric 3290 cm-1), the overlap CdO stretch of the
amide I band (1695-1640 cm-1), the CsN stretch of a
primary aromatic amine (1425 cm-1, 1340-1250 cm-1),
the asymmetric ArNO2 (NdO)2 stretch (1520 cm-1), the
symmetric N(dO)2 stretch (1347 cm-1), and the aro-
matic CsN stretch for ArNO2 (850 cm-1). The IR
spectrum confirms the expected functional groups of
ANOT are present.
Con clu sion . Our results demonstrate that the cy-
clohexene reduction of zoalene using 10% palladium on
carbon coupled with chromatographic isolation methods
is a reliable and efficient means of ANOT synthesis. The
synthesis is a simple one-step process, although an
extensive cleanup was necessary to isolate the ANOT.
The ANOT produced was isolated from the synthesis
products using three chromatographic techniques: dry

Chemical Reduction of Zoalene to ANOT
J. Agric. Food Chem., Vol. 46, No. 9, 1998 3629
Knifton, J . F. Homogeneous catalyzed reduction of nitro
compounds. J . Org. Chem. 1975a , 40, 519.
column, low-pressure liquid, and ion chromatography.
The dry column chromatographic technique isolated the
ANOT isomers from the reaction matrix. Because the
dry column technique did not resolve ANOT from its
5-ANOT isomer, a low-pressure column chromatography
scheme developed from a TLC method effectively iso-
lated ANOT from the 5-ANOT isomer. ANOT was
further purified to an analytical grade material of >99%
purity after an ion exchange chromatographic cleanup
procedure. The synthesis and purification methodology
presented was successful in producing an analytical
grade standard of ANOT.
Knifton, J . F. Selective and sequential hydrogenation of
nitroaromatics catalyzed by dichlorotris(triphenylphosphine)-
ruthenium(II). Tetrahedron Lett. 1975b, 2163.
Loev, B.; Goodman, M. M. Dry column chromatography. Prog.
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Morawski, J .; Kyle, G. Liquid chromatographic determination
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Parks, O. W. Screening tests for sulfa drugs and/or dinitroben-
zamide coccidiostats and their monoamino metabolites in
chicken livers. J . Assoc. Off. Anal. Chem. 1985, 68 (1), 20.
Parks, O. W. Depletion of the monoamino metabolites of
zoalene during frozen storage. J . AOAC Int. 1993, 76 (3),
698.
ACKNOWLEDGMENT
Parks, O. W.; Doerr, R. C. Liquid chromatographic determi-
nation of zoalene and its metabolites in chicken tissue with
electrochemical detection. J . Assoc. Off. Anal. Chem. 1986,
69 (1), 70.
Roth-Maier, D. A.; Kirchgessner, M. Retention of major
elements (calcium, phosphorous, magnesium, sodium, po-
tassium) and trace elements (iron, copper, manganese, zinc)
in broilers by adding coccidiostats. Zentralbl. Veteri-
naermed., Reihe A 1972, 19 (2), 111.
Runnels, T. D.; D’Armi, F. A.; Greene, L. M.; Waller, E. F.;
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and feed efficiency in broilers. Poultry Nutrition Report 5;
University of Delaware: Newark, DE, 1958.
We thank Thomas Miller of Cognis, Inc., for his LC/
MS analysis and data interpretations; Kevin Newman
of Centeon for his assistance with scanning figures for
computer storage and management; Steven Greenwald
of Chemsyn Laboratories for his review of the synthesis
work performed; and Run Wang and Steve Schroeder
of Monsanto, Nutrition and Consumer Sector, Analytical
and Process Chemistry, for their assistance in obtaining
and interpreting the NMR spectra.
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Received for review February 17, 1998. Revised manuscript
received J une 25, 1998. Accepted J uly 2, 1998.
J F9802068