Detection of p-Nitroaniline Released from Degradation of 4,4′-Dinitrocarbanilide in Chicken Breast during Thermal Processing

Article abstract of DOI:10.1021/acs.jafc.9b02259

The diphenylurea 4,4′-dinitrocarbanilide (DNC) is the residue of concern left in edible tissues of broilers fed diets containing the anticoccidial nicarbazin. When chicken meat is submitted to thermal processing, p-nitroaniline (p-NA) is expected from DNC degradation. This work aimed at evaluating whether thermal processing of DNC-containing chicken meat induces p-NA appearance. First, a hydrolysis assay was performed in aqueous solutions at 100 °C in different pH, confirming that DNC cleavage yields p-NA. Then a novel LC-MS/MS method was used to detect traces of this aromatic amine in DNC-containing chicken breast fillets subjected to cooking methods. Our evidence showed p-NA occurrence in such chicken meat samples, which corroborated results from hydrolysis assay. The p-NA appearance in fillets was rather discrete during boiling treatment, but its concentration became pronounced over time for grilling, frying, and roasting, achieving respectively 326.3, 640.0, and 456.9 μg/kg. As far as we are concerned, no other research identified degradation products from DNC residue in heat-processed chicken fillets. Therefore, this study leads to additional approaches to assess impacts on food safety.

Full text of DOI:10.1021/acs.jafc.9b02259

Subscriber access provided by UNIV OF SOUTHERN INDIANA
Food Safety and Toxicology
Detection of p-nitroaniline released from degradation of 4,4´-
dinitrocarbanilide in chicken breast during thermal processing
Danniele Miranda Bacila, Anildo Cunha Jr., Vanessa Gressler, Gerson Neudí Scheuermann,
Arlei Coldebella, Luizinho Caron, Luciana Igarashi Mafra, and Vivian Feddern
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b02259 • Publication Date (Web): 19 Jul 2019
Downloaded from pubs.acs.org on July 22, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the
course of their duties.

Page 1 of 26
Journal of Agricultural and Food Chemistry
Detection of p-nitroaniline released from degradation of 4,4´-
dinitrocarbanilide in chicken breast during thermal processing
Danniele Miranda Bacila,† Anildo Cunha Jr.,‡ Vanessa Gressler,‡
Gerson Neudí Scheuermann,‡ Arlei Coldebella,‡ Luizinho Caron,‡
Luciana Igarashi-Mafra,† and Vivian Feddern*,‡
*Corresponding Author
E-mail: vivian.feddern@embrapa.br. Tel.: 55-49-3441-0400. Fax: 55-49-3441-
0497.
†Departamento de Engenharia Química, Pós-graduação em Engenharia de Alimentos, Universidade Federal do Paraná,
Curitiba, Paraná 80060-000, Brazil
‡Núcleo Temático de Produção de Aves, Embrapa Suínos e Aves, Concórdia, Santa Catarina 89715-899, Brazil
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 2 of 26
2
1
ABSTRACT
2
3
The diphenylurea 4,4´-dinitrocarbanilide (DNC) is the residue of concern left in
edible tissues of broilers fed diets containing the anticoccidial nicarbazin. When
chicken meat is submitted to thermal processing, p-nitroaniline (p-NA) is
expected from DNC degradation. Then, this work aimed at evaluating whether
thermal processing of DNC-containing chicken meat induces p-NA appearance.
First, a hydrolysis assay was performed in aqueous solutions at 100 °C in
different pH, confirming that DNC cleavage yields p-NA. Then, a novel LC-MS/MS
method was used to detect traces of this aromatic amine in DNC-containing
chicken breast fillets subjected to cooking methods. Our evidences showed p-NA
occurrence in such chicken meat samples, which corroborated results from
hydrolysis assay. The p-NA appearance in fillets was rather discrete during
boiling treatment, but its concentration became pronounced over time for grilling,
frying and roasting, achieving respectively 326.3, 640.0 and 456.9 µg/kg. As far
as we are concerned, no other research identified degradation products from
DNC residue in heat-processed chicken fillets. Therefore, this study leads to
further approaches to assess impacts on food safety.
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Keywords: nicarbazin, poultry, chicken fillet, DNC, p-NA, heat treatment,
cooking method, LC-MS/MS
ACS Paragon Plus Environment

Page 3 of 26
Journal of Agricultural and Food Chemistry
3
21
INTRODUCTION
22
23
24
25
Coccidiosis is a common disease in poultry farming caused by protozoa from the
Eimeria genus. The control of this pathogen is achieved by regular use of in-feed
anticoccidials. However, concerns regarding residue deposition of these
additives in chicken meat are often raised.1
26
27
28
29
30
31
Nicarbazin (NCZ) is one of the most commonly used anticoccidials in
poultry feed.2 NCZ comprises an equimolar complex of 4,4´-dinitrocarbanilide
(DNC) and 2-hydroxy-4,6-dimethyl-pyrimidine (HDP).3 When this additive is
administered to broilers, the unchanged DNC accumulates in muscle tissues and
organs4,5, therefore it represents the NCZ marker residue analyzed in monitoring
programs.
32
33
34
35
36
37
38
Although surveillance plans identified non-compliant samples for NCZ
residue (> 200 µg/kg)2,6, studies have shown the disappearance of incurred DNC
in chicken fillets along thermal processing.7,8 However, details behind parent
DNC decay in heat-processed chicken meat remain unclear, especially with
respect to the proper identification of the degradation products. For instance,
while chicken meat is submitted to heat treatment, p-nitroaniline (p-NA) can be
expected from DNC breakdown.7,8
39
40
41
42
43
The p-NA is claimed to cause cancer9,10 and methemoglobinemia.11–13 Its
occurrence in cooked chicken tissues is a matter of concern. Therefore, as a
sequence of the research underway in our laboratory, we focused on revisiting
the DNC-containing cooked chicken samples7 to detect, by liquid
chromatography tandem mass spectrometry (LC-MS/MS), possible p-NA traces
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 4 of 26
4
44
45
released during thermal processing. As a secondary approach, we verified the p-
NA formation from the DNC cleavage in aqueous solution.
46
47
48
EXPERIMENTAL SECTION
Hydrolysis Assay
49
50
51
Reagents. 4,4´-dinitrocarbanilide (97%) and p-nitroaniline (> 99%) were acquired
from Sigma-Aldrich Co. (St Louis, MO, USA). All other reagents were of analytical
grade.
52
53
54
55
56
Solutions. Stock solutions of DNC (600 µmol/L) and p-NA (725 µmol/L) were
prepared in dimethylformamide (DMF). The following buffer solutions were
prepared in ultrapure water: 50 mmol/L phthalate pH 2, 50 mmol/L phthalate pH
4, 50 mmol/L phosphate pH 6, 50 mmol/L phosphate pH 8, and 50 mmol/L borate
pH 10. All buffers contained 1 mol/L KCl.
57
58
59
60
61
62
63
64
65
General procedure. DNC hydrolysis was performed at different pH (2, 4, 6, 8 and
10) at 100 °C during pre-established times (0, 2, 4, 6, 8 and 10 h). For each pH
value, eighteen screw-cap 15 mL test tubes containing a boiling bead were used.
Each tube contained 50 µL of the DNC solution at 600 µmol/L, 250 µL of DMF
and 2.7 mL of the respective buffer solution (initial concentration of DNC in assay
was 10 µmol/L). After vortexing the reaction mixture, the tubes were placed in a
sand-bath, and subsequently heated in an oven at 100 °C. Then, at each set time,
three test tubes were withdrawn and immediately transferred to an ice-water bath
(5–10 °C) for quenching the reaction.
ACS Paragon Plus Environment

Page 5 of 26
Journal of Agricultural and Food Chemistry
5
66
Determination of p-NA. The amount of the aromatic amine released from DNC
hydrolysis was determined on the basis of the Bratton-Marshall diazotization
according to a procedure adapted from previous studies.14,15 Thus, after cooling
the reaction mixture, the following reagents were successively added into the
tubes: 500 µL of 2 mol/L HCl, 50 µL of 2% (w/v) NaNO2 (aq.), 50 µL of 10% (w/v)
sulfamic acid (aq.), and 200 µL of 0.1% (w/v) N-(naphthyl)-ethylene diamine
dihydrochloride monomethanolate (aq.). The volume was adjusted to 5 mL, and
the absorbance was measured at 545 nm on a Varian Cary 50 UV-VIS
spectrophotometer. Based on this same procedure, p-NA calibration curves
within the range of 3–40 µmol/L were prepared in each buffer for quantification of
the amine (R2 > 0.99).
67
68
69
70
71
72
73
74
75
76
77
78
Degradation rate constant. The pseudo first-order rate constants (k1’) for DNC
hydrolytic degradation were detailed in Supporting Information file.
79
Chicken Breast Samples
80
81
82
83
This study was a complementary part of our recent research involving the fate of
NCZ residues in chicken meat. All details upon broiler production, breast
sampling, thermal processing, general cooking procedures, and sample
preparation were previously described.7
84
85
86
87
88
89
DNC-containing chicken breast fillets (Thermal processing experiment). From
that same set of chicken breast samples earlier prepared, a total of 180 portions
from experiment 1 (see subsection Thermal Processing in Bacila et al.7) were
selected for p-NA analysis. Briefly, at that particular experiment, thermal
processing of the chicken breast fillets (portions with 50–60 g) was accomplished
by conventional cooking methods over set times, as follows: boiling at 5, 10, 15,
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 6 of 26
6
90
91
92
93
94
20 and 25 min; grilling at 15, 30, 45, 60 and 75 min; frying at 5, 10, 15, 20 and 25
min; and roasting at 15, 30, 45, 60 and 75 min. A total of six replicates (i.e., 6
breast fillet portions) were prepared for each set time of a cooking method. DNC-
containing raw breast fillets (6 portions per cooking method) represented the
initial condition (zero-time) of thermal processing experiment.
95
96
DNC-free chicken breast fillets (control samples). Four skinless breast fillets
(pectoralis major muscle) were collected from 42-old-day broilers fed diets
without NCZ. Each chicken breast was divided into 6 portions (50–60 g). One by
one, the portions were sorted by chance and assigned to a cooking method. Four
portions, representing the control samples of each cooking method, were
subjected to heating until the core temperature achieved 70 °C (boiling for 15
min; grilling for 60 min; frying for 15 min; roasting for 45 min). The remaining four
raw portions represented the control samples without heat treatment (identical to
the blank samples). All breast portions were freeze-dried according to the
previous procedure of sample preparation.7 Heat-processed control samples
were analyzed to confirm that DNC is the only precursor of p-NA throughout
thermal processing. Raw portions were analyzed to declare breast fillets as free
of p-NA as well.
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
DNC-free chicken breast fillets (blank samples): Six skinless breast fillets
(pectoralis major muscle) were collected from 42-old-day broilers fed diets
without NCZ. Chicken breasts were divided into 6 portions (50–60 g). After
subjecting to the freeze-drying procedure7, these samples declared as p-NA free
were used for method validation.
113
ACS Paragon Plus Environment

Page 7 of 26
Journal of Agricultural and Food Chemistry
7
114
p-NA Determination in Chicken Breast by LC-MS/MS
115
116
117
118
Reagents. LiChrosolv® acetonitrile (ACN) was supplied by Merck (Darmstadt,
Hessen, Germany). p-nitroaniline (> 99%), aniline (> 99.5%), and benzoyl
chloride (99%) were obtained from Sigma-Aldrich Co. (St Louis, MO, USA). All
other reagents were of analytical grade.
119
120
121
122
123
124
125
Solutions. p-NA stock standard solution at 1,000 µg/mL was prepared in ACN.
Working solutions of p-NA were daily prepared at 100 and 10 µg/mL by diluting
the stock solution with ACN. Aniline used as internal standard (IS) was dissolved
in ACN to result in a IS stock solution of 1,000 µg/mL. The IS working solution
was daily prepared at 10 µg/mL by diluting IS stock solution with ACN. The
derivatizing reagent was prepared by diluting 200 µL of benzoyl chloride in 10 mL
of ACN. All solutions were protected from light and stored at -20 °C.
126
127
128
129
130
131
132
133
134
135
136
137
Extraction of chicken meat samples. The Supporting Information (Figure S1)
brings the details of the method steps in flow chart scheme. In order to extract p-
NA, about 2.5 g of ground freeze-dried sample (equivalent to 4–10 g in wet basis)
was weighed into a 50 mL conical polypropylene tube and fortified with 50 µL IS
working solution at 10 µg/mL. Then, 20 mL of 0.5 mol/L perchloric acid solution
(aq.) was added, and the tube was vigorously shaken in a vortex mixer for 30 s.
Further, the tube was shaken for 30 min on a “wrist action” shaker. Centrifugation
was carried out at 3500g and 20 °C for 10 min. The supernatant was filtered
(nylon hydrophilic membrane, 33 mm, 0.45 µm), and collected into another 50
mL conical tube for use in the later steps of liquid-liquid extraction and
derivatization. Blank samples fortified at 500 µg/kg were used as quality control
(QC) in every batch of analysis. A total of 10 QC fortified samples were analyzed.
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 8 of 26
8
138
139
140
141
142
143
144
145
146
147
148
149
Salting-out assisted liquid-liquid extraction (SALLE) and derivatization. First, 5
mL of the extract were transferred into a 15 mL conical polypropylene tube
already containing 1.5 g NaCl and 0.5 g Na-citrate dihydrate. After adding 0.5 mL
of 5 mol/L NaOH (aq.), the tube was vigorously shaken by hand for salt
dissolution. ACN (2 mL) was added, and the tube was shaken again. Phase
separation was achieved by centrifugation at 4370g for 10 min at 20 °C. An aliquot
of the upper ACN phase (500 µL) was transferred to a 2 mL microtube containing
the derivatizing reagent (50 µL) and ACN (450 µL). The solution was incubated
in an oven at 40 °C for at least 15 hours (overnight) and then centrifuged at
15,000g for 5 min at 20 °C. After the derivatization step, 700 µL of the solution
were transferred to a 2 mL vial for injection into the LC-MS/MS. Determinations
were performed in duplicate.
150
151
152
153
154
155
156
157
158
159
160
161
162
LC-MS/MS determination. The analyses were performed using a LC system
Surveyor Plus (Thermo, USA). Separations were carried out in a Kinetex C18 100
Å analytical column (100 mm x 4.6 mm, 5 µm pore size, Phenomenex) combined
with a C18 guard column (SecurityGuard™ Ultra, Phenomenex). Column
temperature: 30 °C. Injection volume: 10 µL. A combination of two mobile phases
(A and B) was used with a constant flow rate at 1.0 mL/min. Mobile phase A:
water with 0.1% formic acid (v/v). Mobile phase B: ACN with 0.1% formic acid
(v/v). Separations were achieved with a gradient program described as follows:
95% A (0–0.5 min), 30% A (0.5–6 min, maintained until 10 min), and 100% B
(10–12 min, held until 13.5 min), 95% A (13.5–15 min). The mobile phase ratio
was changed linearly for each ramp. All compounds were eluted out of the column
within 15 min. The autosampler was operated at 10 °C and rinsed with 50:50
water:ACN (1.2 mL) after each injection.
ACS Paragon Plus Environment

Page 9 of 26
Journal of Agricultural and Food Chemistry
9
163
MS measurements were carried out on a triple-quadrupole mass spectrometer
Quantum Access Max (Thermo, USA). Solutions of the benzoyl-derivatives from
p-NA and aniline (at 10 mg/L in ACN) were directly infused through an integrated
syringe pump at 10 µL/min for tuning the MS spectrometer using electrospray
ionization in positive mode. Under these conditions, the precursor ion and the
respective product ions of each derivative were identified. In addition, the spray
voltage (3.0 kV) and the collision energies (CE) were optimized. For p-NA
derivative, the protonated molecular ion [M+H]+ at m/z 242.9 was selected as the
precursor ion, while the product ions at m/z 105.2 (CE 19 eV) and m/z 77.3 (CE
35 eV) were set for quantification and confirmation, respectively. The protonated
molecular ion [M+H]+ at m/z 198.0 was selected as the precursor ion for aniline
derivative whose ion product at m/z 105.2 (CE 19 eV) was set for quantification
and the other at m/z 77.3 (CE 35 eV) for confirmation. Retention time was also
used for analyte confirmation. By infusing the same solution of derivatives into
the MS spectrometer with mobile phase (50:50 water:ACN, both with 0.1% formic
acid) at 1 mL/min, the source conditions were optimized, as follows: vaporized
temperature at 348 °C; capillary temperature at 350 °C; sheath gas pressure at
50 psi; auxiliary gas pressure at 45 psi. Nitrogen was used as nebulizer gas and
argon as collision gas at a pressure of 1.9 mTorr. The data were processed by
using the Xcalibur™ 2.1 software.
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
Validation. Some performance criteria of the analytical method were evaluated
by an in-house validation, as advised by the Commission Decision
2002/657/EC.16 Specificity was assessed by checking for interferences around
retention time of the benzoyl-derivatives (from p-NA and aniline) on
chromatograms of blank samples (n=20) and heat-processed control samples
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 10 of 26
10
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
(n=4 for each cooking method) before and after spiking the analyte and the IS.
To evaluate false-positive p-NA responses from remnant DNC breakdown during
sample preparation, 100 µL of DNC solution at 100 mg/mL were spiked on seven
blank samples for each day of validation. The relative abundance of the
confirmatory transition in relation to determinative transition was 20%. Linearity
(R2 > 0.98) was evaluated by preparing a matrix-matched calibration curve (using
blank samples) containing the IS (aniline) at six levels of p-NA (100, 500, 1,000,
1,500, 2,000, and 2,500 µg/kg) with three replicates each. Accuracy and precision
were determined using blank samples fortified at four concentration levels: 200,
500, 1,000, and 2,000 µg/kg. Six replicates of each level were analyzed on three
different days. The accuracy was assessed through recovery (80–110%) for each
level. Coefficients of variation (CV < 15%) were calculated to indicate precision
in terms of intra- and inter-day repeatability. The overall matrix effect was
evaluated using heat-processed control samples (of all cooking methods) spiked
at 200, 500 and 1,000 µg/kg (three replicates of each level for grilled samples;
four replicates of each level for boiled, fried or roasted samples). The limits of
detection (LOD) and quantification (LOQ) were determined according to
International Conference on Harmonization guidelines17, as follows: LOD = 3σ/S
and LOQ = 10σ/S, where σ/S is the signal to noise ratio.
RESULTS AND DISCUSSION
209
210
211
212
Method for p-NA Determination. First, p-NA was extracted from freeze-dried
chicken meat samples with an acid aqueous solution at room temperature. After
pH adjustment (pH > 8) for p-NA deprotonation, a salting-out assisted liquid-liquid
extraction with ACN was carried out to isolate the mentioned analyte. Afterward,
ACS Paragon Plus Environment

Page 11 of 26
Journal of Agricultural and Food Chemistry
11
213
214
215
the aromatic amine was derivatized with benzoyl chloride to produce the
respective derivative, determined by LC-MS/MS according to fragmentation
pathway (Figure 1).
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
Validation. Some analytical parameters of the novel method were evaluated.
The specificity was verified since no interference (m/z 242.9 and m/z 105.2) in
blank samples was evidenced around p-NA derivative retention time by checking
LC-MS/MS chromatograms. When these samples were spiked with DNC and
submitted to preparation, no amide signal was observed, indicating the absence
of DNC breakdown along the analytical procedure. This procedure avoids false-
positive responses for p-NA resulting from the remnant DNC in thermally-
processed samples. The method was linear within the range of 100–2,500 µg/kg
in chicken breast (R2 > 0.99) and showed acceptable accuracy (recovery) and
precision (repeatability), according to data in Table 1. Overall recovery complied
with EU guidance within the recommended limits for fortified levels evaluated.16
Coefficients of variation indicated that precision did not exceed the limit of 15%
for inter-day repeatability. Including all heat-processed control samples, the
overall matrix effect was neglected based on following recoveries for the
respective spiking levels: 95–119% (CV < 17%) at 200 µg/kg; 94–103% (CV <
5%) at 500 µg/kg; 92–105% (CV < 8%) at 1,000 µg/kg. The LOD and LOQ for
monitoring the target compound in meat were 10 µg/kg and 30 µg/kg,
respectively. In summary, all minimum requirements were achieved, proving the
suitability of this method. For analytical quality assurance, analysis of QCs
together with real sample batches resulted in a mean recovery of 103% (CV =
6.9%), ensuring a reliable data set.
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 12 of 26
12
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
Findings. Attempts to find p-NA in heat-processed chicken meat were not
conducted without first demonstrating its release from DNC hydrolysis. The
stability of this diphenylurea was assessed in buffered aqueous solutions (pH 2–
10) with p-NA monitoring as the azo-coupling derivative.14,15,18 Surprisingly, the
aromatic amine was not detected when fixing the hydrolysis temperature at 70,
80 or 90 °C. In contrast, DNC was susceptible to cleavage at 100 °C, yielding
free p-NA in both acidic and alkaline media. This hydrolytic decomposition
proceeded slowly at 100 °C, with continuous reduction of the reaction rate at
increasing pH, as indicated by the respective k1' values: 0.066 h-1 at pH 2; 0.061
h-1 at pH 4; 0.057 h-1 at pH 6; 0.050 h-1 at pH 8; and 0.029 h-1 at pH 10 (Table S1
in Supporting Information presents linear regression parameters). Our results are
aligned, in part, with previous reports. For analytical purposes, Nose et al.19
achieved quantitative p-NA formation only when performing the DNC hydrolysis
at 150 °C. Tarbin et al.8 provided further evidence on DNC depletion in aqueous
solution at 100 °C, mentioning that p-NA can be formed. As reported by Audu
and Heyn14, hydrolysis of DNC-like N,N'-diphenylureas, giving rise the
corresponding amines, takes place at very slow rates indeed.
254
255
256
257
258
259
260
261
Laudien and Mitzner proposed mechanisms of acid and basic catalysis for
phenylureas hydrolysis via nucleophilic attack on carbonyl carbon.20,21 These
mechanisms are appropriate to clarify the course of hydrolytic breakdown
investigated herein. Based on a structure-reactivity relation established by the
same authors, the high resistance of DNC to hydrolysis makes sense if the
electron-withdrawing effect promoted by nitro groups is considered. Such
substituents markedly reduce the electron density on the N atoms adjacent to
carbonyl, what hinders the protonation step involved in acid catalysis, and
ACS Paragon Plus Environment

Page 13 of 26
Journal of Agricultural and Food Chemistry
13
262
263
264
265
266
267
adversely affect the reaction velocity. Furthermore, electron-attracting influence
of nitro groups increases the acidity of proton in NH-aryl moiety. This means that
DNC deprotonation is favored in alkaline solutions, leading to formation of its
unreactive conjugate pair in the media. Actually, the predominance of this
competitive equilibrium inhibits basic hydrolysis to the point of making it slower
than acidic medium reaction.
268
269
270
271
272
273
The fact that DNC degrades to p-NA in aqueous solutions did not exclude
other alternatives for residue disappearance in chicken meat subjected to
cooking methods. The suggested pathway towards p-NA would be no more than
speculation unless this aromatic amine was identified in actual heat-processed
fillets previously prepared.7 Thus, we sought for the detection of p-NA traces in
such chicken samples by means of LC-MS/MS to define part of the DNC fate.
274
275
276
277
278
279
280
281
282
283
284
According to chromatograms shown in Figure S2 (see Supporting
Information), the target amine was not detected (< 10 µg/kg) in DNC-containing
raw fillets (samples that represented zero-time in thermal processing
experiment). The absence of p-NA was also verified in DNC-free breast fillets
(control samples) either pre- or post-heating by the cooking methods (Figure S3
in Supporting Information). Both remarks show that p-NA formation in chicken
meat originates only from DNC degradation. Therefore, p-NA occurrence in
breast muscle cannot be related to endogenous deposition resultant from its own
impurity in NCZ consumed by broilers.9,22 In addition, the release of this amine is
not associated to decomposition of another naturally-occurring precursor in
chicken meat matrix.
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 14 of 26
14
285
286
287
288
289
290
291
Meanwhile, typical LC-MS/MS profiles (Figure 2) revealed unequivocally
the occurrence of p-NA (retention time at 9.5 min) in cooked chicken fillets.
Similar chromatographic profiles were observed for the samples heated either by
boiling, grilling, frying, or roasting. In essence, this finding explains into an
acceptable reason why DNC disappears in chicken meat along heat treatment.
As far as we are concerned, no other research has reported this information
before.
292
293
294
295
296
297
298
299
Table 2 shows the p-NA concentration in DNC-containing chicken meat
during thermal processing by different cooking methods. For this set of samples,
some data are missing because relatively low concentrations have been
observed in these assays (values between LOD and LOQ of the analytical
method); nevertheless, this analytical limitation does not diminish the research
merit. The appearance of p-NA in chicken fillets became pronounced in the
course of time for grilling, frying, and roasting. Otherwise, the lowest
concentrations were observed during boiling.
300
301
302
303
304
305
306
307
308
At first glance, such reticent accumulation of the aromatic amine caused
some surprise when crosschecking the earlier remarks7, in which more than 50%
of the initial DNC had already been degraded in the minimum cooking time of all
the applied methods (5 min for boiling, 15 min for grilling, 5 min for frying, and 15
for roasting). In response to this decomposition, a sudden increase in free p-NA
content within the same period was expected, as outlined by the qualitative
scheme in Figure 3. However, this behavior for p-NA concentration was not
confirmed, what suggests a less simplistic reading about the fate of the amine
during chicken meat heating.
ACS Paragon Plus Environment

Page 15 of 26
Journal of Agricultural and Food Chemistry
15
309
310
311
312
313
The p-NA accumulation in thermally-processed breast fillets remains
unclear, but probably it depends on factors beyond degradation itself. Apparently,
the amine levels left in cooked meat were more like a consequence of DNC
cleavage subtracted by losses in concomitant events. One of them, that for now
cannot be ignored, refers to the moisture transport over cooking.
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
For well-stated reasons, interstitial water in the meat is naturally expelled
by heating influence.23,24 This fact gives a reasonable chance for p-NA leaching
through the juices exuded from the fillets. However, besides the exudation
reaching a limit at some point, the water transport does not occur only in liquid
form. A significant moisture loss occurs directly by evaporation24,25, a key-process
for modeling mass transport under grilling26,27, deep-fat frying28–30 and roasting31–
33
conditions. Then, as water escaped from the meat in vapor phase, the p-NA
migration out of the portion was disrupted, leading to impregnation of its traces in
grilled, fried and roasted fillets. By prolonging the exposure time, the increase in
amine concentration may have been a result of interaction between these drying
and retention effects. When a limited screening was accomplished by LC-MS/MS,
amine traces were found in boiling water. Although the quantification was not
done, boiling probably provided p-NA extraction by hot water resulting in the low
concentrations in chicken meat, differently from the other cooking methods.
Further causes that explain this discrete behavior in boiling still need elucidation.
329
330
331
332
333
Besides the issues reported in this study, a relevant feature of DNC such
as its thermal profile could not escape our notice, given its implication on
outcomes. As recently verified by means of thermal analysis techniques, DNC is
a thermo-labile compound whose decomposition occurs at 252 °C and results in
p-NA as well.34 Instead of introducing another perspective, this evidence
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 16 of 26
16
334
335
336
337
indicated that DNC degradation proceeds by hydrolytic breakdown. Thermally
induced decomposition was not considered because the temperature of chicken
fillets (in the core and the boundaries) did not exceed 200 °C in any of the cooking
procedures.7
338
339
340
341
342
343
344
345
Our evidences prove for the first time the p-NA release from incurred DNC
in chicken breast fillets submitted to thermal processing. Thus, the heating effect
on both the DNC content and possible relevant degradation products in chicken
meat should be considered. The findings are a pioneering milestone in
anticoccidial-deriving degradation products. Based on our results, we suggest
further research not only to identify the factors of each cooking method that
determine the net p-NA accumulation, but also to verify whether the levels found
are indeed a matter of concern regarding food safety.
346
347
348
349
350
351
352
353
354
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications
website at DOI:
Additional information on rate constant (Table S1), p-NA extraction
procedure (Figure S1); chromatograms of breast fillets containing DNC (Figure
S2) and free from DNC (Figure S3) (PDF).
ACS Paragon Plus Environment

Page 17 of 26
Journal of Agricultural and Food Chemistry
17
355
356
357
358
359
360
361
362
363
364
Funding
Authors thank Brazilian Agricultural Research Corporation (Embrapa) for
financial support (Project No. 03.16.05.004). This study was financed in part by
the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil
(CAPES) - Finance Code 001, as a scholarship No. 1533827 given to D.M.
Bacila.
Notes
The authors declare no competing financial interest.
365
References
366
367
368
369
370
371
372
373
374
375
376
377
378
379
(1) Clarke, L.; Fodey, T. L.; Crooks, S. R. H.; Moloney, M.; O’Mahony, J.;
Delahaut, P.; O’Kennedy, R.; Danaher, M. Meat Sci. 2014, 97 (3), 358–
374.
(2) Bacila, D. M.; Feddern, V.; Mafra, L. I.; Scheuermann, G. N.; Molognoni,
L.; Daguer, H. Food Res. Int. 2017, 99, 31–40.
(3) Rogers, E. F.; Brown, R. D.; Brown, J. E.; Kazazis, D. M.; Leanza, W. J.;
Nichols, J. R.; Ostlind, D. A.; Rodino, T. M. Science (80-. ). 1983, 222 (10),
630–632.
(4) EFSA. EFSA J. 2003, 16, 1–40.
(5) Lima, A. L. De; Barreto, F.; Rau, R. B.; Resende, G.; Lara, C.; Figueiredo,
T. C. De; Jose, L.; Assis, C. S. De; Canc, S. D. V. 2017, 6–17.
(6) Brasil. Ministério da Agricultura, Pecuária e Abastecimento. [Brazil,
Ministry of Agriculture, Livestock and Food Supply - Results of the National
Plan for the Control of Residues and Contaminants].
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 18 of 26
18
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
(7) Bacila, D. M.; Cunha, A.; Weber, I. F.; Scheuermann, G. N.; Coldebella, A.;
Caron, L.; Molognoni, L.; Daguer, H.; Igarashi Mafra, L.; Feddern, V. J.
Agric. Food Chem. 2018, 66, 8391–8397.
(8) Tarbin, J. A.; Bygrave, J.; Bigwood, T.; Hardy, D.; Rose, M.; Sharman, M.
Food Addit. Contam. 2005, 22 (11), 1126–1131.
(9) EFSA. EFSA J. 2010, 8 (3), 1551:1-40.
(10) Health Council of the Netherlands. p-Nitroaniline; Evaluation of the
carcinogenicity and genotoxicity; Health Council of the Netherlands;
publication no. 2008/08OSH: The Hague, 2008.
(11) Wang, G.; Zhang, X.; Yao, C.; Tian, M. Toxicol. Ind. Health 2011, 27 (2),
167–171.
(12) Nair, R. S.; Auletta, C. S.; Schroeder, R. E.; Johannsen, F. R. Toxicol. Sci.
1990, 15 (3), 607–621.
(13) National Toxicology Program. Natl. Toxicol. Program Tech. Rep. Ser. 1993,
418, 1–203.
(14) Audu, A. A.; Heyn, A. H. A. Water Res. 1988, 22 (9), 1155–1162.
(15) Lee, M.; Anstee, J. H. Anal. Biochem. 1994, 218, 480–482.
(16) EC. Off. J. Eur. Union 2002, L221, 8–36.
(17) ICH. In Proceedings of the International Conference on Harmonisation of
Technical Requirements for Registration of Pharmaceutical for Human
Use; 2005; pp 1–13.
(18) Bratton, C.; Marshall, E. K. J. Biol. Chem. 1939, 128 (5), 537–550.
(19) Nose, N.; Hoshino, Y.; Kikuchi, Y.; Yamada, F.; Kawauchi, S. Food Hyg.
Saf. Sci. 1981, 22 (6), 496–500.
(20) Laudien, R.; Mitzner, R. J. Chem. Soc. Perkin Trans. 2 2001, No. 11, 2226–
ACS Paragon Plus Environment

Page 19 of 26
Journal of Agricultural and Food Chemistry
19
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
2229.
(21) Laudien, R.; Mitzner, R. J. Chem. Soc. Perkin Trans. 2 2001, No. 11, 2230–
2232.
(22) Porter, C. C.; Gilfillan, J. L. Poult. Sci. 1955, 34 (5), 995–1001.
(23) van der Sman, R. G. M. Meat Sci. 2007, 76 (4), 730–738.
(24) Datta, A. K.; Rakesh, V. Compr. Rev. Food Sci. Food Saf. 2013, 12 (1),
24–39.
(25) Pathare, P. B.; Roskilly, A. P. Food Eng. Rev. 2016, 8 (4), 435–447.
(26) Portanguen, S.; Ikonic, P.; Clerjon, S.; Kondjoyan, A. Food Bioprocess
Technol. 2014, 7 (11), 3308–3318.
(27) Wählby, U.; Skjöldebrand, C. J. Food Eng. 2001, 47 (4), 303–312.
(28) Farkas, B. E.; Singh, R. P.; Rumsey, T. R. J. Food Eng. 1996, 29 (2), 211–
226.
(29) Farkas, B. E.; Singh, R. P.; Rumsey, T. R. J. Food Eng. 1996, 29 (2), 227–
248.
(30) Ngadi, M. O.; Watts, K. C.; Correia, L. R. J. Food Eng. 1997, 32 (1), 11–
20.
(31) Feyissa, A. H.; Gernaey, K. V.; Adler-Nissen, J. Meat Sci. 2013, 93 (4),
810–820.
(32) Goñi, S. M.; Salvadori, V. O. J. Food Eng. 2010, 100 (1), 1–11.
(33) Singh, N.; Akins, R. G.; Erickson, L. E. J. Food Process Eng. 1984, 7 (3),
205–220.
(34) Bacila, D. M.; Lazzarotto, M.; Hansel, F. A.; Scheuermann, G. N.; Feddern,
V.; Cunha Jr., A.; Igarashi Mafra, L. J. Therm. Anal. Calorim. 2019, 9, (in
press).
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 20 of 26
20
Table 1. Accuracy and Precision for p-NA Determination in Chicken Breast
Meat
fortified
level
intra-day
inter-day
recovery
(%)a
recovery
(%)b
day
repeatability
(CV, %) a
repeatability
(CV, %)b
(µg/kg)
1
2
3
1
2
3
1
2
3
1
2
3
103.8
97.1
11.0
8.9
200
96.6
9.7
6.6
8.9
14.6
92.7
14.8
7.5
104.2
100.9
98.7
500
7.5
101.2
99.1
4.2
108.7
93.4
8.2
1,000
1.9
97.7
7.5
113.6
89.4
10.9
1.8
2,000
102.2
102.9
14.8
a(n = 6); b(n = 18)
Intra- and inter-day repeatability means precision.
Recovery means accuracy.
ACS Paragon Plus Environment

Page 21 of 26
Journal of Agricultural and Food Chemistry
21
Table 2. Concentration of p-NA in DNC-Containing Chicken Meat Over
Cooking Times
cooking time
p-NA
cooking method
(min)
0 b
5
(µg/kg)a
< LOD
LOD–LOQ
LOD–LOQ
LOD–LOQ
38.4 ± 3.7
51.9 ± 4.9
< LOD
10
15
20
25
0 b
15
30
45
60
75
0 b
5
boiling
LOD–LOQ
60.6 ± 9.0
141.2 ± 31.5
210.4 ± 43.1
326.3 ± 47.6
< LOD
grilling
36.9 ± 8.4
82.5 ± 13.6
78.0 ± 6.5
200.6 ± 52.7
640.0 ± 143.3
< LOD
10
15
20
25
0 b
15
30
45
60
75
frying
< LOD
LOD–LOQ
79.8 ± 18.5
191.7 ± 48.0
456.9 ± 50.7
roasting
a
Average value of 6 replicates for each cooking time. Data were corrected, considering weight
loss during freeze-drying process7.
b The zero-time represents the DNC-containing raw chicken fillets.
LOD: 10 µg/kg; LOQ: 30 µg/kg; LOD–LOQ: among LOD and LOQ values
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 22 of 26
22
Figure Captions
Figure 1. Fragmentation pathway of benzoyl derivatives from p-NA and aniline
(IS).
Figure 2. LC-MS/MS chromatograms showing p-NA traces in DNC-containing
chicken breast fillets submitted to thermal processing by different cooking
methods.
Figure 3. Simplified qualitative scheme to illustrate the behavior of DNC and p-
NA concentrations in chicken breast fillets submitted to different cooking
methods. Straight line represents the DNC and p-NA profiles found
experimentally. Dotted line indicates the expected p-NA profile. The DNC profile
has been adapted from our data previously reported.7
ACS Paragon Plus Environment

Page 23 of 26
Journal of Agricultural and Food Chemistry
23
Figure 1.
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 24 of 26
24
Figure 2.
ACS Paragon Plus Environment

Page 25 of 26
Journal of Agricultural and Food Chemistry
25
Figure 3.
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry Page 26 of 26
RT: 0.00 - 15.00
9.5
100
80
60
40
20
0
Roasting
NL: 2.20E4
242.9 105.2
>
100
80
60
40
20
Frying
NL: 2.85E4
242.9 105.2
>
0
100
Crilling
NL: 1.62E4
242.9 105.2
>
80
60
40
20
0
tHErmAl
ProCEssInG
100
80
60
40
20
0
Boiling
NL: 3.07E3
242.9 105.2
>
100
80
60
40
20
0
Blank sample
NL: 8.20E2
242.9 105.2
>
ACS Paragon Plus Environment
100
80
60
40
20
0
Blank sample fortified at 100 µg/kg
NL: 1.25E4
242.9 105.2
>
0
2
4
6
8
10
12
14
Time (min)