Dinitrocarbanilide Research Articles

Electrochemical Nicarbazin Analysis: Rapid Determination on Bucky Paper Disks Constructed from MWCNT

Nicarbazin, an anticoccidial drug, is a molecular complex comprising 4,4’-dinitrocarbanilide (DNC) and 2-hydroxy-4,6-dimethylpyrimidine (HDP) in a 1:1 molar ratio. The low solubility of DNC in water (below 0.02 mg L−1) necessitates the use of extraction methods for analysis. Traditional DNC determination methods are time consuming, and involve prolonged sample preparation, extraction procedures, and high costs,. This study presents the results of electrochemical analysis of DNC using an innovative electrode platform composed of bucky paper disks made from a blend of multi-walled carbon nanotubes (MWCNT) and polystyrene (MWCNT/PS/BP). The electroanalytical approach includes an initial adsorption stage of DNC on the electrode platform, followed by its subsequent reduction, capitalizing on the reducibility of the nitro groups within DNC. The analytical parameters of this method are favorable, featuring an average recovery value of 100.22 ± 7.87, a linear range of 2.011–15.112 mg·l−1, a sensitivity of 1.58 μA·mg−1 l−1, LOQ (limit of quantification) of 1.33 mg L−1, and LOD (limit of detection) of 0.40 mg·l−1.

Crystal structure of nicarbazin, (C13H10N4O5)(C6H8N2O)

The crystal structure of nicarbazin has been solved and refined using synchrotron X-ray powder diffraction data and optimized using density functional theory techniques. Nicarbazin is a co-crystal of 4,4′-dinitrocarbanilide (DNC) and 2-hydroxy-4,6-dimethylpyrimidine (HDP) molecules. Nicarbazin crystallizes in space group P-1 (#2) with a = 6.90659(8), b = 12.0794(4), c = 13.5040(7) Å, α = 115.5709(11), β = 102.3658(6), γ = 91.9270(4)°, V = 982.466(5) Å3, and Z = 2. The DNC and HDP molecules are linked by two strong N–H⋯O and N–H⋯N hydrogen bonds, and the HDP molecules are linked into centrosymmetric dimers by another N–H⋯O hydrogen bond. These strong hydrogen bonds link the molecules into layers parallel to the ab-plane and parallel stacking of both DNC and HDP molecules is prominent in the structure. The powder pattern has been submitted to ICDD for inclusion in the Powder Diffraction File™ (PDF®).

A Comparable icELISA and Lateral Flow Immunoassay for the Sensitive and Rapid Detection of 4,4'-Dinitrocarbanilide in Chicken.

4,4'-dinitrocarbanilide (DNC) is a key component and marker residue of nicarbazin, which forms residues in edible tissue and then causes nephrotoxicity and hepatotoxicity in humans if used excessively. To simplify sample preparation and monitor the DNC rapidly and accurately, a comparable icELISA and lateral flow immunoassay (LFIA) was developed in this study. Briefly, the reaction parameters were explored for improving the sensitivity of icELISA and LFIA. Under the optimal conditions, methanol was selected as the extracting solvent for DNC in chicken, and 20- and 10-fold dilutions of sample extraction eliminated the matrix effect for icELISA and LFIA, separately. After sample pretreatment, the analysis properties of icELISA and LFIA were compared. The limit of detection of icELISA for DNC was 0.8 μg/kg, and the visual and quantitative limits of detection of LFIA were 8 and 2.5 μg/kg. Compared with icELISA, LFIA showed lower sensitivity but obvious advantages in terms of matrix tolerance and detection time (within 15 min). The sensitivity, specificity, and accuracy of the developed assays satisfied the detection requirement even if using simple sample pretreatment. This comparable icELISA and LFIA provided mutual verifiability methods for the accurate detection of DNC in chicken.

A Europium Nanosphere-Based Time-Resolved Fluorescent Immunochromatographic Assay for the Rapid Screening of 4,4′-Dinitrocarbanilide: Aiming at Improving Strip Method Performance

Considering that the strip method is simple and convenient for users, a Europium nanosphere-based time-resolved fluorescent immunochromatographic assay (TRFICA) for the rapid screening of 4,4'-dinitrocarbanilide (DNC) was developed to improve the performance of strip assays. After optimization, TRFICA showed IC50, the limit of detection, and cut-off values of 0.4, 0.07, and 5.0 ng mL-1, respectively. No significant cross-reactivity (CR < 0.1%) with 15 DNC analogs was observed in the developed method. TRFICA was validated for DNC detection in spiked chicken homogenates, and recoveries ranged from 77.3% to 92.7%, with coefficients of variation of <14.9%. Moreover, the time needed for the detection procedure, including the sample pre-treatment, was less than 30 min for TRFICA, which had never been achieved before in other immunoassays. The newly developed strip test is a rapid, sensitive, quantitative, and cost-effective on-site screening technique for DNC analysis in chicken muscle.

Development of epitopephore-based rational hapten design strategy: A combination of theoretical evidence and experimental validation

Antibody is the key biomolecule that governing the sensitivity and specificity of an immunoassay for chemical compound, also named hapten molecule. Obviously, predication of hapten effectiveness before chemical synthesis is beneficial to boost success, save cost and improve controllability. Here, we proposed and evaluated an epitopephore based rational hapten design (ERHD) to assist antibody production to chemical compound, combining theoretical evidence and then experimental validation by using dinitrocarbanilide (DNC) as a model analyte. Briefly, epitopephores of DNC were firstly generated by HipHop algorithm after features mapping. A homemade drug database also containing reported fragment haptens (HFR) and new designed full hapten (HFU) were constructed, and then was virtually screened by using generated epitopephore followed by structural analysis and visual inspection. The DNC haptens based on the selected hits were further identified by Density Functional Theory before total synthesis. To prove and clarify the usability of the ERHD, two retrieved HFU haptens, one non-retrieved HFU hapten and three non-retrieved HFR haptens were all selected to produce monoclonal antibodies (mAbs) for comparison purpose. A maximal 6000-fold increased affinity of mAb from retrieved HFU than HFR was observed, while, non-retrieved HFU failed to produce antibody to DNC. More importantly, mAbs from HFU haptens provided highly specificity to DNC, while, mAbs from HFR haptens could recognize 15 others analogues. We then constructed antibody structure and investigated molecular recognition of the mAbs to DNC, well supporting the rationality of the ERHD. Lastly, an icELISA was developed for DNC with an IC50 value as low as 0.19 ng mL−1 with high specificity, which has never achieved before.

Nicarbazin Residue in Tissues from Broilers Reared on Reused Litter Conditions.

Nicarbazin (NCZ) is a worldwide used anticoccidial in poultry farming to avoid coccidiosis disease when chickens are reared on conventional poultry litter. If proper dosage and withdrawal time are not followed, the component dinitrocarbanilide (DNC) of NCZ may be present in chicken tissues, therefore posing a risk to consumers if the residues are above 200 µg/kg. Litter reuse is a common and important practice in commercial chicken production. Literature is lacking about the influence of litter reuse on DNC deposition in chicken tissues and its impact on food safety. We aimed to evaluate DNC residues in breast and liver by LC-MS/MS from broilers from an experiment with 10 consecutive flocks during 2 years. The experiment included three treatments containing NCZ in the diet (T1 = 125 mg/kg, 1-21 d; T2 = 125 mg/kg, 1-32 d; T3 = 40 mg/kg, 1-32 d). DNC residues in chicken breast at 21 d in T1 ranged from 648.8-926 µg/kg, at 32 d in T2 and T3 varied, respectively, from 232-667 µg/kg and 52-189 µg/kg. Regarding liver, DNC residues at 21 days in T1 ranged from 11,754-15,281 µg/kg, at 32 days in T2 and T3 varied, respectively, from 10,168-15,021 µg/kg and 2899-4573 µg/kg. When NCZ was withdrawn from feed, DNC residues dropped to &lt;LOQ at 42 d in all treatments. Therefore, the reuse of poultry litter does not compromise food safety regarding DNC residues in chicken tissues, as shown herein up to 10 flocks.

Preparation and identification of an anti-nicarbazin monoclonal antibody and its application in the agriculture and food industries.

BackgroundAs a broad-spectrum drug against chicken coccidiosis, nicarbazine is widely used. The international community has made regulations and requirements on the residue limits of nicarbazine metabolites in chicken. The research reports on the detection methods of nicarbazine residues are mainly based on large-scale instruments such as high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry-mass spectrometry (LC/MS/MS) and so on. However, in the practical monitoring and detection application, the rapid, sensitive, efficient and accurate detection of nicarbazine residues is becoming more and more urgent.MethodsThis study aimed to establish an enzyme-linked immunosorbent assay (ELISA)-based method to detect nicarbazin drug residues with high sensitivity and specificity, and wide applicability. Artificial immunogens were prepared by molecular modification synthesis. Nuclear magnetic resonance (NMR) and ultraviolet analyses were conducted to confirm that the correct product was obtained. Monoclonal antibodies were acquired by immunizing mice and preparing hybridoma cells.ResultsIn this study, 4,4'-dinitrocarbanilide (DNC), a metabolite of nicarbazine, was synthesized and modified to make it have immunogenicity. Fifteen healthy female mice of 6–8 weeks old were immunized in three groups. The successfully immunized mice were screened by serum titer. One mouse with the highest titer was fused and cloned three times, and four positive cell lines were obtained. Nine monoclonal antibodies were obtained from mouse ascites. The best matched antigens and antibodies were screened by an ELISA chessboard method. A detection method of nicarbazine ELISA kit was developed. Our prepared anti-nicarbazin monoclonal antibody had a half-maximal inhibitory concentration (IC50) of 0.825 ng/mL, and the curve range was 0.3–24.3 ng/mL. There was no cross reaction to other six common anti-coccidiosis drugs. The recovery results showed that the fortified recovery of the chicken and duck samples ranged from 74.4–111.7%, the test results of which all met the requirements for veterinary drug residue detection.ConclusionsThis method, which uses a specific antibody against the nicarbazin metabolic product DNC, enables rapid quantitative detection. Our new ELISA-based method should facilitate the development of assays to monitor and detect agricultural and veterinary drug residues.

Determination and Identification of Nicarbazin, Measured as 4,4'-Dinitrocarbanilide (DNC), in Chicken Tissues by Liquid Chromatography With Tandem Mass Spectrometry: Final Action 2013.07.

BackgroundAOAC Method 2013.07 was adopted as First Action in 2013. Since then, the method has been used in numerous residue depletion studies with favorable comments from analysts.ObjectiveTo analyze data from residue depletion studies to support Final Action status.MethodTen residue depletion studies were conducted during May 2014 through May 2019. For each study, harvested incurred tissues were analyzed for nicarbazin using AOAC Method 2013.07 in 1 of 4 laboratories. Each analytical run included one or more fortified quality control test portions. The data from these known fortified matrix test portions were analyzed for reproducibility and repeatability.ResultsFor muscle tissues, relative recovery was 90.4% (95% CI 83.8 to 97.5); RSDr was 5.4% (95% CI 3.8 to 9.2); and RSDR was 7.9%. In the liver, values were 94.5% (95% CI 91.1 to 98.0), 5.8% (95% CI 4.1 to 9.9), and 6.8%, respectively. In the kidney, values were 91.5% (95% CI 85.3 to 98.1), 5.2% (95% CI 3.7 to 8.8), and 9.0%, respectively. In skin with adhering fat, values were 94.5% (95% CI 89.2 to 100.1), 8.9% (95% CI 6.3 to 15.1), and 8.9%, respectively. In all cases, repeatability and reproducibility were within acceptable limits.ConclusionsThe data and positive feedback support the transition of AOAC Method 2013.07 from First Action to Final Action.HighlightsFinal action status is supported by data collected during routine use of the method rather than a traditional multi-laboratory collaborative study. Data were subjected to statistical analysis using the pC-metamer, and then transformed back to the traditional C-metamer.

Design, Synthesis, and Characterization of Tracers and Development of a Fluorescence Polarization Immunoassay for Rapid Screening of 4,4'-Dinitrocarbanilide in Chicken Muscle.

The compound, 4,4′-dinitrocarbanilide (DNC), is the marker residue of concern in edible tissues of broilers fed with diets containing anticoccidial nicarbazin (NIC). In this study, 25 fluorescein-labeled DNC derivatives (tracers) are synthesized and characterized to develop a rapid fluorescence polarization immunoassay (FPIA) for the detection of DNC in chickens using DNC monoclonal antibodies (mAbs). The effect of the tracer structure on the sensitivity of the FPIA is investigated. Our results show that after optimization, the half maximal inhibitory concentrations (IC50) and limit of detection (LOD) of the FPIA in the buffer are 28.3 and 5.7 ng mL−1, respectively. No significant cross-reactivity (CR < 0.89%) with 15 DNC analogues is observed. The developed FPIA is validated for DNC detection in spiked chicken homogenates, and recoveries ranged from 74.2 to 85.8%, with coefficients of variation <8.6%. Moreover, the total time needed for the detection procedure of the FPIA, including sample pretreatment, is <40 min, which has not been achieved in any other immunoassays for DNC from literature. Our results demonstrate that the FPIA developed here is a simple, sensitive, specific, and reproducible screening method for DNC residues in chickens.

Modification of the terms of authorisation regarding the maximum inclusion level of Maxiban® G160 (narasin and nicarbazin) for chickens for fattening.

Following the request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the proposed modification of the terms of the authorisation regarding the maximum inclusion level of Maxiban® G160. The FEEDAP Panel cannot conclude on the safety of Maxiban® G160 at a dose level of 70 + 70 mg/kg feed for the target species. The use of Maxiban® G160 in diets for chickens for fattening at the maximum proposed dose complies with the maximum residue levels (MRLs) in force of narasin and 4,4’‐dinitrocarbanilide (DNC) at 0‐day withdrawal except for DNC in kidney which was slightly above the MRL. Compliance with DNC MRLs was seen in all tissues at 1‐day withdrawal. Based on the available data, the FEEDAP Panel cannot conclude on the safety of Maxiban® G160 for the environment due to the risk identified for the terrestrial organisms due to DNC. Moreover, the high persistence and hydrophobicity of DNC indicate that there might be a risk for bioaccumulation but the risk for secondary poisoning was not identified. The potential of DNC to accumulate in soil over the years should be investigated by monitoring in a field study. The FEEDAP Panel would not be in the position to conclude on the efficacy of Maxiban® G160 in chickens for fattening based on the data provided for the dose of 40 + 40 mg narasin + nicarbazin/kg feed.

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

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.

Thermal profile of 4,4′-dinitrocarbanilide determined by thermogravimetry–differential scanning calorimetry–mass spectrometry (TG–DSC–MS) and pyrolysis–gas chromatography–mass spectrometry (Py–GC–MS)

Nicarbazin is an anticoccidial used in poultry production. Its residue of concern left in chicken meat is 4,4′-dinitrocarbanilide (DNC) whose content can be reduced through meat thermal processing. Thermal analysis techniques can be considered to elucidate how DNC degradation proceeds. This study aimed to investigate the thermal behavior of DNC using TG–DSC–MS and Py–GC–MS. TG–DSC–MS data showed that DNC decomposition occurs in a single step (252–280 °C) as an endothermic event (ΔH of 670 J g−1). Fragments detected at m/z 43, 44 and 65 indicated the release of isocyanic acid, carbon dioxide and 4-nitroaniline as breakdown products. In addition, Py–GC–MS analysis confirmed that DNC is thermally degraded above 250 °C generating 4-nitrophenyl isocyanate and 4-nitroaniline as the major pyrolysis products. Findings obtained from thermal analysis herein are useful to better comprehend the DNC disappearance after submitting chicken meat to heat processing.

Development and optimization of confirmatory liquid chromatography—Orbitrap mass spectrometry method for the determination of 17 anticoccidials in poultry and eggs

A new sensitive and selective multi-residue method based on liquid chromatography - Orbitrap high resolution mass spectrometry (LC-Orbitrap-HRMS) was developed and validated for the determination of 17 anticoccidials in poultry and eggs. Instrumental parameters were optimized by the means of statistical experimental designs to improve the sensitivity, precision, and repeatability of the method. Further optimization of auto-tuned MS parameters led to an increase of signal intensity by 10% to 99% for 16 out of 17 analytes. The sample preparation procedure included extraction from muscle tissue and egg samples with acetonitrile, followed by preconcentration, reconstitution, and filtration. Validation was performed according to the Commission Decision 2002/657/EC. The occurrence of anticoccidials in eggs and poultry was assessed by using the developed analytical procedure within the Latvian national monitoring program, revealing quantifiable residues for 6 analytes (marker residue of nicarbazin - 4,4′-dinitrocarbanilide (DNC), salinomycin, narasin, toltrazuril, and its two metabolites).

Safety and efficacy of Monimax® (monensin sodium and nicarbazin) for chickens for fattening and chickens reared for laying.

The coccidiostat Monimax® (monensin sodium and nicarbazin) is considered safe for chickens for fattening and chickens reared for laying at the highest use level of 50 mg monensin and 50 mg nicarbazin/kg complete feed. This conclusion is extended to chickens reared for laying. For both active substances, the metabolic pathways in the chicken are similar to those in the turkey and rat. Nicarbazin, when ingested, is rapidly split in its two components dinitrocarbanilide (DNC) and 2‐hydroxy‐4,6‐dimethylpyrimidine (HDP) which behave independently. Monimax® does not represent a genotoxic risk. No safety concerns would arise from the nicarbazin impurities p‐nitroaniline and methyl(4‐nitrophenyl) carbamate. The lowest no observed effect level (NOEL) identified for monensin sodium in a developmental study in rabbits is 0.3 mg monensin sodium/kg body weight (bw) per day for maternal toxicity in rabbits. The lowest no observed adverse effect level (NOAEL) identified in a 52‐week study in rat using DNC + HDP was 20 mg DNC + 8 mg HDP/kg bw per day based on the absence of microcrystals in urine and related microscopic renal observations. No significant interaction between monensin sodium and nicarbazin is expected from toxicological studies. The use of Monimax® at the highest proposed dose will not pose a risk to persons consuming animal products from treated chickens for fattening. This conclusion is extended to chickens reared for laying. No withdrawal time is required for Monimax® in chickens for fattening. Residue data comply with the established maximum residue limits (MRLs) for monensin and DNC. Based on the available data, the FEEDAP Panel cannot conclude on the safety of Monimax® for the environment. Monimax® has the potential to control coccidiosis in chickens for fattening at a minimum concentration of 40 mg monensin and 40 mg nicarbazin/kg complete feed.

Degradation of 4,4'-Dinitrocarbanilide in Chicken Breast by Thermal Processing.

Nicarbazin is one of the major anticoccidials used in broiler feeds. The compound 4,4'-dinitrocarbanilide (DNC) is the marker residue of concern left from nicarbazin in chicken meat. The effect of thermal processing on DNC content accumulated in chicken breast was assessed, and samples were analyzed by liquid chromatography-tandem mass spectrometry. Five conventional cooking methods were evaluated: boiling, grilling, microwaving, frying, and roasting. To ensure DNC in meat, broilers were fed nicarbazin without withdrawal period. All heating methods surpassed the 70 °C end point core temperature in chicken breast. Maximum DNC degradation was reached at 10 min for boiling, at 30 min for grilling, and at 2 min for microwaving, and no further reduction was observed for longer thermal processing time. Boiling was more efficient in reducing DNC (69%). Grilling, microwaving, and frying achieved on average 55% of degradation. The outcomes reported herein may be considered in decision-making regarding further review of maximum residue limits.

Safety and efficacy of Coxar® (nicarbazin) for turkeys for fattening.

The coccidiostat Coxar® is safe for turkeys for fattening at the use level of 100 mg nicarbazin/kg complete feed, with a margin of safety of about 1.25. Nicarbazin, when ingested, is rapidly split in its two components 2‐hydroxy‐4,6‐dimethylpyrimidine (HDP) and dinitrocarbanilide (DNC), which behave independently. HDP‐related residues are much lower than those of DNC. DNC is the marker residue. Liver is the target tissue. Nicarbazin is not genotoxic. The primary toxicity resulting from the oral use of nicarbazin is renal toxicity. The lowest no observed adverse effect level (NOAEL) identified in a 52‐week study in rat using DNC+HDP is 20 mg DNC + 8 mg HDP/kg body weight (bw) per day based on the absence of microcrystals in urine and related microscopic renal observations. The use of 100 mg nicarbazin from Coxar®/kg complete feed for turkeys for fattening will not pose a risk to consumers, provided that maximum contents in nicarbazin of 0.1% p‐nitroaniline (PNA) and 0.4% methyl(4‐nitrophenyl) carbamate (M4NPC) would be respected. No withdrawal time is required. Residue data comply with the established maximum residue limits (MRLs). Nicarbazin is not a skin or eye irritant and not a skin sensitiser. These conclusions also apply to the additive Coxar®. Inhalation toxicity of nicarbazin is limited; the granulated additive has a low dusting potential. No risk for users is identified. Based on the available data, the FEEDAP Panel cannot conclude on the safety of Coxar® for the environment. The efficacy of 100 mg nicarbazin from Coxar®/kg feed was demonstrated in three anticoccidial sensitivity tests (AST), but only in one floor pen study. The floor pen study with 75 mg nicarbazin failed to demonstrate evidence of efficacy. No final conclusions on the efficacy of nicarbazin from Coxar® for turkeys for fattening can be drawn.

Safety and efficacy of Monimax® (monensin sodium and nicarbazin) for turkeys for fattening.

The coccidiostat Monimax® (monensin sodium and nicarbazin) is considered safe for turkeys for fattening at the highest use level of 50 mg monensin and 50 mg nicarbazin/kg complete feed. The simultaneous use of Monimax® and certain antibiotic drugs (i.e. tiamulin) is contraindicated. For both active substances, the metabolic pathways in the chicken are similar to those in the turkey and rat. Nicarbazin, when ingested, is rapidly split in its two components dinitrocarbanilide (DNC) and 2‐hydroxy‐4,6‐dimethylpyrimidine (HDP) which behave independently. Monimax® does not represent a genotoxic risk. No safety concerns would arise from the nicarbazin impurities p‐nitroaniline and methyl(4‐nitrophenyl) carbamate. The lowest no observed effect level (NOEL) identified for monensin sodium in a developmental study in rabbits was 0.3 mg monensin sodium/kg body weight (bw) per day for maternal toxicity in rabbits. The lowest no observed adverse effect level (NOAEL) identified in a 52‐week study in rat using DNC + HDP was 20 mg DNC + 8 mg HDP/kg bw per day. No significant interaction between monensin sodium and nicarbazin is expected from toxicological studies. The use of Monimax® at the highest proposed dose will not pose a risk to persons consuming animal products from treated turkeys for fattening. No withdrawal time is required for Monimax® in turkeys for fattening. Residue data comply with the established maximum residue limits for monensin and DNC. Monensin sodium presents a hazard by inhalation and may also be associated with dermal toxicity. Monimax® is not a skin irritant; however, no data are available for the eye irritation potential of monensin. Monimax® is not a skin sensitiser. Based on the available data, the FEEDAP Panel cannot conclude on the safety of Monimax® for the environment. Monimax® has the potential to control coccidiosis in turkeys for fattening at a minimum concentration of 40 mg monensin and 40 mg nicarbazin/kg complete feed.

Investigation of boron mediated reproductive and developmental effects in highly boron exposed population

This review presents up-to-date information about current research on nicarbazin, one of the most used anticoccidials in poultry production. The focus is to elucidate regulation concerning nicarbazin, limits for its residues in food, how maximum residue limits in different countries are calculated regarding edible chicken tissues and the possible implications in human health. Analytical methods to extract and quantify this residue, expressed as dinitrocarbanilide (DNC) are presented and discussed, including qualitative screening and quantitative/confirmatory analytical methods. Monitoring results and occurrence of DNC residues in chicken meat are discussed. Additionally, the causes of eventual chicken meat contamination and possible solutions to reduce or eliminate DNC residue in tissues are also presented. The paper concludes with perspectives, the current state of DNC residue analysis and suggestions for future research, especially considering the gap in the study of residue recycling effect due to continuous chicken litter use.

Determination of the residue levels of nicarbazin and combination nicarbazin-narasin in broiler chickens after oral administration.

The depletion times of the anticoccidial nicarbazin administered individually and of nicarbazin and narasin administered in combination were evaluated by determining the presence and levels of 4,4'-dinitrocarbanilide (DNC), the marker residue for nicarbazin, and narasin residues in the muscle tissues of broiler chickens subjected to a pharmacological treatment. A high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was used. The results showed the presence of all anticoccidial residues; however, the DNC levels were higher when the nicarbazin was administered individually than when it was used in association with narasin throughout the experimental period. After six days of withdrawal, the DNC level following nicarbazin administration alone was lower than the maximum residue level (MRL) of 200 μg kg-1. However, when the nicarbazin was co-administered with narasin, the concentrations of DNC were lower than the MRL after four days of withdrawal. These results may be justified because the dosage of nicarbazin, when administrated individually, is greater than when it is used in combination with narasin. The levels of narasin were lower than the MRL of 15 μg kg-1 throughout the evaluation period. It was concluded that nicarbazin is rapidly metabolized from the broiler muscles up to six days of withdrawal since the DNC levels were lower than the maximum residue level (MRL) and the concentrations of narasin were lower than the MRL throughout the evaluation period.

A novel ion-pairing chromatographic method for the simultaneous determination of both nicarbazin components in feed additives: Chemometric tools for improving the optimization and validation

The development, optimization and validation of an ion-pairing high performance liquid chromatography method for the simultaneous determination of both nicarbazin (NIC) components: 4,4′-dinitrocarbanilide (DNC) and 2-hydroxy-4,6-dimethylpyrimidine (HDP) in bulk materials and feed additives are described. An experimental design was used for the optimization of the chromatographic system. Four variables, including mobile phase composition and oven temperature, were analyzed through a central composite design exploring their contribution to analyte separation. Five responses: peak resolutions, HDP capacity factor, HDP tailing and analysis time, were modelled by using the response surface methodology and were optimized simultaneously by implementing the desirability function. The optimum conditions resulted in a mobile phase consisting of 10.0mmolL−1 of 1-heptanesulfonate, 20.0mmolL−1 of sodium acetate, pH=3.30 buffer and acetonitrile in a gradient system at a flow rate of 1.00mLmin−1. Column was an INERSTIL ODS-3 (4.6mm×150mm, 5μm particle size) at 40.0°C. Detection was performed at 300nm by a diode array detector. The validation results of the method indicated a high selectivity and good precision characteristics, with RSD less than 1.0% for both components, both in intra and inter-assay precision studies. Linearity was proved for a range of 32.0–50.0μgmL−1 of NIC in sample solution. The recovery, studied at three different fortification levels, varied from 98.0 to 101.4 for HDP and from 99.1 to 100.2 for DNC. The applicability of the method was demonstrated by determining DNC and HDP content in raw materials and commercial formulations used for coccidiosis prevention. Assays results on real samples showed that considerable differences in molecular ratio DNC:HDP exist among them.