Determination and confirmation of nicarbazin, measured as 4,4-dinitrocarbanilide (DNC), in chicken tissues by liquid chromatography with tandem mass spectrometry: First Action 2013.07.

A single-laboratory validation (SLV) study was conducted on an LC/MS/MS method for the determination and confirmation of nicarbazin, expressed as 4,4-dinitrocarbanilide (DNC), in chicken tissues, including liver, kidney, muscle, skin with adhering fat, and eggs. Linearity was demonstrated with DNC standard curve solutions using a weighted (1/x) regression and confirmed with matrix-matched standards. Intertrial repeatability precision (relative standard deviation of repeatability; RSD(r) was from 2.5 to 11.3%, as determined in fortified tissues. The precision was verified with incurred tissue, and varied from 0.53 to 2.5%. Average recoveries ranged from 82% in egg to 98% in kidney. Although the average recoveries across all concentrations were within the acceptable range, the method was improved with the inclusion of an internal standard and the use of matrix-matched standards. Accuracy for the improved method in chicken liver varied from 93 to 99% across all concentrations (100-8000 ng/g) compared to recoveries below 80% at concentrations, between 100-400 ng/g in chicken liver for the original method. The limit of detection was estimated to be less than 3.0 ng/g in all tissue types, and the limit of quantitation was validated at 20 ng/g. Based on confirmatory ion ratios and peak retention times, the false-negative rate was estimated as 0.00% (95% confidence limits 0.00, 0.74%) from 484 fortified samples and 12 incurred residue samples analyzed using the U.S. and EU confirmation criteria. Small variations to the method parameters, with the exception of injection volume, did not have a significant effect on recoveries. Stability was determined for fortified tissues, extracts, and standard curve solutions. The data collected in this study satisfy the requirements of SLV studies established by the AOAC Stakeholder Panel for Veterinary Drug Residue and the method was awarded First Action Official Method status by the Expert Review Panel for Veterinary Drug Residues on May 7, 2013.

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.

4,4'-Dinitrocarbanilide (DNC) concentrations in egg shells as a predictor of nicarbazin consumption and DNC dose in goose eggs.

Nicarbazin is being investigated as an infertility agent for the control of non-migratory Canada geese (Branta canadensis L) populations. Nicarbazin is presently registered for use as a coccidiostat for poultry. Geese fed sufficient quantities of nicarbazin will lay non-viable eggs. We established nicarbazin consumption by measuring the concentration of a component of the formulation, 4,4'-dinitrocarbanilide (DNC) in the egg contents (yolk, albumin) in non-viable eggs. To estimate the nicarbazin consumption of birds that laid viable eggs (eggs that hatched or contained an embryo), a high-performance liquid chromatography method was developed to measure the concentration of DNC in egg shells. A statistically significant correlation was established using linear regression between the mean concentrations of DNC in the egg shell and in the egg contents in non-viable eggs. Viable eggs were estimated to contain lower levels of DNC than non-viable eggs. DNC concentrations in both the egg contents and the egg shell increased with increases in nicarbazin dose in feed. Our method allows for the estimation of nicarbazin consumption and DNC dose in eggs under field conditions, which is important in developing an effective infertility agent for over-abundant non-migratory goose populations.