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

Abstract Background AOAC 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. Objective To analyze data from residue depletion studies to support Final Action status. Method Ten 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. Results For 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. Conclusions The data and positive feedback support the transition of AOAC Method 2013.07 from First Action to Final Action. Highlights Final 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.

narasin) exist for use in broilers and turkeys globally (registrations include regions such as the United States, the European Union, Australia, New Zealand, Malaysia, and Canada); however, due to a reduction in reproductive capacity, nicarbazin is not approved for laying hens. Nicarbazin is also approved for control of feral pigeon populations in urban areas (2). Nicarbazin is composed of equimolar amounts of 4,4 0 -dinitrocarbanilide (DNC) and 2-hydroxy-4,6-dimethyl pyrimidine (HDP), with DNC being the more persistent residue (3) and thus the marker residue used for screening of edible poultry tissues. As a result of the wide reach of global approvals for nicarbazin, a robust and reproducible method to enable surveillance across the globe is of significant importance to enable trade and ensure a safe food supply.
Codex Alimentarius and EU Maximum Residue Limits (MRLs) and U.S. tolerances for nicarbazin, measured and expressed as DNC concentrations, in various chicken tissues are presented in Table 1. The U.S. tolerances were updated from 4000 to 52 000 mg/kg in liver tissue in 2018, which resulted in a much higher range than the method was originally intended to cover. A dilution procedure was described in the method to accommodate the expanded range for U.S. tolerance, thereby allowing for continued applicability of this method for use in monitoring nicarbazin residues in the United States.
The AOAC Expert Review Panel (ERP) for Veterinary Drug Residues granted First Action status to AOAC Method 2013.07 in 2013 (4). This report presents data to support the recommendation that the method be ascribed Final Action status.

Analysis of Physiologically Incurred Nicarbazin Drug Residues in Chicken and Eggs Conducted in Four Laboratories
Ten residue depletion studies were conducted to generate data to support drug registration for nicarbazin as a feed additive for chicken. These studies included administering label doses of nicarbazin to the food animals of interest (chicken) and withdrawing the animals following the last drug administration to establish withdrawal periods to demonstrate that when nicarbazin is administered to chicken as per label instructions, nicarbazin drug residues will not be detected above the regulatory safe limits such as MRLs/tolerances established for chicken tissues and eggs.
A fully validated analytical method meeting the requirements of ISO/IEC 17025:2017 (5) and Veterinary International Conference on Harmonization (VICH) criteria (6) was required to be used for the analysis of tissue samples generated in a residue depletion study to support product registration. In that respect, AOAC Method 2013.07, which had been validated under single laboratory conditions and had been accorded AOAC First Action status, was used to measure the concentration of the nicarbazin residues in chicken tissues harvested from experimental chicken over the course of the withdrawal period for each of these 10 studies.
This innovative approach was used in place of the traditional approach, where 10 to 12 laboratories were required to participate in a multi-laboratory study to generate reproducibility precision data.
Data are presented in this article to support Final Action recommendation for AOAC Method 2013.07. The data include standard curves, method quality control (QC) test portion results, nicarbazin residues measured in the residue depletion study, and feedback on the use of the method from the laboratories that conducted the drug residues analysis.
The four laboratories that analyzed the nicarbazin drug residues in the physiologically induced incurred tissue samples and in eggs were the Eurofins Food Integrity and Innovation Laboratory ( [Applicable for the determination and identification of nicarbazin (measured and expressed as 4,4 0 -dinitrocarbanilide; DNC) in chicken liver, kidney, muscle, and skin with adhering fat tissues, and in eggs.] Caution: Solvents employed are common use solvents and reagents. Refer to adequate manuals or safety data sheets to ensure that the safety guidelines are applied before using chemicals. Store in a flammable liquid storage cabinet. Harmful if inhaled, swallowed, or absorbed through the skin. Use appropriate personal protective equipment such as a lab coat, safety glasses, rubber gloves, and a fume hood. Dispose of all materials according to federal, state, and local regulations.

A. Principle
Poultry tissue is cryogenically homogenized with solid sodium sulfate, and then extracted twice with acetonitrile. Extracts are combined, filtered, and diluted accordingly based on the regulatory limits being targeted and the working concentrations of the standards used for LC-MS/MS analysis. Identification is accomplished by comparing the product ions measured in the samples to those present in the standard injections in mass and relative intensity, and comparison of chromatographic retention times between samples and standards. Nicarbazin determination and identification is based on the DNC portion of the molecule as are the regulatory limits and tolerances. Concentrations are determined by LC-MS/MS using a matrix-matched standard curve   When ordered from Eli Lilly and Co., the order will be accompanied by a certificate of analysis that gives details on the DNC purity. Store at 15 to 30 C. Consult the MSDS for safety and handling information.   Note: Different volumes of equivalent concentrations may be substituted.

D. Preparation of Reagents and Standards
Note: Store all stock standards and standard solutions at room temperature protected from light. Stock standards are stable for 3 months and standard solutions for 14 days under these conditions.

E. Sample Preparation
(a) Homogenization and storage of samples.-Initial processing includes grinding or blending of the tissues using cryogenic grinding to produce homogeneous samples. Cryogenic grinding is carried out by freezing the tissue with liquid nitrogen or dry ice and then grinding into a fine powder using a Foss or Robot Coupe grinder or a Waring blender. This process is used to produce a very fine homogeneous powder of the tissue for analysis. Grind a minimum 500 g sample of tissue when possible. Subsamples of 5.00 6 0.05 g tissue (1.00 6 0.05 g for kidney) may be weighed into 50 mL polypropylene tubes and frozen. This will minimize tissue exposure to multiple freeze/thaw cycles. Store all tissues at freezer temperatures (À20 C or below) when not processing or subsampling. It is advisable to store fortified samples of all tissues with experimental samples to verify storage stability. (1) Accurately weigh 5.00 6 0.05 g (1.00 6 0.05 g for kidney) of a representative ground sample of frozen or partially thawed sample into a 50 mL conical polypropylene centrifuge tube.   mass 301.0 amu, Q3 mass 136.7 amu, collision energy À16 V, collision cell exit potential À11 V, entrance potential À6 V; DNC, Q1 mass 301.0 amu, Q3 mass 106.9 amu, collision energy À48 V, collision cell exit potential À7V, entrance potential À4 V; DNC-d 8 , Q1 mass 308.7 amu, Q3 mass 140.6 amu, collision energy À16 V, collision cell exit potential À7 V, entrance potential À6 V.
where A ¼ sample concentration from standard curve (ng/mL); B ¼ extract volume (mL); C ¼ weight of tissue sample (g); and D ¼ dilution factor. (1) Obtain the individual ion chromatograms for the product ions and ensure that the chromatographic retention times for the analytes are 65% relative to the mean retention time of the appropriate analyte in the standard. Extracts may be reinjected if there has been a sudden shift in retention time during the batch analysis exceeding the 5% tolerance. (2) Integrate the area of the DNC peak for each selective reaction monitoring (SRM) trace for the standards and samples. From the integrated area values for DNC, represent the determinative ion as 100% (m/z 301.0!136.7) and calculate the abundance of the identification ion (m/z 301.0!106.9) as a relative percentage for each standard and sample. Using the mean ion abundance percentages (IAP) of the standard solutions within a chromatographic run, calculate the U.S. acceptance range (7) as mean 6 10% arithmetic difference for the samples within that run. For example, at 20% mean IAP of standards the U.S. acceptance range would be 10-30% IAP for samples within that run. For the EU (8), the acceptance range is 6 40% relative to the mean IAP of standards. For example, at 20% mean IAP of standards, the EU acceptance range would be 12-28% for the samples within that run. (h) Standard curve acceptability criteria.-The following criteria will be used for determining curve acceptability: (1) Back-calculated accuracy for any standard curve point must be within 615% of the theoretical value (620% of the theoretical value at the lower limit of quantitation).
(2) Individual data points may be excluded in a given batch provided the curve maintains a minimum of five different concentrations and the standards bracket the QC and unknown test portions. (i) QC acceptability criteria.-The following criteria will be used for determining QC acceptability: (1) Determine recovery of the QC test portions as recovery ¼ (concentration/actual fortification level) Â 100.

Standard Curves
Matrix-matched standard curves were prepared and analyzed according to the method. A representative curve for chicken liver is shown in Figure 1. Weighting and regression data of representative standard curves from each laboratory for each matrix are summarized in Table 2. The standard curve acceptability criteria are found in Section F(g) of the method and include: back-calculated accuracy for any standard curve point must be within 615% of the theoretical value (620% of the theoretical value at the LLOQ); and individual data points may be excluded in a given batch provided the curve maintains a minimum of five different concentrations and the standards bracket the QC and unknown test portions.
In Laboratory 1, the following additional criteria were applied: the coefficient of correlation (r) should be !0.990; 75% of the calibration standards should comply with the backcalculated accuracy requirements; and the calibration curve must have at least one calibration standard at the highest level (ULOQ) and another at the lowest level (LLOQ).
In Laboratory 3, the following additional criteria were applied: the coefficient of correlation (r) should be !0.980; the data should pass the homoscedasticity test (Cochran's test); the data should demonstrate little to no multicollinearity; and the data should show no autocorrelation.
In all cases, the applied criteria were met on each day of testing. It should be noted that the axes and slopes of the standard curves differ based on the concentration units used in the software for calculations. In some cases, the solution concentration (ng/mL) was used, and in other cases the corresponding tissue concentration (mg/kg) was used.

Quality Control Test Portion Samples
QC test portions were prepared and analyzed according to the method, which requires at least one matrix-matched QC test portion fortified at the regulatory level (MRL or tolerance) for each tissue analyzed. QC acceptance criteria are found in Section F(h) of the method and include: the determination of the recovery of the QC test portions as Recovery ¼ (concentration/actual fortification level)*100 and QC test portions must meet the recovery requirements [e.g., 70-110% at !10 mg/kg to <100 mg/kg from the VICH Guideline (6)].
U.S. Food and Drug Administration recovery requirements (5, harmonized with the EU and Japan) were followed and are shown in Table 3. Additional QC sample criteria from the Bioanalytical Method Validation Guidance for Industry (9) were applied in all cases and included the following: !67% of all QC samples must meet the recovery criteria, and at least 50% of QC replicates at each concentration must meet the recovery criteria. Table 4 presents the results of the QC samples analyzed for all 10 studies. Unless otherwise noted, QC replicates were tested on one day (within run) for each tissue type. Not all studies examined all tissue types.
The grand mean recovery from QC test portion sample analyses across all studies were 91.1% for muscle, 95.8% for skin with adhering fat, 95.1% for liver, and 91.8% for kidney. A few test portion analytical results did not meet the recovery criteria. These were one muscle tissue test portion at 200 mg/kg; two kidney tissue test portions at 400 mg/kg and 6000 mg/kg in Depletion Study 1; two liver tissue test portions at 4000 mg/kg in Depletion Study 4; three liver tissue test portions at 4000 mg/kg in Depletion Study 6; and one kidney tissue test portion at 20 mg/kg in Depletion Study 9. When the additional QC sample criteria from the Bioanalytical Method Validation Guidance for Industry (9) were applied, only one failure was noted, and that was with the 4000 mg/kg liver tissue sample in which two-thirds of the replicates were outside the acceptance range of 80-110% recovery at >100 mg/kg.
The QC data from Table 4 were then analyzed by tissue type for reproducibility among the 10 depletion studies. Since the various studies did not use common fortification concentrations for the QC test portions, the data were analyzed under the following conditions and assumptions: the relative recovery value or mean relative recovery at each concentration was used as the method result; each fortification concentration was treated as one "replicate" (n ¼ 1) in each study; each matrix was analyzed separately; the overall study design was unbalanced since the number of fortification concentrations in each study for each matrix varied from 1 to 6. Therefore, the data were analyzed in the pC-metamer (pC ¼ Àlog 10 C, where C is concentration) according to LaBudde (10) using a revised statistical workbook (11).
A summary of the statistical analysis by tissue type is presented in Table 5. Relative standard deviation of repeatability ranged from 5.2 to 8.9%, and relative standard deviation of reproducibility ranged from 6.8 to 8.9%. Thus, both repeatability and reproducibility for all tissue types were well below acceptable precision limits (6). It is interesting to note that for skin with adhering fat the repeatability and reproducibility were equal. Since the reproducibility error comprises error from repeatability and error due to laboratories (in this case studies) with the relationship s 2 r þ s 2 L ¼ s 2 R , it is meaningful to ascribe the   reproducibility error to repeatability error entirely with little or no contribution from laboratory/study error. The intraclass correlation coefficient (ICC) is a ratio of the laboratory variance to the reproducibility variance (s 2 L /s 2 R ) and can be used to assess laboratory (or study) homogeneity. For example, an ICC value of 0.5 would indicate that the laboratory variance accounts for half of the total reproducibility variance. An ICC value close to 0 indicates a lack of correlation among replicates, meaning that there is no difference among laboratories and replicate results are homogeneous and likely to be normally distributed. While three of the four tissue sample types subjected to chemical analysis yielded point estimates of ICC greater than 0, the confidence intervals are too broad to draw any conclusions. More data sets are needed to reduce the size of the confidence intervals and improve the point estimates of ICC. Since there are currently no requirement or acceptance criteria for ICC, these estimates are provided for informational purposes only. Finally, the relative recovery (calculated as the reverse transform of the bias in the pC-metamer) was 90.4% (95% CI 83. 8

Comments From Participating Laboratories
Comments were solicited from the four laboratories at the conclusion of studies regarding the performance and ease of use of the method. The comments are listed here.  Comments/feedback from Laboratory 1.-No difficulties were encountered with the technical conduct of the method. Carry-over occurred in a small number of samples. As carry-over is signal dependent, there were some analytical runs where the signal of some reinjected blanks was slightly higher than 20% of the LLOQ. For the following reasons, the carry-over was deemed to have negligible impact on the analyses: it had no effect on calibration curve accuracy, especially for the lower concentrations; it had no effect on QC accuracy; to mitigate the potential impact of carry-over on sample analysis, samples were injected in ascending order of magnitude of expected concentrations based on pre-slaughter withdrawal times. None of the samples with analyte concentration magnitudes approaching the ULOQ were immediately succeeded by samples with concentrations determined at or above the LLOQ, thus indirectly confirming the insignificance of carry-over on the sample analysis; the use of matrix-matched standards and the stable isotope internal standard supported full confidence in the assay results.
The data support consideration of the transition of the method from Official First Action to Official Final Action.
Comments/feedback from Laboratory 2.-No difficulties were encountered in the technical conduct of the method; the tissue assay results were deemed to reliably reflect the recovery values and calibration plots; the use of matrix-matched standards and the stable isotope internal standard supported full confidence in the assay results.
The data support consideration of the transition of the method from Official First Action to Official Final Action.
Comments/feedback from Laboratory 3.-No difficulties were encountered with the technical conduct of the method; no carryover was detected in any of the matrixes; some matrix effect was notable but without significant impact in all four tissue types.
The data support consideration of the transition of the method from Official First Action to Official Final Action.
Comments/feedback from Laboratory 4.-No difficulties were encountered with the physical performance aspects of the method; the tissue assay results were reliable as reflected in the recovery values and the linearity of the calibration plots; the use of matrix prepared standards and the stable isotope internal standard provide complete confidence in the assay results.
The data support the transition of the method from Official First Action to Official Final Action.

Conclusions
Nontraditional multi-laboratory data analyzed in the pCmetamer and associated calibration curves are presented to support Final Action status of AOAC Method 2013.07. The data analysis demonstrated acceptable repeatability and reproducibility with a very small bias observed in the accuracy of the method.