Determination of Tilmicosin in Bovine, Swine, Chicken, and Turkey Tissues by Liquid Chromatography With Tandem Mass Spectrometry, Single-Laboratory Validation.

BACKGROUND: An LC-MS/MS method was developed for determination and confirmation of tilmicosin in bovine, swine, chicken, and turkey tissues (liver, kidney, muscle, and skin/fat) and bovine milk. OBJECTIVE: The method was subjected to single-laboratory validation to establish method performance parameters. METHOD: Animal tissues and bovine milk were fortified at four concentrations ranging from 0.5 times the lowest maximum residue limit (MRL) or tolerance to 2 times the highest MRL or tolerance considering the Codex and EU MRLs and the US tolerances in the various tissues and milk studied. Incurred tissues were analyzed to verify the precision of the method. RESULTS: The data demonstrated linearity of matrix-matched calibration curves using a weighted (1/×) regression. Recoveries varied from 83.3 to 107.1%. Repeatability precision (RSDr) ranged from 0.465 to 13.4% and intermediate precision (RSDi) ranged from 2.24 to 14.7% in fortified tissue. Repeatability of the method was verified in incurred tissues, ranging from 3.41 to 16.0%. The limits of detection and quantitation of the method are presented and vary by matrix. One confirmatory transition ion was examined across all matrixes and met US and EU criteria for mass spectrometry confirmation. The method was shown to be robust when small changes in method parameters were made, and stability of the analyte in fortified tissues, extracts, standard solutions, and matrix-matched standards was estimated. CONCLUSIONS: The data satisfy the requirements of the AOAC Stakeholder Panel for Veterinary Drug Residue Methods for single-laboratory validation studies and the U.S. Food and Drug Administration Center for Veterinary Medicine Guidance for Industry #208 (VICH GL49). HIGHLIGHTS: The LC-MS/MS method was demonstrated to be suitable for determination and confirmation of tilmicosin residues in bovine, swine, chicken, and turkey tissues and bovine milk based on Codex and EU MRLs and US tolerances.

Quantification of ractopamine residues on and in beef digestive tract tissues.

Ractopamine hydrochloride is a commercial beta-adrenergic agonist commonly used as a dietary supplement in cattle production for improved feed efficiency and growth promotion. Currently, regulatory target tissues (as approved in the New Animal Drug Application with Food and Drug Administration) for ractopamine residue testing are muscle and liver. However, other tissues have recently been subjected to testing in some export markets for U.S. beef, a clear disregard for scientific maximum residue limits associated with specific tissues. The overall goal of this study was to develop and validate an LC-MS/MS assay to determine whether detectable and quantifiable levels of ractopamine in digestive tract-derived edible offal items (i.e., abomasum, omasum, small intestine, and reticulum) of cattle resulted from tissue residues or residual ingesta contamination of exposed surfaces of tissues (rinsates). Tissue samples and corresponding rinsates from 10 animals were analyzed for parent and total ractopamine (tissue samples only). The lower limit of quantitation was between 0.03 and 0.66 ppb depending on the tissue type, and all tissue and rinsate samples tested had quantifiable concentrations of ractopamine. The highest concentrations of tissue-specific ractopamine metabolism (represented by higher total vs. parent ractopamine levels) were observed in liver and small intestine. Contamination from residual ingesta (represented by detectable ractopamine in rinsate samples) was only detected in small intestine, with a measured mean concentration of 19.72 ppb (±12.24 ppb). Taken together, these results underscore the importance of the production process and suggest that improvements may be needed to reduce the likelihood of contamination from residual ractopamine in digestive tract-derived edible offal tissues for market.

Determination of Monensin in Bovine Tissues: A Bridging Study Comparing the Bioautographic Method (FSIS CLG-MON) with a Liquid Chromatography-Tandem Mass Spectrometry Method (OMA 2011.24).

The U.S. Department of Agriculture, Food Safety Inspection Service regulatory method for monensin, Chemistry Laboratory Guidebook CLG-MON, is a semiquantitative bioautographic method adopted in 1991. Official Method of AnalysisSM (OMA) 2011.24, a modern quantitative and confirmatory LC-tandem MS method, uses no chlorinated solvents and has several advantages, including ease of use, ready availability of reagents and materials, shorter run-time, and higher throughput than CLG-MON. Therefore, a bridging study was conducted to support the replacement of method CLG-MON with OMA 2011.24 for regulatory use. Using fortified bovine tissue samples, CLG-MON yielded accuracies of 80-120% in 44 of the 56 samples tested (one sample had no result, six samples had accuracies of >120%, and five samples had accuracies of 40-160%), but the semiquantitative nature of CLG-MON prevented assessment of precision, whereas OMA 2011.24 had accuracies of 88-110% and RSDr of 0.00-15.6%. Incurred residue results corroborated these results, demonstrating improved accuracy (83.3-114%) and good precision (RSDr of 2.6-20.5%) for OMA 2011.24 compared with CLG-MON (accuracy generally within 80-150%, with exceptions). Furthermore, χ2 analysis revealed no statistically significant difference between the two methods. Thus, the microbiological activity of monensin correlated with the determination of monensin A in bovine tissues, and OMA 2011.24 provided improved accuracy and precision over CLG-MON.

Determination of Narasin in Chicken Fat: A Bridging Study Comparing the Bioautographic Method (FSIS CLG Method R22) to a Liquid Chromatography with Tandem Mass Spectrometry Method (AOAC Method 2011.24).

Lilly Method AM-AA-CA-R108-AB-755, which is substantially the same as U.S. Department of Agriculture, Food Safety and Inspection Service (FSIS) Chemistry Laboratory Guidebook (CLG) method R22, is the current regulatory method for determining narasin in cattle and chicken tissues and is based on bioautography, creating a zone of inhibition of bacterial growth, with the size of the zone correlating to the amount of narasin extracted from the tissue. AOAC Method 2011.24 is an LC-tandem mass spectrometry (MS/MS) method for determining narasin content from bovine, swine, or chicken tissues. It has many advantages over the regulatory method, including higher throughput, less solvent use, no use of carbon tetrachloride, a wider method range, inclusion of swine tissues, and it is less labor intensive. In this study, AOAC Method 2011.24 was compared to FSIS CLG method R22 for the determination of narasin in chicken abdominal fat. Fortified chicken-fat samples ranging from 20 to 960 ng/g and incurred chicken-fat samples ranging from 40 to 480 ng/g were assayed by both methods in triplicate. Mean accuracies for the two methods were similar, 77-110% for CLG R22 and 84-96% for AOAC Method 2011.24, and the method results showed a linear correlation. The methods differed in precision, however, with the CLG R22 method yielding 2.6-34% RSD and AOAC Method 2011.24 yielding 0.15-6.4% RSD. It is recommended that AOAC Method 2011.24-granted AOAC Official Method(SM) Final Action status-be adopted as the official U.S. regulatory method.

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.

Determination and confirmation of narasin and monensin in chicken, swine, and bovine tissues by LC/MS/MS: Final Action 2011.24.

A multilaboratory study was conducted to validate the reproducibility of AOAC Official Method 2011.24 for determination of narasin and monensin in chicken, swine, and bovine tissues. This study was intended to satisfy requirements for Final Action status through the AOAC Expert Review Panel process. Ten laboratories participated in the study, analyzing blind duplicates of five incurred residue materials for each analyte. After removal of invalid data sets, the method reproducibility (RSDR 12.8-60.6%, HorRat 0.45-1.47) was within AOAC acceptance criteria. The method was awarded Final Action status by the Official Methods Board on October 4, 2012.

Determination and confirmation of parent and total ractopamine in bovine, swine, and turkey tissues by liquid chromatography with tandem mass spectrometry: Final Action 2011.23.

A multilaboratory study of AOAC Official Method 2011.23 was performed to satisfy requirements for Final Action status through the AOAC expert review panel process. The study included nine collaborating laboratories from the United States, Canada, Brazil, and The Netherlands. Five incurred residue materials (bovine muscle, bovine liver, swine muscle, swine liver, and turkey muscle) were analyzed by each laboratory as blind duplicates for parent and total ractopamine content. After removal of invalid data, the parent and total ractopamine methods demonstrated acceptable reproducibility (RSDR 11.4-42.4%, HorRatR 0.34-2.01) based on AOAC criteria. The method was awarded Final Action status by the Official Methods Board on October 4, 2012.

Determination and confirmation of parent and total ractopamine in bovine, swine, and turkey tissues by liquid chromatography with tandem mass spectrometry: First Action 2011.23.

A candidate method selected by the AOAC Expert Review Panel (ERP) for Ractopamine for determination and confirmation of parent and total ractopamine by LC/MS/MS was validated in a single laboratory for bovine, swine, and turkey tissues. The candidate method utilizes methanol extraction of the tissues, followed by an optional enzymatic hydrolysis for determination of total (parent plus conjugate) ractopamine. A mixed-mode cation exchange SPE cartridge is used to purify the initial extract before LC/MS/MS. Matrix-matched standards and a ractopamine-d6 internal standard are used for quantification of parent and total ractopamine in unknown samples. Validation data demonstrated that mean intertrial recoveries for ractopamine across all concentrations tested ranged from 79.7 to 102.2% for parent ractopamine and from 79.0 to 100.0% when a hydrolysis step was included. Intertrial repeatability precision ranged from 2.44 to 11.1% for parent ractopamine and 4.97 to 15.0% with hydrolysis. Estimated LOD values were below 0.1 ng/g and LOQ values were validated at 0.25x the maximum residue limits. The data satisfy the requirements of the AOAC Stakeholder Panel for Veterinary Drug Residue Methods for single laboratory validation studies. The method was awarded Official Methods of Analysis First Action 2011.23 by the AOAC ERP on Veterinary Drug Residues.

Determination of ractopamine in swine, bovine, and turkey tissues by HPLC with fluorescence detection: first action 2011.22.

A candidate LC method proposed by the Expert Review Panel (ERP) for ractopamine was evaluated in a single-laboratory validation (SLV) study. The matrixes examined included bovine liver, kidney, muscle, and fat; swine liver, kidney, muscle, and fat; and turkey liver and muscle. Solution standards were shown to provide a linear response with an unweighted regression. The method demonstrated acceptable precision (HorRat, values 0.25 to 1.38) and recovery (75.4 to 88.8%) in all fortified matrixes. Method precision was verified with incurred residue tissues (bovine liver, kidney, and muscle; swine liver, kidney, and muscle; and turkey liver and muscle), which yielded RSDr values below 16% for all tissues and below 7% for most tissues. Estimated LOQ values ranged from 1.8 to 20.7 ng/g and support the utility of the method in the range of the maximum residue limits or tolerances for the various tissues. The data satisfy the requirements of the AOAC Stakeholder Panel on Veterinary Drug Residue for SLV studies, and the method was adopted Official Methods ofAnalysis First Action 2011.22 by the AOAC ERP on Veterinary Drug Residues.

Determination of narasin and monensin in bovine, swine, and chicken tissues by liquid chromatography with tandem mass spectrometry: first action 2011.24.

The single-laboratory validation (SLV) of an LC-MS/MS method for determination and confirmation of two ionophores, narasin and monensin, in animal tissues is described. The data demonstrated linearity of matrix-matched calibration curves using a weighted (1/x) regression and selectivity of the method for narasin and monensin in the presence of lasalocid, salinomycin, maduramycin, nicarbazin, and sulfadiazine. Recoveries varied from 86.2 to 103.5% for narasin and 89.1 to 105.1% for monensin. Intertrial repeatability precision [relative standard deviation of repeatability (RSDr)] varied from 3.9 to 13.8% for narasin and 3.3 to 16.3% for monensin in fortified tissue. Precision of the method was verified in incurred tissues. The LOQ of the method was validated and ranged from 0.45 ng/g in milk, to 4.0 ng/g in chicken fat, but was 0.75 ng/g for most tissues. Two confirmatory ions for each analyte were examined across all matrixes, resulting in estimated false-negative rates of 0.00% (95% confidence interval of 0.00-0.68%) for monensin ions (540 samples) compared to the U.S. and European Union (EU) acceptance criteria. The confirmatory ions for narasin demonstrated 0.00% false-negative rates (95% confidence interval of 0.00-0.58%) when compared to either the U.S. or EU criteria in 630 samples. The method was robust when small changes in method parameters were made and stability of fortified tissues, extracts, and calibration solutions were estimated. The data satisfy the requirements of the AOAC Stakeholder Panel on Veterinary Drug Residue for SLV studies, and the method was adopted Official Methods of Analysis First Action 2011.24 by the AOAC Expert Review Panel on Veterinary Drug Residues.

Development of subacute cutaneous lupus erythematosus associated with the use of imiquimod to treat actinic keratoses.

The immunomodulating drug imiquimod is approved by the U.S. Food and Drug Administration (FDA) to treat actinic keratoses, non-facial superficial basal cell carcinomas and genital warts. This drug elicits its immunological response by binding to toll-like receptor 7 (TLR-7) on dendritic cells inducing the production of interferon alpha (IFN-alpha) and other inflammatory cytokines. The authors report the case of a 56-year-old female who developed subacute cutaneous lupus erythematosus (SCLE), as well as severe autoimmune retinitis following a vigorous response to imiquimod 5% cream that was prescribed to treat actinic keratoses. Given the important role of IFN-alpha in the pathogenesis of cutaneous lupus, it is likely that imiquimod either induced or unmasked an autoimmune tendency in this patient.