Marilyn J. Schneider

Multiresidue analysis of fluoroquinolone antibiotics in chicken tissue using liquid chromatography-fluorescence-multiple mass spectrometry.

An efficient liquid chromatographic method for the multiresidue analysis of fluoroquinolone antibiotics in chicken tissue has been developed in which quantitation using fluorescence and confirmation with multiple mass spectrometry (MS(n)) was achieved simultaneously. Using this method, eight fluoroquinolones were analyzed in fortified samples of chicken liver and muscle tissue with recoveries at levels of 10-200 ng/g generally in the range of 60-93%, except for desethylene ciprofloxacin, which consistently gave recoveries >or=45%. Relative standard deviations were excellent in all cases, and the limits of detection in ng/g were determined as follows in liver and (muscle): desethylene ciprofloxacin 0.3 (0.1), norfloxacin 1.2 (0.2), ciprofloxacin 2 (1.5), danofloxacin 0.2 (0.1), enrofloxacin 0.3 (0.2), orbifloxacin 1.5 (0.5), sarafloxacin 2 (0.6), difloxacin 0.3 (0.2). Confirmation of the identities of the fluoroquinolones was achieved by monitoring the ratios of two prominent product ions in MS(2) (desethylene ciprofloxacin) or MS(3) (all others). Levels of confirmation as related to ion ratio variability criteria were established. Enrofloxacin and ciprofloxacin were also determined in enrofloxacin incurred chicken liver and muscle using this method.

Evaluation of a multi-class, multi-residue liquid chromatography – tandem mass spectrometry method for analysis of 120 veterinary drugs in bovine kidney

Traditionally, regulatory monitoring of veterinary drug residues in food animal tissues involves the use of several single-class methods to cover a wide analytical scope. Multi-class, multi-residue methods (MMMs) of analysis tend to provide greater overall laboratory efficiency than the use of multiple methods, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) of targeted drug analytes usually provides exceptional performance even for complicated sample extracts. In this work, an LC-MS/MS method was optimized and validated in a test of 120 drug analytes from 11 different classes in bovine kidney. The method used 10 ml of 4/1 acetonitrile/water for extraction of 2 g samples and cleanup with hexane partitioning. Quantitative and qualitative performance was assessed for the analytes at fortification levels of 10, 50, 100, and 200 ng/g. With the method, 66 drugs gave 70-120% recovery with ≤ 20% RSD at all levels over the course of 3 days. At the 200 ng/g level, 89 drugs met these same standards. Limits of detection were ≤ 10 ng/g for 109 of the analytes in the kidney matrix in validation experiments. Qualitatively, MS/MS identification criteria were set that ion ratios occur within ± 10% (absolute value) from those of the analyte reference standards. At the 10 ng/g level, 57% of the drugs met the identification criteria, which improved to 84% at the 200 ng/g level. The method serves as an efficient and useful additional option among the current monitoring methods available.

Concentrations of antibiotic residues vary between different edible muscle tissues in poultry.

Antibiotics are used by veterinarians and producers to treat disease and improve animal production. The federal government, to ensure the safety of the food supply, establishes antibiotic residue tolerances in edible animal tissues and determines the target tissues (e.g., muscle) for residue monitoring. However, when muscle is selected as the target tissue, the federal government does not specify which type of muscle tissue is used for monitoring (e.g., breast versus thigh). If specific muscle tissues incorporate residues at higher concentrations, these tissues should be selected for residue monitoring. To evaluate this possibility in poultry, chickens were divided into four groups and at 33 days of age were dosed with enrofloxacin (Baytril), as per label directions, at either 25 ppm for 3 days, 25 ppm for 7 days, 50 ppm for 3 days, or 50 ppm for 7 days. Breast and thigh muscle tissues were collected from each bird (n = 5 birds per day per group) during the dosing and withdrawal period, and fluoroquinolone concentrations were determined. The results indicate higher overall enrofloxacin concentrations in breast versus thigh muscle for each treatment group (P < 0.05). These data indicate, at least for enrofloxacin, that not all muscle tissues incorporate antibiotics at the same concentrations. These results may be helpful to regulatory agencies as they determine what tissues are to be monitored to ensure that the established residue safety tolerance levels are not exceeded.

Europium-sensitized luminescence determination of oxytetracycline in catfish muscle

An europium-sensitized time-resolved luminescence (TRL) method was developed to determine oxytetracycline (OTC) in cultivated catfish muscle. Extraction of OTC from fish muscle was performed with pH 4.0 ethylenediaminetetraacetic acid (EDTA)-McIlvaine buffer and clean up with hydrophilic-lipophilic balanced copolymer solid phase extraction (SPE) cartridges. The eluate was used without further concentration for TRL measurement in pH 9.0 micellar tris(hydroxylmethyl)aminomethane (TRIS) buffer. Cetyltrimethylammonium chloride (CTACl) was used as surfactant and EDTA as a co-ligand. The excitation and emission wavelengths were set at 388 and 615nm, respectively. The linear dynamic range was 0-1000ngg(-1) (R(2)=0.9995). The recovery was 92-112% in the fortification range of 50-200ngg(-1) and the limits of detection (LOD) ranged from 3 to 7ngg(-1). Incurred catfish samples were used to demonstrate the performance of the method around 100ngg(-1), the European Union maximum residue level.

Distribution of penicillin G residues in culled dairy cow muscles: implications for residue monitoring.

The U.S. Food and Drug Administration sets tolerances for veterinary drug residues in muscle but does not specify which type of muscle should be analyzed. To determine if antibiotic residue levels are dependent upon muscle type, seven culled dairy cows were dosed with penicillin G (Pen G) from 1 to 3 days and then sacrificed on day 1, 2, or 5 of withdrawal. A variety (9-15) of muscle samples were collected, along with liver and kidney samples. In addition, corresponding muscle juice samples were prepared. All samples were extracted and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine Pen G levels. Results showed that Pen G residue levels can vary between and within different muscles, although no reproducible pattern was identified between cows or withdrawal times. Muscle juice appeared to be a promising substitute for muscle as a matrix for screening purposes. Because of the potential for variation within muscles, all samples taken need to be large enough to be representative.

Expansion of the Scope of AOAC First Action Method 2012.25--Single-Laboratory Validation of Triphenylmethane Dye and Leuco Metabolite Analysis in Shrimp, Tilapia, Catfish, and Salmon by LC-MS/MS.

Prior to conducting a collaborative study of AOAC First Action 2012.25 LC-MS/MS analytical method for the determination of residues of three triphenylmethane dyes (malachite green, crystal violet, and brilliant green) and their metabolites (leucomalachite green and leucocrystal violet) in seafood, a single-laboratory validation of method 2012.25 was performed to expand the scope of the method to other seafood matrixes including salmon, catfish, tilapia, and shrimp. The validation included the analysis of fortified and incurred residues over multiple weeks to assess analyte stability in matrix at -80°C, a comparison of calibration methods over the range 0.25 to 4 μg/kg, study of matrix effects for analyte quantification, and qualitative identification of targeted analytes. Method accuracy ranged from 88 to 112% with 13% RSD or less for samples fortified at 0.5, 1.0, and 2.0 μg/kg. Analyte identification and determination limits were determined by procedures recommended both by the U. S. Food and Drug Administration and the European Commission. Method detection limits and decision limits ranged from 0.05 to 0.24 μg/kg and 0.08 to 0.54 μg/kg, respectively. AOAC First Action Method 2012.25 with an extracted matrix calibration curve and internal standard correction is suitable for the determination of triphenylmethane dyes and leuco metabolites in salmon, catfish, tilapia, and shrimp by LC-MS/MS at a residue determination level of 0.5 μg/kg or below.

Determination of Triphenylmethane Dyes and Their Metabolites in Salmon, Catfish, and Shrimp by LC-MS/MS Using AOAC First Action Method 2012.25: Collaborative Study.

A collaborative study was conducted to evaluate the AOAC First Action 2012.25 LC-MS/MS analytical method for the determination of residues of three triphenylmethane dyes (malachite green, crystal violet, and brilliant green) and their metabolites (leucomalachite green and leucocrystal violet) in seafood. Fourteen laboratories from the United States, Canada, and the European Union member states participated in the study including national and state regulatory laboratories, university and national research laboratories, and private analytical testing laboratories. A variety of LC-MS/MS instruments were used for the analysis. Each participating laboratory received blinded test samples in duplicate of salmon, catfish, and shrimp consisting of negative control matrix; matrix fortified with residues at 0.42, 0.90, and 1.75 μg/kg; and samples of incurred matrix. The analytical results from each participating laboratory were evaluated for both quantitative residue determination and qualitative identification of targeted analytes. Results from statistical analysis showed that this method provided excellent trueness (generally ≥90% recovery) and precision (RSDr generally ≤10%, HorRat<1). The Study Directors recommend Method 2012.25 for Final Action status.

Development and model testing of antemortem screening methodology to predict required drug withholds in heifers.

A simple, cow-side test for the presence of drug residues in live animal fluids would provide useful information for tissue drug residue avoidance programs. This work describes adaptation and evaluation of rapid screening tests to detect drug residues in serum and urine. Medicated heifers had urine, serum, and tissue biopsy samples taken while on drug treatment. Samples were tested by rapid methods and high-performance liquid chromatography (HPLC). The adapted microbial inhibition method, kidney inhibition swab test, was useful in detecting sulfadimethoxine in serum, and its response correlated with the prescribed withdrawal time for the drug, 5 to 6 days posttreatment. The lateral flow screening method for flunixin and beta-lactams, adapted for urine, was useful in predicting flunixin in liver detected by HPLC, 96 h posttreatment. The same adapted methods were not useful to detect ceftiofur in serum or urine due to a lack of sensitivity at the levels of interest. These antemortem screening test studies demonstrated that the method selected, and the sampling matrix chosen (urine or serum), will depend on the drug used and should be based on animal treatment history if available. The live animal tests demonstrated the potential for verification that an individual animal is free of drug residues before sale for human consumption.

Time‐Resolved Luminescence Screening Assay for Tetracyclines in Chicken Muscle

Abstract A time‐resolved luminescence (TRL) assay was developed for effective screening of tetracyclines in chicken muscle at the European Union (EU) maximum residue level of 100 ng g−1. The method involves extraction of the tetracyclines with McIlvaine–ethylenediamine tetraacetic acid (EDTA) buffer, centrifugation, solid‐phase extraction (SPE) clean‐up, formation of an Eu(III) complex, and then measurement of the TRL signal at 615 nm (excitation at 388 nm). Samples fortified with tetracycline (TC), oxytetracycline (OTC) or chlortetracycline (CTC) gave a linear response over the range of 0–1000 ng g−1, with relative sensitivities 5.4×, 2.4×, and 1×, respectively. Limits of detection for TC, OTC, and CTC were 3.5, 8, and 19 ng g−1, respectively. Examination of the least sensitive case, CTC, showed no overlap between the TRL of control chicken extracts and those that had been fortified with 100 ng g−1 CTC. The within‐day variation for these samples averaged 3.1% relative standard deviation (RSD), as did the day‐to‐day variation. The method was tested with blind control and fortified samples over the range of 20–500 ng g−1 CTC to illustrate its utility. Other veterinary drugs approved for chickens in the US (enrofloxacin, nicarbazin, tylosin) did not interfere. This method can provide a useful alternative to microbial screening assays for tetracyclines. #Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.

Terbium-sensitised luminescence screening method for fluoroquinolones in beef serum

Enrofloxacin and danofloxacin are the only fluoroquinolone antibiotics approved for use in cattle in the United States. Microbial screening methods commonly used for monitoring veterinary drug residues are not sensitive or selective for fluoroquinolones. In this work, a luminescence-based screening assay was developed to detect fluoroquinolones in beef serum. This approach takes advantage of the DNA-enhanced luminescence signal of a fluoroquinolone–Tb+3 complex. In this method, serum samples were extracted with acidified acetonitrile in the presence of magnesium sulfate. After centrifugation, evaporation of the supernatant was followed by dissolution of the residue in buffer and filtration. Addition of Tb+3 and DNA then allowed a reading of the luminescence signal. The technique was illustrated using enrofloxacin, and provided good recoveries (73–88%) at 25, 50 and 100 ng ml−1, with reasonable RSDs averaging at 11%. The LOD was 2.5 ng ml−1 based on the variability of response of control serum samples from 18 different steers. The method provided no false-positive or false-negative results while screening blind samples for enrofloxacin and was demonstrated to be quantitative over a range of 0–100 ng ml−1.