Distribution of Flunixin Residues in Muscles of Dairy Cattle Dosed with Lipopolysaccharide or Saline and Treated with Flunixin by Intravenous or Intramuscular Injection.

Twenty dairy cows received flunixin meglumine at 2.2 mg/kg bw, administered once daily by either the intravenous (IV) or intramuscular (IM) route for three consecutive days with either intravenous normal saline (NS) or lipopolysaccharide (LPS) providing a balanced design with five animals per group. Cows were sacrificed after a 4 day withdrawal period, and 13 muscle types were collected and assayed for flunixin by LC-MS/MS. After elimination of sample outliers, the main effects of route of administration (IV or IM), treatment (NS or LPS), and tissue type significantly (P < 0.05) affected flunixin residues, with no interaction (P > 0.05). Intramuscular (nonlabel) flunixin administration produced greater (P < 0.05) flunixin residues in muscle than the IV (label) administration, whereas LPS resulted in lower flunixin levels. Differences among the tissue levels indicate it is necessary to specify the tissue to be used for any monitoring of drug levels for consumer protection.

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.

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.

Validation of a streamlined multiclass, multiresidue method for determination of veterinary drug residues in bovine muscle by liquid chromatography-tandem mass spectrometry.

Multiclass, multiresidue methods are becoming increasingly popular in regulatory monitoring programs due to their increased analytical scope and laboratory efficiency. In this work, we report the development and validation of a new high-throughput analytical method to monitor up to 131 veterinary drug residues, representing at least 13 different classes, in bovine muscle. This novel method streamlined sample preparation to <15 min/sample/analyst, or a batch of 40-60 pre-homogenized samples in <3 h/analyst, through the combination of dispersive solid-phase extraction with in-vial filtration (a new technique known as filter-vial d-SPE). The use of an enhanced sensitivity state-of-the-art tandem mass spectrometer led to <10 ng/g limits of quantification for nearly all drug analytes with injection of 0.17 mg of equivalent sample. Positive and negative switching in electrospray ionization was applied to cover all analytes in an 11-min liquid chromatographic separation. In the 3-day validation study, 100 of the drugs met quantification criteria of 70-120% recoveries and Horwitz Ratio ≤1.0, and the remaining analytes could still be screened at regulatory target levels. In the validation study involving >11,400 analyte results for spiked samples, the rate of false negatives for identification purposes was <5%, and no false positives occurred at appreciable concentrations.

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.

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.

Development and validation of a streamlined method designed to detect residues of 62 veterinary drugs in bovine kidney using ultra-high performance liquid chromatography--tandem mass spectrometry.

In the USA, the US Department of Agriculture's Food Safety and Inspection Service (FSIS) conducts the National Residue Program designed to monitor veterinary drug and other chemical residues in beef and other slaughtered food animals. Currently, FSIS uses a 7-plate bioassay in the laboratory to screen for antimicrobial drugs in bovine kidneys from those animals tested positive by inspectors in the slaughter establishments. The microbial inhibition bioassay has several limitations in terms of monitoring scope, sensitivity, selectivity, and analysis time. Ultra-high performance liquid chromatography - tandem mass spectrometry (UHPLC-MS/MS) has many advantages over the bioassay for this application, and this study was designed to develop, evaluate, and validate a fast UHPLC-MS/MS method for antibiotics and other high-priority veterinary drugs in bovine kidney. Five existing multi-class, multi-residue methods from the literature were tested and compared, and each performed similarly. Experiments with incurred samples demonstrated that a 5-min shake of 2 g homogenized kidney with 10 ml of 4/1 (v/v) acetonitrile/water followed by simultaneous clean-up of the initial extract with 0.5 g C18 and 10 ml hexane gave a fast, simple, and effective sample preparation method for the <10 min UHPLC-MS/MS analysis. An extensive 5-day validation process demonstrated that the final method could be used to acceptably screen for 54 of the 62 drugs tested, and 50 of those met qualitative MS identification criteria. Quantification was not needed in the application, but the method gave ≥ 70% recoveries and ≤ 25% reproducibilities for 30 of the drugs.

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.

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.

Comparison of screening methods for antibiotics in beef kidney juice and serum.

Rapid screening tests can be used as part of an efficient program designed to monitor veterinary drug residues in cattle. In this work, three rapid tests designed to screen samples for the presence of antibiotic residues, the Fast Antimicrobial Screen Test (FAST), Premi and Kidney Inhibition Swab (KIS) tests, were compared using beef kidney juice and serum samples. In order to provide a realistic assessment, potentially incurred samples of beef kidney juice and serum were obtained from 235 carcasses which had been retained by inspectors in a processing plant for further testing. In addition, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was conducted on these samples to identify what antibiotics were present, if any, and their levels. The comparison of the three rapid screening test results with those from LC-MS/MS analysis allowed for a more complete comparison of the relative sensitivity of these analytical methods, as well as valuable information on false positive and negative response rates.

Methods for the analysis of fluoroquinolones in biological fluids.

Methods for the analysis of ten selected fluoroquinolone antibiotics in biological fluids are reviewed. Approaches for sample preparation, detection methods, limits of detection and quantitation, and recovery information are provided for both single analyte and multi-analyte fluoroquinolone methods.

A rapid fluorescence assay for danofloxacin in beef muscle: effect of muscle type on limit of quantitation.

A simple, rapid fluorescence screening assay was applied to the analysis of beef muscle for danofloxacin at the U.S. tolerance level of 200 ng/g. Muscle samples were homogenized in acetic acid-acetonitrile, the resultant mixture centrifuged, and fluorescence of the supernatants was then measured. The significant difference between the fluorescence of control muscle sample extracts and extracts of samples fortified at 200 ng/g allowed for successful discrimination between the samples. Setting a threshold level at the average 200 ng/g fortified sample extract fluorescence -3sigma allowed for identification of potentially violative samples. Successful analysis of a group of blind fortified samples over a range of concentrations was accomplished in this manner, without any false-negative results. The limits of quantitation for danofloxacin, as well as enrofloxacin, using this assay were determined in three types of beef muscle (hanging tenderloin, neck, and eye round steak), as well as in serum. Significant differences in limits of quantitation were found among the three different muscle types examined, with hanging tenderloin muscle providing the lowest value. This work not only shows the potential for use of the fluorescence screening assay as an alternative to currently used microbial or antibody-based assays for the analysis of danofloxacin in beef muscle, but also suggests that assays using beef muscle may vary in performance depending on the specific muscle selected for analysis.

A comparison of the FAST, Premi and KIS tests for screening antibiotic residues in beef kidney juice and serum.

Three microbial inhibition-based screening methods, the fast antimicrobial screening test (FAST), the Premi test, and the kidney inhibition swab (KIS) test, were evaluated using penicillin G, sulfadimethoxine, oxytetracyline, tylosin, danofloxacin, streptomycin, neomycin, and spectinomycin at a range of fortified concentrations in beef kidney juice and beef serum. Each antibiotic was individually tested simultaneously using the different assays in replicate experiments. Detection threshold concentrations for each analyte in each screening assay were determined for the different matrices. Each assay gave a different detectability profile for the different antibiotics, with the largest differences related to neomycin, which was more sensitively detected by the FAST, and penicillin G, which was detected at lower levels by the Premi and KIS tests. In addition to practical considerations, analysts can use the information presented in this study to evaluate each kit for applicability to their monitoring needs.

Simultaneous multiresidue determination of tetracyclines and fluoroquinolones in catfish muscle using high performance liquid chromatography with fluorescence detection.

Efficient methods are needed for analysis of veterinary drug residues in food. A number of methods are available for single analytes. Multiresidue methods are now increasingly available. It is still rare, however, to find methods not involving mass spectrometry which allow for analysis of more than one class of drug residue. An efficient multiresidue method for the simultaneous determination of fluoroquinolones (FQs) and tetracyclines (TCs) in catfish muscle has now been developed. This method involves an extraction of the analytes with a mixture of acetonitrile and citrate buffer containing magnesium chloride. After centrifugation and evaporation of the supernatants, the residues are determined using high performance liquid chromatography with fluorescence detection. With this method, five fluoroquinolones and three tetracyclines were determined in fortified catfish muscle at levels of 20, 50, and 100 ng g(-1). Average recoveries for ciprofloxacin (CIP), sarafloxacin (SAR), danofloxacin (DANO), enrofloxacin (ENRO), difloxacin (DIF), oxytetracycline (OTC), tetracycline (TC), and chlortetracycline (CTC) were in the range of 60-92% with good relative standard deviations. The limits of quantitation ranged from 0.15 to 1.5 ng g(-1). Utilization of the method to successfully analyze catfish muscle samples incurred with enrofloxacin and with oxytetracycline is described.

Simultaneous determination of fluoroquinolones and tetracyclines in chicken muscle using HPLC with fluorescence detection.

A multiresidue method has been developed which allows for the simultaneous determination of both fluoroquinolones and tetracyclines in chicken muscle. Samples were extracted with a mix of acetonitrile and 0.1 M citrate, 150 mM MgCl(2), pH 5.0. After centrifugation and evaporation, the extracts could be analyzed by liquid chromatography with fluorescence detection. Good recoveries (63-95%) were obtained from samples fortified with a mix of five fluoroquinolones and three tetracyclines, with satisfactory relative standard deviations. Limits of detection were 0.5 ng/g (danofloxacin), 1 ng/g (oxytetracycline, ciprofloxacin, enrofloxacin), 1.5 ng/g (tetracycline), 2 ng/g (difloxacin) and 5 ng/g (sarafloxacin, chlortetracycline). Enrofloxacin and its metabolite ciprofloxacin, as well as oxytetracycline were determined in enrofloxacin and oxytetracycline incurred chicken muscle using this method.

Evaluation of serum as a potential matrix for multiresidue determination of fluoroquinolone antibiotics in chicken using liquid chromatography-fluorescence-mass spectrometry(n).

An efficient multiresidue method was successfully applied to the determination of fluoroquinolones (FQs) in chicken serum. In this method, FQs are extracted from matrix with ammoniacal acetonitrile, and the extracts are defatted and then evaporated. After addition of basic phosphate buffer and filtration, the samples are analyzed by liquid chromatography-fluorescence-mass spectrometry(n) (multiple mass spectrometry; MS(n)). This approach allows for simultaneous quantitation (fluorescence) and confirmation (MS(n)) of the FQs. Using this method, 8 FQs were determined in fortified chicken serum at levels of 10, 20, 50, and 100 ng/g. Recoveries ranged from 71-99%, with excellent relative standard deviations (< 10%). Limits of quantitation for the FQs ranged from 0.05-5 ng/g. Confirmation was achieved by comparison of MS2 or MS3 product ion ratios with those of standard FQ samples. These quantitative and confirmatory results were compared with those obtained for muscle using this approach. Serum and muscle samples from enrofloxacin-dosed chickens were also analyzed with this method. The results show that enrofloxacin can be determined in both serum and muscle of chickens dosed at a level formerly approved by the U.S. Food and Drug Administration, for up to at least 48 h after withdrawal from dosing, and suggest that serum can provide an efficient matrix for monitoring FQ levels in chicken.

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.

Multiresidue determination of fluoroquinolones in shrimp by liquid chromatography-fluorescence-mass spectrometry.

An efficient multiresidue method for analysis of fluoroquinolones in shrimp has been developed in which quantitation by fluorescence and confirmation by Multiple Stage Mass Spectrometry (MS) is achieved simultaneously. In this method, shrimp tissue is extracted with ammoniacal acetonitrile and the extract is defatted and then evaporated. After dissolution in basic phosphate buffer, fluoroquinolones in the extract are separated by liquid chromatography and quantitated, taking advantage of their intense fluorescence. Eluate from the fluorescence detector enters the MS, which allows for confirmation by monitoring ratios of 2 prominent product ions in the MS3 or MS2 spectrum. Using this method, 8 fluoroquinolones have been analyzed in shrimp samples fortified at 10, 25, 50, or 100 ppb levels. Recoveries for desethyleneciprofloxacin, norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, orbifloxacin, sarafloxacin, and difloxacin ranged from 75 to 92%, with relative standard deviation values of <6%. The limits of quantitation ranged from 0.1 to 1 ng/g. Enrofloxacin and ciprofloxacin were also successfully determined in enrofloxacin-incurred shrimp using this method.

Rapid fluorescence screening assay for enrofloxacin and tetracyclines in chicken muscle.

A simple, rapid fluorescence assay was developed for screening both enrofloxacin (ENRO) and tetracyclines in chicken muscle at the U.S. tolerance levels (300 ng/g and 2 microg/g, respectively). Screening for both classes of antibiotics is accomplished using one extraction, thus simplifying and expediting the process. The method requires an initial extraction of chicken muscle with 1% acetic acid in acetonitrile, centrifugation, and analysis of the supernatant for ENRO fluorescence. After addition of ammonium hydroxide, magnesium chloride, and methanol, followed by centrifugation and filtration, the supernatant can be measured for tetracycline fluorescence. Chlortetracycline (CTC) was chosen as a representative tetracycline to demonstrate the method, as it displays intermediate sensitivity among the three tetracyclines approved in the U.S. Comparison of the fluorescence of control and tolerance-level-fortified samples of both ENRO and CTC shows no overlap. Setting a threshold as the average fortified fluorescence minus 3sigma allows for successful screening, as illustrated with blind samples as controls or fortified with ENRO and/or CTC over a range of concentrations. This method can provide an alternative or supplemental approach to currently used microbial screening assays.

Rapid fluorescence screening assay for tetracyclines in chicken muscle.

A simple, rapid fluorescence assay was developed for screening tetracyclines in chicken muscle at the U.S. tolerance level (2 mg/kg). The method requires only a homogenization of the tissue in acetonitrile-ammonium hydroxide, centrifugation, addition of Mg+2, and another centrifugation before fluorescence of the supernatant is measured at 505 nm (excitation at 385 nm). Comparison of the fluorescence of control chicken muscle extracts with extracts from muscle fortified with either 2 mg/kg tetracycline, oxytetracycline, or chlortetracycline showed no overlap. A threshold level set at the average fluorescence for a series of fortified 2 mg/kg samples minus 3sigma minimized false-negative responses to provide a successful screening method. The method was tested with blinded samples as controls or samples fortified with tetracycline, oxytetracycline, or chlortetracycline in order to demonstrate its utility. This approach can provide an alternative to microbial screening assays.