Quantitative screening of stilbenes and zeranol and its related residues and natural precursors in veal liver by gas chromatography-mass spectrometry.

An existing gas chromatography-mass spectrometry-based quantitative screening method for the regulatory analysis of the resorcylic acid lactones zeranol, taleranol, and zearalanone and the stilbene anabolic steroids diethylstilbestrol and dienestrol was extended to include natural precursors of zeranol (zearalenone, alpha-zearalenol, and beta-zearalenol) in veal liver. No changes in sample preparation were required; the instrumental conditions were selected to effect a suitable chromatographic separation and detection of the analytes. Validation experiments were performed to verify the performance and applicability of the extended method for the quantitative screening of the original and additional analytes in veal liver in the concentration range from 0.5 to 2.0 microg/kg. The limits of detection were 0.08-0.19 microg/kg. The limits of quantitation were 0.27-0.64 microg/kg. Recoveries were 29-67%. Combined relative measurement uncertainty estimates were 6-21%.

Determination of beta-agonist residues in bovine urine using liquid chromatography-tandem mass spectrometry.

A multiresidue method was developed and validated to screen bovine urine samples for 10 beta-2-adrenergic agonistic drugs--brombuterol, cimaterol, clenbuterol, clenpenterol, isoxsuprine, mabuterol, ractopamine, ritodrine, salbutamol, and tulobuterol--at the 2 microg/L level. The method is also quantitative in the range of 1 to 4 microg/L for all analytes except salbutamol. The procedure uses enzymatic digestion, liquid-liquid extraction, and cleanup on solid-phase extraction columns, followed by detection using a liquid chromatograph-tandem quadrupole mass spectrometer operated in the positive-ion atmospheric pressure chemical ionization multiple-reaction monitoring mode. Method validation included assessment of recoveries, repeatabilities, linearity of responses, decision limits, and detection capabilities. Overall average recoveries ranged from 70-91%; recoveries were generally lower for salbutamol. The decision limits ranged from 0.4-1.0 microg/L, and detection capabilities from 0.6-1.7 microg/L.

Determination of beta-agonists in liver and retina by liquid chromatography-tandem mass spectrometry.

A liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for the determination of bromobuterol, cimaterol, clenbuterol, clenpenterol, hydroxymethylclenbuterol, isoxsuprine, mabuterol, ractopamine, ritrodrine, salbutamol, terbutaline, and tulobuterol residues in bovine liver and retina is reported. This procedure uses enzymatic digestion, liquid-liquid extraction, and cleanup on Oasis HLB solid-phase extraction cartridges, followed by determination of the residues by LC-tandem quadrupole MS using atmospheric pressure chemical ionization in the positive ion mode. Overall average recoveries ranged from 23 to 76% for liver and 34 to 77% for retina. The mean values for samples fortified at levels between 0.5-2.0 microg/kg (liver) and 5-20 microg/kg (retina) agreed within 98-118% of the spiked levels, with coefficients of variation ranging from 6 to 20%. The decision limits, CCalpha, ranged from 0.1 to 0.3 microg/kg for liver, 1-3 microg/kg for retina, and detection capabilities, CCbeta, from 0.2-0.5 microg/kg for liver and 2-5 microg/kg for retina.

Single-laboratory validation of a modified liquid chromatographic method with UV detection for determination of trenbolone residues in bovine liver and muscle.

Trenbolone acetate is a synthetic testosterone analog registered for use in a number of countries as a growth-promoting hormone, applied as an implant in the ears of feedlot cattle. The method is intended for the detection and quantitation of trace amounts of alpha- and beta-trenbolone in bovine tissues (muscle, liver) by liquid chromatography (LC) with UV detection and eliminates the use of the structural analog, 19-nortestosterone, as an internal standard. Trenbolone residues are extracted from tissues that have been homogenized in sodium acetate with a 3-phase liquid-liquid extraction by adding a mixture of water-acetonitrile-dichloromethanehexane, with trenbolone residues preferentially partitioned into the middle acetonitrile layer. The extract is passed through solid-phase extraction cartridges (both C18 and silica gel) using, respectively, methanol-water and acetone-toluene as eluents. Reversed-phase high-performance LC separation is performed, an octadecyl-bonded column with methanol-acetonitrile-water used as mobile phase for sample analysis. The limit of detection is 0.2 ng/g in muscle tissue and 0.6 ng/g in liver tissue, with coefficients of variation of 3.5-12.1% for alpha- and beta-trenbolone at concentrations from 0.2 to 4.0 ng/g fortified in muscle and 3.3-26.0% from liver fortified at 0.6-10.0 ng/g. Absolute recoveries of 40-130% were observed, but the use of fortified matrix curves eliminated recovery correction. Critical control points were identified in a pH adjustment step and an evaporation step during method validation, which included ruggedness testing. Analysis of incurred tissues (bovine liver and muscle) stored at -20 degrees C for over 25 weeks did not identify any significant loss of residues.

Validation of screening method for residues of diethylstilbestrol, dienestrol, hexestrol, and zeranol in bovine urine using immunoaffinity chromatography and gas chromatography/mass spectrometry.

A method was developed, using commercially available immunoaffinity chromatography cleanup cartridges, followed by detection by gas chromatography/mass spectrometry, to screen for residues of the hormone growth promotants diethylstilbestrol, dienestrol, hexestrol, and zeranol in bovine urine. The single-laboratory, in-house validation included assessment of recoveries, repeatability, linearity of response, detection capability, and specificity (cross-reactivity) with a suite of antibiotics and other hormonal growth promotants. The method was validated for screening at a target concentration of 2.0 microg/L in urine. The detection capabilities for the analytes were diethylstilbestrol, 0.24; dienestrol, 0.15; hexestrol, 0.84; and zeranol, 0.28 microg/L.

Laboratory Testing of the Charm Test II Receptor Assays and the Charm Farm Test with Tissues and Fluids from Hogs Fed Sulfamethazine, Chlortetracycline, and Penicillin G.

The potentials of the Charm Test II receptor assays for the detection of residues of sulfonamides, tetracyclines, and ß-lactams and the Charm Farm Test for screening for antimicrobial residues were tested. Market hogs were fed rations containing three times the label level of sulfamethazine, chlortetracycline, and penicillin G for 2 weeks. Groups were killed after 0, 2, 4 and 8 days of withdrawal. Quantitative chemical methods were used to determine residue levels in fluids and tissues. Results were compared with those obtained using the qualitative Charm Test II receptor assays and the Charm Farm Test antimicrobial inhibition assay. For the Charm Test II assays for sulfonamides, tetracyclines and ß-lactams, respectively, 1.6, 5, and 6% of the results were false positive and 7, 5, and 0% were false negative, based on the limits of detection for the test kits and the quantitative results. On a similar basis for the Charm Farm Test, 16% of the results were false positive (6 kidney, 4 muscle, and 6 urine samples) and 1% were false negative (sulfamethazine in one urine sample).

Laboratory Evaluation of the Charm Farm Test for Antimicrobial Residues in Meat.

Charm Farm Tests were applied to 54 muscle and 44 kidney bovine samples, and 95 muscle and 90 kidney porcine samples collected for Canada's national meat inspection program from animals suspected of containing antimicrobial residues. The assays were run in conjunction with Agriculture and Agri-food Canada's routine confirmation analyses for suspect samples collected at federally inspected packing plants. When the bovine and porcine results were combined, 19% of the kidney and 8% of the muscle results were false negatives. Using the Charm Farm Test to screen only the kidney samples would have resulted in 100% identification of the muscle samples that were found to contain violative levels of drug residues. One percent of the kidney and 16% of the muscle results were false positives, on the basis of the results of the confirmatory tests. Minimum detectable levels found in fortified (i.e., artificially contaminated) muscle and kidney with the Charm Farm Test for ceftiofur, chlortetracycline, oxytetracycline, tetracycline, erythromycin, gentamycin, neomycin, penicillin G, streptomycin, sulfamethazine, sulfadimethoxine, tylosin, tilmicosin, and trimethoprim were similar to those claimed by the manufacturer. Minimum detectable levels for the Charm Test II (dihydro) streptomycin and erythromycin receptor assays are reported for fortified muscle and kidney to provide an indication of the relative sensitivities of the Charm Farm Test and the Charm II Tests for these compounds. To further assess the Charm Farm Test's potential as a replacement for existing tests in packing plants, parallel testing needs to be conducted on fresh tissues in a plant environment.