Dietary intake estimates as a means to the harmonization of maximum residue levels for veterinary drugs. II. Proposed application to the Free Trade Agreement between the United States and Canada.

The Free Trade Agreement between the United States and Canada (FTA) went into effect January 1, 1989. To implement certain provisions of the agreement on technical regulations and standards, the United States Food and Drug Administration's Center for Veterinary Medicine, Health and Welfare Canada's Bureau of Veterinary Drugs, and Agriculture Canada established the Working Group on Veterinary Drug Tolerances. The progress of the working group and its continuing efforts to harmonize tolerances for approximately 15 veterinary drugs are discussed. This paper proposes use of the toxicologically determined acceptable daily intake (ADI) for the drug as the safety standard for reaching conclusions on the acceptability of residues in meat for human consumption. Specifically, the "equivalence" of different MRLs for the same veterinary drug would be determined by considering whether they are likely to result in dietary residues that exceed the other country's ADI for the drug. Estimates are made for the veterinary drugs lasalocid, fenbendazole, morantel tartrate, halofuginone, and tilmicosin. Based on these estimates, the U.S. and Canadian MRLs for each drug would be considered equivalent for trade purposes.

High pressure liquid chromatographic determination of nitrofurazone and furazolidone in chicken and pork tissues.

A high pressure liquid chromatographic (HPLC) procedure is presented for the determination of nitrofurazone and furazolidone in chicken and pork tissues in the 2-40 ppb range. Muscle, liver, and kidney are homogenized with cold methanol and water (50 + 50). Following methanol evaporation, the nitrofurans are partitioned into ethyl acetate and cleaned up on an alumina column. After elution with 20% methanol in ethyl acetate and evaporation to dryness, residues are determined by HPLC, using a reverse phase analytical column. Overall average recoveries for nitrofurazone and furazolidone were 65.7 and 73.5%, respectively. Average relative standard deviations of 11.9% (nitrofurazone) and 9.5% (furazolidone) at the 2 ppb level were achieved.

Determination of penicillin G, ampicillin, and cephapirin residues in tissues.

A cylinder plate microbiological method was developed for the rapid, quantitative determination of penicillin G, ampicillin, and cephapirin in animal tissues. The method uses agar plates seeded with stable spores of Bacillus stearothermophilus var. calidolactis and incubated 4 h at 64 degrees C. Standard curves were obtained for the following ranges of concentration of antibiotics in tissues: 0.02-0.32 IU penicillin G/g, 0.0125-0.2 micrograms ampicillin/g, and 0.02-0.32 micrograms cephapirin/g. The proposed method is suitable not only for penicillin residue analysis, for which the sensitivity has been greatly improved compared with the Sarcina lutea method, but also for depletion studies on these antibiotics, which are commonly used to treat diseases in food-producing animals.

Depletion of Brilliant Blue F.C.F., Penicillin G and Dihydrostreptomycin in Milk from Treated Cows with Experimentally Induced Mastitis.

A field trial was set up to compare depletion of Brilliant Blue F.C.F., penicillin and dihydrostreptomycin following three infusions of Streptopen cerate 100 to six cows with experimentally induced staphylococcal mastitis. The presence of dye was noted by milk color at the time of milking, and by the ion exchange resin method in the laboratory. Penicillin residues were determined by a diffusion agar plate microbiological method using Sarcina lutea and by Delvotest P. Dihydrostreptomycin residues were measured by the diffusion agar plate method ( Bacillus subtilis ). Dye extinction times were found to be significantly longer than penicillin and dihydrostreptomycin extinction times in both the infected sides and the healthy sides. No significant differences were observed between healthy and infected sides for dye, penicillin or dihydrostreptomycin extinction.

Microbiological determination of penicillin G, ampicillin, and cloxacillin residues in milk.

A fast cylinder plate microbiological method was developed for the quantitative determination of penicillin G, ampicillin, and cloxacillin in milk. Agar plates seeded with stable spores of Bacillus stearothermophilus var. calidolactis were used and incubated at 64 degrees C for 4 1/2 hr. Standard curves were obtained for the following ranges of concentration of antibiotics: 0.004-0.064 IU penicillin G/mL, 0.0025-0.04 microgram ampicillin/mL, and 0.03-0.48 microgram cloxacillin/mL. The method is suitable for detecting penicillin residues in milk and for quantitative milk-out studies of the above antibiotics used in treatment of bovine mastitis.

Isolation and identification of a fluorophore from ampicillin degradation.

A fluorescent impurity in ampicillin has been isolated and identified as 2-hydroxy-3-phenylpyrazine. The product is formed under acidic conditions similar to those employed in some fluorometric assay procedures. The mechanism of the reaction is proposed to involve cyclization by condensation of the penilloaldehyde of ampicillin. The structure was confirmed by independent synthesis.

High pressure liquid chromatographic determination of nitrofurazone in milk.

A rapid high pressure liquid chromatographic (HPLC) screening method for the quantitative determination of nitrofurazone in milk has been developed. The drug is extracted with ethyl acetate from a 2.0 ml milk serum sample, the organic layer is evaporated to dryness, and the residue is dissolved in the mobile phase and injected into the liquid chromatogarph. A reverse phase muBondapak C18 column is used with monitoring at 365 nm. The detection limit is 5 ppb and recoveries are 57--67%. Mass spectroscopic confirmation of the HPLC nitrofuran peak is described.

Depletion of Brilliant Blue F.C.F. and Penicillin G in Milk from Treated Cows.

A field trial was set up to compare depletion of Brilliant Blue F.C.F. and penicillin following three infusions of Vetspen cerate 100 into six healthly lactating cows. The presence of dye was noted by milk color at the time of milking, and by the ion exchange resin method in the laboratory. Penicillin residues were determined by a diffusion agar plate microbiological method and by Delvotest-P. Dye extinction times were found to be significantly longer than penicillin extinction times (0.05 level of significance). The proportion of cows requiring more milkings for penicillin extinction than for dye extinction was estimated to be 0.17.

Detection of Tracer Dyes in Milk.

An improved ion-exchange resin method to detect low levels of dye marker Brilliant Blue F.C.F. (C.I. No. 42090) in milk was developed. Sensitivity of 0.02 mg of dye per liter (corresponding to 0.008 I.U. of penicillin per milliliter), and semi-quantitative adaptation of the method is described.

Immunological properties of dialysis retentates from penicillin.

Retentates from benzylpenicillin preparations marketed in Canada were used in immunological studies in order to determine if they were immunogenic in experimental animals with respect to anaphylactic antibody production. The retentates obtained consisted mainly of penicillin polymers. Impurities of protein nature were either insignificant or absent. The penicillin polymers were found to be non-immunogenic in Albino Wistar rats, Wistar Furth rats and Hartley guinea pigs. Furthermore, they were unable to effect positive PCA tests in rats passively sensitized to the penicilloyl determinant.