G. O. Korsrud

Determination of penicillin G in bovine plasma by high-performance liquid chromatography after pre-column derivatization

A simple, selective, and sensitive liquid chromatographic method with ultraviolet detection was developed for the analysis of penicillin G in bovine plasma. The assay utilizes a simple extraction of penicillin G from plasma (with a known amount of penicillin V added as internal standard) with water, dilute sulphuric acid and sodium tungstate solutions, followed by concentration on a conditioned C18 solid-phase extraction column. After elution with 500 microliters of elution solution, the penicillins are derivatized with 500 microliters of 1,2,4-triazole-mercuric chloride solution at 65 degrees C for 30 min. The penicillin-mercury mercaptide complexes are separated by reversed-phase liquid chromatography on a C18 column. The method, which has a detection limit of 5 ng/ml (ppb) in bovine plasma, was used to quantitatively measure the concentrations of penicillin G in plasma of steers at a series of intervals after the intramuscular administration of a commercial formulation of procaine penicillin G.

Residue depletion in tissues and fluids from swine fed sulfamethazine, chlortetracycline and penicillin G in combination

Twenty-four hogs were fed a ration for 14 days containing three times the recommended label dose of a combination drug which included sulfamethazine, chlortetracycline and penicillin G. Groups of six hogs were slaughtered 0, 2, 4, or 8 days after withdrawal. Six untreated control hogs were slaughtered 5 days before the first group, of six treated hogs, were slaughtered. Residue concentrations were determined in kidney, liver, muscle, serum and urine. At zero withdrawal the kidney from one hog contained 0.018 mg penicillin G per kg and the serum from the same hog contained 0.016 mg penicillin G per litre. Penicillin G was not detected in any other samples that were analysed. Chlortetracycline concentrations in tissues at zero withdrawal time were below accepted Canadian Maximum Residue Limits (MRL) for chlortetracycline of 1 mg/kg in muscle, 2 mg/kg in liver and 4 mg/kg in kidney and were below the limit of quantitation in all tissues 4 days after withdrawal. Sulfamethazine persisted in the tissues longer than penicillin G or chlortetracycline. Sulfamethazine concentrations were above the Canadian MRL of 0.1 mg/kg at zero withdrawal time and did not decrease to below the MRL until 8 days after withdrawal. Our results suggest that, if the label withdrawal period of 10 days is observed, an increase in the dosage of up to three times the recommended rate is unlikely to increase significantly the risk that residues would occur in the tissues of treated hogs at concentrations which exceed MRLs. Sulfamethazine concentrations in all matrices decreased after storage at -76 degrees C for 6 months.

Evaluation of the Swab Test on Premises, the Calf Antibiotic and Sulfa Test, and a Microbial Inhibitor Test with Standard Solutions of 22 Antibiotics

The sensitivities of the Swab Test on Premises (STOP), the Calf Antibiotic and Sulfa Test (CAST) and a laboratory Microbial Inhibitor Test (MIT) previously used for confirmation of STOP results were compared. Different media were compared for the STOP and MIT. The CAST was more sensitive than the STOP for the 22 antibiotics tested. The sensitivity of the STOP procedure increased for 15 of the 22 antibiotics tested when the standard STOP medium, Antibiotic Medium No. 5 (AM-5), was replaced with Saskatoon Antibiotic Medium-3 (SAM-3), the medium used for thin-layer bioautography. When Antibiotic Medium No. 2 (AM-2) was used in the STOP procedure, the overall sensitivity was intermediate between that obtained using AM-5 and SAM-3. For the MIT, Micrococcus luteus ATCC 9341 provided greater sensitivity as a test organism than Bacillus cereus ATCC 11778 for 16 antibiotics and was less sensitive for 4 antibiotics, particularly chlortetracycline, oxytetracycline and tetracycline. M. luteus ATCC 9341 was notably more sensitive as a test organism for bacitracin and lincomycin than was B. cereus ATCC 11778.

Performance of five screening tests for the detection of penicillin G residues in experimentally injected calves

Three calves were each injected with a single intramuscular (IM) dose of penicillin G procaine at either 3750, 7500, or 15000 IU per kg of body weight and killed at 24 h postinjection, along with a control calf that had not received penicillin. Tissues were tested by the Swab Test on Premises (STOP), the Calf Antibiotic and Sulfa Test (CAST), the Brilliant Black Reduction Test (BBRT), the Charm Test II, thin layer chromatography - bioautography (TLC/BA), and high performance liquid chromatography (HPLC). Samples of muscle, liver, and kidney from all injected calves contained detectable penicillin residues when analyzed by HPLC. The BBRT and Charm Test II were the most sensitive test kits for penicillin G in muscle, while the Charm Test II also detected residues in livers and kidneys from all injected animals. The STOP and CAST were less sensitive, although improved performance was observed for the STOP using a modified growth medium. Penicillin residues were detected in all livers and kidneys from injected animals using TLC/BA. Urine collected from injected animals 12 and 24 h postinjection was positive by the Live Animal Swab Test (LAST). All urine and tissue samples from the control animal were negative. The BBRT and Charm Test II appear to offer greater sensitivity for penicillin G residues than such currently used procedures as STOP and CAST but should be confirmed by a suitable laboratory method, such as the HPLC procedure used in this study.

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 β-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 β-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).