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Screening method for identification of β-lactams in bovine urine by use of liquid chromatography and a microbial inhibition test

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  • 1 College of Veterinary Medicine, Department of Farm Animal Health and Resource Management, North Carolina State University, Raleigh, NC 27606.
  • | 2 Present address is Department of Veterinary Pathobiology, Texas Veterinary Medical Center, Texas A&M University, College Station, TX 77843-4467.
  • | 3 College of Veterinary Medicine, Department of Farm Animal Health and Resource Management, North Carolina State University, Raleigh, NC 27606.
  • | 4 USDA, Agricultural Research Service, Beltsville Agricultural Research Center, Beltsville, MD 20705.

Abstract

Objective—To develop a multiple-residue screening method for the detection of β-lactams in bovine urine.

Animals—6 clinically normal Holstein cows and 6 calves.

Procedure—Pooled urine obtained from cows was used as a negative-control sample or spiked with varying concentrations of 6 β-lactam antibiotics. Urine samples were prepared for liquid chromatography by diluting 1 ml of urine with 9 ml of 0.01M KH2PO4, 0.01M Na2PO4, and filtering. Filtrate (2,000 ml) was eluted with a mobile phase in a gradient program. A fraction corresponding to each β-lactam of interest was collected and evaporated to < 1 ml, and water then was added to achieve a 1ml volume. The collected fraction was tested, using a microbial inhibition test. Then, calves were fed milk spiked with a mixture of 5 β-lactam antibiotics at a concentration 40X the FDA tolerance in milk. Three hours following the feeding, urine samples were obtained from the calves and tested, as described for the urine samples for the cows.

Results—The lowest concentrations of amoxicillin, ampicillin, cephapirin, cloxacillin, desfuroylceftiofurcysteine, and penicillin G that were consistently detected in urine were 100, 10, 100, 250, 1,000, and 10 ng/ml, respectively. Amoxicillin, ampicillin, cephapirin, cloxacillin, desacetylcephapirin, and penicillin G were detected in urine samples of 6/6, 5/6, 0/6, 6/6, 2/6, and 3/6 calves respectively, fed antibiotic- spiked milk.

Conclusions and Clinical Relevance—The integrated method described can be used to detect or identify β-lactam antibiotics in bovine urine. This method can be used to test cattle for β-lactam residues. (Am J Vet Res 2001;62:326–330)

Abstract

Objective—To develop a multiple-residue screening method for the detection of β-lactams in bovine urine.

Animals—6 clinically normal Holstein cows and 6 calves.

Procedure—Pooled urine obtained from cows was used as a negative-control sample or spiked with varying concentrations of 6 β-lactam antibiotics. Urine samples were prepared for liquid chromatography by diluting 1 ml of urine with 9 ml of 0.01M KH2PO4, 0.01M Na2PO4, and filtering. Filtrate (2,000 ml) was eluted with a mobile phase in a gradient program. A fraction corresponding to each β-lactam of interest was collected and evaporated to < 1 ml, and water then was added to achieve a 1ml volume. The collected fraction was tested, using a microbial inhibition test. Then, calves were fed milk spiked with a mixture of 5 β-lactam antibiotics at a concentration 40X the FDA tolerance in milk. Three hours following the feeding, urine samples were obtained from the calves and tested, as described for the urine samples for the cows.

Results—The lowest concentrations of amoxicillin, ampicillin, cephapirin, cloxacillin, desfuroylceftiofurcysteine, and penicillin G that were consistently detected in urine were 100, 10, 100, 250, 1,000, and 10 ng/ml, respectively. Amoxicillin, ampicillin, cephapirin, cloxacillin, desacetylcephapirin, and penicillin G were detected in urine samples of 6/6, 5/6, 0/6, 6/6, 2/6, and 3/6 calves respectively, fed antibiotic- spiked milk.

Conclusions and Clinical Relevance—The integrated method described can be used to detect or identify β-lactam antibiotics in bovine urine. This method can be used to test cattle for β-lactam residues. (Am J Vet Res 2001;62:326–330)