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OBJECTIVE: To develop a multiple-residue screening method for the detection of beta-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 beta-lactam antibiotics. Urine samples were prepared for liquid chromatography by diluting 1 ml of urine with 9 ml of 0.01M KH2PO4, 0.01 M Na2PO4, and filtering. Filtrate (2,000 ml) was eluted with a mobile phase in a gradient program. A fraction corresponding to each beta-lactam of interest was collected and evaporated to < 1 ml, and water then was added to achieve a 1 ml volume. The collected fraction was tested, using a microbial inhibition test. Then, calves were fed milk spiked with a mixture of 5 beta-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 beta-lactam antibiotics in bovine urine. This method can be used to test cattle for beta-lactam residues.

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The potential for antibiotic residues in calves from consuming milk containing penicillin G or amoxicillin was investigated. Six calves were fed milk replacer, 6% body weight twice daily, containing 0.293, 2.92, or 5.85 microg of penicillin/ml (ppm) G or 0.25, 1.0, or 2.0 microg of amoxicillin/ml for three consecutive feedings. Urine and blood samples were collected after each feeding. Serum and urine samples were tested with a microbial receptor assay and a microbial growth inhibition assay to indicate potential drug residues. Penicillin G and amoxicillin were detected in the serum and urine of several calves 3 h after drinking spiked milk replacer. Possible violative drug residues in the calves were detected by the microbial growth inhibition assay up to 15 h after drinking spiked milk replacer. Penicillin G, but not amoxicillin, could be detected in urine 24 h after the final feeding of spiked milk replacer. Subsequently, six calves were fed milk replacer containing 11.7 microg of penicillin G/ml (ppm) twice daily, 6% body weight per feeding. Calves were slaughtered 3 h after the final feeding. Mean (+/-SD) concentrations of penicillin G measured by high-pressure liquid chromatography in liver, kidney, muscle, and serum were 0.409 (+/-0.167) microg/g, 0.031 (+/-0.012) microg/g 0.008 (+/-0.002) microg/g, and 0.013 (+/-0.006) mg/ml, respectively. This study indicates that calves fed milk with amoxicillin or penicillin G could possibly have violative residues if slaughtered within 24 h after feeding. Violative drug residues in liver tissue were found in calves slaughtered 3 h after consuming milk replacer containing 11.7 microg of penicillin G/ml (ppm).

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A simplified procedure was developed for determination of tetracycline antibiotics in tissues which improved stability of these compounds in sample extracts and eliminated the need for troublesome cleanup procedures. Tissues were homogenized in water. Acetonitrile (16 mL) and then 1 mL of 0.1 M H(3)PO(4) were added to 4 mL of homogenate and the clear supernatant was filtered. The filtrate was mixed with hexane and dichloromethane and the resulting water layer was collected, evaporated to 1-2 mL, and filtered into autosampler vials. Ion-pairing liquid chromatography was used to separate tetracyclines from interferences in sample extracts, eliminating the need for further cleanup. Analysis was isocratic using a Phenomonex Prodigy ODS(3) column with a mobile phase of 4 mM oxalic acid, 4 mM sodium oxalate, 10 mM sodium decanesulfonate-acetonitrile (70 + 30 for oxytetracycline and tetracycline; 66 + 34 for chlortetracycline). Recoveries were generally in the 90-100% range with limits of quantitation of 0. 05-0.1 ppm. The procedure was evaluated with beef and pork muscle, liver, and kidney.

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Cephapirin is one of six beta-lactam antibiotics approved for use in the treatment of food-producing animals in the United States. When used for treatment of mastitis by intramammary infusion, it is partially converted to a microbiologically active metabolite identified as deacetylcephapirin (DACEP). The degradation was followed in four cows with naturally acquired mastitis which were treated with cephapirin. DACEP persisted longer than the parent compound in the milk. When a calf was treated with cephapirin by intramuscular injection, the compound was almost completely converted to DACEP in tissues. The deacetyl form must be considered in the determination of residues in treated animals.

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Fifty-four milk samples from commercial sources that tested positive for beta-lactam antibiotics were analyzed by a multiresidue liquid chromatographic (LC) procedure based on LC fractionation. Penicillin G and cephapirin were the beta-lactam antibiotics found most frequently. Some samples did not contain detectable beta-lactam antibiotics. In a few, the presence of a beta-lactam antibiotic was suspected because certain LC fractions tested positive for antimicrobial activity, which was no longer present in a replicate treated with beta-lactamase. However, the unknowns could not be identified by LC analysis.

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Heat stability of antibiotics in foods to cooking has been determined by a variety of methods. These include heating in such liquid media as milk, water, buffers and meat extracts, and in solids such as buffered meat homogenates and various sausages. Inactivation of incurred residues in tissues and eggs was also studied. Time and temperature of heating were more easily controlled in liquid media, but results in actual meat products are more indicative of actual cooking processes. Ordinary cooking procedures for meat, even to "well-done", cannot be relied on to inactivate even the more heat sensitive compounds such as penicillins and tetracyclines. More severe heating as for canning or prolonged cooking with moist heat can inactivate the more heat sensitive compounds. The relevance to food safety is uncertain since the nature of the degradation products is unknown in most cases.

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Antibiotic residues in animal tissues can be detected by various screening tests based on microbial inhibition. In the 7-plate assay used by the U.S. Department of Agriculture's Food Safety and Inspection Service (FSIS), penicillinase is incorporated into all but one plate to distinguish beta-lactam antibiotics from other types. However, beta-lactams such as cloxacillin and the cephalosporins are resistant to degradation by penicillinase. They may not be identified as beta-lactams by this procedure, and thus, they may be identified as unidentified microbial inhibitors (UMIs). However, these penicillinase-resistant compounds can be degraded by other beta-lactamases. The present study describes an improved screening protocol to identify beta-lactam antibiotics classified as UMIs. A multiresidue liquid chromatographic procedure based on a method for determining beta-lactams in milk was also used to identify and quantitate residues. The 2 methods were tested with 24 tissue FSIS samples classified as containing UMIs. Of these, 3 contained penicillin G, including one at a violative level, and 5 contained a metabolite of ceftiofur. The others were negative for beta-lactam antibiotics.

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OBJECTIVE: To compare results of 6 commercially available milk antimicrobial screening tests with results of liquid chromatography (LC) when testing milk samples from individual cows treated for mild clinical mastitis by intramammary (IMM) infusion with amoxicillin or penicillin G. ANIMALS: 6 cows with noninduced clinical mastitis: 3 treated by IMM infusion with amoxicillin and 3 treated by IMM infusion with penicillin G. PROCEDURE: Composite milk samples were collected before, during, and after treatment. Samples were assayed by use of the screening tests and their results and those of LC were compared. The LC results were assumed to represent the true result. RESULTS: Results of screening tests compared well with results of LC, with agreement of 94%. Positive screening test results for samples containing drug values below the established tolerance or safe level, as evaluated by LC, were obtained from 2 cows in which abnormal milk, as well as marked increases in composite milk somatic cell count, were observed. With the exception of 1 test in 1 cow, all screening tests had negative results at the end of the labeled milk-withholding time. CONCLUSIONS AND CLINICAL IMPLICATIONS: On the basis of results of the limited sample reported, the screening tests appeared to provide good agreement overall, compared with LC results, when testing milk of individual cows treated by IMM infusion with amoxicillin or penicillin G. Positive screening test results for milk samples containing amoxicillin or penicillin G at values below the established tolerance or safe level, as evaluated by LC, may occasionally be obtained.

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Screening of milk shipments for beta-lactam antibiotic residues is mandatory in the USA and is widely used in other countries. Interpretation of positive screening test results has been difficult. Only six beta-lactam antibiotics are approved for use in food-producing animals in the USA but many others are used in other countries. A multiresidue procedure was developed for identification and quantitation of unknown beta-lactam antibiotics. The residues were extracted with acetonitrile and tetraethylammonium chloride. The extract was concentrated by evaporation and filtered. The concentrated extract was then loaded onto an HPLC column in 100% 0.01 M KH2PO4 and eluted with an acetonitrile gradient. Fractions corresponding to analytes of interest were collected and tested for antibiotics using rapid milk screening tests. Fractions testing positive were analyzed by HPLC. The identity of beta-lactams was confirmed by treating a replicate with beta-lactamase.

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Ceftiofur, a recently introduced antibiotic of the beta-lactam group, is rapidly converted to both free and protein-bound metabolites when administered by the prescribed procedure, intramuscular injection. Free metabolites retain antimicrobial activity and may be detected by antibiotic-screening tests based on antimicrobial activity. It is thus desirable to distinguish ceftiofur metabolites from other antibiotics. A multiresidue procedure for beta-lactams in milk was adapted for determination of free ceftiofur metabolites by collecting appropriate liquid chromatographic fractions during cleanup. Analysis of tissues from treated animals showed that the principal free metabolite was the desfuroylceftiofurcysteine conjugate.

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OBJECTIVE: A microbial receptor assay method (MRAM; Charm II test) for beta-lactam antibiotics and a liquid chromatography (LC) method with a detection limit of 2 to 5 ppb were evaluated for detection of ampicillin or amoxicillin residues in milk samples from individual cows. DESIGN: The MRAM was compared to the LC in 2 respects. Measured concentrations of drugs were compared, as well as the classification of samples relative to the FDA tolerance value of 10 ppb. ANIMALS: A total of 6 clinically normal lactating Holstein cows were used per drug. PROCEDURE: Ampicillin trihydrate or amoxicillin trihydrate was administered at an extra-label dosage of 22 mg/kg of body weight, IM, once to each of 6 cows/drug. Milk samples were collected at milkings prior to and for 156 hours after drug administration. Drug concentrations in milk samples from individual cows were determined by use of the MRAM and LC tests. Additionally, the classification of milk samples relative to the presence or absence of residues above the FDA tolerance value was determined. Pharmacokinetic analysis was performed on derived milk drug concentrations. RESULTS: Concentration of ampicillin in milk samples from all cows was < 10 ppb by the MRAM and LC methods by the fourth milking (48 hours) after treatment with ampicillin. Values were < 10 ppb by both methods for all cows treated with amoxicillin by the sixth milking (72 hours) after treatment. For individual milk samples, significant differences were found between test methods in the proportion of positive (failing) tests; the MRAM had a higher proportion of presumptive positives. CONCLUSIONS: Even at an extra-label dosage of 22 mg/kg, IM, milk residues > 10 ppb (the FDA tolerance value) were not detected beyond the label milk withholding times for ampicillin (48 hours) and amoxicillin (96 hours). When used for testing milk of individual cows by the control point procedure, the MRAM had a tendency to give presumptive positive test results for milk samples containing < 10 ppb ampicillin or amoxicillin as determined by LC.

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A procedure for identifying and quantitating violative beta-lactams in milk is described. This procedure integrates beta-lactam residue detection kits with the multiresidue automated liquid chromatographic (LC) cleanup method developed in our laboratory. Spiked milk was deproteinized, extracted, and subjected to reversed-phase LC using a gradient program that concentrated the beta-lactams. Amoxicillin, ampicillin, cephapirin, ceftiofur, cloxacillin, and penicillin G were, thus, separated into 5 fractions that were subsequently tested for activity by using 4 kits. beta-lactams in the positive fractions were quantitated by analytical LC methods developed in our laboratory. The LC cleanup method separated beta-lactam antibiotics from each other and from interferences in the matrix and also concentrated the antibiotics, thus increasing the sensitivity of the kits to the beta-lactam antibiotics. The procedure facilitated the task of identifying and measuring the beta-lactam antibiotics that may be present in milk samples.

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A radioimmunoassay test for tetracyclines (Charm II) was compared with high-pressure liquid chromatography (HPLC) for detection of oxytetracycline (OTC) residues in milk samples from individual lactating cows. Oxytetracycline was administered by 1 of 3 routes (IV, IM, or intrauterine) to 21 lactating dairy cows. A total of 292 duplicate milk samples were collected from milkings before and through 156 hours after OTC administration. Concentration of OTC in these samples was determined by use of the Charm II test and an HPLC method with a lower limit of quantitation, approximately 2 ng of OTC/ml. Samples were also classified with respect to presence of OTC residues relative to the FDA safe concentration (< or = 30 ng/ml), using the Charm II (by control point determination) and HPLC methods. There was a significant (P < or = 0.05) difference between test methods in classification of milk samples with respect to presence or absence of OTC at the FDA safe concentration. A total of 48 of the 292 test results (16.4%) did not agree. Using the HPLC test results as the standard with which Charm II test results were compared, 47 false presumptive-violative test results and 1 false presumptive-nonviolative Charm II test result (a sample containing 31 ng of OTC/ml, as evaluated by HPLC) were obtained. The samples with false presumptive-violative Charm II results contained < 30 ng of OTC/ml, as evaluated by HPLC. In some respects, the Charm II test performed appropriately as a screening test to detect OTC residues in milk samples from individual cows.(ABSTRACT TRUNCATED AT 250 WORDS)

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In the United States, testing of all milk for residues of beta-lactam antibiotics is now mandatory. Although a number of screening tests for determination of beta-lactam antibiotic residues have been proposed, few reference methods of the required sensitivity (< 10 ppb) are available. Methods for determination of several beta-lactam antibiotics using an automated liquid chromatography (LC) cleanup have been described recently. This paper describes the integration of these methods into a single extraction and cleanup procedure. Milk was deproteinized with 0.2M Et4NCI and acetonitrile. The resulting filtrate was evaporated to about 1 mL, made to 4 mL, and filtered through a disposable filter cartridge. For cleanup, 2 mL filtrate was loaded onto a bonded C18 LC column in 0.01M KH2PO4 (A) and eluted with an acetonitrile (B) gradient using a program of 100 A:0 B(0-3 min) to 60 A:40 B (30 min). The beta-lactams were concentrated into narrow bands and separated from each other. A fraction corresponding to each compound of interest was collected and rechromatographed for analysis. The procedure has been applied successfully to determination of ampicillin, amoxicillin, cephapirin, penicillin G, penicillin V, ceftiofur, and cloxacillin. In principle it can be applied to the determination of any beta-lactam antibiotic or metabolite thereof by collecting the appropriate fractions.

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Milk antimicrobial residues are a serious concern for the dairy industry. Residues of the tetracycline family of antimicrobials have been reported in market milk by investigators, using radioimmunoassay and microbial receptor technology (hereafter referred to as the Charm II test). In response to these reports, an investigation was conducted to determine the potential of 3 extra-label routes of oxytetracycline (OTC) administration to cause milk residues above the Food and Drug Administration safe value of 30 parts per billion (ppb). Lactating Holstein cows were administered OTC once by use of 1 of 3 routes: IV at 16.5 mg/kg of body weight (n = 6); IM at 11 mg/kg (n = 6); and intrauterine (IU) at 2 g in 500 ml of saline solution/cow (n = 6). Duplicate milk samples were collected at the milking prior to drug administration and for the next 13 milkings at 12-hour intervals. Concentrations of OTC in milk samples were analyzed by use of the Charm II tes for tetracyclines (limit of OTC detection, approx 5 ppb) and were compared with concentrations determined by use of a high-performance liquid chromatography (HPLC) method (lower limit of OTC quantitation, approx 2 ppb). The potential for milk OTC residues above the Food and Drug Administration safe value of 30 ppb after treatment was considerably greater for the IV and IM routes, compared with the IU route.(ABSTRACT TRUNCATED AT 250 WORDS)

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This paper reviews recent developments in the liquid chromatographic (LC) methods of analysis for the residues of antibiotics (aminoglycosides, chloramphenicol, sulfonamides, tetracyclines, macrolides, beta-lactams, etc.) in food products of animal origin. The review also covers clean-up procedures, such as, ultrafiltration, liquid-liquid partition, solid-phase extraction, immunoaffinity, and matrix solid-phase dispersion, for use as extraction, deproteination, and concentration steps. The LC methods offer considerable potential for rapid automated analysis, and some may be used as direct screening for residues in meat and milk.

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A simple and sensitive method was developed for determination of cephapirin and its metabolite, desacetylcephapirin, in milk. For extraction/deproteinization, 2 mL 0.2M tetraethylammonium chloride and 28 mL acetonitrile are added to 10 mL milk, and 20 mL clear filtrate (= 5 mL milk) is collected. Filtrate is evaporated to 1-2 mL and made up to 4 mL with water, filtered, and transferred to 4 mL autosampler vials. For cleanup, 2 mL filtrate is loaded onto a Supelcosil LC-18 column in 0.01M KH2PO4 (A) using a Waters WISP autosampler. The column is eluted with an acetonitrile (B) gradient from 100% A (0-3 min) to 70%A:30%B (24 min). Fractions corresponding to cephapirin and desacetylcephapirin are collected. A 0.02M pH 2.26 buffer of 0.01M decanesulfonate-acetonitrile was used for cephapirin (80 + 20) and 0.02M pH 1.96 buffer of 0.01M decanesulfonate-acetonitrile was used for desacetylcephapirin (84 + 16) on a Polymer Laboratories PLRP-S column. Recoveries at 0.01-10 ppm were 91-98%; estimated detection limits were near 2 ppb and were comparable to sensitive screening tests.

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A liquid chromatographic (LC) method was developed for the simultaneous identification and quantitation of oxytetracycline, tetracycline, and chlortetracycline in milk. Milk samples (5 mL) were deproteinized by adding 1 mL 1N HCl and 24 mL acetonitrile, and filtering. Dichloromethane and hexane were added to 15 mL filtrate to separate the water layer. The organic layer was washed with 1 mL deionized water, and the combined water layers were diluted to 4 mL. Sample aliquots of 1000 microL were then injected directly and analyzed on an LC system. The sensitivity limit of the method is 5 ppb for each antibiotic; no interferences are present at their retention times. Mean recoveries from milk spiked at 0.01-1 ppm ranged from 87 to 99%, and precision was good.

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The objective was to develop confirmatory high-performance liquid chromatographic methods for penicillin residues in animal tissues with detection limits of less than or equal to 10 ng/g. A previously described procedure was modified by using a larger sample size and isocratic analysis. Tissues (15 g) were blended with 45 ml of water and 20 ml of homogenate were mixed with 40 ml acetonitrile and filtered. The filtrate (30 ml) was mixed with 10 ml of 0.2 M H3PO4 and extracted with methylene chloride. The combined methylene chloride layers were mixed with acetonitrile and hexane, washed with two 4-ml portions of water and then extracted with four 1-ml portions of 0.01 M phosphate buffer (pH 7). The combined buffer extracts were concentrated to 1 ml under reduced pressure. Analysis was isocratic during 0.01 M phosphate buffer (pH 7)-acetonitrile with proportions 85:15 (penicillin G), 82:18 (penicillin V) or 78:22 (cloxacillin). A polystyrene-divinylbenzene copolymer column, 150 x 4.6 mm I.D. (Polymer Labs. PLRP-S), was used with a flow-rate of 1 ml/min and detection at 210 nm. The presence of penicillins was confirmed by treating a duplicate sample with penicillinase. Recoveries were greater than 90% in most instances. Detection limits were 5 ng/g in muscle and higher in liver and kidney. The procedure is a simple and sensitive method for confirming the presence of penicillins in animal tissues.

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