The SL3 -Lactam Test is a 3 min, receptor-based lateral flow Rapid One Step Assay (ROSA) that detects 5 of 6 -lactam drugs approved for dairy cattle in the United States. The method was evaluated through the AOAC Research Institute Performance-Tested Method program following a U.S. Food and Drug Administration protocol. Three combined lots detected penicillin G 4.2 parts per billion (ppb), ampicillin 8.7 ppb, amoxicillin 7.8 ppb, cephapirin 16.0 ppb, and ceftiofur (total metabolites) 51 ppb at least 90 of the time, with 95 confidence as determined by dose response probit analysis. These detection levels are less than safe level/tolerances but not more than 50less. Lot repeatability was within 20. Incurred residues were detected comparably or more sensitively to fortified samples due to the cumulative effect of biologically active metabolites. There were no interferences from somatic cells at 1 M/mL, bacterial cells 500 000 colony-forming units/mL, or 30 other non--lactam drugs. These performances met approval conditions of the National Conference on Interstate Milk Shipments. Ruggedness conditions were incorporated into public health procedures for annual laboratory proficiency and certification.

The Charm Safe-Level β-Lactam Test was evaluated by a U.S. Food and Drug Administration (FDA) test protocol administered by the AOAC–Research Institute. The sensitivity and selectivity of the test were evaluated with >800 negative raw commingled and drug-fortified milk samples by the manufacturer and an independent laboratory. Probit analysis by the independent laboratory determined the following 90% positive levels with 95% confidence: amoxicillin, 5.6 ppb; ampicillin, 8.5 ppb; cephapirin, 13.7 ppb; ceftiofur, 46.2 ppb; and penicillin G, 3.6 ppb. These values were within a range of ±20% of the manufacturer's data. Selection of negative samples met confidence specifications. Ruggedness parameters were studied and defined, and the stability of frozen milk was verified. There were no interferences from somatic cells (1 000 000 somatic cell count/mL) or bacteria (300 000 colony-forming units/mL), or from 27 other non-β-lactam animal drugs. Test performance with raw milk samples containing incurred penicillin, ampicillin, and amoxicillin was consistent with the dose responses determined with fortified milk samples. Incurred cephalosporin in raw milk samples was detected at lower levels than was cephalosporin in fortified milk samples, presumably because of the presence of metabolite, as verified by other test methods. Quality control data support consistency in manufacture between batches and the stability of refrigerated test reagents for up to 1 year. Successful fulfillment of these criteria led to FDA certification of the test when used with a reader in U.S. milk testing programs.

A qualitative 3 minute one step assay for detecting beta-lactam, sulfonamide and tetracycline antibiotics was validated following milk screening test guidelines developed by FDA-CVM, AOAC-RI and IDF. The validated 90% detection levels with 95% confidence were penicillin G 2 part per billion (ppb); amoxicillin 4 ppb; ampicillin 9 ppb; ceftiofur plus metabolites 50 ppb; cloxacillin 9 ppb; cephapirin 15 ppb; sulfadimethoxine 8 ppb; sulfamethazine 9 ppb; chlortetracycline 34 ppb; oxytetracycline 53 ppb; and tetracycline 42 ppb. Detection levels were lower than US and Canadian allowable limits for milk and were consistent with most European Maximum Residue Limits. Tests of raw commingled cows’ milk indicated a low positive error rate of < 0.3% with no interferences demonstrated by 1.08 MM/mL somatic cells, Gram – or + bacteria < 300K/mL, freeze/thawing, or non-targeted drugs. Detection of incurred residues were similar to, or more sensitive to, fortified samples. Some cross reactivity across drug families occurred in interference studies and therefore initial positive samples should be confirmed with drug family specific screening methods. The National Conference of Interstate Milk Shipments approval as a bulk tank/tanker screening test was completed in three stages for each drug family, including a tetracycline confirmation procedure to target the USA tolerance levels. Detection and robustness were found to be appropriate for multiple countries’ regulatory requirements for screening tests. The method development, validation and approval was intended to diversify and increase the verification tools for the control of the major antibiotic drug families used in managing cows’ health and welfare.

Testing milk for antibiotics before acceptance into dairies is required by the U.S. Pasteurized Milk Ordinance. Technological advances in tests have reduced screening times and improved detection accuracy. This work describes the validation of the Charm Rapid One Step Assay Beta-Lactam 30 Second Test according to the U.S. Food and Drug Administration Center for Veterinary Medicine protocol for raw commingled milk. Milk is added to the lateral flow test strip in an incubator/reader to deliver a 30 second result. Independent laboratory validation followed sensitivity, interference, and incurred residue protocols. Sensitivity, in parts per billion (ppb = µg/kg), using a probit curve determined 90% percent detection with 95% confidence, which met National Conference of Interstate Milk Shipments (NCIMS) specifications. Six U.S. approved beta-lactam drugs were detected below, but within 50% of, target/tolerance levels for penicillin G 2.9 ppb, ampicillin 5.9 ppb, amoxicillin 5.8 ppb, cephapirin 13 ppb, cloxacillin 8.1 ppb, and ceftiofur metabolites 73 ppb. No interferences were observed from 33 animal drugs at 100 ppb, somatic cells at 1.2 million/mL, or bacterial levels of >300 000 CFU/mL. Incurred residue detection levels were similar to levels determined with the spiked parent compound. The data support NCIMS that the BL30SEC method met U.S. criteria for testing milk for beta-lactams.