BL30SEC Method for Detection of Beta-Lactams in Raw Commingled Cow Milk: AOAC Performance Tested MethodSM 061902.

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.

Validation of the charm 3 SL3 beta-lactam test for screening raw milk in compliance with the U.S. pasteurized milk ordinance. Performance Tested Method 071002.

The Charm 3 SL3 beta-Lactam Test is a 3 min receptor-based lateral-flow Rapid One-Step Assay (ROSA) that detects the six beta-lactam drugs of concern approved for dairy cattle in the United States. The method is a biochemical formulation change of the SL3 beta-Lactam Test evaluated and approved in 2007. The Charm 3 SL3 was evaluated under the AOAC Research Institute Performance Tested Method (PTM) program following the protocol of the U.S. Food and Drug Administration, Center for Veterinary Medicine. The method was approved as PTM 071002 on May 8, 2009. The following drugs were detected in three combined lots: penicillin G at 3.8 ppb, ampicillin at 8.0 ppb, amoxicillin at 8.4 ppb, cephapirin at 20.0 ppb, ceftiofur (total metabolites) at 79 ppb, and cloxacillin at 8.6 ppb > or = 90% of the time with 95% confidence. These detection levels are lower than, but within 75% of, the U.S. Safe Level/Tolerances. Lot-to-lot repeatability was typically within 20% of these determined levels. The test kit was found to be suitable for testing thawed frozen samples. It was also found to respond with equal or better sensitivity to samples that contained incurred analytes, i.e., both the microbiologically active parent drug and its active metabolites. There were no interferences from somatic cells at 1.1 million/mL, bacterial cells at 300 000 CFU/mL, or 32 other non-beta-lactam drugs at 100 ppb. Ruggedness experiments indicated that the test procedure is robust. These results meet the fit-for-purpose approval criteria for inclusion in the National Conference for Interstate Milk Shipments milk testing program.

Validation of the SL3 beta-Lactam Test for screening milk in compliance with U.S. pasteurized milk ordinance: performance tested method 040701.

The SL3 beta-Lactam Test is a 3 min, receptor-based lateral flow Rapid One Step Assay (ROSA) that detects 5 of 6 beta-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 50% less. 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-beta-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.