Validation of the AnticFast® Beta-Lactams & Tetracyclines Combo Test Kit for Detection of Residues of Beta-Lactams (Penicillins and Cefalosporins) and Tetracyclines in Raw Cows’ Milk: AOAC Performance Tested MethodSM 032403
Authors: Broekaert Katrien , Ooghe Sigrid , Yang Meng , Tian Xiumei , Reybroeck Wim 
Published: June 2024

Abstract

Background
The AnticFast® Beta-lactams & Tetracyclines Combo Test Kit is a qualitative two-step (2 min + 5 min) rapid lateral flow assay to detect β-lactam (penicillins and cefalosporins) and tetracycline antibiotic residues in raw commingled cows’ milk.

Objective
The method performance was evaluated according to Commission Implementing Regulation 2021/808 and Community Reference Laboratories Residues Guidelines for the Validation of Screening Methods for Residues of Veterinary Medicines and submitted for AOAC Performance Tested MethodsSM certification.

Methods
The AnticFast Beta-lactams & Tetracyclines Combo Test Kit was evaluated for detection capability (CCβ), selectivity, false-positive results, repeatability, robustness, suitability for various milk types and milk compositions, milks from various species, and test kit consistency and stability. Samples included milks spiked at concentrations bracketing the EU maximum residue limits (MRLs) for β-lactams and tetracyclines as well as bulk farm and tanker milks.

Results
The AnticFast Beta-lactams & Tetracyclines Combo Test Kit is specific for the detection of residues of β-lactams and tetracyclines in milk and does not detect residues from other antibiotic families. Interference was seen with clavulanic acid, a β-lactamase inhibitor, which was expected. The test can detect (minimum a 95% detection) all residues of β-lactams (penicillins and cepfalosporins) and tetracyclines (parent drugs and their 4-epimers) present on the EU-MRL list for milk at their respective MRL except for desfuroylceftiofur and cefalexin, with a 95% detection only above the MRL. No false positives were detected in 599 blank samples (out of 305 farm and 301 tanker load samples) tested on both channels. Five real positives were detected and confirmed on the tetracycline channel for the farm milk samples, and two positives were detected and confirmed on the β-lactam channel for the tanker samples. Robustness testing indicated that the detection in high-protein raw cows’ milk and heat-treated milk types (UHT, sterilized, and reconstituted milk powder) may be slightly hampered. For substances with a detection capability well below the MRL, this interference does not cause problems since detection at MRL remains guaranteed, but care should be taken for substances with a CCβ at or near their MRL. Diminished sample flow was seen for high-fat raw cows’ milk and for all other cows’ milk types other than raw milk and blank ewes’ milk, so sample flow should always be verified for these milk types.

Conclusions
Results of this validation show that the AnticFast Beta-lactams & Tetracyclines Combo Test Kit is a reliable test for rapid screening of raw cows’ milk for residues of β-lactam and tetracycline antibiotics.

Highlights
The AnticFast Beta-lactams & Tetracyclines Combo Test Kit is an easy, reliable, robust, and highly specific test for screening of β-lactam (penicillins and cefalosporins) and tetracycline antibiotic residues in milk with incubation at room temperature. In raw cows’ milk, all tetracyclines are detected below 10 µg/kg.