Purpose: Evaluate tetracycline test method with dilution confirmation according to FDA-CVM criteria.
Methods: Rapid One Step Assay (ROSA) Tetracycline (Charm Sciences, Inc.) method used at CODEX cumulative tetracycline maximum residue levels (100 ppb) was adapted with a tetracycline dilution buffer to confirm US tolerance (300 ppb) without being overly sensitive (<150 ppb chlortetracycline [CTC], < 120 ppb oxytetracycline [OTC], < 70 ppb tetracycline [TC]). The adapted ROSA Tetracycline-SL (Dilution Confirmation) method was evaluated by Q-laboratories, Cincinnati, OH for 90% detection with 95% confidence using n=30 replicates at 5 concentrations per drug and n=60 negative. Somatic and bacterial interference samples n=60 negative and n=30 positives were evaluated by Eurofin, DQCI Mounds View, MN. Data on chemical interferences, freeze thawing, and assay perturbations were submitted by manufacturer. Proficiency study using 16 NCIMS laboratories was performed with 2 zero samples, 2 samples each at 300 ppb CTC or OTC and 1 sample each at 100 ppb CTC or OTC.
Results: Q-laboratory detection CTC=289 ppb, OTC=294 ppb, and TC=126 ppb compared to manufacture data CTC= 205 ppb, OTC=181 ppb, and TC= 81 ppb. There were no interferences from somatic cells (1.1 M/mL) or bacteria (>300,000 CFU/mL) or from animal drugs (36 non-tetracycline drugs at 100 ppb) or from other assay perturbations. Sixteen laboratories successfully performed the method to detect 300 ppb CTC and OTC, 64 positives of 64 replicates, while not being overly sensitive to 100 ppb CTC, 88% positive, or 100 ppb OTC, 50% positive.
Significance: The ROSA Tetracycline-SL (Dilution Confirmation) method for detecting tetracyclines in commingled milk at US Tolerances meets FDA-CVM and NCIMS criteria for acceptance.