SIMULTANEOUS DETERMINATION OF MULTIPLE TETRACYCLINE RESIDUES IN MILK BY METAL CHELATE AFFINITY-CHROMATOGRAPHY - COLLABORATIVE STUDY

To meet federal and state regulatory needs, a liquid chromatographic ( LC) method with ultraviolet (UV) detection was developed for determina tion of 7 tetracyclines at 30 ng/mL in milk. Raw milk samples are defa tted, acidified, and centrifuged to remove proteins, and tetracyclines are specifically absorbed from the milk by chelation with metal ions bound to small Chelating Sepharose Fast Flow columns. Tetracyclines ar e removed from these columns with EDTA-containing buffer, and extracts are further cleaned by ultrafiltration. Finally, extracts are concent rated and analyzed simultaneously by using on-line concentration. This method was validated in a collaborative study that involved 11 labora tories, including the authors' laboratory. Each laboratory was asked t o prepare and analyze known control and fortified milk samples, as wel l as 18 coded blind samples. Eight laboratories completed all analyses . Average interlaboratory recoveries for the known fortified samples r anged from 59% (methacycline at 15 ng/mL) to 78% (oxytetracycline at 6 0 ng/mL). Average recovery for each of 7 residues at 30 ng/mL were bet ween 60 and 110%, meeting single-residue guidelines for accuracy set b y the U.S. Food and Drug Administration. Reproducibility relative stan dard deviation (RSD(R)) for the known fortified samples varied from 11 to 39%, with 6 of 7 residues at the 30 ng/mL level having RSD(R) valu es at or below 20%. Seven of 8 laboratories correctly identified blind control milk samples and all 28 residues present in blind samples. Th e metal chelate affinity-LC method for determination of multiple tetra cycline residues in milk has been adopted first action by AOAC INTERNA TIONAL.