Mc. Carson et W. Breslyn, SIMULTANEOUS DETERMINATION OF MULTIPLE TETRACYCLINE RESIDUES IN MILK BY METAL CHELATE AFFINITY-CHROMATOGRAPHY - COLLABORATIVE STUDY, Journal of AOAC International, 79(1), 1996, pp. 29-42
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.