Development of an ASE-GC-MS/MS method for detecting dinitolmide and its metabolite 3-ANOT in eggs.

An accelerated solvent extraction coupled with gas chromatography-tandem mass spectrometry (ASE-GC-MS/MS) method for detecting dinitolmide residue and its metabolite (3-amino-2-methyl-5-nitrobenzamide, 3-ANOT) in eggs was developed and optimized. The samples were extracted using ASE with acetonitrile as the extractant and were purified by passage through a neutral alumina solid-phase extraction column. Then, the samples were analyzed using the GC-MS/MS method. The optimized method parameters were validated according to the requirements set forth by the European Union and the Food and Drug Administration. The average recoveries of dinitolmide and 3-ANOT from eggs (egg white, egg yolk, and whole egg) at the limit of quantification (LOQ), 0.5 maximum residue limit (MRL), 1 MRL, and 2 MRL were 82.74% to 87.49%, the relative standard deviations (RSDs) were less than 4.63%, and the intra-day RSDs and the inter-day RSDs were 2.96% to 5.21% and 3.94% to 6.34%, respectively. The limits of detection and the LOQ were 0.8 to 2.8 µg/kg and 3.0 to 10.0 µg/kg, respectively. The decision limits (CCα ) were 3001.69 to 3006.48 µg/kg, and the detection capabilities (CCß ) were 3001.74 to 3005.22 µg/kg. Finally, the new method was successfully applied to the quantitative determination of dinitolmide and 3-ANOT in 50 commercial eggs from local supermarkets.

Depletion of the monoamino metabolites of Zoalene during frozen storage.

3-Amino-5-nitro-o-toluamide and 5-amino-3-nitro-o-toluamide, the principal metabolites in the tissues of chickens fed a diet containing Zoalene (3,5-dinitro-o-toluamide), were shown to deplete in frozen liver tissues stored up to 1 year at -20 degrees C, but not at -70 degrees C. A slight loss of 5-amino-3-nitro-o-toluamide also occurred in thigh muscle at -20 degrees C. Evidence is presented that demonstrates that the depletion of the metabolites was partially the result of their transformation to glucopyranosyl derivatives; both the alpha- and beta-anomers of the 5-amino-3-nitro-o-toluamide conjugate were observed in liquid chromatograms of tissue extracts.

Liquid chromatographic determination of zoalene and its metabolites in chicken tissues with electrochemical detection.

A procedure is described for the quantitation of Zoalene (3,5-dinitro-o-toluamide) and its 2 major monoamino metabolites in chicken tissues. The method includes blender extraction of tissue with chloroformethyl acetate (1 + 1), adsorption of the drug and metabolites on neutral alumina, and subsequent elution of the residues with pH 3.5 formate buffer-methanol (6.5 + 3.5). Recovered residues were separated on a 5 micron C18 column with the alumina eluting solvent as the LC mobile phase. The parent drug and metabolites were detected and quantitated with an electrochemical detector in the reductive mode with a minimum level of reliable measurement of 0.1 ppm. Overall mean recoveries greater than 85% were obtained with Zoalene and its 2 monoamino metabolites in breast, thigh, and liver tissues fortified with 0.25-2.00 ppm. The results on tissues from chickens fed a diet containing 0.0125% Zoalene are presented.

Liquid chromatographic determination of zoalene in medicated feeds.

A rapid, reliable separation and quantitation of zoalene (3,5-dinitro-o-toluamide) from feeds is accomplished by using reverse phase liquid chromatography (LC) and ultraviolet detection. An extraction technique which is similar to the present AOAC official colorimetric method is used before chromatographic analysis. This extraction is followed by an activated alumina cleanup and LC to separate zoalene from feed matrix. The methodology was applied to a variety of spiked feed matrices, and yielded good recoveries. Liquid chromatographic results were shown to correlate with colorimetric determinations.