Feed Additive Residues, Isolation and Identification of Amino-nitro-o-toluamide Formed by Biological Reduction of 3,5-Dinitro-o-tuamide

Abstract

Biological reduction of 3,5-dinitro-o-toluamide results in the formation of amino-nitro-otoluamide. The isomer isolated from tissue homogenates was identified by chemical and physical tests as the 3-amino-5-nitro-o-toluamide. The isomer which is generally obtained by the chemical reduction of 3,5-dinitro-o-toluamide is the 5-amino-3-nitro-otoluamide.

Chemical Reduction of Zoalene to ANOT for Use in Zoalene Residue Studies

“…The identity of the fractions as ANOT and 5-ANOT was supported by this analysis. Colorimetric analysis of preparative LC fractions of the prepared ANOT fraction solutions showed maximum absorbencies that were consistent with ANOT absorbance values from the literature (Figure 3) accord-ing to Smith et al (1963). Slight shifting of the absorbance maxima from the literature values was noted (Table 3) and attributed to normal instrumental error.…”

“…Liquid chromatography/mass spectrometry (LC/MS) analysis of the synthesis reaction product yielded results that are consistent with the molecular weight of ANOT. Positive ion thermospray mass spectra for the ANOT The FTIR spectra of the preparative LC fraction produced the characteristic IR stretches associated with the functional groups of ANOT (Figure 5) according to Smith et al (1963). The figure illustrates the important response of the ANOT functional groups (Shriner et al, 1980;Silverstein et al, 1991): the NsH stretch of the primary amine coupled doublet (asymmetric 3373 cm -1 ; symmetric 3290 cm -1 ), the overlap CdO stretch of the amide I band (1695-1640 cm -1 ), the CsN stretch of a primary aromatic amine (1425 cm -1 , 1340-1250 cm -1 ), the asymmetric ArNO 2 (NdO) 2 stretch (1520 cm -1 ), the symmetric N(dO) 2 stretch (1347 cm -1 ), and the aromatic CsN stretch for ArNO 2 (850 cm -1 ).…”

“…Colorimetric Analysis of Preparative LC Fractions. Colormetric analysis of preparative chromatography ANOT fractions was performed using a Beckman DU-64 spectrophotometer according to the method of Smith et al (1963). The analysis solution was prepared by adding 50 µL of a 9 µg/µL ANOT fraction solution in methanol to 2.5 mL of 0.25 N HCl and 125 µL of 0.1% NaNO 2 solution.…”

“…A biological synthesis of ANOT from zoalene in chicken liver tissue (Smith et al, 1963) resulted primarily in the formation of ANOT. The other isomer formed was 5-amino-3-nitro-o-toluamide (5-ANOT).…”