A novel chemical synthesis and purification scheme to produce ANOT (3-amino-5-nitro-o-toluamide) was developed to yield an analytical standard grade material for zoalene residue investigations. An original conversion of zoalene (3,5-dinitro-o-toluamide) to ANOT was developed. Several literature references were available for the partial reduction of dinitro aryl compounds, but none detailed the conversion of zoalene to ANOT. A chemical synthesis was chosen over a biological synthesis due to the latter's complex reaction matrix, difficult recovery, and low batch yields. Using general partial reduction of dinitro aryl schemes, it was found that 10% palladium on carbon coupled to a cyclohexene reducing agent provided an optimum ANOT yield. Chromatographic techniques were utilized to isolate ANOT from the product mixture and to purify it to a standard grade material (>99% pure). The chemically synthesized ANOT product was characterized using colorimetric, TLC, FTIR, NMR, and LC/MS techniques. Keywords: ANOT; zoalene; zoaline residue; chemical reduction

A more time efficient and simpler method than methods based upon AOAC procedure 979.24 was developed for the analysis of concentrated microencapsulated vitamin D3. The improved efficiency and simplicity begins with the use of a 10% water in DMSO solution to dissolve the sample matrix. A 2.0 mL DMSO volume is combined with 200 mg of microencapsulated vitamin D3 in a flask or bottle. The mixture stands without agitation for 10 min when testing a starch matrix sample and for 30−60 min when testing a cellulose matrix sample. Five minutes of sonication or heating is required for vitamin A/D combinations. Next, 2,2,4-trimethylpentane (isooctane) is pipetted into the DMSO solution−sample mixture. The isooctane volume used was proportional to the sample concentration; specific isooctane volumes relative to sample concentrations are provided. The mixture is stirred for ca. 5 min using a magnetic stirrer. The supernatant is filtered through a 0.2 μm PTFE membrane into a suitable LC vial and chromatographed. Validation data which support equivalence to traditional AOAC 979.24-based methodology are presented. Keywords: Vitamin D; DMSO extraction; vitamin analysis