II. MATERIALS AND METHODS 



PREPARATION AND 

 CHARACTERIZATION OF 

 FORMULATED DIETS 



Formulated diets were prepared by adding a dry 

 premix (approximately equal amounts of feed 

 and nitrofurantoin) to the appropriate amount of 

 feed (Table 1). The mixture was blended for 15 

 minutes. The homogeneity of diet mixtures for- 

 mulated at the analytical chemistry and study 

 laboratories was evaluated by extracting feed 

 samples (taken from three positions in the blen- 

 der) with acetonitrile and determining the ab- 

 sorption at 365 nm (analytical chemistry labora- 

 tory) or from 360 to 380 nm (study laboratory). 

 Further studies indicated that nitrofurantoin 

 was stable in feed at 2,000 ppm when stored for 2 

 weeks in the dark at temperatures up to 45" C. 

 In these studies, samples were extracted with 

 acetonitrile:acetic acid (99:1) and analyzed by 

 high-performance liquid chromatography with a 



TABLE 1 



pBondapak Cig column, a water/acetonitrile 

 (80:20) mobile phase, and ultraviolet detection 

 at 254 nm. In the 13- week and 2-year studies, 

 formulated diets were stored at 5° C for no longer 

 than 14 days. 



Periodic analyses of feed mixtures by the same 

 analytical methods used for the homogeneity 

 studies were conducted by the study and analyti- 

 cal chemistry laboratories to determine if the 

 formulated diets contained the correct concen- 

 trations of nitrofurantoin. Formulated diets 

 were analyzed once during the 13-week studies 

 The results ranged from 93% to 103% of the tar- 

 get concentrations (Table 2) During the 2-year 

 studies, feed mixtures were analyzed every 1-2 

 months, and concentrations varied from 80% to 

 11 3% of the target concentrations (Table 3). Be- 

 cause 78/83 formulated diets analyzed were 

 within 10% of the target concentrations, it is es- 

 timated that the formulated diets were prepared 



PREPARATIO.N AND STORAGE OF FORMULATED DIETS IN THE FEED STUDIES OF 



NITROFURANTOIN 



Fourteen-Day Studies 



Thirteen-Week Studies 



TwoYear Studies 



Preparation 



Premix prepared by mixing weighed amounts 

 of nitrofurantoin and feed in a specimen cup 

 for 1 min; remaining feed mixed with premix 

 in a 16-qt blender for 15 min 



Maximum Storage Time 



14d 



Storage Conditions 



Room temperature in the dark 



Same as 14-d studies 



14d 



5° C in the darlc 



Same as 14-d studies 



14d 



5° C in the dark before use and 

 then at room temperature 



TABLE 2. RESULTS OF ANALYSIS OF FORMULATED DIETS IN THE THIRTEEN WEEK FEED 



STUDIES OF NITROFURANTOIN (a) 



Determined as a 

 Percent of Target 



95.7 

 93.3 

 103.1 

 100.8 

 99.8 

 97.2 



(a) Mix dale; 4/29/80 



(bl Results of duplicate analysis 



(c) Average of values obtained from three locations in the blender 



27 



Nitrofurantoin, NTPTR341 



