II. MATERIALS AND METHODS 



PROCURKMKNT AND 

 CHARACTKRIZATION OF 

 NITROFURANTOIN 



Nitrofurantoin was obtained in one lot (lot no. 

 03540) IVoin Norwich Katon Pharmaceuticals 

 (Norwich, NY). Purity and identity determina- 

 tions were conducted by Midwest Research Insti- 

 tute (MRU (Kansas City, MO). MRI reports on 

 analyses performed in support of the nitrofuran- 

 toin studies are on file at the National Institute 

 of Environmental Health Sciences. 



Lot no. 03540 was obtained as a yellow, micro- 

 crystalline powder with a melting point of 251°- 

 255° C. The identity of nitrofurantoin was con- 

 firmed by infrared (P'igure 1), ultraviolet/visible, 

 and nuclear magnetic resonance (F'igure 2) spec- 

 troscopy. The infrared and nuclear magnetic 

 resonance spectra were consistent with litera 

 ture spectra (Analytical Profiles of Drug Sub 

 stances, 1976). 



The purity of nitrofurantoin was determined by 

 elemental analysis, water analysis, titration of 

 the imide group, thin-layer chromatography, 

 and high-performance liquid chromatography. 

 Cumulative data indicated that lot no. 03540 

 was greater than 99% pure Results of elemen- 

 tal analyses for carbon, hydrogen, and nitrogen 

 agreed with the theoretical values. Water con- 

 tent by Karl Fischer titration was less than 

 0.02%. Titration of the imide group with tetra- 

 butylammonium hydroxide indicated a purity of 

 99.6%. Thin layer chromatography on silica gel 

 plates with either a cyclohexane acetone: 

 methanol:acetic acid (45:45:5:5) or a toluene: 

 2-butanone:acetic acid (40:60:1) solvent system 

 detected a single spot with visualization by 



ultraviolet and visible light and a sodium hy- 

 droxide-saturated methanol spray. No impuri- 

 ties with a peak area greater than 0.1% of the 

 major peak area were detected by high-perform- 

 ance liquid chromatography on a pBondapak 

 Ci8 column with a water:acetonitrile (88:12) mo- 

 bile phase at a fiow rate of 1 ml/minute and 

 ultraviolet detection at 365 nm Analysis of lot 

 no 03540 by the same high-performance liquid 

 chromatographic system (with a slightly dif 

 ferent solvent ratio) did not detect nitrofurazone, 

 5-nitro-2-furaldehyde, or 3-(f(5-nitro-2-furanyl)- 

 methylene|amino|-2,4-imidizolidinedione at 

 concentrations equal to or greater than the 

 minimal detectable concentrations (0.03%, 2.4%, 

 andO.2% w/v). 



Stability studies performed by high-performance 

 liquid chromatography with a pBondapak Cih 

 column, a wateriacetonitrile (70:30) mobile 

 phase at a flow rate of 1.5 ml/minute, and ultra- 

 violet detection at 254 nm indicated that nitro- 

 furantoin was stable for 2 weeks at tempera- 

 tures up to 60° C. Further confirmation of the 

 stability of the bulk chemical (stored at 5° C) 

 during the toxicity studies was obtained by titra- 

 tion with tetrabutylammonium hydroxide and 

 high-performance liquid chromatography with a 

 Hewlett-Packard RP-8 column or a Perkin- 

 Elmer ODS Sil-X column, ultraviolet detection 

 at 365 nm, and a wateracelonitrile mobile phase 

 at a flow rate of 1 ml/minute. The acetonitrile 

 concentration was increased from 30% to 50% 

 over 20 minutes or from 5% to 45% over 15 min- 

 utes. No degradation of the bulk chemical was 

 seen over the course of the studies. Identity of 

 the chemical at the study laboratory was con- 

 firmed by infrared spectroscopy. 



Nitrofurantoin, NTPTR341 



24 



