Triethylamine buffer— Transfer 100 mL of triethylamine to a 1000-mL volumetric flask. Add about 800 mL of water, and cautiously add 80 mL of phosphoric acid. Mix, allow to cool to ambient temperature, dilute with water to volume, mix, and pass through a nylon filter having a 0.5-µm or finer porosity.

Mobile phase— Prepare a filtered and degassed mixture of water, acetonitrile, and Triethylamine buffer (790:200:10). Make any necessary adjustments (see System Suitability under Chromatography 621).

Standard preparation— Transfer about 50 mg of USP Nitrofurazone RS, accurately weighed, to a 50-mL low-actinic volumetric flask, add 10 mL of dimethylformamide, and swirl to dissolve. Dilute with alcohol to volume, and mix. Transfer 10.0 mL of this solution to a 100-mL low-actinic volumetric flask, dilute with alcohol to volume, and mix. Transfer 10.0 mL of this solution to a 100-mL low-actinic volumetric flask containing 15 mL of alcohol, dilute with water to volume, and mix. This solution contains about 0.01 mg of USP Nitrofurazone RS per mL.

Assay preparation— Transfer an accurately weighed portion of Ointment, equivalent to about 1 mg of nitrofurazone, to a 100-mL low-actinic volumetric flask. Add 0.2 mL of dimethylformamide and about 25 mL of alcohol, and sonicate for about 35 minutes. Dilute with water to volume, mix, and pass through a nylon filter having a 0.5-µm or finer porosity. Use the filtrate.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 365-nm detector and a 3.9-mm × 30-cm column that contains packing L1. The flow rate is about 2 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the column efficiency determined from the nitrofurazone peak is not less than 1500 theoretical plates, and the relative standard deviation for replicate injections is not more than 2%.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of nitrofurazone (C6H6N4O4) in the portion of Ointment taken by the formula: in which C is the concentration, in mg per mL, of USP Nitrofurazone RS in the Standard preparation; and rU and rS are the nitrofurazone peak responses obtained from the Assay preparation and the Standard preparation, respectively.