A: Infrared Absorption 197K.

B: Ultraviolet Absorption 197U—

Test solution: 10 mg per mL, in chloroform.

Diluent— Prepare a mixture of water and acetonitrile (6:1).

Mobile phase— Dissolve 4.0 g of ammonium acetate and 7.0 g of sodium perchlorate in 1300 mL of water, adjust with phosphoric acid to a pH of 2.2, and mix. Prepare a filtered and degassed mixture of this solution and 240 mL of acetonitrile. Make adjustments if necessary (see System Suitability under Chromatography 621).

System suitability solution— Transfer 10.0 mg of USP Ofloxacin Related Compound A RS and 10.0 mg of USP Ofloxacin RS to a 100-mL volumetric flask, dissolve in and dilute with Diluent to volume, and mix. Dilute 10.0 mL of this solution with Diluent to 50.0 mL. Dilute 1.0 mL of this solution with Diluent to 50.0 mL.

Standard solution— Quantitatively dissolve an accurately weighed quantity of USP Ofloxacin RS in Diluent to obtain a solution that contains 0.0004 mg per mL of ofloxacin.

Test solution— Quantitatively dissolve an accurately weighed quantity of Ofloxacin in Diluent to obtain a solution containing about 0.2 mg per mL.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 294-nm detector and a 4.6-mm × 15-cm column that contains packing L1. The column temperature is maintained at 45. The flow rate is about 0.5 mL per minute. Chromatograph the System suitability solution, and record the peak responses as directed for Procedure: the resolution, R, between ofloxacin and ofloxacin related compound A is not less than 2.0; and the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Inject equal volumes (about 10 µL) of the Test solution and the Standard solution into the chromatograph, record the chromatograms for a period of time that is about 2.5 times the retention time of the ofloxacin peak, and measure the areas for all of the peaks except the solvent peak. Calculate the percentage of each impurity with an area greater than 0.1 times the average area of the ofloxacin peak obtained from the Standard solution by the formula: in which C is the concentration, in mg per mL, of USP Ofloxacin RS in the Standard solution; CT is the concentration, in mg per mL, of Ofloxacin in the Test solution; ri is the peak area for an individual impurity; and rS is the average area of the ofloxacin peak obtained from the Standard solution: not more than 0.3% of any individual impurity is found; and the sum of all impurities found is not more than 0.5%.

Internal standard solution— Prepare a solution in sodium hydroxide solution (1 in 100) containing 0.7 µL of n-propyl alcohol per mL. Transfer 2.0 mL of this solution to a 250-mL volumetric flask, dilute with the same sodium hydroxide solution (1 in 100) to volume, and mix.

Standard solution— Prepare a solution in Internal standard solution containing 10.0 µg each of methanol and dehydrated alcohol per mL. Transfer 2.0 mL of this solution to a vial fitted with a septum and crimp cap, and seal. Heat the sealed vial at 90 for 2 minutes, and shake for 6 minutes.

Test solution— Transfer 40 mg of Ofloxacin, accurately weighed, to a vial fitted with a septum and a crimp cap, add 2.0 mL of Internal standard solution, and seal the vial. Heat the sealed vial at 90 for 2 minutes, and shake for 6 minutes.

Blank— Transfer 2.0 mL of the Internal standard solution to a vial fitted with a septum and crimp cap, and seal. Heat the sealed vial at 90 for 2 minutes, and shake for 6 minutes.

Chromatographic system (see Chromatography 621)— The gas chromatograph is equipped with a flame-ionization detector, a 0.53-mm × 30-m fused silica column coated with a 3.0-µm film of stationary phase G43, and a fused silica precolumn. Helium is used as the carrier gas at a flow rate of about 7 mL per minute. The injection port and detector temperatures are maintained at about 170 and 250, respectively. Condition the column with the helium flowing at 200 for 2 hours or until a stable baseline is obtained. For analysis, the column temperature is programmed according to the following steps. It is maintained at 35 for 3 minutes, then increased to 90 at a rate of 20 per minute, then increased further to 200 at a rate of 40 per minute, and then maintained for 2 minutes. Chromatograph the headspace of the Standard solution, and record the peak responses as directed for Procedure: the relative retention times are about 0.5 for methanol, 0.6 for ethanol, and 1.0 for n-propyl alcohol; the resolution, R, between the methanol peak and the ethanol peak is not less than 2.0; and the relative standard deviation for replicate injections is not more than 5%.

Procedure— Use a heated gas tight syringe to make injections of the headspace into the chromatograph. Separately inject equal volumes (about 1 mL) of the headspace of the Standard solution, the Blank, and the Test solution into the chromatograph, record the chromatograms, and measure the peak area responses. Calculate the percentage of methanol and ethanol in the Ofloxacin taken by the formula: in which W is the weight, in mg, of Ofloxacin taken to prepare the Test solution; and RU, RB, and RS are the peak response ratios of the relevant alcohol peak to the internal standard peak obtained from the Test solution, the Blank, and the Standard solution, respectively: not more than 0.005% of methanol and not more than 0.05% of ethanol are found.