A: Thin-Layer Chromatographic Identification Test 201BNP: meets the requirements.

B: It meets the requirements of the liquid chromatographic procedure in the test for Composition.

Standard preparations— Transfer 3.11 g of zinc oxide, accurately weighed, to a 250-mL volumetric flask, add 80 mL of 1 N hydrochloric acid, warm to dissolve, cool, dilute with water to volume, and mix. This solution contains 10 mg of zinc per mL. Further dilute this solution with 0.001 N hydrochloric acid to obtain Standard preparations containing 0.5, 1.5, and 2.5 µg of zinc per mL, respectively.

Test preparation— Transfer about 200 mg of Bacitracin Zinc, accurately weighed, to a 100-mL volumetric flask. Dissolve in 0.01 N hydrochloric acid, dilute with the same solvent to volume, and mix. Pipet 2 mL of this solution into a 200-mL volumetric flask, dilute with 0.001 N hydrochloric acid to volume, and mix.

Procedure— Concomitantly determine the absorbances of the Standard preparations and the Test preparation at the zinc resonance line of 213.8 nm, with a suitable atomic absorption spectrophotometer (see Spectrophotometry and Light-scattering 851), equipped with a zinc hollow-cathode lamp and an air–acetylene flame, using 0.001 N hydrochloric acid as the blank. Plot the absorbances of the Standard preparations versus concentration, in µg per mL, of zinc, and draw the straight line best fitting the three plotted points. From the graph so obtained, determine the concentration, in µg per mL, of zinc in the Test preparation. Calculate the content of zinc, in percent, in the portion of Bacitracin Zinc taken by the formula: in which C is the concentration in µg per mL, of zinc in the Test preparation; and W is the weight, in mg, of the portion of Bacitracin Zinc taken.

Buffer— Dissolve 34.8 g of potassium phosphate, dibasic, in 1 L of water. Adjust with 27.2 g of potassium phosphate, monobasic, dissolved in 1 L of water, to a pH of 6.0.

Mobile Phase— Prepare a mixture of methanol, water, Buffer, and acetonitrile (26:15:5:2). Mix well, and degas.

Diluent— Dissolve 40 g of sodium edetate in 1 L of water. Adjust with dilute sodium hydroxide to a pH of 7.0.

System suitability solution— Dissolve an accurately weighed quantity of USP Bacitracin Zinc RS in Diluent to obtain a solution with a nominal concentration of about 2.0 mg per mL.

Reporting threshold solution— Dilute quantitatively, with water, a suitable volume of System suitability solution to obtain a solution with a known concentration of 0.01 mg per mL.

Peak identification solution— Dissolve a weighed quantity of USP Bacitracin Zinc RS in a suitable volume of Diluent to obtain a solution with a nominal concentration of about 2.0 mg per mL. Heat in boiling water bath for 30 minutes. Cool to room temperature.

Test solution— Dissolve an accurately weighed quantity of Bacitracin Zinc in Diluent to obtain a solution with a nominal concentration of about 2.0 mg per mL.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with an absorbance detector and an end-capped 4.6- × 250-mm column that contains 5-µm packing L1. The flow rate is 1.0 mL per minute. Set the wavelength of the detector at 300 nm. Inject about 100 µL of the Peak identification solution, and identify the location of bacitracin F, which is a known impurity, using the relative retention time shown in Table 1. Change the wavelength of the detector and set it to 254 nm. Chromatograph the System suitability solution, and record the peak responses as directed for Procedure: identify the peaks of the most active components of bacitracin (bacitracins A, B1, B2 and B3), early eluting peptides (those eluting before the peak due to bacitracin B1) and the impurity, bacitracin F, using the relative retention time values given in Table 1. Calculate the peak-to-valley ratio using the formula: in which Hp is the height above the baseline of the peak due to bacitracin B1; and HV is the height above the baseline of the lowest point of the curve separating the bacitracin B1 peak from the peak due to bacitracin B2. The peak-to-valley ratio is not less than 1.2.

Procedure— Separately inject equal volumes (100 µL) of Diluent, Test solution, and Reporting threshold solution. Record the chromatograms for about three times the retention time of bacitracin A. Identify the peaks using the relative retention times shown in Table 1. Measure the peak areas of all peaks in the Test solution. [note—Disregard any peak in the Test solution having an area less than the area of the bacitracin A peak in the Reporting threshold solution; disregard any peak observed in the Diluent.]