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.

Buffer— Dissolve 34.8 g of dibasic potassium phosphate in 1 L of water. Adjust with 27.2 g of monobasic potassium phosphate 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.

System suitability solution— Dissolve an accurately weighed quantity of USP Bacitracin Zinc RS in water, add diluted hydrochloric acid, using about 2% of the final volume, and dilute with water to obtain a solution with a nominal concentration of about 2.0 mg per mL.

Reporting threshold solution— Dilute quantitatively, with Mobile phase, a suitable volume of System suitability solution to obtain a solution having a known concentration of 0.01 mg per mL. This solution is used to determine the reporting threshold.

Peak identification solution— Dissolve a suitable quantity of USP Bacitracin Zinc RS in a suitable volume of a 40 g per L solution of sodium edetate (pH adjusted to 7.0 with dilute sodium hydroxide), to obtain a solution having a nominal concentration of about 2.0 mg per mL. Heat in a boiling water bath for 30 minutes. Cool to room temperature.

Test solution— Dissolve an accurately weighed quantity of Bacitracin in Mobile phase to obtain a solution having a known 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-mm × 25-cm 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 given 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 by the formula: where 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 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 (about 100 µL) of the Mobile phase, the Test solution, and the Reporting threshold solution. Record the chromatograms for about 3 times the retention time of bacitracin A. Identify the peaks using the relative retention time values given 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 bacitracin A in the Reporting threshold solution; and disregard any peak observed in the blank run.]