Bacitracin Zinc

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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Bacitracins, zinc complex;

Bacitracin zinc complex

CAS RN^®: 1405-89-6; UNII: 89Y4M234ES.

1 DEFINITION

Bacitracin Zinc is the zinc complex of bacitracin, which consists of a mixture of antimicrobial polypeptides, the main components being bacitracins A, B1, B2, and B3. It has a potency of NLT 65 Bacitracin Units/mg, calculated on the dried basis. It contains NLT 4.0% and NMT 6.0% of zinc (Zn), calculated on the dried basis.

2 IDENTIFICATION

A. Meets the requirements of the test for Composition of Bacitracin

B. Meets the requirements of the test for Zinc Content

3 ASSAY

3.1 PROCEDURE

(See Antibiotics-Microbial Assays (81)..)

Analysis: Proceed as directed in the chapter.

Acceptance criteria: NLT 65 Bacitracin Units/mg on the dried basis

4 SPECIFIC TESTS

4.1 COMPOSITION OF BACITRACIN

Diluent: 40 g/L of edetate disodium in water adjusted with 8 N sodium hydroxide to a pH of 7.0

Solution A: 34.8 g/L of dibasic potassium phosphate in water

Solution B: 27.2 g/L of monobasic potassium phosphate in water

Solution C: Solution B and Solution A (9:2). The pH of the mixture is about 6.

Solution D: 0.1 mM edetate disodium in a mixture of Solution C and water (1:3)

Solution E: Methanol and acetonitrile (27:2)

Mobile phase: Solution E and Solution D (63:37)

System suitability solution: 2 mg/mL of USP Bacitracin Zinc RS in Diluent

Reporting threshold solution: 0.01 mg/mL of USP Bacitracin Zinc RS from System suitability solution in water

Peak identification solution: 2 mg/mL of USP Bacitracin Zinc RS in Diluent. Heat in a boiling water bath for 30 min, and cool to room

temperature.

Sample solution: 2 mg/mL of Bacitracin Zinc in Diluent

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 254 nm and 300 nm. Quantitative analysis is performed at 254 nm; 300 nm is only used to identify the location of bacitracin F.

Column: 4.6-mm x 25-cm; end-capped 5-µm packing L1

Flow rate: 1 mL/min

Injection volume: 100 µL

Run time: NLT 3 times the retention time of bacitracin A

System suitability

Samples: System suitability solution and Peak identification solution

Analyze the Peak identification solution at 300 nm. Identify bacitracin F, a known impurity, using the relative retention time provided in

Table 1. Analyze the System suitability solution at 254 nm. 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 in Table 1.

Table 1

Suitability requirements

Peak-to-valley ratio: NLT 1.2

The Peak-to-valley ratio is calculated as follows:

Result = H_P/H_V

H_P = height above the baseline of the peak due to bacitracin B1

H_V = height above the baseline of the lowest point of the curve separating the bacitracin B1 peak from the bacitracin B2 peak

Samples: Diluent, Reporting threshold solution, and Sample solution

Quantitative analysis is performed at 254 nm.

Content of bacitracin A

Calculate the percentage of bacitracin A in the portion of Bacitracin Zinc taken:

Result = (r_A/r_T) x 100

r_A = peak area of bacitracin A from the Sample solution

r_T = sum of all peak areas above the reporting threshold from the Sample solution

Content of active bacitracin

Calculate the percentage of active bacitracin (bacitracin A, B1, B2, and B3) in the portion of Bacitracin Zinc taken:

Result = [(r_A1 + r_B1 + r_B2 + r_B3)/г_T) х 100

r_A1 = peak area of bacitracin A from the Sample solution 

r_B1 = peak area of bacitracin B1 from the Sample solution 

r_B2 = peak area of bacitracin B2 from the Sample solution

r_B3 = peak area of bacitracin B3 from the Sample solution

г_T = sum of all peak areas above the reporting threshold from the Sample solution

Limit of early eluting peptides

Calculate the percentage of early eluting peptides (peaks eluting before bacitracin B1) in the portion of Bacitracin Zinc taken:

Result = (r_P/r_T) x 100

r_P = sum of peak areas for all peaks before bacitracin B1 from the Sample solution

r_T = sum of all peak areas above the reporting threshold from the Sample solution

Limit of bacitracin F

Calculate the percentage of bacitracin F in the portion of Bacitracin Zinc taken:

Result = (r_F/r_A) x 100

r_F = peak area of bacitracin F from the Sample solution

r_A = peak area of bacitracin A from the Sample solution

Acceptance criteria: See Table 2. Disregard any peaks from the Sample solution that are observed in the Diluent chromatogram. Disregard any peaks from the Sample solution having a peak area less than bacitracin A in the Reporting threshold solution.

Table 2

4.2 ZINC CONTENT

[NOTE-The Standard solutions and the Sample solution may be quantitatively diluted with 1 mM hydrochloric acid, if necessary, to obtain solutions of suitable concentrations, adaptable to the linear or working range of the instrument.]

Standard stock solution: 10 mg/mL of zinc from Zinc oxide in 1 N hydrochloric acid. Prepare as follows. Transfer a suitable amount of zinc oxide to a suitable volumetric flask, add 1 N hydrochloric acid using 32% of the final volume, warm to dissolve, cool and dilute with water volume.

Standard solutions: 0.5, 1.5, and 2.5 µg/mL of zinc from Standard stock solution in 0.001 N hydrochloric acid

Sample stock solution: 2 mg/mL of Bacitracin Zinc in 0.01 N hydrochloric acid

Sample solution: 0.02 mg/mL of Bacitracin Zinc from Sample stock solution in 0.001 N hydrochloric acid

Instrumental conditions

(See Atomic Absorption Spectroscopy (852).)

Mode: Atomic absorption spectrophotometry

Analytical wavelength: 213.8 nm

Lamp: Zinc hollow-cathode

Flame: Air-acetylene

Blank: 0.001 N hydrochloric acid

Analysis

Samples: Standard solutions, Sample solution, and Blank

Plot the absorbances of the Standard solutions versus concentration, in µg/mL, of zinc, and draw the straight line best fitting the three plotted points. From the graph, determine the concentration, in µg/mL, of zinc in the Sample solution.

Calculate the percentage of zinc in the portion of Bacitracin Zinc taken:

Result = C x D x (V/W) x F x 100

C = concentration of zinc in the Sample solution obtained from the curve (µg/mL)

D = dilution factor for the Sample solution, 100 mL/mL

V = volume of Sample stock solution (mL)

W = weight of Bacitracin Zinc used to prepare the Sample stock solution (mg)

F = conversion factor, 0.001 mg/µg

Acceptance criteria: 4.0%-6.0% on the dried basis

4.3 PH (791)

Sample solution: A saturated solution in water containing about 100 mg/mL

Acceptance criteria: 6.0-7.5

4.4 LOSS ON DRYING (731)

Sample: 100 mg

Analysis: Dry the Sample in a capillary-stoppered bottle under vacuum at 60° for 3 h.

Acceptance criteria: NMT 5.0%

4.5 STERILITY TESTS (71)

Where the label states that it is sterile, it meets the requirements of the chapter. If the membrane filtration test is used, add 20 g of edetate disodium to each L of Fluid A.

5 ADDITIONAL REQUIREMENTS

PACKAGING AND STORAGE: Preserve in tight containers, and store below 25°.

LABELING: Label it to indicate that it is to be used in the manufacture of nonparenteral drugs only. Where it is packaged for prescription compounding, label it to indicate that it is not sterile and that the potency cannot be assured for longer than 60 days after opening, and to state the number of Bacitracin Units/mg. Where it is intended for use in preparing sterile dosage forms, the label states that it is sterile or must be subjected to further processing during the preparation of sterile dosage forms.

USP REFERENCE STANDARDS (11)

USP Bacitracin Zinc RS