Bacitracin

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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Bacitracin

CAS RN®: 1405-87-4; UNII: 58H6RWO52I.

1 DEFINITION

Bacitracin is a mixture of polypeptides produced by the growth of an organism of the licheniformis group of Bacillus subtilis (Fam. Bacillacaea),

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.

2 IDENTIFICATION

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

Change to read:

B.

Sample: 0.2 g

Analysis: Ignite the Sample. Allow to cool. Dissolve the residue in 0.1 mL of dilute hydrochloric acid. Add 5 mL of water and 0.2 mL of sodium hydroxide TS 3.

Acceptance criteria: No white precipitate is formed.

3 ASSAY

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 IMPURITIES

Residue on Ignition 〈281〉: NMT 0.5%

5 SPECIFIC TESTS

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 A and Solution B (2:9). 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 D and Solution E (37:63)

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 in Mobile phase

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 254 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 × 25-cm; end-capped 5-µm packing L1

Flow rate: 1 mL/min

Injection volume: 100 µL

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 bacitracin B₁ peak), and the impurity (bacitracin F) using the relative retention times in Table 1.

Suitability requirements

Peak-to-valley ratio: NLT 1.2, System suitability solution

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

Result = Hₚ/Hᵥ

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

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

Analysis

Samples: Diluent, Reporting threshold solution, and Sample solution

Content of bacitracin A

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

Result = (rₐ/rₜ) × 100

rₐ = peak area of bacitracin A from the Sample solution

rₜ = 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 taken:

Result = [(rₐ + r_B₁ + r_B₂ + r_B₃)/rₜ] × 100

rₐ = 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

rₜ = 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 taken:

Result = (rₑ/rₜ) × 100

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

rₜ = 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 taken:

Result = (r_F/rₐ) × 100

r_F= peak area of bacitracin F from the Sample solution

rₐ = 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 that have a peak area less than bacitracin A in the Reporting threshold solution.

pH 〈791〉

Sample solution: 10,000 Bacitracin units/mL in water

Acceptance criteria: 5.5–7.5

Loss on Drying 〈731〉

Sample: 100 mg

Analysis: Dry the Sample in a capillary-stoppered bottle under vacuum at a pressure not exceeding 5 mm of mercury at 60° for 3 h.

Acceptance criteria: NMT 5.0%

Sterility Tests 〈71〉: Where the label states that the Bacitracin is sterile, it meets the requirements.

Change to read:

Bacterial Endotoxins Test 〈85〉: The level of bacterial endotoxins is such that the requirement under the relevant dosage form monograph(s) in which Bacitracin is used can be met. Where the label states that Bacitracin must be subjected to further processing during the preparation of injectable dosage forms, the level of bacterial endotoxins is such that the requirement under the relevant dosage form monograph(s) in which Bacitracin is used can be met.

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in tight containers, and store below 8°.

Change to read:

Labeling: 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 Bacitracin must be subjected to further processing during the preparation of injectable dosage forms to ensure acceptable levels of bacterial endotoxins, it is so labeled.

Change to read:

USP Reference Standards 〈11〉

USP Bacitracin Zinc RS