Gramicidin

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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Gramicidin

CAS RN®: 1405-97-6; UNII: 5IE62321P4.

1 DEFINITION

Gramicidin is an antibacterial substance produced by the growth of Bacillus brevis Dubos (Fam. Bacillaceae). It may be obtained from Tyrothricin. It has a potency of NLT 900 µg of gramicidin per mg, calculated on the dried basis.

2 IDENTIFICATION

Delete the following:

Spectroscopic Identification Tests, 〈197〉Ultraviolet-Visible Spectroscopy (USP 1-Dec-2023)

Add the following:

A. Spectroscopic Identification Tests 〈197〉, Infrared Spectroscopy: 197K (USP 1-Dec-2023)

Add the following:

B. The retention times of the major peaks of the Sample solution correspond to those of the Standard solution, as obtained in the test for Composition. (USP 1-Dec-2023)

3 ASSAY

Change to read:

Antibiotics—Microbial Assays 〈81〉: Determine the median concentration (S3), starting with the concentration suggested in 〈81〉, Table 7, to obtain the optimum concentration–response relationship. (USP 1-Dec-2023)

Acceptance criteria: It has a potency of NLT 900 µg of gramicidin per mg calculated on the dried basis.

4 IMPURITIES

Residue on Ignition 〈281〉: NMT 1.0%, the charred residue being moistened with 2 mL of nitric acid and 5 drops of sulfuric acid Add the following:

Organic Impurities

Mobile phase: Methanol and water (71:29)

Sensitivity solution: 2 µg/mL of USP Gramicidin RS in Mobile phase

Standard solution: 1 mg/mL of USP Gramicidin RS in Mobile phase

Sample solution: 1 mg/mL of Gramicidin in Mobile phase

4.1 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 282 nm

Column: 4.6-mm × 25-cm; 5-µm packing L1

Column temperature: 50°

Flow rate: 1.0 mL/min

Injection volume: 20 µL

4.2 System suitability

Samples: Sensitivity solution and Standard solution

[Note—The relative retention times in Table 1 are provided as information that could aid in peak assignment.]

Table 1

^a N-Formyl-l-valylglycyl-l-alanyl-d-leucyl-l-alanyl-d-valyl-l-valyl-d-valyl-l-tryptophyl-d-leucyl-l-tyrosyl-d-leucyl-l-tryptophyl-d-leucyl-N-(2- hydroxyethyl)-l-tryptophanamide.

^b N-Formyl-l-isoleucylglycyl-l-alanyl-d-leucyl-l-alanyl-d-valyl-l-valyl-d-valyl-l-tryptophyl-d-leucyl-l-tyrosyl-d-leucyl-l-tryptophyl-d-leucyl-N-(2- hydroxyethyl)-l-tryptophanamide.

^c N-Formyl-l-valylglycyl-l-alanyl-d-leucyl-l-alanyl-d-valyl-l-valyl-d-valyl-l-tryptophyl-d-leucyl-l-tryptophyl-d-leucyl-l-tryptophyl-d-leucyl-N-(2- hydroxyethyl)-l-tryptophanamide.

^d N-Formyl-l-isoleucylglycyl-l-alanyl-d-leucyl-l-alanyl-d-valyl-l-valyl-d-valyl-l-tryptophyl-d-leucyl-l-tryptophyl-d-leucyl-l-tryptophyl-d-leucyl-N- (2-hydroxyethyl)-l-tryptophanamide.

^e N-Formyl-l-valylglycyl-l-alanyl-d-leucyl-l-alanyl-d-valyl-l-valyl-d-valyl-l-tryptophyl-d-leucyl-l-phenylalanyl-d-leucyl-l-tryptophyl-d-leucyl-N-(2- hydroxyethyl)-l-tryptophanamide.

Suitability requirements

Resolution: NLT 1.5 between gramicidin A1 and gramicidin A2, Standard solution

Signal-to-noise ratio: NLT 10 for the gramicidin A1 peak, Sensitivity solution

4.3 Analysis

Sample: Sample solution

Calculate the percentage of each impurity peak (the peak other than gramicidins A1, A2, B1, C1, and C2):

Result = (r_U /r_T ) × 100

r_U = peak response of the individual impurity

r_T = total response of all the peaks

Acceptance criteria: Disregard any impurity with a peak area less than that of gramicidin A1 in the Sensitivity solution. NMT 1 impurity exceeds 1.0%.

Any individual impurity: NMT 2.0 _{(USP 1-Dec-2023)}

5 SPECIFIC TESTS

Melting Range or Temperature 〈741〉, Procedures, Procedure for Class Ia: NLT 229°, determined after drying

Crystallinity 〈695〉: Meets the requirements

Loss on Drying 〈731〉

Sample: About 100 mg

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

Acceptance criteria: NMT 3.0%

Add the following:

Composition

Mobile phase, Sensitivity solution, Standard solution, Sample solution, Chromatographic system, and System suitability: Proceed as directed in the Organic Impurities.

Analysis

Sample: Sample solution

Calculate the percentage of each of the components (gramicidins A1, A2, B1, C1, and C2):

Result = (r_U /r_T) × 100

r_U = peak response of the individual component

r_T = total response of all the peaks

Acceptance criteria: See Table 2.

Table 2

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in tight containers.

USP Reference Standards 〈11〉

USP Gramicidin RS.