Neomycin Sulfate

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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C₂₃H₄₆N₆O₁₃ (free base)          614.65 (free base)

Neomycin B

2-Deoxy-4-O-(2,6-diamino-2,6-dideoxy-α-d-glucopyranosyl)-5-O-[3-O-(2,6-diamino-2,6-dideoxy-β-l-idopyranosyl)-β-d-ribofuranosyl]-d-streptamine (free base) CAS RN: 119-04-0. _{(USP 1-Aug-2023)}

Neomycin sulfate CAS RN: 1405-10-3; UNII: 057Y626693.

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1 DEFINITION

Neomycin Sulfate is the sulfate salt of a kind of neomycin, an antibacterial substance produced by the growth of Streptomyces fradiae Waksman (Fam. Streptomycetaceae), or a mixture of two or more such salts, the main component being neomycin B sulfate. _{(USP 1-Aug-2023)} It has a potency equivalent to NLT 600 µg of neomycin/mg, calculated on the dried basis.

2 IDENTIFICATION

A. It meets the requirements for neomycin under Thin-Layer Chromatographic Identification Test (201BNP).

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B.

Mobile phase, System suitability solution, Sensitivity solution, Standard solution, Chromatographic system, and System suitability: Proceed as directed in the test for Composition of Neomycin Sulfate.

Sample identification solution: 0.025 mg/mL of Neomycin Sulfate in Mobile phase

Acceptance criteria: The retention time of the neomycin B peak of the Sample identification solution corresponds to that of the Standard solution, as obtained in the test for Composition of Neomycin Sulfate (USP 1-Aug-2023)

C. IDENTIFICATION TESTS-GENERAL (191), Chemical Identification Tests, Sulfate

Sample solution: 50 mg/mL

Acceptance criteria: Meets the requirements

3 ASSAY

PROCEDURE

(See Antibiotics-Microbial Assays (81).)

Analysis: Proceed as directed in the chapter.

Acceptance criteria: NLT 600 µg of neomycin/mg on the dried basis

Add the following:

4 IMPURITIES

ORGANIC IMPURITIES

Mobile phase, System suitability solution, Sensitivity solution, Standard solution, Sample solution, Chromatographic system, and System suitability: Proceed as directed in the test for Composition of Neomycin Sulfate.

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of each individual impurity in the portion of Neomycin Sulfate taken:

Result = (r_u /r_s ) × (C_s /C_u ) × 100

r_u = peak response of each individual impurity from the Sample solution

r_s = peak response of neomycin B from the Standard solution

C_s = concentration of USP Neomycin B RS in the Standard solution (mg/mL)

C_u = concentration of neomycin sulfate in the Sample solution (mg/mL)

Acceptance criteria: See Table 1. The reporting threshold is 1.0%.

Table 1

^a 3-N-Acetyl-2-deoxy-4-O-(2,6-diamino-2,6-dideoxy-α-d-glucopyranosyl)-5-O-[3-O-(2,6-diamino-2,6-dideoxy-β-l-idopyranosyl)-β-dribofuranosyl]-d-streptamine.

_{(USP 1-Aug-2023)}

5 SPECIFIC TESTS

Add the following:

COMPOSITION OF NEOMYCIN SULFATE

Mobile phase: 20 mL of trifluoroacetic acid, 6 ml of 50% sodium hydroxide TS, and 500 mL of water, diluted with water to 1000 ml.

System suitability solution: 0.1 mg/mL of USP Neomycin Sulfate System Suitability Mixture RS (contains neomycin B and neomycin C) in Mobile phase

Sensitivity solution: 0.005 mg/mL of USP Neomycin B RS in Mobile phase

Standard solution: 0.025 mg/mL of USP Neomycin B. RS and 0.01 mg/mL of USP Neomycin A RS in Mobile phase

Sample solution: 0.5 mg/mL of Neomycin Sulfate in Mobile phase

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: Pulsed amperometric electrochemical

Electrodes

Indicator: Gold

Reference: Silver-silver chloride

Auxillary: Stainless steel (cell body)

Waveform: See Table 2.

Table 2

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

Flow rate: 0.7 mL/min

Post-column reagent: 20 g/L of sodium hydroxide (carbonate-free), prepared from 50% sodium hydroxide TS in water, carbon dioxide-free.

This solution is added pulselessly to the column euent using a 375-µL polymeric mixing coil.¹

Flow rate of post-column reagent: 0.5 mL/min

[Note—Adjust accordingly to meet the System suitability requirements.]

Injection volume: 10 µL

Run time: NLT 1.5 times the retention time of neomycin B

System suitability

Samples: System suitability solution, Sensitivity solution, and Standard solution

Suitability requirements

Resolution: NLT 2.0 between the neomycin C and neomycin B peaks, System suitability solution

Relative standard deviation: NMT 5.0% each for neomycin A and neomycin B, Standard solution

Signal-to-noise ratio: NLT 10 for the neomycin B peak, Sensitivity solution

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of neomycin A in the portion of Neomycin Sulfate taken:

Result = (r_u /r_s ) × (C_s /C_u ) × 100

r_u = peak response of neomycin A from the Sample solution

r_s = peak response of neomycin A from the Standard solution

C_s = concentration of USP Neomycin A RS in the Standard solution (mg/mL)

C_u = concentration of neomycin sulfate in the Sample solution (mg/mL)

Calculate the percentage of neomycin C in the portion of Neomycin Sulfate taken:

Result = (r_u /r_s ) × (C_s /C_u ) × 100

r_u = peak response of neomycin C from the Sample solution

r_s = peak response of neomycin B from the Standard solution

C_s = concentration of USP Neomycin B RS in the Standard solution (mg/mL)

C_u = concentration of neomycin sulfate in the Sample solution (mg/mL)

Acceptance criteria: See Table 3.

Table 3

^a 2-Deoxy-4-O-(2,6-diamino-2,6-dideoxy-α-d-glucopyranosyl)-5-O-[3-O-(2,6-diamino-2,6-dideoxy-α-d-glucopyranosyl)-β-d-ribofuranosyl]-dstreptamine.

_{(USP 1-Aug-2023)}

LOSS ON DRYING (731)

Sample: About 100 mg

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

Acceptance criteria: NMT 8.0%

PH (791)

Sample solution: 33 mg/mL of neomycin

Acceptance criteria: 5.0-7.5

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STERILITY TESTS (71): _{(USP 1-Aug-2023)} Where the label states that Neomycin Sulfate is sterile, it meets the requirements under the relevant _{(USP 1-Aug-2023)} dosage form.

_{(USP 1-Aug-2023)}

Change to read:

BACTERIAL ENDOTOXINS TEST (85) _{(USP 1-AUG-2023)} Where the label states that Neomycin Sulfate is sterile or that it 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 neomycin is used can be met.

_{(USP 1-Aug-2023)}

6 ADDITIONAL REQUIREMENTS

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PACKAGING AND STORAGE: Preserve in tight, light-resistant containers. Store at controlled room temperature _{(USP 1-Aug-2023)}

LABELING: Where it is intended for use in preparing injectable or other sterile dosage forms, the label states that it is sterile or must be subjected to further processing during the preparation of injectable or other sterile dosage forms.

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USP REFERENCE STANDARDS (11)

USP Neomycin Sulfate RS

USP Neomycin AF RS

2-Deoxy-4-O-(2,6-diamino-2,6-dideoxy-α-d-glucopyranosyl)-d-streptamine (free base).

C₁₂H₂₆N₄O₆  (free base)      322.36 (free base)

USP Neomycin Sulfate System Suitability Mixture RS

A mixture of neomycin 8 sulfate and neomycin C sulfate. _{(USP 1-Aug-2023)}

¹ A suitable mixing coil is the knitted reaction coil, part #043700, available from Dionex Corporation.