Nystatin, Neomycin Sulfate, Gramicidin, and Triamcinolone Acetonide Ointment

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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1 DEFINITION

Nystatin, Neomycin Sulfate, Gramicidin, and Triamcinolone Acetonide Ointment contains NLT 90.0% and NMT 140.0% of the labeled amounts of nystatin, neomycin, and gramicidin, and NLT 90.0% and NMT 110.0% of the labeled amount of triamcinolone acetonide (C₂₄H₃₁FO₆).

2 IDENTIFICATION

2.1 A. Thin-Layer Chromatography

Standard solution: 100 µg/mL of USP Triamcinolone Acetonide RS in chloroform

Sample solution: Transfer 2 g of Ointment into a conical flask, add 5.0 mL of chloroform, and shake for 10 min. Add 15 mL of alcohol, and shake for an additional 10 min. Filter the solution into a centrifuge tube, and evaporate the filtrate to dryness. Dissolve the residue in alcohol to obtain a solution containing about 250 µg/mL of triamcinolone acetonide.

Chromatographic system

(See Chromatography 〈621〉, General Procedures, Thin-Layer Chromatography.)

Mode: TLC

Adsorbent: 0.25-mm layer of chromatographic silica gel

Application volume: 10 µL

Developing solvent system: Chloroform, benzene, and methanol (5:2:1)

Spray reagent: A mixture of a 1-in-5 solution of sodium hydroxide and a 1-in-500 solution of blue tetrazolium in methanol (1:1)

Analysis

Samples: Standard solution and Sample solution

Apply the Standard solution and the Sample solution on a line parallel to and about 1.5 cm from the bottom edge of the TLC plate. Proceed as directed in the chapter and develop in the Developing solvent system until the solvent front has moved about 12 cm above the application line. Remove the plate, allow the solvent to evaporate, and spray with Spray reagent.

Acceptance criteria: The intensity of the blue color and the R_f of the spot of the Sample solution corresponds to that of the Standard solution.

3 ASSAY

3.1 Nystatin

(See Antibiotics—Microbial Assays 〈81〉.)

Standard: USP Nystatin RS

Sample stock solution: Transfer a suitable, accurately weighed portion of Ointment into a blender jar. Add a sufficient, accurately measured volume of dimethylformamide and blend at high speed for 3–5 min to obtain a convenient concentration. Dilute quantitatively an accurately measured volume of the solution so obtained with dimethylformamide to obtain a stock solution containing about 400 USP Nystatin Units/mL.

Sample solution: Dilute an accurately measured volume of the Sample stock solution quantitatively with Buffer B.6 to obtain a nominal concentration of nystatin equal to the median concentration of the Standard.

Analysis: Proceed as directed for nystatin in the chapter.

Acceptance criteria: 90.0%–140.0% of the labeled amount of nystatin

3.2 Neomycin

(See Antibiotics—Microbial Assays 〈81〉.)

Standard: USP Neomycin Sulfate RS

Sample stock solution: Transfer an accurately weighed portion of Ointment, equivalent to about 3.5 mg of neomycin, to a separator, add about 50 mL of ether, and shake well. Extract with four 20-mL portions of Buffer B.3. Combine the aqueous extracts, and dilute with Buffer B.3 to an appropriate volume to obtain a stock solution of convenient concentration.

Sample solution: Dilute the Sample stock solution quantitatively and stepwise with Buffer B.3 to obtain a nominal concentration of neomycin equal to the median concentration of the Standard.

Analysis: Proceed as directed for neomycin in the chapter.

Acceptance criteria: 90.0%–140.0% of the labeled amount of neomycin

3.3 Gramicidin

(See Antibiotics—Microbial Assays 〈81〉.)

Standard: USP Gramicidin RS

Sample stock solution: Dissolve an accurately weighed portion of Ointment in 50 mL of hexanes, transfer into a separator, and extract with four 20-mL portions of 80% alcohol. Combine the extracts in a suitable volumetric flask, dilute with alcohol to volume, and mix.

Sample solution: Dilute an accurately measured volume of the Sample stock solution quantitatively and stepwise with alcohol to obtain a nominal concentration of gramicidin equal to the median concentration of the Standard.

Analysis: Proceed as directed for gramicidin in the chapter.

Acceptance criteria: 90.0%–140.0% of the labeled amount of gramicidin

3.4 Triamcinolone Acetonide

Mobile phase: Acetonitrile and water (approximately 30:70)

Internal standard solution: 50 µg/mL of fluoxymesterone in isopropyl alcohol

Standard stock solution: 75 µg/mL of USP Triamcinolone Acetonide RS in the Internal standard solution

Standard solution: Mix an accurately measured volume of the Standard stock solution with an equal volume of Mobile phase to obtain a solution containing about 37.5 µg/mL of USP Triamcinolone Acetonide RS.

Sample solution: Transfer an accurately weighed quantity of Ointment, equivalent to about 1.5 mg of triamcinolone acetonide, to a screw-capped tube. Add 20.0 mL of the Internal standard solution, and cap securely. Heat at 60° for 5 min, and then swirl vigorously for NLT 30 s. Repeat the heating and swirling sequence 3 times. Cool in a methanol-ice bath for 15–20 min, and then centrifuge at −5° for 15 min. Dilute an accurately measured volume of the supernatant with an equal volume of Mobile phase. Cool in a methanol-ice bath for 10–15 min, with occasional agitation. Pass first through a pledget of glass wool or a prefilter disk, and then pass through a membrane of 0.45-µm pore size to obtain a clear solution.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

[Note—Adjust the operating parameters with Mobile phase, such that the separation of triamcinolone acetonide and the internal standard is optimized, with a retention time of about 14.5 min for triamcinolone acetonide.]

Mode: LC

Detector: UV 254 nm

Column: 4-mm × 30-cm; packing L1

Column temperature: Room temperature

Injection volume: 15–25 µL of equal volumes of the Standard solution and Sample solution

System suitability

Sample: Standard solution

Suitability requirements

Resolution: NLT 2.0 between the triamcinolone acetonide and fluoxymesterone peaks

Relative standard deviation: NMT 3.0% for 5 replicate injections

Analysis

Samples: Standard solution and Sample solution

Measure the peak heights of the internal standard and triamcinolone acetonide at the same retention times obtained from the Standard solution and Sample solution.

Calculate the percentage of the labeled amount of triamcinolone acetonide (C₂₄H₃₁FO₆) in the portion of Ointment taken:

Result = (R_U/R_S) × (C_S/C_U) × 100

R_U = peak height ratio of triamcinolone acetonide to the internal standard from the Sample solution

R_S = peak height ratio of triamcinolone acetonide to the internal standard from the Standard solution

C_S = concentration of USP Triamcinolone Acetonide RS in the Standard solution (mg/mL)

C_U = nominal concentration of triamcinolone acetonide in the Sample solution (mg/mL)

(USP 1-Dec-2024)

Acceptance criteria: 90.0%–110.0%

4 SPECIFIC TESTS

Minimum Fill 〈755〉: Meets the requirements

Water Determination 〈921〉, Method I

[Note—Use 20 mL of a mixture of toluene and methanol (7:3) in place of methanol in the titration vessel.]

Acceptance criteria: NMT 0.5%

5 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in tight containers.

USP Reference Standards 〈11〉

USP Gramicidin RS

USP Neomycin Sulfate RS

USP Nystatin RS

USP Triamcinolone Acetonide RS