Nystatin, Neomycin Sulfate, Thiostrepton, 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, Thiostrepton, and Triamcinolone Acetonide Ointment contains NLT 90.0% and NMT 130.0% of the labeled amounts of nystatin, neomycin, and thiostrepton, 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

Change to read:

3.1 Nystatin

[Note—Protect solutions that contain Nystatin from ambient light. Store at 8° until they can be injected.]

Solution A: Dissolve 10.8 ± 1.0 g of ammonium acetate in 2500 mL of water. Adjust with acetic acid to a pH of 6.50 ± 0.05.

Solution B: 1.0 mg/mL of butylated hydroxytoluene in methanol

Mobile phase: Acetonitrile, methanol, and Solution A (2:1:5).

[Note—Pass through a 0.45-µm nylon filter.]

System suitability solution: Transfer about 50 mg of USP Nystatin RS into a 50-mL low-actinic volumetric flask. Add 0.5 mL of 0.01 N sodium hydroxide, and allow to sit for 1 min. Add 5 mL of Solution A. Add about 25 mL of methanol, and sonicate to dissolve. Dilute with methanol to volume, and store in low-actinic glassware.

Standard solutions: 5400 USP Nystatin Units/mL of USP Nystatin RS in Solution B, in duplicate. Store in low-actinic glassware.

Sample solutions: Thoroughly mix Ointment prior to sampling. In duplicate, transfer about 1.0 g of Ointment having a known density, accurately weighed, into a low-actinic sample bottle. Add 20.0 mL of Solution B, and insert a polytef-coated magnetic stir bar with dimensions of about 12.7 mm × 7.9 mm. Clamp the bottles onto a suitable mixer mill,¹ and mix for a minimum of 5 min at about 30 Hz. Centrifuge at about 1350 × g for 5 min, or until the supernatant is clear. Transfer the supernatant to low-actinic glassware.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 304 nm

Column: 3.9-mm × 15-cm; 4-µm packing L1.

[Note—After each analysis, rinse the column with a mixture of acetonitrile and water (85:15) until the baseline is stable, and store in this solution. At the beginning of the next run, rinse with Mobile phase until the baseline is stable.]

Column temperature: 40°

Flow rate: 2.0 mL/min

Injection volume: 15 µL

System suitability

Sample: Standard solutions

[Note—The relative retention times, using the System suitability solution, for nystatin A1 and nystatin A2 are about 1.0 and 1.4, respectively. The relative retention times are provided as information that could aid in peak assignment.]

Suitability requirements

Column efficiency: NLT 1200 theoretical plates for the nystatin A1 peak

Tailing factor: NMT 2.0

Relative standard deviation: NMT 3.0% for replicate injections

Analysis

Samples: Standard solutions and Sample solutions

Separately inject equal volumes of the duplicate Standard solutions and duplicate Sample solutions, record the chromatograms, and measure the peak areas for nystatin A1 and nystatin A2.

Calculate the percentage of the labeled amount of nystatin in the portion of Ointment taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = average peak areas of the sum of nystatin A1 and nystatin A2 from the Sample solutions

r_S = average peak areas of the sum of nystatin A1 and nystatin A2 from the Standard solutions

C_S = concentration of USP Nystatin RS in the Standard solutions (USP Nystatin Units/mL)

C_U = nominal concentration of nystatin in the Sample solutions (USP Nystatin Units/mL) (USP 1-Dec-2024)

Acceptance criteria: 90.0%–130.0%

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 2.5 mg of neomycin, into a 250-mL conical flask. Add 50 mL of hexanes and shake to disperse the Ointment. Transfer the mixture to a 250-mL separator. Wash the flask with 50 mL of 0.01 N hydrochloric acid, with shaking, and transfer the washing to a separator. Stopper the separator, shake, and allow the layers to separate. Draw off the lower aqueous layer, collecting it in a 250-mL volumetric flask. Repeat the extraction of the hexanes layer remaining in the separator with two or more 50-mL portions of 0.01 N hydrochloric acid, combining the aqueous extracts in the 250-mL volumetric flask. Dilute the contents of the volumetric flask with 0.01 N hydrochloric acid to volume, and mix.

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 the turbidimetric assay for neomycin in the chapter.

Acceptance criteria: 90.0%–130.0% of the labeled amount of neomycin

3.3 Thiostrepton

(See Antibiotics—Microbial Assays 〈81〉.)

Standard: USP Thiostrepton RS

Sample stock solution: Transfer a suitable, accurately weighed portion of Ointment into a blender jar. Add a sufficient, accurately measured volume of dimethyl sulfoxide, blend on high speed to obtain a convenient concentration, and filter.

Sample solution: Dilute an accurately measured volume of the Sample stock solution quantitatively with Dimethyl sulfoxide to obtain a nominal concentration of thiostrepton equal to the median concentration of the Standard.

Analysis: Proceed as directed for thiostrepton in the chapter.

Acceptance criteria: 90.0%–130.0% of the labeled amount of thiostrepton

Change to read:

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

5 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in tight containers.

Labeling: Label it to indicate that it is for veterinary use only.

USP Reference Standards 〈11〉

USP Nystatin RS

USP Neomycin Sulfate RS

USP Thiostrepton RS

USP Triamcinolone Acetonide RS

¹ A suitable mixer mill can be obtained from Retsch Inc., product no. MM 301.