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Nystatin, Neomycin Sulfate, Thiostrepton, and Triamcinolone Acetonide Ointment
» Nystatin, Neomycin Sulfate, Thiostrepton, and Triamcinolone Acetonide Ointment contains not less than 90.0 percent and not more than 130.0 percent of the labeled amounts of nystatin, neomycin, and thiostrepton, and not less than 90.0 percent and not more than 110.0 percent of the labeled amount of triamcinolone acetonide (C24H31FO6).
Packaging and storage— Preserve in tight containers.
Labeling— Label it to indicate that it is for veterinary use only.
Identification— Place 2 g of Ointment in a conical flask, add 5.0 mL of chloroform, and shake for 10 minutes. Add 15 mL of alcohol, and shake for an additional 10 minutes. 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 of triamcinolone acetonide per mL. Proceed as directed in the Identification test under Triamcinolone Acetonide Cream, beginning with “Apply 10 µL of this solution”: the specified result is observed.
Minimum fill 755: meets the requirements.
Residual solvents 467: meets the requirements.
(Official January 1, 2007)
Assay for nystatin— [NOTE—Protect solutions that contain nystatin from ambient light.]
Ammonium acetate buffer— 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.
Mobile phase— Mix 2500 mL of Ammonium acetate buffer, 1000 mL of acetonitrile, and 500 mL of methanol. Pass through a 0.45-µm nylon filter.
BHT solution— Weigh about 1.0 g of butylated hydroxytoluene, and transfer to a 1000-mL volumetric flask. Dilute with methanol to volume, and mix.
Standard preparation— In duplicate, dissolve an accurately weighed quantity of USP Nystatin RS in BHT solution to obtain a solution having a known concentration of about 5400 USP Nystatin Units per mL. Store in low-actinic glassware.
System suitability solution— Weigh about 50 mg of USP Nystatin RS, and transfer to a 50-mL low-actinic volumetric flask. Add 0.5 mL of 0.01 N sodium hydroxide, and allow to sit for 1 minute. Add 5 mL of Ammonium acetate buffer. Add about 25 mL of methanol, and sonicate to dissolve. Dilute with methanol to volume, and store in low-actinic glassware.
Assay preparation— Thoroughly mix the Ointment prior to sampling. In duplicate, accurately weigh about 1.0 g of Ointment having a known density into a low-actinic sample bottle. Add 20.0 mL of BHT solution, and insert a polytef-coated magnetic stir bar having dimensions of about 12.7 × 7.9 mm. Clamp the bottles onto a suitable mixer mill,1 and mix for a minimum of 5 minutes at about 30 Hz. Centrifuge at about 1350 × g for 5 minutes, or until the supernatant is clear. Transfer the supernatant to low-actinic glassware.
Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 304-nm detector and a 3.9-mm × 15-cm column that contains 4-µm packing L1. The column temperature is maintained at 40, and the flow rate is about 2.0 mL per minute. [NOTE—Solutions containing nystatin should be stored at 8 until they can be injected into the chromatograph.] Chromatograph the Standard preparation and the System suitability solution, and record the peak areas as directed for Procedure: using the System suitability solution, the relative retention times for the nystatin A1 and nystatin A2 peaks are about 1.0 and 1.4, respectively; the column efficiency, using the nystatin A1 peak, is not less than 1200 theoretical plates; the tailing factor is not more than 2.0; and the relative standard deviation for replicate injections is not more than 3.0%. [NOTE—After the conclusion of the run, 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.]
Procedure— Separately inject equal volumes (about 15 µL) of the duplicate Standard preparation and Assay preparation into the chromatograph, record the chromatograms, and measure the peak areas for nystatin A1 and nystatin A2. Calculate the quantity, in USP Nystatin Units, of nystatin in the portion of Ointment taken by the formula:
20(CS / WU)(ru / rs),
in which CS is the concentration of USP Nystatin RS, in USP Nystatin Units per mL, of the Standard preparation; WU is the weight, in g, of Ointment taken to prepare the Assay preparation; and ru and rs are the average peak areas of the sum of nystatin A1 and nystatin A2 obtained from the Assay preparation and the Standard preparation, respectively.
Assay for neomycin— Proceed as directed for the turbidimetric assay for neomycin under Antibiotics—Microbial Assays 81, placing an accurately weighed portion of Ointment, equivalent to about 2.5 mg of neomycin, in a 250-mL conical flask, and treating it as follows. 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. Dilute this solution quantitatively and stepwise with Buffer No. 3 to obtain a Test Dilution having a concentration assumed to be equal to the median dose of the Standard.
Assay for thiostrepton— Proceed as directed for thiostrepton under Antibiotics—Microbial Assays 81, blending a suitable, accurately weighed portion of Ointment in a high-speed blender with a sufficient, accurately measured volume of dimethyl sulfoxide to give a convenient concentration, and filter. Quantitatively dilute an accurately measured volume of the filtrate so obtained with dimethyl sulfoxide to obtain a Test Dilution having a concentration of thiostrepton assumed to be equal to the median dose of the Standard.
Assay for triamcinolone acetonide— Proceed with Ointment as directed in the Assay under Triamcinolone Acetonide Cream, but read “Ointment” in place of “Cream” throughout.
1  A suitable mixer mill can be obtained from Retsch Inc., 74 Walker Lane, Newtown, PA 18940 (www.retsch-us.com; 267-757-0351), product number MM 301.
Auxiliary Information— Staff Liaison : Ian DeVeau, Ph.D., Associate Director
Expert Committee : (VET05) Veterinary Drugs 05
USP29–NF24 Page 1571
Pharmacopeial Forum : Volume No. 30(6) Page 2020
Phone Number : 1-301-816-8178