Tetracycline Hydrochloride for Topical Solution »Tetracycline Hydrochloride for Topical Solution is a dry mixture of Tetracycline Hydrochloride and Epitetracycline Hydrochloride with Sodium Metabisulfite packaged in conjunction with a suitable aqueous vehicle.It contains not less than 90.0percent and not more than 130.0percent of the labeled amount of tetracycline hydrochloride,when constituted as directed. Packaging and storage— Preserve in tight,light-resistant containers. USP Reference standards á11ñ USP Tetracycline Hydrochloride RS. Identification— Dissolve a suitable quantity in methanol to obtain a solution containing 1mg of tetracycline hydrochloride per mL,and filter,if necessary,to obtain a clear solution.Using the clear solution as the Test Solution,proceed as directed under Identification—Tetracyclines á193ñ:the specified result is obtained. pHá791ñ: between 1.9and 3.5,in the solution constituted as directed in the labeling. Loss on drying á731ñ Dry the contents of 1container,accurately weighed,in a capillary-stoppered bottle in vacuum at a pressure not exceeding 5mm of mercury at 60for 3hours:it loses not more than 5.0%of its weight. Content of epitetracycline hydrochloride— Standard solution— Use the Standard preparationprepared as directed under the Assay. Test solution— Open the column stopcock of the Chromatographic columnremaining from the Assay preparationin the Assay,and collect the eluate in a low-actinic,50-mLvolumetric flask until the column runs dry.Use this as the Test solution. Procedure— Proceed as directed for Procedurein the Assay.Calculate the quantity,in mg,of epitetracycline hydrochloride (C22H24N2O8·HCl)in each mLof the constituted Topical Solution taken by the formula: 0.002(WP/V)(AU/AS), in which AUis the absorbance of the solution from the Test solution,and the other terms are defined therein:the quantity of epitetracycline hydrochloride is between 115.0%and 140.0%of the quantity of tetracycline hydrochloride found as determined in the Assay. Assay— Edetate disodium solution— Dissolve 74.4g of edetate disodium in about 1800mLof water,adjust with ammonium hydroxide to a pHof 7.0,dilute with water to 2000mL,and mix. Stationary phase— Mix 95mLof Edetate disodium solutionand 5mLof a mixture of glycerine and polyethylene glycol 400(4:1). Alkaline methanol solution— On the day of use,prepare a mixture of methanol and ammonium hydroxide (19:1). Column support— Suspend 300g of chromatographic siliceous earth in 2000mLof 6Nhydrochloric acid in a suitable vessel,and mix for about 15minutes.Filter,and wash the siliceous earth with water until the last washing is neutral to moistened litmus paper.Suspend the washed siliceous earth in 2000mLof a mixture of ethyl acetate and methanol (1:1),and mix for about 15minutes.Filter,and dry the siliceous earth in vacuum at 60for about 16hours.Weigh,add 0.5mLof Stationary phasefor each g of dried siliceous earth,and shake until the column packing is uniformly moist.Store in a tight container. Chromatographic column— Prepare as directed under Column Partition Chromatography á621ñ,using a 10-×300-mm chromatographic tube equipped with a solvent reservoir on the top and a stopcock on the bottom and packed with 8±0.1g of Column support. Standard preparation— Transfer about 22mg of USP Tetracycline Hydrochloride RS,accurately weighed,to a 25-mLvolumetric flask,add 1mLof methanol,and swirl to dissolve.Dilute with Stationary phaseto volume,and mix.Transfer 2.0mLof this solution to a 10-mLvolumetric flask,dilute with Stationary phaseto volume,and mix.Pipet 2.0mLof the resulting solution into the Chromatographic column,and allow it to permeate the Column support.Add 20mLof benzene to the solvent reservoir,and collect the eluate at the rate of about 1mLper minute,using a 50-mLgraduated cylinder as a receiver.When the benzene level reaches the top of Column support,add 60mLof chloroform to the solvent reservoir,and continue collecting the eluate until 30mLhas been collected.Discard this eluate,and continue collecting the eluate in a low-actinic,50-mLvolumetric flask.When the chloroform level reaches the top of the Column support,add 10mLof a mixture of butyl alcohol and chloroform (1:1)to the solvent reservoir,and replace the low-actinic,50-mLvolumetric flask with a 10-mLgraduated cylinder.Collect 8mLof the eluate,close the column stopcock,and transfer the eluate to the low-actinic,50-mLvolumetric flask.Rinse the graduated cylinder with 2mLof chloroform,and add the rinsing to the volumetric flask.The eluate in the 50-mLvolumetric flask is the Standard preparation. Assay preparation— Constitute the Topical Solution as directed in the labeling.Transfer an accurately measured volume of the constituted Topical Solution,equivalent to about 4.4mg of tetracycline hydrochloride,to a 25-mLvolumetric flask,dilute with Stationary phaseto volume,and mix.Pipet 2.0mLof this solution into the Chromatographic column,and allow it to penetrate the Column support.Add 20mLof benzene to the solvent reservoir,and collect the eluate at the rate of about 1mLper minute,using a 50-mLgraduated cylinder as a receiver.When the benzene level reaches the top of the Column support,add 60mLof chloroform to the solvent reservoir,and continue collecting the eluate until 30mLhas been collected.Discard this eluate,and continue collecting the eluate in a low-actinic,50-mLvolumetric flask.When the chloroform level reaches the top of the Column support,add 50mLof a mixture of butyl alcohol and chloroform (1:1)to the solvent reservoir,and replace the low-actinic,50-mLvolumetric flask with a 10-mLgraduated cylinder.Collect 8mLof the eluate,close the column stopcock,and transfer the eluate to the low-actinic,50-mLvolumetric flask.Rinse the graduated cylinder with 2mLof chloroform,and add the rinsing to the volumetric flask.The eluate in the 50-mLvolumetric flask is the Assay preparation.Retain the column for the test for Content of epitetracycline hydrochloride. Procedure— Add 2.0mLof Alkaline methanol solutionto the Standard preparation,and the Assay preparation,dilute each with chloroform to volume,and mix.Concomitantly,within 10minutes of preparation,determine the absorbances of these solutions at the wavelength of maximum absorbance at about 366nm,with a suitable spectrophotometer,using chloroform as the blank.Calculate the quantity,in mg,of tetracycline hydrochloride in each mLof the constituted Topical Solution taken by the formula: 0.0002(WP/V)(AU/AS), in which Wis the weight,in mg,of USP Tetracycline Hydrochloride RStaken,Pis the potency,in µg per mg,of USP Tetracycline Hydrochloride RS,Vis the volume,in mL,of constituted Topical Solution taken,and AUand ASare the absorbances of the solutions from the Assay preparationand the Standard preparation,respectively. Auxiliary Information— Staff Liaison:William W.Wright,Ph.D.,Scientific Fellow Expert Committee:(PA7)Pharmaceutical Analysis 7 USP28–NF23Page 1888 Phone Number:1-301-816-8335