Standard solution— Use the Standard preparation prepared as directed under the Assay.

Test solution— Open the column stopcock of the Chromatographic column remaining from the Assay preparation in the Assay, and collect the eluate in a low-actinic, 50-mL volumetric flask until the column runs dry. Use this as the Test solution.

Procedure— Proceed as directed for Procedure in the Assay. Calculate the quantity, in mg, of epitetracycline hydrochloride (C22H24N2O8·HCl) in each mL of the constituted Topical Solution taken by the formula: in which AU is 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.

(Official January 1, 2007)

Edetate disodium solution— Dissolve 74.4 g of edetate disodium in about 1800 mL of water, adjust with ammonium hydroxide to a pH of 7.0, dilute with water to 2000 mL, and mix.

Stationary phase— Mix 95 mL of Edetate disodium solution and 5 mL of 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 300 g of chromatographic siliceous earth in 2000 mL of 6 N hydrochloric acid in a suitable vessel, and mix for about 15 minutes. Filter, and wash the siliceous earth with water until the last washing is neutral to moistened litmus paper. Suspend the washed siliceous earth in 2000 mL of a mixture of ethyl acetate and methanol (1:1), and mix for about 15 minutes. Filter, and dry the siliceous earth in vacuum at 60 for about 16 hours. Weigh, add 0.5 mL of Stationary phase for 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 621, 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 ± 0.1 g of Column support.

Standard preparation— Transfer about 22 mg of USP Tetracycline Hydrochloride RS, accurately weighed, to a 25-mL volumetric flask, add 1 mL of methanol, and swirl to dissolve. Dilute with Stationary phase to volume, and mix. Transfer 2.0 mL of this solution to a 10-mL volumetric flask, dilute with Stationary phase to volume, and mix. Pipet 2.0 mL of the resulting solution into the Chromatographic column, and allow it to permeate the Column support. Add 20 mL of benzene to the solvent reservoir, and collect the eluate at the rate of about 1 mL per minute, using a 50-mL graduated cylinder as a receiver. When the benzene level reaches the top of Column support, add 60 mL of chloroform to the solvent reservoir, and continue collecting the eluate until 30 mL has been collected. Discard this eluate, and continue collecting the eluate in a low-actinic, 50-mL volumetric flask. When the chloroform level reaches the top of the Column support, add 10 mL of a mixture of butyl alcohol and chloroform (1:1) to the solvent reservoir, and replace the low-actinic, 50-mL volumetric flask with a 10-mL graduated cylinder. Collect 8 mL of the eluate, close the column stopcock, and transfer the eluate to the low-actinic, 50-mL volumetric flask. Rinse the graduated cylinder with 2 mL of chloroform, and add the rinsing to the volumetric flask. The eluate in the 50-mL volumetric 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 4.4 mg of tetracycline hydrochloride, to a 25-mL volumetric flask, dilute with Stationary phase to volume, and mix. Pipet 2.0 mL of this solution into the Chromatographic column, and allow it to penetrate the Column support. Add 20 mL of benzene to the solvent reservoir, and collect the eluate at the rate of about 1 mL per minute, using a 50-mL graduated cylinder as a receiver. When the benzene level reaches the top of the Column support, add 60 mL of chloroform to the solvent reservoir, and continue collecting the eluate until 30 mL has been collected. Discard this eluate, and continue collecting the eluate in a low-actinic, 50-mL volumetric flask. When the chloroform level reaches the top of the Column support, add 50 mL of a mixture of butyl alcohol and chloroform (1:1) to the solvent reservoir, and replace the low-actinic, 50-mL volumetric flask with a 10-mL graduated cylinder. Collect 8 mL of the eluate, close the column stopcock, and transfer the eluate to the low-actinic, 50-mL volumetric flask. Rinse the graduated cylinder with 2 mL of chloroform, and add the rinsing to the volumetric flask. The eluate in the 50-mL volumetric flask is the Assay preparation. Retain the column for the test for Content of epitetracycline hydrochloride.

Procedure— Add 2.0 mL of Alkaline methanol solution to the Standard preparation, and the Assay preparation, dilute each with chloroform to volume, and mix. Concomitantly, within 10 minutes of preparation, determine the absorbances of these solutions at the wavelength of maximum absorbance at about 366 nm, with a suitable spectrophotometer, using chloroform as the blank. Calculate the quantity, in mg, of tetracycline hydrochloride in each mL of the constituted Topical Solution taken by the formula: in which W is the weight, in mg, of USP Tetracycline Hydrochloride RS taken, P is the potency, in µg per mg, of USP Tetracycline Hydrochloride RS, V is the volume, in mL, of constituted Topical Solution taken, and AU and AS are the absorbances of the solutions from the Assay preparation and the Standard preparation, respectively.