Diluting solvent , Mobile phase, Resolution solution, and Chromatographic system—Prepare as directed in the Assay under Tetracycline Hydrochloride.

Assay preparation— Transfer an accurately weighed portion of Ointment, equivalent to about 300 mg of tetracycline hydrochloride, to a glass-stoppered conical flask, add 20 mL of cyclohexane, and shake. Add 35 mL of methanol, and sonicate for about 20 minutes. Filter this solution into a 100-mL volumetric flask, and rinse the sides of the conical flask with 40 mL of methanol, filtering the rinsing through the filter into the volumetric flask. Dilute with methanol to volume, and mix. Transfer 2.0 mL of this solution to a 50-mL volumetric flask, dilute with Diluting solvent to volume, and mix.

Standard preparation— Dissolve an accurately weighed quantity of USP Tetracycline Hydrochloride RS quantitatively in methanol to obtain a solution having a known concentration of about 1 mg per mL. Transfer 6.0 mL of this solution to a 50-mL volumetric flask, dilute with Diluting solvent to volume, and mix. This solution contains about 0.12 mg of USP Tetracycline Hydrochloride RS per mL.

Procedure— Proceed as directed in the Assay under Tetracycline Hydrochloride. Calculate the quantity, in mg, of C22H24N2O8·HCl in the portion of Ointment taken by the formula: in which the terms are as defined therein.