Mobile phase, Resolution solution, and Chromatographic system— Proceed as directed in the Assay.

Test solution 1— Transfer about 25 mg of Minocycline Hydrochloride, accurately weighed, to a 100-mL volumetric flask, dilute with water to volume, and mix. Protect this solution from light, store in a refrigerator, and use within 3 hours.

Test solution 2— Transfer 1.0 mL of Test solution 1 to a 50-mL volumetric flask, dilute with water to volume, and mix. Protect this solution from light, store in a refrigerator, and use within 3 hours.

Test solution 3— Transfer 6.0 mL of Test solution 2 to a 10-mL volumetric flask, dilute with water to volume, and mix. Protect this solution from light, store in a refrigerator, and use within 3 hours.

Procedure— Separately inject equal volumes (about 20 µL) of Test solution 1, Test solution 2, and Test solution 3 into the chromatograph, record the chromatograms, and measure the areas for all the peaks. [NOTE—Record the chromatogram of Test solution 1 for a period of time that is about 2.6 times the retention time of minocycline.] Calculate the percentage of epiminocycline in the portion of Minocycline Hydrochloride taken by the formula: in which rE1 is the peak response for epiminocycline obtained from Test solution 1; and rM3 is the peak response for minocycline obtained from Test solution 3. Not more than 1.2% is found. Calculate the total percentage of impurities other than epiminocycline in the portion of Minocycline Hydrochloride taken by the formula: in which rs is the sum of the responses of all impurity peaks other than epiminocycline obtained from Test solution 1; and rM2 is the peak response for minocycline obtained from Test solution 2. Not more than 2.0% of other impurities is found.

(Official January 1, 2007)

Mobile phase— Prepare a mixture of 0.2 M ammonium oxalate, 0.01 M edetate disodium, dimethylformamide, and tetrahydrofuran (600:180:120:80). Adjust with ammonium hydroxide to a pH of 7.2, and pass through a filter having a 0.5-µm or finer porosity. Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve an accurately weighed quantity of USP Minocycline Hydrochloride RS in water to obtain a solution having a known concentration of about 500 µg of minocycline (C23H27N3O7) per mL. Use this solution within 3 hours.

Resolution solution— Transfer 10 mg of USP Minocycline Hydrochloride RS to a 25-mL volumetric flask, add 20 mL of 0.2 M ammonium oxalate, and swirl to dissolve. Heat on a water bath at 60 for 180 minutes, and allow to cool. Dilute with water to volume, and mix.

Assay preparation— Transfer an accurately weighed quantity of Minocycline Hydrochloride, equivalent to about 50 mg of minocycline (C23H27N3O7), to a 100-mL volumetric flask, add water to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 280-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L1, and is maintained at a constant temperature of about 40. The flow rate is about 1.5 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the capacity factor, k¢, is not less than 5.0 and not more than 11.5; the tailing factor for the analyte peak is not less than 0.9 and not more than 2.0; and the relative standard deviation for replicate injections is not more than 2.0%. Chromatograph the Resolution solution and record the peak responses as directed for Procedure: the relative retention times are about 0.7 for epiminocycline and 1.0 for minocycline; and the resolution, R, between epiminocycline and minocycline is not less than 4.6.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in µg per mg, of minocycline (C23H27N3O7), in the portion of Minocycline Hydrochloride taken by the formula: in which C is the concentration, in µg per mL, of minocycline (C23H27N3O7) in the Standard preparation; W is the weight, in mg, of Minocycline Hydrochloride taken; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.