Mobile phase and Diluent— Prepare as directed in the Assay.

System suitability solution— Prepare as directed for Resolution solution in the Assay.

Methacycline standard stock solution— Dissolve an accurately weighed quantity of USP Methacycline Hydrochloride RS in Diluent, and dilute quantitatively, and stepwise if necessary, to obtain a solution having a known concentration of about 1.2 mg per mL.

Standard solution 1— Prepare as directed for the Standard preparation in the Assay.

Standard solution 2— Transfer 2.0 mL of Standard solution 1 and 2.0 mL of the Methacycline standard stock solution to a 100-mL volumetric flask, dilute with Diluent to volume, and mix. This solution contains about 0.024 mg each of USP Doxycycline Hyclate RS and USP Methacycline Hydrochloride RS per mL.

Test solution— Use the Assay preparation, prepared as directed in the Assay.

Chromatographic system (see Chromatography 621)— Prepare as directed in the Assay. Chromatograph the System suitability solution, and record the peak responses as directed for Procedure: the relative retention times are about 0.4 for 4-epidoxycycline (the main degradation product), 0.6 for methacycline, 0.7 for 6-epidoxycycline, and 1.0 for doxycycline; the resolution, R, between 4-epidoxycycline and doxycycline is not less than 3.0; and the tailing factor is not more than 2.0. Chromatograph Standard solution 1, and record the peak responses as directed for Procedure: the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 20 µL) of Standard solution 2 and the Test solution into the chromatograph, record the chromatograms for a period of time that is 1.7 times the retention time of doxycycline, and measure the peak areas. Calculate the percentage of methacycline in the portion of Doxycycline Hyclate taken by the formula: in which CM is the concentration, in mg per mL, of USP Methacycline Hydrochloride RS in Standard solution 2; W is the weight, in mg, of Doxycycline Hyclate taken to prepare the Test solution; and rU and rM are the methacycline peak responses obtained from the Test solution and Standard solution 2, respectively. Not more than 2% of methacycline is found. Calculate the percentage of each related compound, other than methacycline, in the portion of Doxycycline Hyclate taken by the formula: in which CS is the concentration, in mg per mL, of USP Doxycycline Hyclate RS in Standard solution 2; W is the weight, in mg, of Doxycycline Hyclate taken to prepare the Test solution; ri is the peak response for each impurity obtained from the Test solution; and rS is the doxycycline peak response obtained from Standard solution 2. Not more than 0.5% of any impurity eluting before methacycline is found; not more than 2% of 6-epidoxycycline is found; and not more than 0.5% of any impurity eluting after the main doxycycline peak is found.

Mobile phase— Transfer 2.72 g of monobasic potassium phosphate, 0.74 g of sodium hydroxide, 0.50 g of tetrabutylammonium hydrogen sulfate, and 0.40 g of edetate disodium to a 1000-mL volumetric flask. Add about 850 mL of water, and stir to dissolve. Add 60 g of tertiary butyl alcohol with the aid of water, dilute with water to volume, and adjust with 1 N sodium hydroxide to a pH of 8.0 ± 0.1. Pass this solution through a filter having a porosity of 0.5 µm or finer, and degas before using. Make any necessary adjustments (see System Suitability under Chromatography 621). Decreasing the proportion of tertiary butyl alcohol results in a longer retention time of doxycycline and improved separation of doxycycline from the related compounds.

Diluent— Use 0.01 N hydrochloric acid.

Resolution solution— Prepare a solution of USP Doxycycline Hyclate RS in Diluent containing about 6 mg of doxycycline per mL. Transfer 5 mL of this solution to a 25-mL volumetric flask, heat on a steam bath for 60 minutes, and evaporate to dryness on a hot plate, taking care not to char the residue. Dissolve the residue in 0.01 N hydrochloric acid, dilute with Diluent to volume, and mix. Pass a portion of this solution through a filter having a porosity of 0.5 µm or finer, and use the filtrate as the Resolution solution. This solution contains a mixture of 4-epidoxycycline, 6-epidoxycycline, and doxycycline. When stored in a refrigerator, this solution may be used for 14 days. [note—Throughout the following sections, protect the Standard preparation and the Assay preparation from light.]

Standard preparation— Transfer about 12 mg of USP Doxycycline Hyclate RS, accurately weighed, to a 10-mL volumetric flask, add about 6 mL of Diluent, sonicate for about 5 minutes or until dissolved, dilute with Diluent to volume, and mix.

Assay preparation— Transfer about 120 mg of Doxycycline Hyclate, accurately weighed, to a 100-mL volumetric flask, dissolve in and dilute with Diluent to volume, and mix. Pass through a membrane filter having a porosity of 0.5 µm or finer.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 270 nm detector, and a 4.6-mm × 25-cm column that contains packing L21 and is maintained at 60 ± 1. The flow rate is about 1 mL per minute. Chromatograph the Resolution solution, and record the peak responses as directed for Procedure: the relative retention times are about 0.4 for 4-epidoxycycline (the main degradation product), 0.7 for 6-epidoxycycline, and 1.0 for doxycycline, the resolution, R, between the 4-epidoxycycline peak and the doxycycline peak is not less than 3.0, and the tailing factor for the doxycycline peak is not more than 2.0. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms for a period of time that is 1.7 times the retention time of doxycycline, and measure the responses for the major peaks. Calculate the potency, in µg of doxycycline (C22H24N2O8) per mg, of the Doxycycline Hyclate taken by the formula: in which C is the concentration, in mg per mL, of USP Doxycycline Hyclate RS in the Standard preparation; P is the designated potency, in µg of doxycycline (C22H24N2O8) per mg, of USP Doxycycline Hyclate RS; W is the quantity, in mg, of Doxycycline Hyclate taken to prepare the Assay preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.