Mobile phase andDiluent— Prepare as directed in the Assay.

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

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

Standard solution 1— Prepare as directed for the Standard preparationin the Assay.

Standard solution 2— Transfer 2.0mLof Standard solution 1and 2.0mLof the Methacycline standard stock solutionto a 100-mLvolumetric flask,dilute with Diluentto volume,and mix.This solution contains about 0.024mg each of USP Doxycycline Hyclate RSand USP Methacycline Hydrochloride RSper mL.

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

Chromatographic system (see Chromatography á621ñ)—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 0.4for 4-epidoxycycline (the main degradation product),0.6for methacycline,0.7for 6-epidoxycycline,and 1.0for 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 20µL)of Standard solution 2and the Test solutioninto the chromatograph,record the chromatograms for a period of time that is 1.7times 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 CMis the concentration,in mg per mL,of USP Methacycline Hydrochloride RSin Standard solution 2;Wis the weight,in mg,of Doxycycline Hyclate taken to prepare the Test solution;and rUand rMare the methacycline peak responses obtained from the Test solutionand 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 CSis the concentration,in mg per mL,of USP Doxycycline Hyclate RSin Standard solution 2;Wis the weight,in mg,of Doxycycline Hyclate taken to prepare the Test solution;riis the peak response for each impurity obtained from the Test solution;and rSis 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 2.72g of monobasic potassium phosphate,0.74g of sodium hydroxide,0.50g of tetrabutylammonium hydrogen sulfate,and 0.40g of edetate disodium to a 1000-mLvolumetric flask.Add about 850mLof water,and stir to dissolve.Add 60g of tertiary butyl alcohol with the aid of water,dilute with water to volume,and adjust with 1Nsodium hydroxide to a pHof 8.0±0.1.Pass this solution through a filter having a porosity of 0.5µm or finer,and degas before using.Make any necessary adjustments (see System Suitabilityunder Chromatography á621ñ).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 0.01Nhydrochloric acid.

Resolution solution— Prepare a solution of USP Doxycycline Hyclate RSin Diluentcontaining about 6mg of doxycycline per mL.Transfer 5mLof this solution to a 25-mLvolumetric flask,heat on a steam bath for 60minutes,and evaporate to dryness on a hot plate,taking care not to char the residue.Dissolve the residue in 0.01Nhydrochloric acid,dilute with Diluentto volume,and mix.Pass a portion of this solution through a filter having a porosity of 0.5µm 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 14days.[NOTE—Throughout the following sections,protect the Standard preparationand the Assay preparationfrom light.]

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

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

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 270nm detector,and a 4.6-mm ×25-cm column that contains packing L21and is maintained at 60±1.The flow rate is about 1mLper minute.Chromatograph the Resolution solution,and record the peak responses as directed for Procedure:the relative retention times are about 0.4for 4-epidoxycycline (the main degradation product),0.7for 6-epidoxycycline,and 1.0for 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 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms for a period of time that is 1.7times 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 Cis the concentration,in mg per mL,of USP Doxycycline Hyclate RSin the Standard preparation;Pis the designated potency,in µg of doxycycline (C22H24N2O8)per mg,of USP Doxycycline Hyclate RS;Wis the quantity,in mg,of Doxycycline Hyclate taken to prepare the Assay preparation;and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.