A: Ultraviolet Absorption á197Uñ—

Solution: 20µg per mL.

Medium: 0.1Nhydrochloric acid.

B: To 1mg add 2mLof sulfuric acid:a light red color is produced.

Tetrabutylammonium hydrogen sulfate solution— Dissolve 1g of tetrabutylammonium hydrogen sulfate in 100mLof water.Adjust with 1Nsodium hydroxide to a pHof 7.5.

Edetate disodium solution— Dissolve 0.04g of edetate disodium in 100mLof water.Adjust with 1Nsodium hydroxide to a pHof 7.5.

pH7.5Phosphate buffer— Prepare a mixture of 0.33Mdibasic potassium phosphate and 0.33Mmonobasic sodium phosphate (85:15).Adjust,if necessary,by adding more of the appropriate component to a pHof 7.5.

Mobile phase— Transfer,with the aid of 200mLof water,50g of tertiary butyl alcohol to a 1000-mLvolumetric flask.Add 60mLof pH7.5Phosphate buffer,50mLof Tetrabutylammonium hydrogen sulfate solution,and 10mLof Edetate disodium solution,and dilute with water to volume.Degas before use.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Dissolve an accurately weighed quantity of USP Oxytetracycline RSin 0.01Nhydrochloric acid to obtain a solution having a known concentration of about 0.22mg per mL.

System suitability solution— Prepare a solution of tetracycline hydrochloride in 0.01Nhydrochloric acid containing about 0.2mg per mL.Mix 3mLof this solution and 1.5mLof the Standard preparation,and dilute with water to 25mL.

Assay preparation— Transfer about 44mg of Oxytetracycline to a 200-mLvolumetric flask,add about 25mLof 0.01Nhydrochloric acid,swirl to dissolve,dilute with 0.01Nhydrochloric acid to volume,and mix.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm ×25-cm column that contains packing L21and is maintained at 60±2.The flow rate is about 1mLper minute.Chromatograph the System suitability solution,and record the peak responses as directed for Procedure:the relative retention times are about 0.6for oxytetracycline and 1.0for tetracycline;and the resolution,R,between the oxytetracycline peak and the tetracycline peak is not less than 5.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the tailing factor is not more than 1.25;and the relative standard deviation for replicate injections is not more than 1.0%.

Procedure— [NOTE—Use peak areas where peak responses are indicated.]Separately inject equal volumes (about 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in µg,of C22H24N2O9in each mg of the Oxytetracycline taken by the formula: in which Cis the concentration,in mg per mL,of USP Oxytetracycline RSin the Standard preparation;Pis the assigned potency,in µg per mg,of USP Oxytetracycline RS;Wis the weight,in mg,of the Oxytetracycline taken to prepare the Assay preparation;and rUand rSare the oxytetracycline peak responses obtained from the Assay preparationand the Standard preparation,respectively.