Solution: 20µg per mL.

Medium: hydrochloric acid in methanol (1in 1200).

Mobile phase— Prepare a mixture of 0.2Mammonium oxalate,dimethylformamide,and 0.1Medetate disodium (11:5:4),adjust with tetrabutylammonium hydroxide,40percent in water,to a pHof 7.0,and filter.Make adjustments,if necessary (see System Suitabilityunder Chromatography á621ñ).

System suitability preparation— Prepare a solution of USP Methacycline Hydrochloride RSand USP Doxycycline Hyclate RSin Mobile phasecontaining about 0.5mg of each per mL.

Standard preparation— Quantitatively dissolve an accurately weighed quantity of USP Methacycline Hydrochloride RSin Mobile phaseto obtain a solution having a known concentration of about 0.5mg per mL.

Assay preparation— Transfer about 50mg of Methacycline Hydrochloride,accurately weighed,to a 100-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 354-nm detector and a 4.6-mm ×15-cm column that contains 3.5-µm packing L1.The flow rate is about 1mLper minute.Chromatograph the System suitability preparation,and record the peak responses as directed for Procedure:the relative retention times are about 0.75for methacycline and 1.0for doxycycline;and the resolution,R,between methacycline and doxycycline is not less than 1.5.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the tailing factor is not more than 1.5;and the relative standard deviation for replicate injections is not more than 1.0%.

Procedure— Separately inject equal volumes (about 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the areas for the major peaks.Calculate the quantity,in µg,of methacycline (C22H22N2O8)in each mg of Methacycline Hydrochloride taken by the formula: in which Cis the concentration,in mg per mL,of USP Methacycline Hydrochloride RSin the Standard preparation;Eis the methacycline content,in µg per mg,of USP Methacycline RS;Wis the quantity,in mg,of Methacycline Hydrochloride taken to prepare the Assay preparation;and rUand rSare the methacycline peak areas obtained from the Assay preparationand the Standard preparation,respectively.