A: Extract a quantity of finely powdered Tablets,equivalent to about 250mg of methazolamide,with about 50mLof acetone.Filter,and add solvent hexane until a heavy white precipitate is formed.Collect the solid on a filter,and dry:the IRabsorption spectrum of a potassium bromide dispersion of the methazolamide so obtained exhibits maxima only at the same wavelengths as that of a similar preparation of USP Methazolamide RS.

B: Dissolve about 100mg of the dried solid obtained in Identificationtest Ain 5mLof 1Nsodium hydroxide,and add 5mLof a mixture of 1g of hydroxylamine hydrochloride and 500mg of cupric sulfate in 100mLof water.Heat the solution on a steam bath for 15minutes:the solution turns dark amber,then a black precipitate is formed.

C: The retention time of the major peak in the chromatogram of the Assay preparationcorresponds to the chromatogram of the Standard preparation,as obtained in the Assay.

Medium: pH4.5acetate buffer,prepared by mixing 2.99g of sodium acetate and 1.66mLof glacial acetic acid with water to obtain 1000mLof a solution having a pHof 4.5;900mL.

Apparatus 2: 75rpm.

Time: 45minutes.

Procedure— Determine the amount of C5H8N4O3S2dissolved from UVabsorbances at the wavelength of maximum absorbance at about 252nm of filtered portions of the solution under test,suitably diluted with pH4.5acetate buffer,in comparison with a Standard solution having a known concentration of USP Methazolamide RSin the same Medium.

Tolerances— Not less than 75%(Q)of the labeled amount of C5H8N4O3S2is dissolved in 45minutes.

pH2.5Buffer— Transfer 16.8mLof dibutylamine to a beaker containing 70mLof water.Adjust with phosphoric acid to a pHof 2.5,dilute with water to 100mL,and mix.

Mobile phase— Prepare a mixture of water,methanol,and pH2.5Buffer(375:15:6).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

pH4.5Acetate buffer— Dissolve 2.99g of sodium acetate and 1.66mLof glacial acetic acid in water,dilute with water to 1000mL,and mix.Adjust,if necessary,with glacial acetic acid or sodium hydoxide to a pHof 4.5.

Standard preparation— Dissolve an accurately weighed quantity of USP Methazolamide RSin methanol to obtain a solution having a concentration of about 0.5mg per mL.Dilute this solution quantitatively,and stepwise if necessary,with pH4.5Acetate bufferto obtain a solution having a known concentration of about 50µg per mL.

Assay preparation— Weigh and finely powder not fewer than 20Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 50mg of methazolamide,to a 250-mLvolumetric flask,add 65mLof pH4.5Acetate buffer,and sonicate to dissolve.Add 65mLof methanol,and sonicate again until dissolved.Dilute with pH4.5Acetate bufferto volume,mix,and filter.Dilute an accurately measured volume of the filtrate with pH4.5Acetate bufferto obtain a solution having a concentration of about 50µg of methazolamide per mL.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 252-nm detector and an 8-mm ×10-cm column that contains packing L10.The flow rate is about 2mLper minute.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 3.0%.

Procedure— Separately inject equal volumes (about 25µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the areas for the major peaks.Calculate the quantity,in mg,of methazolamide (C5H8N4O3S2)in the portion of Tablets taken by the formula: in which Cis the concentration,in µg per mL,of USP Methazolamide RSin the Standard preparation;and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.