A: The RFvalue of the principal spot observed in the chromatogram of the Test solutioncorresponds to those of the principal spots observed in the chromatograms of the Standard solutions,as obtained in the test for Chromatographic purity.

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

Medium: pH4.0acetate buffer,prepared by transferring 2g of sodium hydroxide to a 1-Lvolumetric flask,dissolving in 450mLof water,adjusting with glacial acetic acid to a pHof 4.0,and diluting with water to volume;900mL.

Apparatus 1: 100rpm.

Time: 45minutes.

Procedure— Determine the amount of C18H19ClN4dissolved by employing UVabsorption at the wavelength of maximum absorbance at about 290nm on filtered portions of the solution under test,suitably diluted with Dissolution Medium,if necessary,in comparison with a Standard solution having a known concentration of USP Clozapine RSin the same Medium.

Tolerances— Not less than 85%(Q)of the labeled amount of C18H19ClN4is dissolved in 45minutes.

Adsorbent: 0.25-mm layer of chromatographic silica gel mixture.

Diluting solution— Prepare a mixture of chloroform and methanol (4:1).

Test solution— Weigh and finely powder not fewer than 20Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 125mg of clozapine,to a 25-mLvolumetric flask,dissolve in 20mLof Diluting solution,shake by mechanical means for 15minutes,dilute with Diluting solutionto volume,filter,and mix.

Standard solutions— Dissolve an accurately weighed quantity of USP Clozapine RSin Diluting solution,and mix to obtain a solution having a known concentration of 5.0mg per mL.Dilute portions of this solution quantitatively with Diluting solutionto obtain the following solutions:

Application volume: 20µL.

Developing solvent system: a mixture of n-heptane,chloroform,dehydrated alcohol,and ammonium hydroxide (30:30:30:1).

Procedure— Proceed as directed for Thin-Layer Chromatographyunder Chromatography á621ñ.Examine the plate under short-wavelength UVlight,and compare the intensities of any secondary spots observed in the chromatogram of the Test solutionwith those of the principal spots in the chromatograms of the Standard solutions:no secondary spot from the chromatogram of theTest solutionis larger or more intense than the principal spot obtained from Standard solution A(0.5%),and the sum of the intensities of the secondary spots obtained from the Test solutioncorresponds to not more than 2.0%.

Mobile phase— Prepare a filtered and degassed mixture of methanol,water,and triethylamine (800:200:0.75).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Dissolve an accurately weighed quantity of USP Clozapine RSin methanol,and dilute quantitatively,and stepwise if necessary,with water to obtain a solution having a known concentration of about 0.125mg per mL.[NOTE—The final solvent composition of methanol and water is about 8:2.]

Resolution solution— Transfer about 10mg of clozapine,accurately weighed,to a suitable container.Add 5mLof 0.1Nhydrochloric acid,and heat for 2hours at 90.Transfer this solution to a 100-mLvolumetric flask,add 15mLof water,dilute with methanol to volume,and mix.Transfer about 10mLof this solution to a suitable container,add about 10mLof Standard preparation,and mix.

Assay preparation— Weigh and finely powder not fewer than 20Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 125mg of clozapine,to a 1-Lvolumetric flask,dissolve in 640mLof methanol,sonicate for 10minutes,dilute with water to volume,mix,and filter.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 257-nm detector and a 4.0-mm ×25-cm column that contains packing L7.The flow rate is about 1mLper minute.Chromatograph the Resolution solution,and record the peak responses as directed forProcedure:the resolution,R,between the clozapine peak and any other peak is not less than 1.5.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the column efficiency is not less than 1500theoretical plates,and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of clozapine (C18H19ClN4)in the portion of Tablets taken by the formula: in which Cis the concentration,in mg per mL,of USP Clozapine RSin the Standard preparation;and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.