» Clozapine Tablets contain not less than 90.0 percent and not more than 110.0 percent of the labeled amount of clozapine (C18H19ClN4).
Packaging and storage Preserve in well-closed containers.
USP Reference standards 11
A: The RF value of the principal spot observed in the chromatogram of the Test solution corresponds 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 preparation corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay.
Medium: pH 4.0 acetate buffer, prepared by transferring 2 g of sodium hydroxide to a 1-L volumetric flask, dissolving in 450 mL of water, adjusting with glacial acetic acid to a pH of 4.0, and diluting with water to volume; 900 mL.
Apparatus 1: 100 rpm.
Time: 45 minutes.
Procedure Determine the amount of C18H19ClN4 dissolved by employing UV absorption at the wavelength of maximum absorbance at about 290 nm 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 RS in the same Medium.
Tolerances Not less than 85% (Q) of the labeled amount of C18H19ClN4 is dissolved in 45 minutes.
Uniformity of dosage units 905: meets the requirements.
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 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 125 mg of clozapine, to a 25-mL volumetric flask, dissolve in 20 mL of Diluting solution, shake by mechanical means for 15 minutes, dilute with Diluting solution to volume, filter, and mix.
Standard solutions Dissolve an accurately weighed quantity of USP Clozapine RS in Diluting solution, and mix to obtain a solution having a known concentration of 5.0 mg per mL. Dilute portions of this solution quantitatively with Diluting solution to obtain the following solutions:
Application volume: 20 µL.
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 Chromatography under Chromatography 621. Examine the plate under short-wavelength UV light, and compare the intensities of any secondary spots observed in the chromatogram of the Test solution with those of the principal spots in the chromatograms of the Standard solutions: no secondary spot from the chromatogram of the Test solution is 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 solution corresponds 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 Suitability under Chromatography 621).
Standard preparation Dissolve an accurately weighed quantity of USP Clozapine RS in methanol, and dilute quantitatively, and stepwise if necessary, with water to obtain a solution having a known concentration of about 0.125 mg per mL. [noteThe final solvent composition of methanol and water is about 8:2. ]
Resolution solution Transfer about 10 mg of clozapine, accurately weighed, to a suitable container. Add 5 mL of 0.1 N hydrochloric acid, and heat for 2 hours at 90. Transfer this solution to a 100-mL volumetric flask, add 15 mL of water, dilute with methanol to volume, and mix. Transfer about 10 mL of this solution to a suitable container, add about 10 mL of Standard preparation, and mix.
Assay preparation Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 125 mg of clozapine, to a 1-L volumetric flask, dissolve in 640 mL of methanol, sonicate for 10 minutes, dilute with water to volume, mix, and filter.
Chromatographic system (see Chromatography 621) 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 1 mL per minute. Chromatograph the Resolution solution, and record the peak responses as directed for Procedure: 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 1500 theoretical plates, and the relative standard deviation for replicate injections is not more than 2.0%.
Procedure Separately inject equal volumes (about 10 µL) of the Standard preparation and the Assay preparation into 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:
1000C(rU/rS)in which C is the concentration, in mg per mL, of USP Clozapine RS in the Standard preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.
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USP35NF30 Page 2750Pharmacopeial Forum: Volume No. 34(3) Page 589