0.05 M Tris buffer— Dissolve 60.5 g of tris(hydroxymethyl)aminomethane in 6 L of water. Dilute with water to 10 L, and adjust with phosphoric acid to a pH of 9.0 ± 0.05. Dissolve 100 g of sodium dodecyl sulfate in about 6 L of the prepared buffer, transfer this solution to the remaining buffer solution, and mix.

Medium: 0.05 M Tris buffer; 900 mL.

Apparatus 2: 100 rpm.

Time: 120 minutes.

Triethylamine solution, Mobile phase, and Chromatographic system— Proceed as directed in the Assay.

Standard solution— Dissolve an accurately weighed quantity of USP Phenytoin RS in methanol to obtain a solution having a known concentration of 3.0 mg per mL. Transfer a portion of this solution to a suitable container, and dilute quantitatively, and stepwise if necessary, with Medium to obtain a concentration of 0.06 mg per mL. Transfer 10.0 mL of this solution to a 50-mL volumetric flask, dilute with Mobile phase to volume, and mix.

Test solution— Withdraw a portion of the solution under test, and filter, discarding the first 3 mL of the filtrate. Pipet 10.0 mL of this solution into a 50-mL volumetric flask, dilute with Mobile phase to volume, and mix.

Procedure— Separately inject equal volumes (about 25 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the areas of the peak responses. Determine the amount of C15H12N2O2 dissolved by comparison of the peak responses obtained from the Standard solution and the Test solution.

Tolerances— Not less than 70% (Q) of the labeled amount of C15H12N2O2 is dissolved in 120 minutes.

Triethylamine solution— Transfer 1 mL of triethylamine to a 100-mL volumetric flask, dilute with water to volume, and mix.

Mobile phase— Prepare a filtered and degassed mixture of water, methanol, acetonitrile, Triethylamine solution, and acetic acid (500:270:230:5:1). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve an accurately weighed quantity of USP Phenytoin RS in Mobile phase, and dilute quantitatively, and stepwise if necessary, with Mobile phase to obtain a solution having a known concentration of about 0.5 mg per mL.

Assay preparation— Weigh and finely powder not fewer than 20 Chewable Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 250 mg of phenytoin, to a 500-mL volumetric flask, dissolve in and dilute with Mobile phase to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 25-cm column that contains packing L1. The flow rate is about 1.5 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the column efficiency is not less than 6500 theoretical plates; the tailing factor is not more than 1.5; and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 25 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 C15H12N2O2 in the portion of Chewable Tablets taken by the formula: in which C is the concentration, in mg per mL, of USP Phenytoin RS in the Standard preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.