Mobile phase— Prepare as directed in the Assay.

Standard solution— Dissolve an accurately weighed quantity of USP Phenytoin RS in methanol, and dilute quantitatively, and stepwise if necessary, with methanol to obtain a solution having a known concentration of about 10 µg per mL.

Test solution— Use Assay preparation A, prepared as directed in the Assay.

Resolution solution— Prepare a solution of benzoin in methanol having a concentration of about 10 µg per mL. Dissolve 10 mg of USP Phenytoin RS in 10.0 mL of the benzoin solution.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 220-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L1. The flow rate is about 1.5 mL per minute. Chromatograph the Resolution solution, and record the peak responses as directed for the Procedure: the relative retention times are about 0.75 for phenytoin and 1.0 for benzoin; and the resolution, R, is not less than 1.5.

Procedure— [note—Use peak areas where peak responses are indicated.] Separately inject equal volumes (about 20 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the responses for all of the peaks. Calculate the percentage of each impurity peak in the Test solution taken by the formula: in which C is the concentration, in µg per mL, of USP Phenytoin RS in the Standard solution; D is the concentration, in µg per mL, of phenytoin in the Test solution; ri is the peak response for each impurity; and rS is the peak response for phenytoin in the Standard solution: not more than 0.9% total impurities is found, excluding benzophenone.

Mobile phase and Test solution—Prepare as directed in the test for Chromatographic purity.

Standard solution— Dissolve an accurately weighed quantity of benzophenone in methanol, and dilute quantitatively, and stepwise if necessary, with methanol to obtain a solution having a known concentration of about 1.0 µg per mL.

Chromatographic system (see Chromatography 621)— Use the same system as directed in the test for Chromatographic purity except to chromatograph three injections of the Standard solution, and record the peak responses as directed for Procedure: the relative standard deviation is not more than 5.0%.

Procedure— [note—Use peak areas where peak responses are indicated.] Separately inject equal volumes (about 20 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the responses for all of the peaks. The relative retention times are about 0.25 for phenytoin and 1.0 for benzophenone. Calculate the percentage of benzophenone in the portion of Phenytoin taken by the formula: in which C is the concentration, in µg per mL, of benzophenone in the Standard solution; D is the concentration, in µg per mL, of phenytoin in the Test solution; and rU and rS are the benzophenone peak responses obtained from the Test solution and the Standard solution, respectively: not more than 0.1% of benzophenone is found.

Mobile phase— Prepare a filtered and degassed mixture of methanol and water (55:45). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve, with the aid of sonication if necessary, 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 100 µg per mL.

Assay preparation— Transfer about 100 mg of Phenytoin, accurately weighed, to a 100-mL volumetric flask, dissolve in and dilute with methanol to volume, and mix (Assay preparation A). Transfer 10.0 mL of this solution to a second 100-mL volumetric flask, dilute with Mobile phase to volume, and mix (Assay preparation B).

Resolution solution— Prepare a solution of benzoin in Mobile phase having a concentration of about 1.5 mg per mL. Mix 1.0 mL of this solution and 9.0 mL of the Standard preparation.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 220-nm detector and a 4.6-mm × 25-cm column that contains 5-µm 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 relative standard deviation is not more than 1.0%. Chromatograph the Resolution solution. The relative retention times are about 0.75 for phenytoin and 1.0 for benzoin; the resolution, R, is not less than 1.5; and the tailing factor for the phenytoin peak is not more than 1.5.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and Assay preparation B 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 Phenytoin 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.