A: Dissolve about 300 mg of Phenytoin Sodium, accurately weighed, in about 50 mL of water in a separator. Add 10 mL of 3 N hydrochloric acid, and extract with three successive portions, measuring 100, 60, and 30 mL, respectively, of a 1 in 2 mixture of ether and chloroform. Evaporate the combined extracts, and dry the residue of phenytoin at 105 for 4 hours: the IR absorption spectrum of a potassium bromide dispersion of the residue so obtained exhibits maxima only at the same wavelengths as that of a similar preparation of USP Phenytoin RS.

B: It responds to the flame test for Sodium 191.

Mobile phase, Standard stock preparation, System suitability stock solution, and System suitability solution— Prepare as directed in the Assay.

Standard solution— Dissolve accurately weighed quantities of benzophenone, USP Phenytoin RS, USP Phenytoin Related Compound A RS, and USP Phenytoin Related Compound B RS in Mobile phase, and dilute quantitatively, and stepwise if necessary, with Mobile phase to obtain a solution having known concentrations of 0.5 µg per mL, 1 µg per mL, 9 µg per mL, and 9 µg per mL, respectively.

Test solution— Use the Assay stock preparation.

Chromatographic system— Proceed as directed in the Assay, except to inject the Standard solution instead of the Standard preparation: the relative standard deviation for replicate injections is not more than 5.0% for each compound.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the peak responses. Calculate the percentage of phenytoin related compound A, phenytoin related compound B, and benzophenone in the portion of Phenytoin Sodium taken by the formula: in which C is the concentration, in µg per mL, of the respective analyte in the Standard solution; D is the concentration, in µg per mL, of Phenytoin Sodium in the Test solution; and ri and rS are the peak responses for phenytoin related compound A, phenytoin related compound B, or benzophenone obtained from the Test solution and the Standard solution, respectively: not more than 0.9% each of phenytoin related compound A and phenytoin related compound B is found, and not more than 0.1% of benzophenone is found. Calculate the percentage of every other impurity in the portion of Phenytoin Sodium taken by the formula: in which C is the concentration, in µg per mL, of USP Phenytoin RS in the Standard solution; 274.25 and 252.27 are the molecular weights of phenytoin sodium and phenytoin, respectively; ri and rS are the peak responses of each impurity obtained from the Test solution and the Standard solution, respectively; and the other term is as defined above. Not more than 0.9% of total impurities is found, excluding benzophenone.

Mobile phase— Prepare a filtered and degassed mixture of 0.05 M monobasic ammonium phosphate buffer, adjusted to a pH of 2.5 with phosphoric acid, acetonitrile, and methanol (45:35:20). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard stock preparation— Transfer about 100 mg of USP Phenytoin RS, accurately weighed, to a 100-mL volumetric flask, dissolve in Mobile phase, and sonicate, if necessary, to dissolve.

System suitability stock solution— Transfer 5.0 mL of the Standard stock preparation to a 50-mL volumetric flask, and dilute with Mobile phase to volume to obtain a solution having a known concentration of about 100 µg per mL.

System suitability solution— Transfer about 75.0 mg of benzoin to a 50-mL volumetric flask, dissolve in 10 mL of methanol, and dilute with a mixture of 0.05 M monobasic ammonium phosphate buffer, previously adjusted with phosphoric acid to a pH of 2.5 and acetonitrile (45:35), to volume. Transfer 1.0 mL of the solution so obtained to a 10-mL volumetric flask, and dilute with the System suitability stock solution to volume.

Standard preparation— Transfer 5 mL of the Standard stock preparation to a 100-mL volumetric flask, and dilute with Mobile phase to volume.

Assay stock preparation— Transfer about 100 mg of Phenytoin Sodium, accurately weighed, to a 100-mL volumetric flask, dissolve in and dilute with Mobile phase to volume, and mix.

Assay preparation— Transfer 5.0 mL of the Assay stock preparation to a 100-mL volumetric flask, dilute with Mobile phase to volume, and mix.

Chromatographic system— 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 responses as directed for Procedure: the relative standard deviation for replicate injections is not more than 1.0%. Chromatograph the System suitability solution, and record the peak responses as directed for Procedure: the relative retention times are 1.0 for phenytoin and about 1.3 for benzoin; the column efficiency is not less than 9400 theoretical plates for the phenytoin peak; the tailing factor is not more than 1.5; and the resolution, R, between phenytoin and benzoin is not less than 1.5.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the peak responses for the major peaks. Calculate the quantity, in mg, of C15H11N2NaO2 in the portion of Phenytoin Sodium taken by the formula: in which C is the concentration, in mg per mL, of USP Phenytoin RS in the Standard preparation; 274.25 and 252.27 are the molecular weights of phenytoin sodium and phenytoin, respectively; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.