Mobile phase— Prepare as directed in the Assay.

Standard solution— Dissolve an accurately weighed quantity of USP Phenytoin RSin methanol,and dilute quantitatively,and stepwise if necessary,with methanol to obtain a solution having a known concentration of about 10µg 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 10µg per mL.Dissolve 10mg of USP Phenytoin RSin 10.0mLof the benzoin solution.

Chromatographic system (see Chromatography á621ñ)—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 1.5mLper minute.Chromatograph the Resolution solution,and record the peak responses as directed for the Procedure:the relative retention times are about 0.75for phenytoin and 1.0for 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 20µL)of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the responses for all of the peaks.Calculate the percentage of each impurity peak in the Test solutiontaken by the formula: in which Cis the concentration,in µg per mL,of USP Phenytoin RSin the Standard solution;Dis the concentration,in µg per mL,of phenytoin in the Test solution;riis the peak response for each impurity;and rSis 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 1.0µg per mL.

Chromatographic system (see Chromatography á621ñ)— Use the same system as directed in the test for Chromatographic purityexcept 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 20µL)of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the responses for all of the peaks.The relative retention times are about 0.25for phenytoin and 1.0for benzophenone.Calculate the percentage of benzophenone in the portion of Phenytoin taken by the formula: in which Cis the concentration,in µg per mL,of benzophenone in the Standard solution;Dis the concentration,in µg per mL,of phenytoin in the Test solution;and rUand rSare the benzophenone peak responses obtained from the Test solutionand the Standard solution,respectively:not more than 0.1%of benzophenone is found.

Solvent— Use dimethyl sulfoxide.

Mobile phase— Prepare a filtered and degassed mixture of methanol and water (55:45).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Dissolve,with the aid of sonication if necessary,an accurately weighed quantity of USP Phenytoin RSin Mobile phase,and dilute quantitatively,and stepwise if necessary,with Mobile phaseto obtain a solution having a known concentration of about 100µg per mL.

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

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

Chromatographic system (see Chromatography á621ñ)—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 1.5mLper 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 0.75for phenytoin and 1.0for 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 20µL)of the Standard preparationand Assay preparation Binto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of C15H12N2O2in the portion of Phenytoin taken by the formula: in which Cis the concentration,in mg per mL,of USP Phenytoin RSin the Standard preparation;and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.