A: Shake a volume of Oral Suspension, equivalent to about 100 mg of phenytoin, with 50 mL of a mixture of ether and chloroform (1 in 2) in a separator, evaporate the extract to dryness, and dry in vacuum at 105 for 4 hours: the phenytoin so obtained melts between 292 and 299 with some decomposition, the procedure for Class I being used (see Melting Range or Temperature 741).

B: Dissolve 50 mg of the residue obtained in Identification test A in 50 mL of chloroform, with slight warming if necessary. To 5 mL of this solution add 0.2 mL of a freshly prepared solution of cobaltous acetate in methanol (1 in 100) and 1 mL of a freshly prepared solution of isopropylamine in methanol (1 in 20), and mix: a violet to red-violet color is produced.

0.05 M Tris buffer— Dissolve 36.3 g of tris(hydroxymethyl)aminomethane and 60 g of sodium lauryl sulfate in 6 L of water, adjust with hydrochloric acid to a pH of 7.5 ± 0.05, and degas.

Medium: 0.05 M Tris buffer; 900 mL.

Apparatus 2: 35 rpm.

Time: 60 minutes.

Dissolution procedure— Shake the sample suspension well, about 100 shakes. Using a 5-mL syringe, collect approximately 5 mL of suspension, and record the weight. With the paddles lowered, gently empty the contents of each syringe into the bottom of each vessel containing the Medium. Start rotating the paddles. Reweigh each syringe, and determine the amount of suspension delivered into each vessel. At the end of 60 minutes, remove 4 mL from each vessel, and pass through a 0.45-µm nylon filter presaturated with Medium.

0.02 M Sodium phosphate buffer— Dissolve 2.76 g of monobasic sodium phosphate in 1 L of water.

Mobile phase— Prepare a filtered and degassed mixture of 0.02 M Sodium phosphate buffer, methanol, and acetonitrile (50:27:23), and mix. Adjust with phosphoric acid to a pH of 3.0. Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard solution— Transfer about 70 mg of USP Phenytoin RS, accurately weighed, to a 500-mL volumetric flask. Dissolve in 15 mL of methanol, dilute with Medium to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 240-nm detector and a 4.6-mm × 15-cm column that contains packing L1. The flow rate is about 1 mL per minute. Chromatograph the Standard solution, and record the responses as directed for Procedure: the column efficiency is not less than 5400 theoretical plates; the tailing factor is not more than 2.0; 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 solution and the solution under test into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of C15H12N2O2 dissolved. [note—The density of Oral Suspension must be determined and used in calculating the quantity, in mg, of phenytoin dissolved.]

Tolerances— Not less than 80% (Q) of the labeled amount of C15H12N2O2 is dissolved in 60 minutes.

for oral suspension packaged in single-unit containers: meets the requirements.

for oral suspension packaged in multiple-unit containers: meets the requirements.

Mobile phase— Prepare a filtered and degassed mixture of water, methanol, acetonitrile, 0.5% triethylamine in water, and 1.74 N acetic acid (191:100:40:1.3:1). Make adjustments if necessary.

Standard preparation— Dissolve an accurately weighed quantity of USP Phenytoin RS in methanol, and dilute quantitatively, and stepwise if necessary, with Mobile phase to obtain a solution having a known concentration of about 0.625 mg per mL.

Assay preparation— Transfer a quantity of Oral Suspension, equivalent to about 125 mg of phenytoin, into a 200-mL volumetric flask, rinse the pipet with 40 mL of methanol, and add the rinsings to the flask. Add about 50 mL of Mobile phase, dilute with methanol to volume, mix, sonicate, and filter.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 229-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 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 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 phenytoin (C15H12N2O2) in the portion of Oral Suspension 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.