Medium: water; 900 mL.

Apparatus 2: 50 rpm.

Time: 20 minutes.

Phosphate buffer— Transfer 8.7 g of dibasic potassium phosphate to a 1 L volumetric flask, dilute with water to volume, and adjust with phosphoric acid to a pH of 6.8.

Mobile phase— Prepare a filtered and degassed mixture of Phosphate buffer, methanol, and acetonitrile (96:4:3). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Transfer about 10 mg of USP Zalcitabine RS, accurately weighed, to a 250-mL volumetric flask. Add about 200 mL of Medium, and sonicate until complete solution is effected. Dilute with Medium to volume, and mix.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 270-nm detector and a 3.9-mm × 15-cm column that contains 10-µm packing L1. The flow rate is about 1 mL per minute. Chromatograph the Standard preparation, and record the peak responses as described for Procedure: the retention time is about 5 minutes; the tailing factor is not more than 2; and the relative standard deviation is not more than 2%.

Procedure— Separately inject equal volumes (about 150 µL) of the Standard preparation and a filtered portion of the solution under test into the chromatograph, record the chromatograms, and measure the responses of the major peaks. Calculate the quantity, in mg, of zalcitabine in the Tablets taken by the formula: in which C is the concentration, in mg per mL, of USP Zalcitabine RS in the Standard preparation; and rU and rS are the zalcitabine peak responses obtained from the solution under test and the Standard preparation, respectively.

Tolerances— Not less than 80% (Q) of the labeled amount of C9H13N3O3 is dissolved in 20 minutes.

(Official January 1, 2007)

Buffer solution— Dissolve 3.4 g of monobasic potassium phosphate in sufficient water to make 1 L. Using a suitable pH meter, adjust with phosphoric acid to a pH of 2.2. [NOTE—If too much phosphoric acid is added, yielding a pH below 2.2, the pH may be adjusted to 2.2 with 0.025 M monobasic potassium phosphate.] Add 1.08 g of sodium 1-octanesulfonic acid, and mix.

Mobile phase— Prepare a filtered and degassed mixture of Buffer solution and acetonitrile (85:15).

Diluent— Prepare a mixture of water and acetonitrile (17:3).

Resolution solution— Dissolve weighed quantities of USP Zalcitabine RS and USP Zalcitabine Related Compound A RS in Diluent, and dilute quantitatively, and stepwise if necessary, with the same solvent to obtain a solution containing about 0.02 mg per mL of zalcitabine and 0.002 mg per mL of zalcitabine related compound A.

Standard preparation— Dissolve an accurately weighed quantity of USP Zalcitabine RS in Diluent, and dilute quantitatively, and stepwise if necessary, with the same solvent to obtain a solution having a known concentration of about 0.008 mg per mL.

Assay preparation— Transfer 5 Tablets to a volumetric flask suitable to obtain a solution containing about 0.008 mg zalcitabine per mL. Add a volume of Diluent that is about six-tenths of the volume of the flask, sonicate for 15 minutes, and shake by mechanical means for 10 minutes. Dilute with Diluent to volume, and mix. Filter before use.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 280-nm detector, a precolumn cartridge that contains packing L1, and a 4.6-mm × 25-cm analytical 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 Procedure: the resolution, R, between the zalcitabine and zalcitabine related compound A peaks is not less than 1.1, and the tailing factor for the zalcitabine peak is not more than 1.5. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative standard deviation for replicate injections is not more than 2%.

Procedure— Separately inject equal volumes (about 50 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses of the major peaks. Calculate the quantity, in mg, of C9H13N3O3 in the portion of Tablets taken by the formula: in which C is the concentration, in mg per mL, of USP Zalcitabine RS in the Standard preparation; V is the volume, in mL, of the volumetric flask used to prepare the Assay preparation; and rU and rS are the zalcitabine peak responses obtained from the Assay preparation and the Standard preparation, respectively.