A: Infrared Absorption 197K.

B: The retention time of the major peak in the chromatogram of the Assay preparation corresponds to that of the Standard preparation, as obtained in the Assay.

C: Prepare a test solution of it in a mixture of methanol and water (1:1) containing 50 mg per mL. Similarly prepare a Standard solution, using USP Zalcitabine RS. Separately apply 10 µL portions of the test solution and the Standard solution to a suitable thin-layer chromatographic plate (see Chromatography 621) coated with a 0.25-mm layer of chromatographic silica gel mixture. Place the plate in a paper-lined chromatographic chamber saturated with a solvent system consisting of the clear lower layer of a mixture of alcohol, dichloromethane, and water (3:2:2), and develop the chromatogram. When the solvent front has moved about three-fourths of the length of the plate, remove the plate from the chamber, mark the solvent front, and allow to dry. Locate the spots on the plate by examination under short-wavelength UV light: the RF value of the principal spot obtained from the test solution corresponds to that obtained from the Standard solution.

Test solution: 7 mg per mL, in water.

Phosphate buffer , Mobile phase, Resolution solution, Standard preparation, and Assay preparation—Prepare as directed in the Assay.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 270-nm detector and a 4.6-mm × 15-cm column that contains 5-µm packing L1. The flow rate is about 1 mL per minute. Chromatograph the Resolution solution, and record the peak responses as directed for Procedure: the resolution, R, between zalcitabine and zalcitabine related compound A is not less than 2.0, and the tailing factor for zalcitabine is not greater than 1.5. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative standard deviation is not more than 2%.

Procedure— Inject a volume (about 20 µL) of the Assay preparation into the chromatograph, record the chromatograms, and measure the responses of the major peaks. Inject a volume of acetonitrile in water (3 in 100) as a chromatographic blank. Calculate the percentage of each impurity in the portion of zalcitabine taken by the formula: in which ri is the peak response for each impurity, and rs is the sum of the responses of all of the peaks: not more than 0.3% of any individual impurity is found, and the sum of all impurities is not more than 2.0%.

Test solution: 50 mg per mL, in a mixture of methanol and water (1:1).

Standard solution: a mixture of methanol and water (1:1).

Eluant: the lower layer of a mixture of alcohol, dichloromethane, and water (3:2:2).

Visualization: 1.

(Official January 1, 2007)

Phosphate buffer— Dissolve 6.8 g of monobasic potassium phosphate and 8.7 g of dibasic potassium phosphate in 2000 mL of water. Adjust, if necessary, with dilute phosphoric acid or potassium hydroxide solution (1 in 10) to a pH of 6.8 ± 0.05.

Mobile phase— Prepare a filtered and degassed mixture of Phosphate buffer and acetonitrile (97:3). Make adjustments if necessary (see System Suitability under Chromatography 621).

Resolution solution— Dissolve USP Zalcitabine RS and USP Zalcitabine Related Compound A RS in a mixture of acetonitrile in water (3 in 100), and dilute quantitatively, and stepwise if necessary, with the same solvent to obtain a solution containing about 0.024 mg of each per mL.

Standard preparation— Dissolve an accurately weighed quantity of USP Zalcitabine RS in a mixture of acetonitrile and water (3 in 100) to obtain a solution having a known concentration of about 0.5 mg per mL.

Assay preparation— Transfer about 100 mg of Zalcitabine, accurately weighed, to a 200-mL volumetric flask. Dissolve in a mixture of acetonitrile and water (3 in 100), dilute with the same solvent to volume, and mix.

Chromatographic system— The liquid chromatograph is equipped with a 270-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 preparation, and record the peak responses as directed for Procedure: the tailing factor for the zalcitabine peak is not greater than 1.5, and the relative standard deviation is not more than 2.0%. Chromatograph the Resolution solution: the resolution, R, between zalcitabine and zalcitabine related compound A is not less than 2.

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 responses for the major peaks. Calculate the quantity, in mg, of C9H13N3O3 in the portion of Zalcitabine 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 peak responses obtained from the Assay preparation and the Standard preparation, respectively.