A: Infrared Absorption 197K.

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

0.01 M Ammonium acetate— Prepare as directed in the Assay.

Solution A— Prepare a filtered and degassed mixture of 0.01 M Ammonium acetate and acetonitrile (96.5:3.5).

Solution B— Prepare a filtered and degassed mixture of 0.01 M Ammonium acetate and acetonitrile (75:25).

Mobile phase— Use variable mixtures of Solution A and Solution B as directed for Chromatographic system. Make adjustments if necessary (see System Suitability under Chromatography 621).

System suitability solution— Prepare a 0.50 mg per mL solution of USP Stavudine System Suitability Mixture RS in water.

Test solution— Prepare a solution of Stavudine, accurately weighed, in water, and having a concentration of about 0.5 mg per mL.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L1. The flow rate is about 2.1 mL per minute. The chromatograph is programmed as follows. Chromatograph the System suitability solution, and record the peak responses as directed for Procedure: the retention time of the main stavudine peak is 10.5 ± 2 minutes; the relative retention times are about 1.0 for stavudine and 0.28 for thymine; the resolution, R, between thymidine epimer and thymidine is greater than or equal to 1.15, and that between stavudine and -stavudine is greater than or equal to 1.0; the capacity factor, k ¢, is greater than 4; and the column efficiency is greater than 9500 theoretical plates.

Procedure— Inject equal volumes (about 10 µL) of the System suitability solution and the Test solution into the chromatograph, record the chromatograms for twice the retention time of the major peak, or at least until the last impurity has eluted, and measure the area of the responses for all the peaks. Determine the percentage of thymine in the portion of Stavudine taken by the formula: in which F is the relative response factor and is equal to 0.69; rU is the peak response of thymine obtained from the Test solution; and rs is the sum of the responses of all the related peaks in the chromatogram of the Test solution, including that of the main stavudine peak: not more than 0.5% of thymine is found. Calculate the percentage of all other impurities in the portion of Stavudine taken by the formula: in which rU is the peak area response of each impurity obtained from the Test solution; and rs is the sum of the area responses of all the related peaks in the chromatogram of the Test solution, including that of the main stavudine peak and disregarding any peak observed in the blank: not more than 0.1% of any impurity is found; and not more than 1.0% of total impurities is found, including thymine. The quantitation limit is 0.03% of the total sample related peak areas.

(Official January 1, 2007)

0.01 M Ammonium acetate— Dissolve 0.77 g of ammonium acetate in about 900 mL of water in a 1000-mL volumetric flask. Dilute with water to volume, and mix.

Mobile phase— Prepare a filtered and degassed mixture of 0.01 M Ammonium acetate and acetonitrile (95:5).

Standard preparation— Transfer about 10 mg of USP Stavudine RS, accurately weighed, to a 100-mL volumetric flask, and dissolve in and dilute with water to volume. Pipet 10.0 mL of this solution into a 50-mL volumetric flask, dilute with water to volume, and mix.

Assay preparation— Transfer about 10 mg of the Stavudine to a 100-mL volumetric flask, dissolve in and dilute with water to volume, and mix. Pipet 10.0 mL of this solution into a 50-mL volumetric flask, dilute with water to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 3.3-cm column that contains 3-µm packing L1. The flow rate is about 0.7 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the retention time of the stavudine peak is between 2.8 and 5.0 minutes; the column efficiency is not less than 800 theoretical plates; the tailing factor is less than or equal to 1.6; and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure — Separately inject equal volumes (about 25 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 C10H12N2O4 in the portion of Stavudine taken by the formula: in which C is the concentration, in mg per mL, of USP Stavudine RS in the Standard preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.