A: Thin-Layer Chromatographic Identification Test 201—

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.

Medium: water; 900 mL.

Apparatus 2: 75 rpm.

Time: 30 minutes.

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

Standard solution— Dissolve an accurately weighed quantity of USP Stavudine RS in water, and dilute quantitatively, and stepwise if necessary, with water to obtain a solution having a known concentration corresponding to that of the solution under test.

Chromatographic system (see Chromatography 621)— Proceed as directed in the Assay except that the liquid chromatograph is equipped with a 254-nm detector. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the column efficiency is not less than 800 theoretical plates; the tailing factor is not more than 2; and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Determine the amount of C10H12N2O4 dissolved, employing the procedure set forth in the Assay, making any necessary modifications. The injection volume is about 10 µL.

Tolerances— Not less than 80% (Q) of the labeled amount of C10H12N2O4 is dissolved in 30 minutes.

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

Resolution solution— Proceed as directed in the Assay.

Standard solution— Using sonication, dissolve an accurately weighed quantity of thymine in water, and dilute quantitatively, and stepwise if necessary, with water, to obtain a solution having a known concentration of about 1 µg per mL.

Test solution— Use the Assay preparation.

Chromatographic system— Proceed as directed in the Assay. The relative standard deviation for replicate injections of the Standard solution is not more than 3.0%.

Procedure— Proceed as directed in the Assay, recording the chromatograms for a period of time that is 2.5 times the retention time of stavudine, and measure the responses of all the peaks. Calculate the quantity of thymine in each Capsule taken by the formula: in which C is the concentration, in mg per mL, of the Standard solution; V is the volume, in mL, used to prepare the Test solution; D is the dilution factor of the Test solution; N is the number of Capsules taken to prepare the Test solution; and rU and rS are the peak responses obtained from the Test solution and the Standard solution, respectively. Not more than 1.0% of thymine is found. Calculate the percentage of unknown impurities, not including thymine, in the portion of Capsules taken by the formula: in which ri is the peak response for each impurity; and rs is the sum of the responses of all the peaks: not more than 0.2% of any individual impurity is found; and not more than 2.0% of total impurities, including thymine, is found. The quantitation limit is 0.05% of the total sample related peak response.

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).

Resolution solution— Dissolve accurately weighed quantities of thymine and thymidine in water, and dilute quantitatively, and stepwise if necessary, with water to obtain a solution having a known concentration of 0.1 µg of each component per mL.

Standard preparation— Using sonication, dissolve an accurately weighed quantity of USP Stavudine RS in water, and dilute quantitatively, and stepwise if necessary, with water to obtain a solution having a concentration of about 0.1 mg per mL.

Assay preparation— Open not fewer than 3 Capsules, and dissolve the contents quantitatively in water. Dilute quantitatively, and stepwise if necessary, with water to obtain a solution having a concentration of about 0.1 mg of stavudine per mL.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 268-nm detector and a 4.6-mm × 3.3-cm column that contains packing L1. The flow rate is about 0.7 mL per minute. Chromatograph the Resolution solution, and record the peak responses as directed for Procedure: the resolution, R, between thymine and thymidine is not less than 2.0, and thymine is resolved from the void volume. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the retention time for 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 not more than 1.8; and the relative standard deviation for replicate injections is not more than 2.0%.

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 stavudine (C10H12N2O4) in each Capsule taken by the formula: in which C is the concentration, in mg per mL, of USP Stavudine RS in the Standard preparation; V is the volume, in mL, used to prepare the Assay preparation; N is the number of Capsules taken to prepare the Assay preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.