Identification—
A: Thin-Layer Chromatographic Identification Test 
201
—
Test solution—
Using sonication, dissolve a portion of Capsule contents in enough water to obtain a solution having a concentration of 0.2 mg of stavudine per mL, filter, and mix. Use the filtrate as the Test solution.
Application volume:
10 µL, applied in two 5-µL portions.
Developing solvent system:
a mixture of chloroform, alcohol, and water (100:50:2).
Procedure—
Proceed as directed in the chapter. Allow the spots to dry, and develop the chromatogram in the Developing solvent system until the solvent front has moved about 10 cm from the origin. Remove the plate from the developing chamber, mark the solvent front, and allow to air dry for 5 to 10 minutes.
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.
Specific rotation 781S:
between –40
and –45
, determined in a solution in water containing 10 mg of stavudine per mL. Disperse a sufficient quantity of Capsule content, equivalent to 200 mg of stavudine, in 50 mL of acetone. Bring to a boil, and pass through a fine-porosity filter. Precipitate the stavudine with 150 mL of heptane, filter the crystals, wash with heptane, and dry in air.
Dissolution 711—
Medium:
water; 900 mL.
Apparatus 2:
75 rpm.
Time:
30 minutes.
Determine the amount of C10H12N2O4 dissolved by employing the following method.
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.
Related compounds—
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:
(CVD/N)(rU / rS),
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:
100(ri / rs),
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.
Assay—
[NOTE—All solutions must be prepared immediately prior to use and remain refrigerated until use.
]
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 per mL of each component.
Standard preparation—
Transfer about 50 mg of
USP Stavudine RS, accurately weighed, to a 500-mL volumetric flask, and dissolve in about 200 mL of water, sonicate, dilute with water to volume, and mix.
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 (C
10H
12N
2O
4) in each Capsule taken by the formula:
C(V/N)(rU / rS),
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.
