Related compounds—
[NOTE—All testing solutions must be prepared immediately prior to use and remain refrigerated until use.
]
Solution A, Solution B, Resolution solution, and Chromatographic system—
Proceed as directed in the Assay.
Standard solution—
Prepare as directed for Standard preparation in the Assay.
Test solution—
Prepare as directed for Assay preparation.
Procedure—
Noting the retention times of the impurities relative to that of stavudine, calculate the percentage of all other impurities in the portion of Stavudine for Oral Solution taken by the formula:
100(Fri / rs),
in which
F is the relative response factor and is equal to 0.69 for thymine (relative retention time of about 0.24) and equal to 1.0 for all other peaks;
ri 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 sample-related peaks in the chromatogram including that of the main stavudine peak: not more than 1.0% of thymine is found, not more than 0.2% of any other individual impurity is found, and not more than 1.5% of total impurities is found.
Assay—
[NOTE—All testing solutions must be prepared immediately prior to use and remain refrigerated until use.
]
25 mM Ammonium acetate—
Dissolve 1.93 g of ammonium acetate in about 900 mL of water in a 1000-mL volumetric flask. Dilute with water to volume, and mix.
Solution A—
Prepare a filtered and degassed mixture of 25 mM Ammonium acetate and methanol (94:6).
Solution B—
Prepare a filtered and degassed mixture of 25 mM Ammonium acetate and methanol (1:1).
Resolution solution—
Prepare a solution in water of thymidine and thymine containing 2.5 µg of each per mL.
Standard preparation—
Prepare a solution of
USP Stavudine RS in water having a concentration of 0.1 mg per mL.
Assay preparation—
Transfer to a suitable volumetric flask an accurately measured volume of Stavudine for Oral Solution, constituted as directed in the labeling, and dilute quantitatively, and stepwise if necessary, with water to obtain a solution having a concentration of 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 and a 4-mm × 20-mm guard column (L1). The flow rate is about 1 mL per minute. The chromatograph is programmed as follows.
Time
(minutes) |
Solution A
(%) |
Solution B
(%) |
Elution |
| 0 |
100 |
0 |
equilibration |
| 0–12 |
100 |
0 |
isocratic |
| 12.1 |
100®0 |
0®100 |
step gradient |
| 12.1–17 |
0 |
100 |
isocratic |
| 17.1 |
0®100 |
100®0 |
step gradient |
| 17.1–35 |
100 |
0 |
re-equilibration |
Chromatograph the
Resolution solution, and record the peak responses as directed for
Procedure: the resolution,
R, between thymine and thymidine is not less than 8.4. Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the column efficiency is not less than 2000 theoretical plates; the tailing factor for the stavudine peak is not more than 2; 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 areas for the major peaks. Calculate the quantity, in mg, of stavudine (C
10H
12N
2O
4) in each mL of Stavudine for Oral Solution taken by the formula:
(L/D)(C)(rU / rS),
in which
L is the labeled quantity, in mg, of stavudine (C
10H
12N
2O
4) in each mL of the Stavudine for Oral Solution;
D is the concentration, in mg, of stavudine per mL of the
Assay preparation, based on the labeled quantity of stavudine in the portion of Stavudine for Oral Solution taken;
C is the concentration, in mg per mL, of
USP Stavudine RS in the
Standard preparation; and
rU and
rS are the peak area responses obtained from the
Assay preparation and the
Standard preparation, respectively.
