Delete the following:
Ordinary impurities 
466
—
Test solution:
methanol.
Standard solution:
methanol.
Application volume:
10 µL.
Eluant:
a mixture of toluene and isopropyl alcohol (90:10), in a nonequilibrated chamber.
Visualization:
5.
1S (USP31)
Add the following:
Related compounds—
Solution A:
acetonitrile.
Solution B:
water.
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).
Blank solution—
Prepare a mixture of Solution A and Solution B (50:50).
Standard solution—
Dilute 1.0 mL of the Standard stock solution with 4.0 mL of acetonitrile and 5.0 mL of water, and mix.
Test solution—
Weigh accurately 40 mg of Oxandrolone into a 10-mL volumetric flask, dissolve in 5.0 mL of acetonitrile using an ultrasonic bath, dilute with water to volume, and mix. [Note—The Test solution, the Standard solution, and the Blank solution are made up fresh and injected immediately.]
Chromatographic system—
The liquid chromatograph is equipped with a 210-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L1. The column temperature is maintained at 40
. The flow rate is about 0.7 mL per minute. The chromatograph is programmed as follows.
Time
(minutes) |
Solution A
(%) |
Solution B
(%) |
 Elution |
| 0 |
50 |
50 |
equilibration |
| 0–30 |
50®100 |
50®0 |
linear gradient |
| 30–32 |
100®50 |
0®50 |
linear gradient |
| 32–40 |
50 |
50 |
re-equilibration |
Chromatograph the
Standard solution, and record the peak responses as directed for
Procedure: the resolution,
R, between oxandrolone related compound A and oxandrolone related compound B is not less than 1.5, and the resolution,
R, between oxandrolone related compound B and oxandrolone is not less than 2.0; the tailing factor is not more than 1.5; and the relative standard deviation for replicate injections is not more than 5.0%.
Procedure—
Separately inject equal volumes (about 50 µL) of the
Blank solution, the
Standard solution, and the
Test solution into the chromatograph, record the chromatograms, and measure the peak responses. Calculate the percentage of oxandrolone related compound A in the portion of Oxandrolone taken by the formula:
(C/W)(rU / rS)
in which
C is the concentration, in µg per mL, of oxandrolone related compound A in the
Standard solution; W is the weight, in mg, of Oxandrolone taken to prepare the
Test solution; rU is the peak area of oxandrolone related compound A in the chromatogram of the
Test solution; and
rS is the peak area obtained for oxandrolone related compound A in the chromatogram of the
Standard solution.
Calculate the percentage of oxandrolone related compound C, methyltestosterone, D1-mestalone, specified unknown impurity 1, and each impurity eluting at a relative retention time greater than or equal to 2.2 (relative to retention time of oxandrolone) by the formula:
(1/F)(C/W)(rU / rS)
in which F is the relative response factor (see accompanying table for values); C is the concentration, in µg per mL, of oxandrolone related compound C in the Standard solution; W is the weight, in mg, of Oxandrolone taken to prepare the Test solution; rU is the peak area of oxandrolone related compound C, methyltestosterone, D1-mestalone, specified unknown impurity 1, or each impurity eluting at a relative retention time greater than or equal to 2.2 in the chromatogram of the Test solution; and rS is the peak area obtained for oxandrolone related compound C in the chromatogram of the Standard solution.
Calculate the percentage of each impurity, except oxandrolone related compound A, oxandrolone related compound C, methyltestosterone, D1-mestalone, specified unknown impurity 1, and other impurities eluting at relative retention times greater than or equal to 2.2 by the formula:
(1/F)(C/W)(rU / rS)
in which
F is the relative response factor for each impurity (see accompanying table for values);
C is the concentration, in µg per mL, of
USP Oxandrolone RS in the
Standard solution; W is the weight, in mg, of Oxandrolone taken to prepare the
Test solution; rU is the peak area of each impurity, in the chromatogram of the
Test solution, other than peak areas of oxandrolone related compound A, oxandrolone related compound C, methyltestosterone,
D1-mestalone, specified unknown impurity 1, and other impurities eluting at relative retention times greater than or equal to 2.2; and
rS is the peak area obtained for oxandrolone in
Standard solution. Disregard any peak observed in the chromatogram obtained from the
Blank solution. Disregard any impurity peak that is less than 0.05%. The impurities meet the requirements specified in the accompanying table.
| Compound |
Relative Retention Time |
Relative Response Factor (F) |
Limit (%) |
| Secodicarboxylic acid1 |
0.46 |
4.1 |
0.1 |
7,8-Didehydro-oxandrolone2 (Oxandrolone related
compound A) |
0.90 |
— |
0.1 |
4-Oxa-isomer3 (Oxandrolone
related compound B) |
0.94 |
1.4 |
0.3 |
| Oxandrolone |
1.00 |
— |
— |
| Oxandrolone open lactone methyl ester4 |
1.09 |
1.5 |
0.1 |
| Secoacid anhydride5 |
1.12 |
2.5 |
0.1 |
| Methyltestosterone6 |
1.25 |
0.8a |
0.1 |
| 17-epi-Oxandrolone7 |
1.33 |
1.0 |
0.3 |
| D1-Mestalone8 |
1.48 |
1.3a |
0.1 |
| 4-Oxa-isomer (beta epimer)9 |
1.52 |
1.4 |
0.3 |
| Specified unknown impurity 1 |
1.63 |
0.6a |
0.1 |
| Oxandrolone-17-acetate10 |
2.14 |
1.9 |
0.1 |
Anhydro-oxandrolone11
(Oxandrolone related
compound C) |
3.29 |
— |
0.5 |
| Individual unknown impurity |
— |
1.0 |
0.1 |
| Total impurities |
— |
— |
1.0 |
1
17  -Hydroxy-17  -methyl-2-nor-5 -androstan-1,3-dioic acid.
|
2
17 -Hydroxy-17 -methyl-2-oxa-5 -androst-7-en-3-one.
|
3
17 -Hydroxy-17 -methyl-4-oxa-5 -androstan-3-one.
|
4
Methyl-(1,17 -dihydroxy-17 -methyl-1,3-seco-2-nor-5 -androstane-3-oate.
|
5
17 -Hydroxy-17 -methyl-2-oxa-5 -androstan-1,3-dione.
|
6
17 -Hydroxy-17 -methyl-5 -androst-4-ene-3-one.
|
7
17 -Hydroxy-17 -methyl-2-oxa-5 -androstan-3-one.
|
8
17 -Hydroxy-17 -methyl-5 -androst-1-ene-3-one.
|
9
17 -Hydroxy-17 -methyl-4-oxa-5 -androstan-3-one.
|
10
17 -Hydroxy-17 -methyl-2-oxa-5 -androstan-3-one 17-acetate.
|
11
17,17-Dimethyl-18-nor-2-oxa-5 -androst-13-en-3-one.
|
|
a
F values relative to oxandrolone related compound C.
|
1S (USP31)
Change to read:
Assay—
Mobile phase—
Prepare a filtered and degassed mixture of water and acetonitrile (50:50). Make adjustments if necessary (see
System Suitability under
Chromatography 621).
1S (USP31)
Standard preparation—
Dissolve an accurately weighed quantity of
USP Oxandrolone RS in acetonitrile, and dilute quantitatively, and stepwise if necessary, with acetonitrile to obtain a solution having a known concentration of about 3 mg per mL.
[NOTE—Sonicate if necessary to dissolve.
]
Assay preparation—
Transfer to a suitable volumetric flask an accurately weighed quantity of Oxandrolone, and dissolve in and dilute with acetonitrile to volume to obtain a solution having a concentration of about 3 mg per mL.
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with a 210-nm detector and a 4.6-mm × 25-cm column that contains packing L1. The flow rate is about 0.8 mL per minute. Chromatograph the
Standard preparation, 1S (USP31) and record the peak responses as directed for
Procedure: the column efficiency is not less than 2000 theoretical plates; the tailing factor is not more than 2.0; 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 C
19H
30O
3 in the portion of Oxandrolone taken by the formula:
VC(rU / rS)
in which
V is the final volume, in mL, of the
Assay preparation; C is the concentration, in mg per mL, of
USP Oxandrolone RS in the
Standard preparation; and
rU and
rS are the peak responses obtained from the
Assay preparation and the
Standard preparation, respectively.
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