Identification
Dilute a volume of Injection with acetone to provide a solution containing approximately 5 mg of nandrolone decanoate per mL. This solution responds to
Identification test
C under
Nandrolone Decanoate, 5-µL portions of the test solution and the Standard solution being used.
Limit of nandrolone
Standard preparation
Dissolve 25.0 mg of
USP Nandrolone RS in 50.0 mL of acetone. Dilute 5.0 mL of this solution with acetone to 50.0 mL, and mix.
Test preparation
Transfer an accurately measured volume of Injection, equivalent to about 50 mg of nandrolone decanoate, to a 10-mL volumetric flask, dilute with acetone to volume, and mix.
Procedure
Apply 10 µL each of the
Standard preparation and of the
Test preparation to a suitable thin-layer chromatographic plate (see
Chromatography 621) coated with a 0.25-mm layer of chromatographic silica gel mixture. Allow the spots to dry, and develop the chromatogram in a solvent system consisting of a mixture of
n-heptane and acetone (3:1) until the solvent front has moved about three-fourths of the length of the plate. Remove the plate from the developing chamber, mark the solvent front, and allow the solvent to evaporate. Return the dry plate to the developing chamber containing the same solvent system, and again develop the chromatogram until the solvent front has moved the same distance from the origin. Remove the plate from the developing chamber, and allow the solvent to evaporate. Locate the spots on the plate by lightly spraying with a 4 in 10 solution of sulfuric acid in methanol and heating at about 100
for 10 minutes. Cool, and examine under long-wavelength UV light: any yellow fluorescent spot from the
Test preparation at an
Rf value of about 0.2 is not greater in size or intensity than that produced by the
Standard preparation at the same
Rf value, corresponding to not more than 1.0% of nandrolone.
Assay
0.02 M Ammonium acetate solution
Transfer about 1.6 g of ammonium acetate to a 1-liter volumetric flask. Dissolve in and dilute with water to volume.
Mobile phase
Prepare a filtered and degassed mixture of alcohol and
0.02 M Ammonium acetate solution (66:34). Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Standard preparation
Dissolve an accurately weighed quantity of
USP Nandrolone Decanoate RS with tetrahydrofuran, and dilute quantitatively and stepwise if necessary, with tetrahydrofuran to obtain a solution having a known concentration of about 0.2 mg per mL.
Assay preparation
Transfer an accurately measured volume of Injection, equivalent to about 400 mg of nandrolone decanoate to a 200-mL volumetric flask, dilute with tetrahydrofuran to volume, and mix. Transfer 10.0 mL of this solution to a 100-mL volumetric flask, dilute with tetrahydrofuran to volume, and mix.
Chromatographic system (see Chromatography 621)
The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 15-cm column containing 5-µm packing L1. The flow rate is about 1.5 mL per minute. The column temperature is maintained at 40
. Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the capacity factor,
k¢, for nandrolone decanoate is not less than 5.3; the tailing factor for the Nandrolone Decanoate peak is not more than 1.4; and the relative standard deviation for replicate injections is not more than 2.0%.
Procedure
Separately inject equal volumes (about 10 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
28H
44O
3 in each mL of the Injection taken by the formula:
2000(C/V) (rU / rS),
in which
C is the concentration, in mg per mL, of
USP Nandrolone Decanoate RS in the
Standard preparation; V is the volume, in mL, of the injection taken to prepare the
Assay preparation, and
rU and
rS are the peak responses obtained from the
Assay preparation and the
Standard preparation, respectively.