(Ph. Eur. monograph 1568)
C21H32N2O 328.5 10418-03-8
Action and use
Anabolic steroid; androgen.
DEFINITION
17-Methyl-2′H-5α-androst-2-eno[3,2-c]pyrazol-17β-ol.
Content
98.5 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance
White or almost white, hygroscopic, crystalline powder.
Solubility
Practically insoluble in water, soluble in dimethylformamide, slightly soluble in ethanol (96 per cent), very slightly soluble in methylene chloride.
It shows polymorphism (5.9).
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
Comparison: stanozolol CRS.
If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in the minimum volume of methylene chloride R, evaporate to dryness at room temperature under an air-stream and record new spectra using the residues.
B. Liquid chromatography (2.2.29) as described in the test for related substances with the following modification.
Injection: Test solution and reference solution (c).
Results: The principal peak in the chromatogram obtained with the test solution is similar in retention time and size to the principal peak in the chromatogram obtained with reference solution (c).
TESTS
Specific optical rotation (2.2.7)
+ 37 to + 41 (dried substance).
Dissolve 60.0 mg in methanol R and dilute to 20.0 mL with the same solvent.
Impurities A and B
Thin-layer chromatography (2.2.27).
Solvent mixture: methanol R1, methylene chloride R (10:90 V/V).]
Test solution: Dissolve 20 mg of the substance to be examined in 1.0 mL of the solvent mixture.
Reference solution: Dissolve 2 mg of stanozolol CRS, 2.0 mg of stanozolol impurity A CRS and 2.0 mg of stanozolol impurity B CRS in 1.0 mL of the solvent mixture. Dilute 0.1 mL of the solution to 2.0 mL with the solvent mixture.
Plate: TLC silica gel plate R.
Mobile phase: glacial acetic acid R, ethyl acetate R, cyclohexane R (2:48:50 V/V/V).
Application: 10 μL.
Development: Over 3/4 of the plate.
Drying: In air.
Detection: Spray with vanillin reagent R and heat at 120 °C.
System suitability: Reference solution:
— the chromatogram shows 3 clearly separated spots, due to stanozolol, impurity A and impurity B, in order of increasing RF value.
Limits:
— impurity A: any spot due to impurity A is not more intense than the corresponding spot in the chromatogram obtained with the reference solution (0.5 per cent);
— impurity B: any spot due to impurity B is not more intense than the corresponding spot in the chromatogram obtained with the reference solution (0.5 per cent).
Related substances
Liquid chromatography (2.2.29).
Test solution: Dissolve 15.0 mg of the substance to be examined in methanol R and dilute to 5.0 mL with the same solvent.
Reference solution (a): Dilute 1.0 mL of the test solution to 100.0 mL with methanol R. Dilute 1.0 mL of this solution to 10.0 mL with methanol R.
Reference solution (b): Dissolve 1 mg of stanozolol CRS and 1 mg of stanozolol impurity B CRS in methanol R and dilute to 20 mL with the same solvent.
Reference solution (c): Dissolve 15.0 mg of stanozolol CRS in methanol R and dilute to 5.0 mL with the same solvent.
Column:
— size: l = 0.15 m, Ø = 4.6 mm;
— stationary phase: end-capped octadecylsilyl silica gel for chromatography R1 (5 μm).
Mobile phase: 1 g/L solution of sodium dihydrogen phosphate R adjusted to pH 3.0 with phosphoric acid R, methanol R1 (30:70 V/V).
Flow rate: 1.5 mL/min.
Detection: Spectrophotometer at 228 nm.
Injection: 25 μL of the test solution and reference solutions (a) and (b).
Run time: 3 times the retention time of stanozolol.
Identification of impurities: Use the chromatogram obtained with reference solution (b) to identify the peak due to
impurity B.
Relative retention: With reference to stanozolol (retention time = about 12 min): impurity B = about 1.3.
System suitability: Reference solution (b):
— resolution: minimum 4.0 between the peaks due to stanozolol and impurity B.
Limits:
— unspecified impurities: for each impurity, not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.10 per cent);
— total: not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent);
— disregard limit: 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.05 per cent).
Loss on drying (2.2.32)
Maximum 1.0 per cent, determined on 1.000 g by drying at 105 °C at a pressure not exceeding 0.7 kPa.
ASSAY
Dissolve 0.250 g in 50 mL of anhydrous acetic acid R. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20).
1 mL of 0.1 M perchloric acid is equivalent to 32.85 mg of C21H32N2O.
STORAGE
In an airtight container, protected from light.
IMPURITIES
Specified impurities A, B.
A. 17β-hydroxy-17-methyl-5α-androstan-3-one (mestanolone),
B. 17β-hydroxy-2-(hydroxymethylene)-17-methyl-5α-androstan-3-one (oxymetholone).
