(Ph. Eur. monograph 0975)
C24H31FO6 434.5 987-24-6
Action and use
Glucocorticoid.
DEFINITION
9-Fluoro-11β,17-dihydroxy-16β-methyl-3,20-dioxopregna-1,4-dien-21-yl acetate.
Content
97.0 per cent to 103.0 per cent (anhydrous substance).
CHARACTERS
Appearance
White or almost white, crystalline powder.
Solubility
Practically insoluble in water, freely soluble in acetone, soluble in ethanol (96 per cent) and in methylene chloride. It shows polymorphism (5.9).
IDENTIFICATION
First identification: A, B.
Second identification: B, C.
A. Infrared absorption spectrophotometry (2.2.24).
Comparison betamethasone acetate 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 methanol R, evaporate to dryness on a water-bath and record new spectra using the residues.
B. Thin-layer chromatography (2.2.27).
Test solution: Dissolve 10 mg of the substance to be examined in the mobile phase and dilute to 10.0 mL with the mobile phase.
Reference solution: Dissolve 10 mg of betamethasone acetate CRS in the mobile phase and dilute to 10.0 mL with the mobile phase.
Plate: TLC silica gel F254 plate R.
Mobile phase methanol R, methylene chloride R (10:90 V/V).
Application: 5 μL.
Development: Over 3/4 of the plate.
Drying: In air.
Detection: Spray with a solution prepared as follows: dissolve 0.25 g of 2,4-dihydroxybenzaldehyde R in glacial acetic acid R, dilute to 50 mL with the same solvent and add a mixture of 12.5 mL of sulfuric acid R and 37.5 mL of glacial acetic acid R; heat at 90 °C for 35 min or until the spots appear, allow to cool and examine in daylight and in ultraviolet light at 365 nm.
Results: The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution.
C. Add about 2 mg to 2 mL of sulfuric acid R and shake to dissolve. Within 5 min, a deep brown colour develops. Add this solution to 10 mL of water R and mix. The colour is discharged and a clear solution remains.
TESTS
Specific optical rotation (2.2.7)
+ 120 to + 128 (anhydrous substance).
Dissolve 0.250 g in dioxan R and dilute to 25.0 mL with the same solvent.
Related substances
Liquid chromatography (2.2.29).
Test solution: Dissolve 25.0 mg of the substance to be examined in 4 mL of acetonitrile R and dilute to 10.0 mL with the same solvent.
Reference solution (a): Dissolve 2 mg of betamethasone acetate CRS and 2 mg of dexamethasone acetate CRS (impurity B) in the mobile phase and dilute to 100 mL with the mobile phase.
Reference solution (b): Dilute 1.0 mL of the test solution to 100.0 mL with the mobile phase.
Column:
— size: l = 0.25 m, Ø = 4.6 mm;
— stationary phase: end-capped octadecylsilyl silica gel for chromatography R (5 μm).
Mobile phase: Mix 380 mL of acetonitrile R and 550 mL of water for chromatography R and allow to equilibrate; dilute to 1000 mL with water for chromatography R and mix.
Flow rate: 1 mL/min.
Detection: Spectrophotometer at 254 nm.
Injection: 20 μL.
Run time: 2.5 times the retention time of betamethasone acetate.
Retention time: Betamethasone acetate = about 19 min; impurity B = about 22 min.
System suitability: Reference solution (a):
— resolution: minimum 3.3 between the peaks due to betamethasone acetate and impurity B; if necessary, adjust slightly the concentration of acetonitrile in the mobile phase.
Limits:
— impurities A, B, C, D: for each impurity, not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent);
— total: not more than 1.25 times the area of the principal peak in the chromatogram obtained with reference solution (b) (1.25 per cent);
— disregard limit: 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.05 per cent).
Water (2.5.12)
Maximum 4.0 per cent, determined on 0.100 g.
ASSAY
Dissolve 0.100 g in ethanol (96 per cent) R and dilute to 100.0 mL with the same solvent. Dilute 2.0 mL of the solution to 100.0 mL with ethanol (96 per cent) R. Measure the absorbance (2.2.25) at the absorption maximum at 240 nm.
Calculate the content of C24H31FO6 taking the specific absorbance to be 350.
STORAGE
Protected from light.
IMPURITIES
Specified impurities A, B, C, D.
A. 9-fluoro-11β,17,21-trihydroxy-16β-methylpregna-1,4-diene-3,20-dione (betamethasone),
B. 9-fluoro-11β,17-dihydroxy-16α-methyl-3,20-dioxopregna-1,4-dien-21-yl acetate (dexamethasone acetate),
C. 9-fluoro-17-hydroxy-16β-methyl-3,20-dioxopregna-1,4-diene-11β,21-diyl diacetate (betamethasone 11,21-diacetate),
D. 9,11β-epoxy-17-hydroxy-16β-methyl-3,20-dioxo-9β-pregna-1,4-dien-21-yl acetate.
