(Ph Eur monograph 0312)
C22H29FO5 392.5 378-44-9
Betamethasone contains not less than 97.0 per cent and not
more than the equivalent of 103.0 per cent of
9-fluoro-11b,17,21-trihydroxy-16b-methylpregna-1,4-diene-3,20-dione, calculated with
reference to the dried substance.
A white or almost white, crystalline powder, practically
insoluble in water, sparingly soluble in ethanol, very slightly soluble in methylene
A, C, D, E.
10.0 mg in ethanol R and dilute to
100.0 ml with the same solvent. Place 2.0 ml of the solution in a stoppered tube, add 10.0
ml of phenylhydrazine-sulphuric acid solution R, mix and heat in a water-bath at 60C
for 20 min. Cool immediately. The absorbance (2.2.25) of the solution measured at 419 nm
is not greater than 0.10.
by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained
with betamethasone CRS. If the spectra obtained in the solid state with the
substance to be examined and the reference substance show differences, dissolve the
substance to be examined and the reference substance separately in the smallest necessary
quantity of methylene chloride R and evaporate to dryness on a water-bath. Using
the residues, record the spectra again.
by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica
gel with a fluorescent indicator having an optimal intensity at 254 nm.
Dissolve 10 mg of the substance to be examined in a mixture of
1 volume of methanol R and 9 volumes
of methylene chloride R and dilute to 10 ml with the same mixture of solvents.
Reference solution (a)
Dissolve 20 mg of betamethasone CRS in a mixture of 1
volume of methanol R and 9 volumes of methylene
chloride R and dilute to 20 ml with the same mixture of solvents.
Reference solution (b)
Dissolve 10 mg of dexamethasone CRS in reference
solution (a) and dilute to 10 ml with the same solution.
Apply separately to the plate 5 ml of each solution. Develop
over a path of 15 cm using a mixture of 5 volumes of butanol R saturated with water R, 10 volumes of toluene R and 85
volumes of ether R. Allow the plate to
dry in air and examine in ultraviolet light at 254 nm. The principal spot in the
chromatogram obtained with the test solution is similar in position and size to the
principal spot in the chromatogram obtained with reference solution (a). Spray with alcoholic
solution of sulphuric acid R. Heat at 120C for 10 min or until the spots appear.
Allow to cool. Examine the chromatograms in daylight and in ultraviolet light at 365 nm.
The principal spot in the chromatogram obtained with the test solution is similar in
position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the
principal spot in the chromatogram obtained with reference solution (a). The test is not
valid unless the chromatogram obtained with reference solution (b) shows two spots which
may however not be completely separated.
about 5 mg with 45 mg of heavy magnesium oxide R and ignite in a crucible until an
almost white residue is obtained (usually less than 5 min). Allow to cool, add 1 ml of water R, 0.05 ml of phenolphthalein solution R1
and about 1 ml of dilute hydrochloric acid R to render the solution colourless.
Filter. Add 1.0 ml of the filtrate to a freshly prepared mixture of 0.1 ml of alizarin
S solution R and 0.1 ml of zirconyl nitrate solution R. Mix, allow to stand for
5 min and compare the colour of the solution with that of a blank prepared in the same
manner. The test solution is yellow and the blank is red.
about 2 mg to 2 ml of sulphuric acid R and shake to dissolve. Within 5 min, a deep
reddish-brown colour develops. Add the solution to 10 ml of water R and mix. The colour is discharged and a
clear solution remains.
Specific optical rotation (2.2.7)
Dissolve 0.125 g in methanol
R and dilute to 25.0 ml with the same solvent. The specific optical rotation is
+118 to +126, calculated with reference to the dried substance.
Examine by liquid chromatography (2.2.29).
Dissolve 25.0 mg of the substance to be examined in a mixture
of equal volumes of acetonitrile R and methanol
R and dilute to 10.0 ml with the same solvent.
Reference solution (a)
Dissolve 2 mg of betamethasone CRS and 2 mg of methylprednisolone
CRS in mobile phase A and dilute to 100.0 ml with the same mobile phase.
Reference solution (b)
Dilute 1.0 ml of the test solution to 100.0 ml with mobile
The chromatographic procedure may be carried out using:
a stainless steel column 0.25 m long and 4.6 mm in internal
diameter packed with octadecylsilyl silica gel for chromatography R (5 mm),
as mobile phase at a flow rate of 2.5 ml/min, a
linear-gradient programme using the following conditions:
Mobile phase A In a 1000 ml volumetric
flask mix 250 ml of acetonitrile R with 700 ml of water R and allow to equilibrate; adjust the volume
to 1000 ml with water R and mix again,
Mobile phase B Acetonitrile R,
as detector a spectrophotometer set at 254 nm,
maintaining the temperature of the column at 45C.
Equilibrate the column with mobile phase B at a flow rate of
2.5 ml/min for at least 30 min and then with mobile phase A for 5 min. For subsequent
chromatograms, use the conditions described from 40 min to 46 min.
Adjust the sensitivity of the system so that the height of the
principal peak in the chromatogram obtained with 20 ml of reference solution (b) is not
less than 50 per cent of the full scale of the recorder.
Inject 20 ml of reference solution (a). When the chromatograms
are recorded in the conditions described above, the retention times are:
methylprednisolone, about 11.5 minutes and betamethasone, about 12.5 minutes. The test is
not valid unless the resolution between the peaks corresponding to methylprednisolone and
betamethasone is at least 1.5; if necessary, adjust the concentration of acetonitrile in
mobile phase A.
Inject separately 20 ml of the mixture of equal volumes of acetonitrile
R and methanol R as a blank, 20 ml of the test solution and 20 ml of reference
solution (b). In the chromatogram obtained with the test solution: the area of any peak,
apart from the principal peak, is not greater than the area of the principal peak in the
chromatogram obtained with reference solution (b) (1.0 per cent) and not more than one
such peak has an area greater than half the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.5 per cent); the sum of the areas of all the
peaks, apart from the principal peak, is not greater than twice the area of the principal
peak in the chromatogram obtained with reference solution (b) (2.0 per cent). Disregard
any peak due to the blank and any peak with an area less than 0.05 times the area of the
principal peak in the chromatogram obtained with reference solution (b).
Loss on drying (2.2.32)
Not more than 0.5 per cent, determined on 0.500 g by drying in
an oven at 100C to 105C.
Dissolve 0.100 g in alcohol
R and dilute to 100.0 ml with the same solvent. Dilute 2.0 ml of the solution
to 100.0 ml with alcohol R. Measure
the absorbance (2.2.25) at the maximum at 238.5 nm.
Calculate the content of C22H29FO5 taking
the specific absorbance to be 395.
Store protected from light.
Action and use