EUROPEAN PHARMACOPOEIA 5.

0 Betamethasone

Heavy metals (2.4.8). 12 ml of solution S complies with limit examined and the reference substance separately in the
test A for heavy metals (20 ppm). Prepare the standard using smallest necessary quantity of methylene chloride R and
lead standard solution (2 ppm Pb) R. evaporate to dryness on a water-bath. Using the residues,
Water (2.5.12). Not more than 2.0 per cent, determined on record the spectra again.
0.50 g by the semi-micro determination of water. C. Examine by thin-layer chromatography (2.2.27), using
as the coating substance a suitable silica gel with a
ASSAY fluorescent indicator having an optimal intensity at
Dissolve 0.140 g in 50 ml of a mixture of 1 volume 254 nm.
of anhydrous acetic acid R and 7 volumes of acetic Test solution. Dissolve 10 mg of the substance to be
anhydride R. Titrate with 0.1 M perchloric acid, determining examined in a mixture of 1 volume of methanol R and
the end-point potentiometrically (2.2.20). 9 volumes of methylene chloride R and dilute to 10 ml
1 ml of 0.1 M perchloric acid is equivalent to 16.42 mg of with the same mixture of solvents.
C10H20N2O6S2. Reference solution (a). Dissolve 20 mg of
betamethasone CRS in a mixture of 1 volume of
STORAGE methanol R and 9 volumes of methylene chloride R and
Store in an airtight container. dilute to 20 ml with the same mixture of solvents.
IMPURITIES 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 µl 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
A. 2-ethenylpyridine. 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
01/2005:0312 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
BETAMETHASONE acid R. Heat at 120 °C for 10 min or until the spots
appear. Allow to cool. Examine the chromatograms in
Betamethasonum 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.
D. Mix about 5 mg with 45 mg of heavy magnesium oxide R
and ignite in a crucible until an almost white residue is
C22H29FO5 Mr 392.5 obtained (usually less than 5 min). Allow to cool, add
1 ml of water R, 0.05 ml of phenolphthalein solution R1
DEFINITION and about 1 ml of dilute hydrochloric acid R to render
Betamethasone contains not less than 97.0 per cent and the solution colourless. Filter. Add 1.0 ml of the filtrate
not more than the equivalent of 103.0 per cent of 9-fluoro- to a freshly prepared mixture of 0.1 ml of alizarin S
11β,17,21-trihydroxy-16β-methylpregna-1,4-diene-3,20-dione, solution R and 0.1 ml of zirconyl nitrate solution R.
calculated with reference to the dried substance. Mix, allow to stand for 5 min and compare the colour of
the solution with that of a blank prepared in the same
CHARACTERS manner. The test solution is yellow and the blank is red.
A white or almost white, crystalline powder, practically E. Add about 2 mg to 2 ml of sulphuric acid R and shake
insoluble in water, sparingly soluble in ethanol, very slightly to dissolve. Within 5 min, a deep reddish-brown colour
soluble in methylene chloride. develops. Add the solution to 10 ml of water R and mix.
The colour is discharged and a clear solution remains.
IDENTIFICATION
First identification : B, C. TESTS
Second identification : A, C, D, E. Specific optical rotation (2.2.7). Dissolve 0.125 g in
methanol R and dilute to 25.0 ml with the same solvent. The
A. Dissolve 10.0 mg in ethanol R and dilute to 100.0 ml specific optical rotation is + 118 to + 126, calculated with
with the same solvent. Place 2.0 ml of the solution in a reference to the dried substance.
stoppered tube, add 10.0 ml of phenylhydrazine-sulphuric
acid solution R, mix and heat in a water-bath at 60 °C for Related substances. Examine by liquid chromatography
20 min. Cool immediately. The absorbance (2.2.25) of the (2.2.29).
solution measured at 419 nm is not greater than 0.10. Test solution. Dissolve 25.0 mg of the substance to be
B. Examine by infrared absorption spectrophotometry examined in a mixture of equal volumes of acetonitrile R
(2.2.24), comparing with the spectrum obtained with and methanol R and dilute to 10.0 ml with the same solvent.
betamethasone CRS. If the spectra obtained in the solid Reference solution (a). Dissolve 2 mg of betamethasone CRS
state with the substance to be examined and the reference and 2 mg of methylprednisolone CRS in mobile phase A and
substance show differences, dissolve the substance to be dilute to 100.0 ml with the same mobile phase.

General Notices (1) apply to all monographs and other texts 1087
Betamethasone EUROPEAN PHARMACOPOEIA 5.0

Reference solution (b). Dilute 1.0 ml of the test solution to ASSAY

100.0 ml with mobile phase A. Dissolve 0.100 g in alcohol R and dilute to 100.0 ml with the
The chromatographic procedure may be carried out using : 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
— a stainless steel column 0.25 m long and 4.6 mm in at 238.5 nm.
internal diameter packed with octadecylsilyl silica gel for
Calculate the content of C22H29FO5 taking the specific
chromatography R (5 µm),
absorbance to be 395.
— as mobile phase at a flow rate of 2.5 ml/min, a
linear-gradient programme using the following STORAGE
conditions : Store protected from light.
Mobile phase A. In a 1000 ml volumetric flask mix 250 ml IMPURITIES
of acetonitrile R with 700 ml of water R and allow to
equilibrate ; adjust the volume to 1000 ml with water R A. dexamethasone,
and mix again,
Mobile phase B. Acetonitrile R,
Time Mobile phase A Mobile Comment
(min) (per cent V/V) phase B
(per cent V/V)
0 100 0 isocratic
15 100 0 begin linear gradient
40 0 100 end chromatogram, B. 21-chloro-9-fluoro-11β,17-dihydroxy-16β-methylpregna-
return to 100A 1,4-diene-3,20-dione,
41 100 0 begin equilibration
with A
46 = 0 100 0 end equilibration, begin
next chromatogram

— as detector a spectrophotometer set at 254 nm,

maintaining the temperature of the column at 45 °C.
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 C. 17,21-dihydroxy-16β-methylpregna-1,4,9(11)-triene-3,20-
phase A for 5 min. For subsequent chromatograms, use the dione,
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 µl of
reference solution (b) is not less than 50 per cent of the full
scale of the recorder.
Inject 20 µl 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 D. 9-fluoro-11β,17-dihydroxy-16β-methyl-3,20-dioxopregna-1,
corresponding to methylprednisolone and betamethasone 4-dien-21-yl ethoxycarboxylate,
is at least 1.5 ; if necessary, adjust the concentration of
acetonitrile in mobile phase A.
Inject separately 20 µl of the mixture of equal volumes of
acetonitrile R and methanol R as a blank, 20 µl of the
test solution and 20 µl 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 E. 9,11β-epoxy-17,21-dihydroxy-16β-methyl-9β-pregna-1,4-
one such peak has an area greater than half the area of the diene-3,20-dione,
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 100 °C to F. 17,21-dihydroxy-16β-methylpregna-1,4,11-triene-3,20-
105 °C. dione,

1088 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 5.0 Betamethasone acetate

CHARACTERS
A white or almost white, crystalline powder, practically
insoluble in water, freely soluble in acetone, soluble in
alcohol and in methylene chloride.
It shows polymorphism.
IDENTIFICATION
First identification : B, C.
G. 11α,17,21-trihydroxy-16β-methylpregna-1,4-diene-3,20- Second identification : A, C, D, E, F.
dione, A. Dissolve 10.0 mg in ethanol R and dilute to 100.0 ml
with the same solvent. Place 2.0 ml of this solution
in a ground-glass-stoppered tube, add 10.0 ml of
phenylhydrazine-sulphuric acid solution R, mix and heat
in a water-bath at 60 °C for 20 min. Cool immediately.
The absorbance (2.2.25) of the solution measured at
419 nm is not more than 0.10.
B. Examine by infrared absorption spectrophotometry
(2.2.24), comparing with the spectrum obtained with
betamethasone acetate CRS. If the spectra obtained in
H. 14-fluoro-11β,17,21-trihydroxy-16β-methyl-8α,9β,14β- the solid state show differences, dissolve the substance to
pregna-1,4-diene-3,20-dione, 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.
C. Examine 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.
Test solution. 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
I. 8-fluoro-11β,17,21-trihydroxy-16β-methyl-8α,9β-pregna- with the same mixture of solvents.
1,4-diene-3,20-dione,
Reference solution (a). Dissolve 20 mg of betamethasone
acetate 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 prednisolone
acetate CRS in reference solution (a) and dilute to 10 ml
with the same solution.
Apply to the plate 5 µl of each solution. Prepare the
mobile phase by adding a mixture of 1.2 volumes of
J. 17,21-dihydroxy-16β-methylpregna-1,4-diene-3,20-dione. water R and 8 volumes of methanol R to a mixture of
15 volumes of ether R and 77 volumes of methylene
chloride R. Develop over a path of 15 cm. Allow the plate
to dry in air and examine in ultraviolet light at 254 nm.
01/2005:0975 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
BETAMETHASONE ACETATE solution (a). Spray with alcoholic solution of sulphuric
acid R. Heat at 120 °C for 10 min or until the spots
Betamethasoni acetas appear. Allow to cool. Examine the plate 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 clearly separated spots.
D. Add about 2 mg to 2 ml of sulphuric acid R and shake
to dissolve. Within 5 min, a deep reddish-brown colour
C24H31FO6 Mr 434.5 develops. Add the solution to 10 ml of water R and mix.
The colour is discharged and a clear solution remains.
DEFINITION E. Mix about 5 mg with 45 mg of heavy magnesium oxide R
Betamethasone acetate contains not less than 97.0 per and ignite in a crucible until an almost white residue is
cent and not more than the equivalent of 103.0 per cent obtained (usually less than 5 min). Allow to cool, add 1 ml
of 9-fluoro-11β,17-dihydroxy-16β-methyl-3,20-dioxopregna- of water R, 0.05 ml of phenolphthalein solution R1 and
1,4-diene-21-yl acetate, calculated with reference to the about 1 ml of dilute hydrochloric acid R to render the
anhydrous substance. solution colourless. Filter. To a freshly prepared mixture

General Notices (1) apply to all monographs and other texts 1089