EUROPEAN PHARMACOPOEIA 7.0 principal spot in the chromatogram obtained with reference

EUROPEAN PHARMACOPOEIA 7.0
Methylprednisolone acetate
Results A : 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).
Detection B : spray with alcoholic solution of sulfuric acid R.
Heat at 120 °C for 10 min or until the spots appear. Allow to
cool. Examine in daylight and in ultraviolet light at 365 nm.
Results B : the principal spot in the chromatogram obtained
with the test solution is similar in position, colour in daylight,
D. (E)- and (Z)-11β,20-dihydroxy-6α-methylpregna-1,4,17(20)fluorescence in ultraviolet light at 365 nm and size to the
triene-3,21-dione.
principal spot in the chromatogram obtained with reference
solution (a).
01/2008:0933
System suitability : reference solution (b):
corrected 6.0
— the chromatogram shows 2 spots which, when examined
in ultraviolet light at 365 nm, may not be completely
METHYLPREDNISOLONE ACETATE
separated.
C. Thin-layer chromatography (2.2.27).
Methylprednisoloni acetas
Test solution (a). Dissolve 25 mg of the substance to be
examined in methanol R and dilute to 5 mL with the same
solvent (solution A). Dilute 2 mL of this solution to 10 mL
with methylene chloride R.
Test solution (b). Transfer 2 mL of solution A to a 15 mL glass
tube with a ground-glass stopper or a polytetrafluoroethylene
cap. Add 10 mL of saturated methanolic potassium
hydrogen carbonate solution R and immediately pass
a current of nitrogen R through the solution for 5 min.
Stopper the tube. Heat in a water-bath at 45 °C protected
C24H32O6
Mr 416.5
from light for 1 h. Allow to cool.
[53-36-1]
Reference solution (a). Dissolve 25 mg of
DEFINITION
methylprednisolone acetate CRS in methanol R
11β,17-Dihydroxy-6α-methyl-3,20-dioxopregna-1,4-dien-21-yl
and dilute to 5 mL with the same solvent (solution B). Dilute
acetate.
2 mL of this solution to 10 mL with methylene chloride R.
Content : 97.0 per cent to 103.0 per cent (dried substance).
Reference solution (b). Transfer 2 mL of solution B to
a 15 mL glass tube with a ground-glass stopper or a
CHARACTERS
polytetrafluoroethylene cap. Add 10 mL of saturated
Appearance : white or almost white, crystalline powder.
methanolic potassium hydrogen carbonate solution R
and immediately pass a current of nitrogen R through the
Solubility : practically insoluble in water, sparingly soluble in
solution for 5 min. Stopper the tube. Heat in a water-bath at
acetone and in ethanol (96 per cent).
45 °C protected from light for 1 h. Allow to cool.
IDENTIFICATION
Plate : TLC silica gel GF254 plate R.
First identification : A, B.
Mobile phase : add a mixture of 1.2 volumes of water R and
Second identification : C, D, E.
8 volumes of methanol R to a mixture of 15 volumes of
ether R and 77 volumes of methylene chloride R.
A. Infrared absorption spectrophotometry (2.2.24).
Application : 5 μL.
Comparison : methylprednisolone acetate CRS.
Development : over a path of 15 cm.
If the spectra obtained in the solid state show differences,
dissolve the substance to be examined and the reference
Drying : in air.
substance separately in the minimum volume of acetone R,
Detection A : examine in ultraviolet light at 254 nm.
evaporate to dryness and record new spectra using the
Results A : the principal spot in each of the chromatograms
residues.
obtained with the test solutions is similar in position and
B. Thin-layer chromatography (2.2.27).
size to the principal spot in the chromatogram obtained with
Solvent mixture : methanol R, methylene chloride R
the corresponding reference solution.
(1:9 V/V).
Detection B : spray with alcoholic solution of sulfuric acid R.
Test solution. Dissolve 10 mg of the substance to be
Heat at 120 °C for 10 min or until the spots appear. Allow to
examined in the solvent mixture and dilute to 10 mL with the
cool. Examine in daylight and in ultraviolet light at 365 nm.
solvent mixture.
Results B : the principal spot in each of the chromatograms
Reference solution (a). Dissolve 10 mg of
obtained with the test solutions is similar in position, colour
methylprednisolone acetate CRS in the solvent
in daylight, fluorescence in ultraviolet light at 365 nm and
mixture and dilute to 10 mL with the solvent mixture.
size to the principal spot in the chromatogram obtained with
Reference solution (b). Dissolve 10 mg of prednisolone
the corresponding reference solution. The principal spot in
acetate CRS and 10 mg of methylprednisolone acetate CRS
each of the chromatograms obtained with test solution (b)
in the solvent mixture, then dilute to 10 mL with the solvent
and reference solution (b) has an RF value distinctly lower
mixture.
than that of the principal spot in each of the chromatograms
obtained with test solution (a) and reference solution (a).
Plate : TLC silica gel GF254 plate R.
Mobile phase : butanol R, toluene R, ether R (5:10:85 V/V/V). D. Add about 2 mg to 2 mL of sulfuric acid R and shake to
dissolve. Within 5 min, an intense red colour develops. When
Application : 5 μL.
examined in ultraviolet light at 365 nm, a reddish-brown
Development : over a path of 15 cm.
fluorescence is seen. Add this solution to 10 mL of water R
Drying : in air.
and mix. The colour fades and there is a greenish-yellow
Detection A : examine in ultraviolet light at 254 nm.
fluorescence in ultraviolet light at 365 nm.
General Notices (1) apply to all monographs and other texts
2483
Methylprednisolone acetate
EUROPEAN PHARMACOPOEIA 7.0
E. About 10 mg gives the reaction of acetyl (2.3.1).
TESTS
Specific optical rotation (2.2.7) : + 97 to + 105 (dried 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 20.0 mg of the substance to be examined
in 5 mL of tetrahydrofuran R and dilute to 10.0 mL with
water R.
Reference solution (a). Dissolve 4 mg of methylprednisolone
acetate CRS and 4 mg of dexamethasone acetate CRS in the
mobile phase, then dilute to 20.0 mL with the mobile phase.
Dilute 2.0 mL of this solution to 10.0 mL with the mobile phase.
Reference solution (b). Dilute 1.0 mL of the test solution to
50.0 mL with the mobile phase.
Column :
— size : l = 0.25 m, Ø = 4.6 mm ;
— stationary phase : octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : in a 1000 mL volumetric flask mix 260 mL
tetrahydrofuran R and 700 mL of water R, then allow to
equilibrate ; dilute to 1000 mL with water R and mix again.
Flow rate : 1 mL/min.
Detection : spectrophotometer at 254 nm.
Equilibration : with the mobile phase for about 45 min.
Injection : 20 μL.
Run time : 1.5 times the retention time of methylprednisolone
acetate.
Retention time: methylprednisolone acetate = about 43 min ;
dexamethasone acetate = about 57 min.
System suitability : reference solution (a) :
— resolution : minimum 6.5 between the peaks due to
methylprednisolone acetate and dexamethasone acetate ; if
necessary, adjust the concentration of water R in the mobile
phase.
Limits :
— total : not more than 0.5 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(1.0 per cent) ;
— disregard limit : 0.025 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.05 per cent).
Loss on drying (2.2.32) : maximum 0.5 per cent, determined on
1.000 g by drying in an oven at 105 °C.
B. 11β,17,21-trihydroxy-6α-methylpregna-1,4-diene-3,20-dione
(methylprednisolone),
C. R = OH : 11β,17-dihydroxy-6α-methylpregna-1,4-diene3,20,21-trione,
D. R = H : 11β-hydroxy-6α-methylpregna-1,4-diene-3,20,21-trione,
E. 11β,17-dihydroxy-3,20-dioxopregna-1,4-dien-21-yl acetate
(prednisolone acetate),
F. 6α-methyl-3,11,20-trioxopregna-1,4-dien-21-yl acetate,
ASSAY
Dissolve 0.100 g in ethanol (96 per cent) R and dilute to
100.0 mL with the same solvent. Dilute 1.0 mL of this solution
to 100.0 mL with ethanol (96 per cent) R. Measure the
absorbance (2.2.25) at the absorption maximum at 243 nm.
Calculate the content of C24H32O6 taking the specific absorbance
to be 355.
STORAGE
Protected from light.
G. 11β,17-dihydroxy-6α-methyl-3,20-dioxopregn-4-en-21-yl
acetate,
IMPURITIES
A. (20RS)-11β,17,20-trihydroxy-6α-methyl-3-oxopregna-1,4-dien21-yl acetate,
2484
H. 11β-hydroxy-6α-methyl-3-oxopregna-1,4,17(20)-trien-21-yl
acetate.
See the information section on general monographs (cover pages)
Methylprednisolone hydrogen succinate
EUROPEAN PHARMACOPOEIA 7.0
01/2008:1131
corrected 6.0
System suitability : reference solution (b):
— the chromatogram shows 2 spots which may, however,
not be completely separated.
METHYLPREDNISOLONE HYDROGEN C. Thin layer chromatography (2.2.27).
SUCCINATE
Test solution (a). Dissolve 25 mg of the substance to be
examined in methanol R with gentle heating and dilute to
Methylprednisoloni hydrogenosuccinas
5 mL with the same solvent (solution A). Dilute 2 mL of this
solution to 10 mL with methylene chloride R.
Test solution (b). Transfer 2 mL of solution A to a 15 mL glass
tube with a ground-glass stopper or a polytetrafluoroethylene
cap. Add 10 mL of a 0.8 g/L solution of sodium hydroxide R
in methanol R and immediately pass a stream of nitrogen R
through the solution for 5 min. Stopper the tube. Heat in a
water-bath at 45 °C, protected from light, for 30 min. Allow
to cool.
Reference solution (a). Dissolve 25 mg of
C26H34O8
Mr 474.6
methylprednisolone hydrogen succinate CRS in
[2921-57-5]
methanol R with gentle heating and dilute to 5 mL with the
same solvent (solution B). Dilute 2 mL of this solution to
DEFINITION
10 mL with methylene chloride R.
4-[(11β,17-Dihydroxy-6α-methyl-3,20-dioxopregna-1,4-dien-21Reference solution (b). Transfer 2 mL of solution B to
yl)oxy]-4-oxobutanoic acid.
a 15 mL glass tube with a ground-glass stopper or a
Content : 97.0 per cent to 103.0 per cent (dried substance).
polytetrafluoroethylene cap. Add 10 mL of a 0.8 g/L solution
CHARACTERS
of sodium hydroxide R in methanol R and immediately
pass a stream of nitrogen R through the solution for 5 min.
Appearance : white or almost white, hygroscopic powder.
Stopper the tube. Heat in a water-bath at 45 °C, protected
Solubility : practically insoluble in water, slightly soluble
from light, for 30 min. Allow to cool.
in acetone and in anhydrous ethanol. It dissolves in dilute
Plate : TLC silica gel F254 plate R.
solutions of alkali hydroxides.
Mobile phase : add a mixture of 1.2 volumes of water R and
IDENTIFICATION
8 volumes of methanol R to a mixture of 15 volumes of
First identification : A, B.
ether R and 77 volumes of methylene chloride R.
Second identification : C, D.
Application : 5 μL.
A. Infrared absorption spectrophotometry (2.2.24).
Development : over a path of 15 cm.
Comparison : methylprednisolone hydrogen succinate CRS.
Drying : in air.
B. Thin layer chromatography (2.2.27).
Detection A : examine in ultraviolet light at 254 nm.
Solvent mixture : methanol R, methylene chloride R
Results A : the principal spot in each of the chromatograms
(1:9 V/V).
obtained with the test solutions is similar in position and
Test solution. Dissolve 10 mg of the substance to be
size to the principal spot in the chromatogram obtained with
examined in the solvent mixture and dilute to 10 mL with the
the corresponding reference solution.
solvent mixture.
Detection B : spray with alcoholic solution of sulfuric acid R.
Reference solution (a). Dissolve 20 mg of
Heat at 120 °C for 10 min or until the spots appear. Allow to
methylprednisolone hydrogen succinate CRS in
cool. Examine in daylight and in ultraviolet light at 365 nm.
the solvent mixture and dilute to 20 mL with the solvent
Results B : the principal spot in each of the chromatograms
mixture.
obtained with the test solutions is similar in position, colour
Reference solution (b). Dissolve 10 mg of hydrocortisone
in daylight, fluorescence in ultraviolet light at 365 nm and
hydrogen succinate CRS in reference solution (a) and dilute
size to the principal spot in the chromatogram obtained with
to 10 mL with reference solution (a).
the corresponding reference solution. The principal spot in
Plate : TLC silica gel F254 plate R.
each of the chromatograms obtained with test solution (b)
Mobile phase : anhydrous formic acid R, anhydrous
and reference solution (b) has an RF value distinctly higher
ethanol R, methylene chloride R (0.1:1:15 V/V/V).
than that of the principal spot in each of the chromatograms
Application : 10 μL.
obtained with test solution (a) and reference solution (a).
Development : over a path of 15 cm.
D. Add about 2 mg to 2 mL of sulfuric acid R and shake to
dissolve. Within 5 min a reddish-brown colour develops. Add
Drying : in air.
this solution to 10 mL of water R and mix. The colour fades
Detection A : examine in ultraviolet light at 254 nm.
and a precipitate is formed.
Results A : the principal spot in the chromatogram obtained
with the test solution is similar in position and size to the
TESTS
principal spot in the chromatogram obtained with reference
Appearance of solution. The solution is clear (2.2.1).
solution (a).
Dissolve 0.100 g in 5 mL of sodium hydrogen carbonate
Detection B : spray with alcoholic solution of sulfuric
solution R.
acid R ; heat at 120 °C for 10 min or until the spots appear
and allow to cool ; examine in daylight and in ultraviolet light Specific optical rotation (2.2.7) : + 87 to + 95 (dried substance).
at 365 nm.
Dissolve 0.250 g in dioxan R and dilute to 25.0 mL with the
Results B : the principal spot in the chromatogram obtained same solvent.
with the test solution is similar in position, colour in daylight,
Related substances. Liquid chromatography (2.2.29).
fluorescence in ultraviolet light at 365 nm and size to the
principal spot in the chromatogram obtained with reference Test solution. Dissolve 25.0 mg of the substance to be examined
in the mobile phase and dilute to 10.0 mL with the mobile phase.
solution (a).
General Notices (1) apply to all monographs and other texts
2485