Glycyrrhetic Acid (Enoxolone) CAS 471-53-4 Assay 98.0~101.0% (Potentiometry)

General Notices
(Ph Eur monograph 1511)
C30H46O4 470.7 471-53-4
Action and use
Treatment of benign peptic ulcer disease.
Ph Eur
DEFINITION
(20β)-3β-Hydroxy-11-oxo-olean-12-en-29-oic acid.
Content
98.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance
White or almost white crystalline powder.
Solubility
Practically insoluble in water, soluble in ethanol, sparingly soluble in methylene chloride.
It shows polymorphism (5.9).
IDENTIFICATION
First identification A.
Second identification B, C.
A. Examine by infrared absorption spectrophotometry (2.2.24).
Comparison Enoxolone CRS.
If the spectra obtained in the solid state show differences, dissolve 0.2 g of the substance to be examined and 0.2 g of the reference substance separately in 6 ml of ethanol R. Boil under
a reflux condenser for 1 h and add 6 ml of water R. A precipitate is formed. Cool to about 10℃ and filter with the aid of vacuum. Wash the precipitate with 10 ml of alcohol R, dry in an oven at 80℃ and record new spectra.
B. Thin-layer chromatography (2.2.27).
Test solution Dissolve 10 mg of the substance to be examined in methylene chloride R and dilute to 10 ml with the same solvent.
Reference solution Dissolve 10 mg of enoxolone CRS in methylene chloride R and dilute to 10 ml with the same solvent.
Plate TLC silica gel plate R.
Mobile phase glacial acetic acid R, acetone R, methylene chloride R (5:10:90 V/V/V).
Application 5 µl.
Development Over 2/3 of the plate.
Drying In air for 5 min.
Detection Spray with anisaldehyde solution R and heat at 100-105℃ for 10 min. 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. Dissolve 50 mg in 10 ml of methylene chloride R. To 2 ml of this solution, add 1 ml of acetic anhydride R and 0.3 ml of sulphuric acid R. A pink colour is produced.
TESTS
Appearance of solution
The solution is clear (2.2.1) and not more intensely coloured than reference solution Y6 (2.2.2, Method II).
Dissolve 0.1 g in ethanol R and dilute to 10 ml with the same solvent.
Specific optical rotation (2.2.7) + 145 to + 154 (dried substance).
Dissolve 0.50 g in dioxan R and dilute to 50.0 ml with the same solvent.
Related substances
Liquid chromatography (2.2.29).
Test solution Dissolve 0.10 g of the substance to be examined in the mobile phaseand dilute to 100.0 ml with the mobile phase.
Reference solution (a) Dilute 2.0 ml of the test solution to 100.0 ml with the mobile phase.
Reference solution (b) Dilute 5.0 ml of reference solution (a) to 100.0 ml with the mobile phase.
Reference solution (c) Dissolve 0.1 g of 18α-glycyrrhetinic acid R in tetrahydrofuran R and dilute to 100.0 ml with the same solvent. To 2.0 ml of the solution, add 2.0 ml of the test solution and dilute to 100.0 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),
-temperature: 30℃.
Mobile phase Mix 430 volumes of tetrahydrofuran R and 570 volumes of a 1.36 g/l solution of sodium acetate R adjusted to pH 4.8 with glacial acetic acid R.
Flow rate 0.8 ml/min.
Detection Spectrophotometer at 250 nm.
Injection 20 µl loop injector; inject the test solution and the reference solutions.
Run time 4 times the retention time of Enoxolone.
System suitability:
-resolution: minimum of 2.0 between the peaks due to enoxolone and to 18α-glycyrrhetinic acid in the chromatogram obtained with reference solution (c).
Limits:
-any impurity: not more than 7 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.7 per cent),
-total: not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (2.0 per cent),
-disregard limit: 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.05 per cent).
Heavy metals (2.4.8) Maximum 20 ppm.
1.0 g complies with limit test F. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32) Maximum 0.5 per cent, determined on 1.000 g by drying in an oven at 105℃ for 4 h.
Sulphated ash (2.4.14) Maximum 0.2 per cent, determined on 1.0 g.
ASSAY
Dissolve 0.330 g in 40 ml of dimethylformamide R. Titrate with 0.1 M tetrabutylammonium hydroxide, determining the end-point potentiometrically (2.2.20). Carry out a blank titration. 1 ml of 0.1 M tetrabutylammonium hydroxide is equivalent to 47.07 mg of C30H46O4
Crown Copyright 2006 330 46 4
STORAGE
Protected from light.
IMPURITIES
A. (20β)-3β-hydroxy-11-oxo-18α-olean-12-en-29-oic acid,
B. (4β,20β)-3β,23-dihydroxy-11-oxo-olean-12-en-29-oic acid.
Ph Eur