欧洲药典植物甾醇检测方法

PHYTOSTEROL

Phytosterolum

DEFINITION

Natural mixture of sterols obtained from plants of the genuses Hypoxis. Pin us and Picea.

Content: minimum 70.0 per cent of p-sitosterol (dried substance).

CHARACTERS

Appearance : white or almost white powder.

Solubility: practically insoluble in water, soluble in tetrahydrofuran, sparingly soluble in ethyl acetate・

IDENTIFICATION

A.Mix 1 ml of acetic anhydride R with 0.5 ml of solution S (see

Tests). After the addition of 0.1 ml of sulphuric acid R a

red colour is produced which changes rapidly to violet then to blue and finally to green.

B.Examine the chromatograms obtained in the assay. Results: the

principal peak in the chromatogram obtained with the test

solution is similar in retention time to

the peak in the chromatogram obtained with reference

solution (b)・

TESTS

Solution S. Dissolve 1.0 gin tetrahydrofuran R and dilute to 20 ml with the same solvent

Appearance of solution. Solution S is clear (227) and not more intensely coloured than reference solution Y6(222、Method II}. Acidity or alkalinity. Shake 0.20 g with a mixture of 4.0 ml of ethyl acetate R and 10.0 ml oi carbon dioxide-free water R for 3 min. Allow the layers to separate・ To the aqueous layer add 0」ml of bromothymol bluo solution RI. If the solution is yellow not more than 0.5 ml of 0.01 M sodium hydroxide is required to change the colour to blue・ If the solution is blue not more than 0.5 ml of 0.01 Mhydrochloric acid is required to change the colour to yellow.

Specific optical rotation (227):■ 15 to -28 (dried substance).

Dissolve 0.500 g in ethyl acetate R and dilute to 10.0 ml with the same solvent

Acid value (25・刀：maximum 1A determined on 2,0 g

Peroxide value (25・5)： maximum 10 A

Saponification value (25®: maximum 3.

Other sterols. Examine the chromatogram obtained with the test solution in the assay (Figure 191L-1).

Composition of ihe other sterols;

一cholesterol: maximum 0.5 per cent,

-brassicasterol: maximum 0.5 per cent,

-campesterol: maximum 15.0 per cent,

-campestanol: maximum 5,0 per cent,

一stigmasterol\ maximum 5.0 per cent,

一sitostanoh maximum 15.0 per cent,

-L7-stigmastenoL maximum 5.0 per cent

Loss on drying (2.232): maximum 4.0 per cent, determined on 0.250 g by drying in an oven at 100-105 Q C for 2 h・

Total ash (2476): maximum 0.5 per cent, determined on 1.0 g.

ASSAY

Gas chromatography (2.2.28} \ use the normalisation procedure. Test solution. Dissolve 0.100 g in tetrahydro furan R and dilute to 10.0 ml with the same solvent. Introduce 100 p) of this solution into a 3 ml flask and evaporate to dryness under nitrogen R. Add 100 pl of a freshly prepared mixture of 50 pl of 1-methylimidazole R and 1.0 ml of heptafluor(>N-

methyl^lrimethi/ls[lyl)butanamide R, close the flask tightly and heat to 10() °C for 15 min. Allow to cook Reference solution (a 丿.Dissolve 25 mg of ^sitosterol R and 25 mg of sitostanoi R in tetrahydrofuran R and dilute to 10.0 ml with the same solvent. Introduce 100 pl of this solution into a 3 ml flask and evaporate to dryness under nitrogen R. Add 100 pl of a freshly prepared mixture of 50 pl of l・m€thy【imidQ2olQ and L0 ml of h0ptdfluoFdN・m凶hyLNJtYimgthylsilyUbutaYiamidoR. Close the flask tightly and heat to 100 °C for 15 min. Allow to cool. Reference solution ⑹.Dissolve 0.100 g of J^sitost^ol R in tetrahydrofuran R and dilute to 10>0 ml with the same solvent Introduce 100 pl of this solution into a 3 ml flask and evaporate to dryness under nitrogen R. Add 100 pl of a freshly prepared mixture of 50 pl of 1 -methylimidazote R and 1.0 ml of heDtafluoFO・N・7nethyl・N・(trimethykily【)bulcmanii(l€ R. Close the flask tightly and heat to 100 °C for 15 min. Allow to cool

Column:

- material: quartz,

-stze\ f= 25 m, 0 = 0.3 mm,

-stationary phase: poli/(dimethyl}(dip虑砂)(小加・nyl)siloxane R (1 pm).

Carrier gas\ hydrogen for chromatography幷.

Flow rate: 2 ml/min.

Split ratio: 1:20.

Temperature;

-column: 280 °C,

一injection port and detector: 300 C C.

Detection: flame ionisation・

Injection: 1 pl.