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 QC 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 CC.
Detection: flame ionisation・
Injection: 1 pl. Relative retentions with reference to p-sitosterol (retention
time 二 about 16 min): cholesterol = about 0.7; brassicasterol
= about 077; campe$terol = about 0.84； campestanol = about
0.86; stigmasterol = about 0.9; sitostanol = about 1.02; A7-stigmastenol about 1.1.
System suitability\ reference solution (a):
— resolution; minimum of 1.0 between the peaks due to
^sitosterol and sitostanol.
STORAGE
In an airtight container, protected from light.
2. brassicasterol 6. |3-sitos terol
3. campes⑹ol
4. campestanol 7. sitostanol
8. A7-5ti^mastenol
Figure 1911.-1.・ -Assay of phytosterol (trtmethylsilane
derivatives) 1.
cholesterol
5. stigmasterol