Sommige vette hulpstoffen bevatten vrije of veresterde sterolen. Na verzeping maken deze deel uit van de zogenaamde onverzeepbare rest. De Pharmacopoeia Europea geeft voor de analyse hiervan het voorschrift 2.4.23. STEROLS IN FATTY OILS.Separation of the sterol fraction Prepare the unsaponifiable matter and then isolate the sterol fraction of the fatty oil by thin-layer chromatography (2.2.27), using silica gel g R in a 0.3 mm to 0.5 mm layer as the coating substance.
Test solution (a).
In a 150 ml flask fitted with a reflux condenser, place a volume of a 2 g/l solution of betulin R in methylene chloride R containing betulin corresponding to about 10 per cent of the sterol content of the sample used for the determination (e.g. in the case of olive oil add 500 �l, in the case of other vegetable oils add 1500 �l of the betulin solution). If the monograph requires the content of the individual sterols as a percentage of the sterol fraction, the addition of betulin may be omitted. Evaporate to dryness under a current of nitrogen R. Add 5.00 g (m g) of the substance to be examined. Add 50 ml of 2 M alcoholic potassium hydroxide R and heat on a water-bath for l h, swirling frequently. Cool to a temperature below 25 �C and transfer the contents of the flask to a separating funnel with 100 ml of water R. Shake the liquid carefully with three quantities, each of 100 ml, of peroxide-free ether R. Combine the ether layers in another separating funnel containing 40 ml of water R, shake gently for a few minutes, allow to separate and reject the aqueous phase. Wash the ether phase with several quantities, each of 40 ml, of water R, until the aqueous phase is no longer alkaline to phenolphthalein. Transfer the ether phase to a tared flask, washing the separating funnel with peroxide-free ether R. Distil off the ether with suitable precautions and add 6 ml of acetone R. Carefully remove the solvent in a current of nitrogen R. Dry to constant mass at 100 �C to 105 �C. Allow to cool in a desiccator and weigh. Dissolve the residue in a minimal volume of methylene chloride R.
Test solution (b).
Treat 5.00 g of rapeseed oil R as prescribed for the substance to be examined, beginning at the words "Add 50 ml of 2 M alcoholic potassium hydroxide R".
Test solution (c).
Treat 5.00 g of sunflower oil R as prescribed for the substance to be examined, beginning at the words "Add 50 ml of 2 M alcoholic potassium hydroxide R".
Reference solution.Dissolve 25 mg of cholesterol R and 10 mg of betulin R in 1 ml of methylene chloride R.
Use a separate plate for each test solution. Apply separately as a band 20 mm by 3 mm 20 �l of the reference solution and as a band 40 mm by 3 mm 0.4 ml of test solution (a), test solution (b) or test solution (c). Develop over a path of 18 cm using a mixture of 35 volumes of ether R and 65 volumes of hexane R. Dry the plates in a current of nitrogen R. Spray the plates with a 2 g/l solution of dichlorofluorescein R in ethanol R and examine in ultraviolet light at 254 nm.
The chromatogram obtained with the reference solution shows bands corresponding to cholesterol and betulin.
The chromatograms obtained with the test solutions show bands with similar Rf values due to sterols.From each of the chromatograms, remove an area of coating corresponding to the area occupied by the sterol bands and additionally the area of the zones 2 mm to 3 mm above and below the visible zones corresponding to the reference solution. Place separately in three 50 ml flasks. To each flask add 15 ml of hot methylene chloride R and shake. Filter each solution through a sintered-glass filter (40) or suitable filter paper and wash each filter with three quantities, each of 15 ml, of methylene chloride R. Place the combined filtrate and washings from each filter separately in three tared flasks, evaporate to dryness under a stream of nitrogen R and weigh.
Determination of the sterols
Examine by gas chromatography (2.2.28). Carry out the operations protected from humidity and prepare the solutions immediately before use.
Test solution.
To the sterols separated from the substance to be examined by thin-layer chromatography add, per milligram of residue, 0.02 ml of a freshly prepared mixture of 1 volume of chlorotrimethylsilane R, 3 volumes of hexamethyldisilazane R and 9 volumes of anhydrous pyridine R. Shake carefully until the sterols are completely dissolved. Allow to stand in a desiccator over diphosphorus pentoxide R for 30 min. Centrifuge if necessary and use the supernatant liquid.
Reference solution (a).
To 9 parts of the sterols separated from rapeseed oil R by thin-layer chromatography add 1 part of cholesterol R. To the mixture add, per milligram of residue, 0.02 ml of a freshly prepared mixture of l volume of chlorotrimethylsilane R, 3 volumes of hexamethyldisilazane R and 9 volumes of anhydrous pyridine R. Shake carefully until the sterols are completely dissolved. Allow to stand in a desiccator over diphosphorus pentoxide R for 30 min. Centrifuge if necessary and use the supernatant liquid. Reference solution (b).
To the sterols separated from sunflower oil R by thin-layer chromatography add, per milligram of residue, 0.02 ml of a freshly prepared mixture of 1 volume of chlorotrimethylsilane R, 3 volumes of hexamethyldisilazane R and 9 volumes of anhydrous pyridine R. Shake carefully until the sterols are completely dissolved. Allow to stand in a desiccator over diphosphorus pentoxide R for 30 min. Centrifuge if necessary and use the supernatant liquid. The chromatographic procedure may be carried out using:maintaining the temperature of the column at 260 �C, that of the injection port at 280 �C and that of the detector at 290 �C.
- a fused-silica column 20 m to 30 m long and 0.25 mm to 0.32 mm in internal diameter, coated with poly[methyl (95)phenyl(5)]siloxane R or of poly[methyl(94)phenyl(5) vinyl(1)]siloxane R (film thickness 0.25 �m),
- hydrogen for chromatography R at a velocity of 30 cm to 50 cm per second or helium for chromatography R at a velocity of 20 cm to 35 cm per second as the carrier gas. Measure the velocity as follows: maintaining the indicated operating conditions for the determination of the sterols, inject 1 �l to 3 �l of methane or propane. Measure the time in seconds required by the gas to pass through the column from the moment of the injection to the appearance of the peak (tm). The velocity is given by L/tm, where L is the length of the column in centimetres,
- a flame-ionisation detector,
- a split injector (1/50 or 1/l00),
Inject l �l of each solution.The chromatogram obtained with reference solution (a) shows four principal peaks corresponding to cholesterol, brassica-sterol, campesterol and b-sitosterol and the chromatogram obtained with reference solution (b) shows four principal peaks corresponding to campesterol, stigmasterol, b-sitosterol and D7-stigmastenol. The retention times of the sterols relative to b-sitosterol are given in the Table . The peak of the internal standard (betulin) must be clearly separated from the peaks of the sterols to be determined. For the chromatogram obtained with the test solution, identify the peaks and calculate the percentage content of each sterol in the sterol fraction of the substance to be examined using the following expression: A = area of the peak corresponding to the component to be determined, S = sum of the areas of the peaks corresponding to the components indicated in Table 2.4.23.-1. If required in the monograph, calculate the content of each sterol in milligrams per 100 grams of the substance to be examined using the following expression: A = area of the peak corresponding to the component to be determined, AS = area of the peak corresponding to betulin, m = mass of the sample of the substance to be examined in grams, mS = mass of betulin R added in milligrams.
Table Retention times of sterols relative to b-sitosterol for two different columns
Poly[methyl-(95)phenyl(5)]siloxane
Poly[methyl(94)-phenyl(5)vinyl-(1)]siloxane
Cholesterol
0.63
0.67
Brassicasterol
0.71
0.73
24-Methylenecholesterol
0.80
0.82
Campesterol
0.81
0.83
Campestanol
0.82
0.85
Stigmasterol
0.87
0.88
7-Campesterol
0.92
0.93
5,23-Stigmastadienol
0.95
0.95
Clerosterol
0.96
0.96
-Sitosterol
1
1
Sitostanol
1.02
1.02
5-Avenasterol
1.03
1.03
5,24-Stigmastadienol
1.08
1.08
7-Stigmastenol
1.12
1.12
7-Avenasterol
1.16
1.16
Betulin
1.4
1.6
Farmaceutische Analyse 5e-jaar |
10 september 1999