De Pharmacopoeia Europea geeft voor de gaschromatografische analyse van vetzuren in vette oliën het volgende voorschrift:
2.4.22. FOREIGN OILS IN FATTY OILS BY GAS CHROMATOGRAPHY
The test for foreign oils is carried out on the methyl esters of the fatty acids contained in the oil to be examined. This method is not applicable to oils that contain glycerides of fatty acids with an epoxy-, hydroepoxy-, cyclopropanic or cyclopropenic group, or those that contain a large proportion of fatty acids of chain length less than eight carbon atoms or to oils with an acid value greater than 2.0.Examine by gas chromatography (2.2.28).
Test solution.
When prescribed in the monograph, dry the oil to be examined before the methylation step. Weigh 1.0 g of the oil into a 25 ml round-bottomed flask with a ground-glass neck fitted with a reflux condenser and a gas port into the flask. Add 10 ml of anhydrous methanol R and 0.2 ml of a 60 g/l solution of potassium hydroxide R in methanol R. Attach the reflux condenser, pass nitrogen R through the mixture at a rate of about 50 ml/min, shake and heat to boiling. When the solution is clear (usually after about 10 min), continue heating for a further 5 min. Cool the flask under running water and transfer the contents to a separating funnel. Rinse the flask with 5 ml of heptane R and transfer the rinsings to the separating funnel and shake. Add 10 ml of a 200 g/l solution of sodium chloride R and shake vigorously. Allow to separate and transfer the organic layer to a vial containing anhydrous sodium sulphate R. Allow to stand then filter.
Reference solution (a).
Prepare 0.50 g of the mixture of calibrating substances with the composition described in Table 2.4.22.-1; dissolve in heptane R and dilute to 50.0 ml with the same solvent.
Reference solution (b).
Dilute 1.0 ml of reference solution (a) to 10.0 ml with heptane R.The chromatographic procedure may be carried out using:
maintaining the temperature of the column at 180 �C and that of the injection port and of the detector at 200 �C. If necessary or where prescribed, raise the temperature of the column at a rate of 5 �C per minute from 120 �C to 200 �C.
- a glass or stainless steel column 2 m to 3 m long and 2 mm to 4 mm in internal diameter packed with diatomaceous earth for gas chromatography R (125 �m to 200 �m), impregnated with 5 per cent to 15 per cent of polyethyleneglycol succinate R or polyethyleneglycol adipate R,
- nitrogen for chromatography R as the carrier gas at a flow rate of 25 ml/min,
- a flame-ionisation detector,
The chromatographic procedure may also be carried out using:
maintaining the temperature of the column at 160 �C to 200 �C, according to the length and type of column used (for a column 30 m long and coated with a layer of macrogol 20 000 R, 200 �C), and that of the injection port and of the detector at 250 �C.
- a capillary glass or quartz column (preferably a wall-coated open tube) 10 m to 30 m long and 0.2 mm to 0.8 mm in internal diameter the inner surface of which is coated with a layer of poly[(cyanopropyl)(methyl)][(phenyl) (methyl)]siloxane R or of macrogol 20 000 R (0.1 �m to 0.5 �m thick) or some other suitable stationary phase,
- helium for chromatography R or hydrogen for chromatography R as the carrier gas at a flow rate of 1.3 ml/min (for a column 0.32 mm in internal diameter),
- a flame-ionisation detector,
- a split ratio of 1:100 or less according to the internal diameter of the column used (1:50 for a 0.32 mm column),
If necessary or where prescribed, raise the temperature of the column at a rate of 3 �C per minute from 170 �C to 230 �C (for the macrogol 20 000 R column).Inject 0.5 �l of reference solution (a). Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained is 50 per cent to 70 per cent of the full scale of the recorder.
Determine the retention times of the various constituent fatty acids. Inject 1 �l of reference solution (b) and check the signal-to-noise ratio of the peak corresponding to methyl myristate.
Inject from 0.5 �l to 1.0 �l of the test solution. Record the chromatograms for 2.5 times the retention time of methyl oleate. Evaluate the chromatogram as indicated below.
The test is not valid unless:
- in the chromatogram obtained with reference solution (a), the number of theoretical plates (n) (2.2.28), calculated for the peak corresponding to methyl stearate, is at least 2000 for packed and 30 000 for capillary columns,
- in the chromatogram obtained with reference solution (a), the resolution (Rs) (2.2.28) between the peaks corresponding to methyl oleate and methyl stearate is at least 1.25 for packed and 1.8 for capillary columns, and, where prescribed in the monograph, the resolution between the peaks corresponding to methyl linolenate (C18:3) and methyl arachidate (C20:0) or methyl arachidate (C20:0) and methyl eicosenoate (C20:1) is at least 1.8,
- in the chromatogram obtained with reference solution (b) the signal-to-noise ratio (2.2.28) of the peak corresponding to methyl myristate is at least five.
Table 2.4.22.-1. � Calibrating substances*
Composition (per cent m/m)
Mixture of the following substances
Equivalent chain
length **Isothermal
Linear temperature programme
Methyl laurate R
12.0
5
10
Methyl myristate R
14.0
5
15
Methyl palmitate R
16.0
10
15
Methyl stearate R
18.0
20
20
Methyl arachidate R
20.0
40
20
Methyl oleate R
18.6
20
20
* For GC with capillary column and split inlet system, it is recommended that the component with the longest chain length of the mixture to be examined is added to the calibration mixture.
** This value, which is to be calculated using calibration curves, is given as an example for a column of polyethyleneglycol succinate R.Evaluation of chromatograms
Avoid working conditions tending to give masked peaks (presence of constituents with small differences between retention times, e.g. linolenic acid and arachidic acid).Qualitative analysis.
Draw calibration curves using the chromatogram obtained with the calibration solution and the information in Table 2.4.22.-1.
- using isothermal operating conditions giving the logarithms of reduced retention time as a function of the number of carbon atoms of the fatty acid; identify the peaks by means of the straight line thus obtained and the �equivalent chain lengths� of the different peaks. The calibration curve of the saturated acids is a straight line. The logarithms of reduced retention times of unsaturated acids are situated on this line at points corresponding to non-integral numbers known as �equivalent chain lengths�;
- using linear temperature programming giving the retention time according to the number of carbon atoms of the fatty acid; identify by reference to the calibration curve.
Quantitative analysis.
The normalisation procedure is used in which the sum of the areas of the peaks in the chromatogram, except that of the solvent, is set at 100 per cent.
The use of an electronic integrator is recommended.
The content of a constituent is calculated by determining the area of the corresponding peak as a percentage of the sum of the area of all the peaks.
Disregard any peak with an area less than 0.05 per cent of the total area.
Farmaceutische Analyse 5e-jaar |
10 september 1999