1994R0086 — EN — 03.02.1999 — 001.001 — 1
This document is meant purely as a documentation tool and the institutions do not assume any liability for its contents
►B COMMISSION REGULATION (EC) No 86/94
of 19 January 1994
establishing a reference method for the determination of sitosterol and stigmasterol in butter
(OJ L 17, 20.1.1994, p. 7)
Amended by:
Official Journal
No page date
►M1Commission Regulation (EC) No 175/1999 of 26 January 1999 L 20 22 27.1.1999
1994R0086 — EN — 03.02.1999 — 001.001 — 2
▼B
COMMISSION REGULATION (EC) No 86/94
of 19 January 1994
establishing a reference method for the determination of sitosterol
and stigmasterol in butter
THE COMMISSION OF THE EUROPEAN COMMUNITIES,
Havingreg ard to the Treaty establishingthe European Community,
Having regard to Council Regulation (EEC) No 804/68 of 27 June 1968
on the common organization of the market in milk and milk prod-ucts(
1), as last amended by Regulation (EEC) No 2071/92 (2), and in
particular Article 6 (7) thereof,
Whereas butter may be traced and the traced products have to be
controlled accordingto Articles 3 and 6 of Commission Reg ulation(EEC) No 570/88 (
3), as last amended by Regulation (EC) No 3049/
93 (4);
Whereas strict compliance with the conditions relatingto the tracingof
butter is essential in order to prevent the risk of unauthorized use ofsubsidized butter;
Whereas takingaccount of the importance of tracingin the proper
functioningof this scheme it is necessary to establish common methodsfor detection of all tracers required by this scheme applicable in thesame way throughout the Community; this would in particular ensureequal treatment of all operators havingaccess to this scheme andeliminate unequal conditions of competition which may result atpresent, as a consequence of different national methods of analysis;
Whereas a reference method for the determination of stigmasterol and
sitosterol in butteroil has been established by Commission Regulation(EEC) No 3942/92 (
5); whereas this method can be applied in a slightly
modified form for the determination of sterol tracers in butter;
Whereas the accuracy of the method and the tolerance limits had to be
determined;
Whereas the measures provided for in this Regulation are in accordance
with the opinion of the Management Committee for Milk and MilkProducts,
HAS ADOPTED THIS REGULATION:
Article 1
The reference method of analysis to determine the stigmasterol or
β-sitosterol content of butter accordingto Article 6 of
►M1Regulation (EC) No 2571/97 ◄is specified in the Annex.
Butter has been traced correctly if the results obtained are in accordance
with the requirements specified in paragraph 8 of this Annex.
Article 2
This Regulation shall enter into force on the third day following its
publication in the Official Journal of the European Communities .
It shall apply from 1 April 1994.
This Regulation shall be binding in its entirety and directly applicable in
all Member States.
(1) OJ No L 148, 28. 6. 1968, p. 13.
(2) OJ No L 215, 30. 7. 1992, p. 64.
(3) OJ No L 55, 1. 3. 1988, p. 31.
(4) OJ No L 273, 5. 11. 1993, p. 7.
(5) OJ No L 399, 31. 12. 1992, p. 29.
1994R0086 — EN — 03.02.1999 — 001.001 — 3
▼B
ANNEX
DETERMINATION OF SITOSTEROL OR STIGMASTEROL IN
BUTTER BY CAPILLARYCOLUMN GAS CHROMATOGRAPHY
1. SCOPE AND FIELD OF APPLICATION
The method describes a procedure for the quantitative determination of
sitosterol or stigmasterol in butter. Sitosterol is taken to be the sum ofB-sitosterol and 22 dihydro B sitosterol, other sitosterols are assumed tobe insignificant. It is applicable to samples received under Regulation(EEC) No 570/88.
2. PRINCIPLE
The butter is saponified with potassium hydroxide in ethanolic solution
and the unsaponifiables are extracted with diethyl ether.
The sterols are transformed into trimethyl-silyl ethers and are analysed
by capillary-column gas chromatography with reference to an internalstandard betulin.
3. APPARATUS
3.1. 150 ml saponification flask fitted with a reflux condenser having
ground-glass joints.
3.2. 500 ml separatingfunnels.3.3. 250 ml flasks.3.4. Pressure equalizingfunnels, 250 ml or similar, to collect waste diethyl
ether.
3.5. Glass column, 350 mm × 20 mm fitted with sintered glass plug.3.6. Waterbath or isomantle.3.7. Reaction vials, 2 ml.3.8. Gas chromatograph suitable for use with a capillary column, provided
with a splittingsystem consistingof:
3.8.1. a thermostatic chamber for columns capable of maintainingthe desired
temperature with an accuracy of ± 1 °C;
3.8.2. a temperature-adjustable vaporization unit;
3.8.3. a flame ionization detector and converter amplifier;3.8.4. an integrator-recorder suitable for use with the converter-amplifier
(3.8.3).
3.9. A fused-silica capillary column entirely coated with BP1 or equivalent
in a uniform thickness 0,25 µm; the column must be capable ofresolvingtrimethyl silyl derivatives of lanosterol and sitosterol, a BP1,length 12 m, internal diameter 0,2 mm is suitable.
3.10. A 1 µl gas chromatography microsyringe with hardened needle.
4. REAGENTS
All reagents must be of recognized analytical grade. The water used
must be distilled water or water of at least equivalent purity.
4.1. Ethanol, at least 95 % pure.4.2. Potassium hydroxide, 60 % solution, dissolve 600 gpotassium
hydroxide (minimum 85 %) in water and make up to one litre withwater.
4.3. Betulin of at least 99 % purity.
4.3.1. Internal standard solutions. Betulin in diethyl ether (4.4).
4.3.1.1. The concentration of betulin solution used for sitosterol determination
should be 1,0 mg/ml.
1994R0086 — EN — 03.02.1999 — 001.001 — 4
▼B
4.3.1.2. The concentration of betulin solution used for stigmasterol deter-
mination should be 0,4 mg/ml.
4.4. Diethyl ether, analytical purity (free from peroxides or residue).4.5. Sodium sulphate, anhydrous, granular previously dried at 102 °C for two
hours.
4.6. Silylatingreag ent, for example TRI-SIL (available from Pierce Chem-
ical Co, Cat No 49001) or equivalent. (WARNING: TRI-SIL is flam-mable toxic, corrosive and a possible carcinogen. Laboratory personnelmust be familiar with TRI-SIL safety data and take the appropriateprecautions.)
4.7. Lanosterol.4.8. Sitosterol, of known purity not less than 90 % pure (P).
Note 1: The purity of standard materials used for calibration must be
determined usingthe method of normalization. Assume that all sterolspresent in the sample are represented on the chromatogram the total areaof the peaks represents 100 % of the sterol constituents and that thesterols give the same detector response. Linearity of the system must bevalidated over the concentration ranges of interest.
4.8.1. Sitosterol standard solution — prepare a solution containing, to the
nearest 0,001 mg/ml, approximately 0,5 mg/ml (W
1) sitosterol (4.8) in
diethyl ether 4.4).
4.9. Stigmasterol, of known purity not less than 90 % pure (P).4.9.1. Stigmasterol standard solution — prepare a solution containing, to the
nearest 0,001 mg/m, approximately 0,2 mg/ml (W
1) stigmasterol (4.9) in
diethyl ether (4.4).
4.10. Resolution test mixture. Prepare a solution containing0,05 mg /ml lanos-
terol (4.7) and 0,5 mg/ml sitosterol (4.8) in diethyl ether (4.4).
5. PROCEDURE
5.1. Preparation of standard solutions for chromatography. The internal
standard solution (4.3.1) must be added to the appropriate sterolstandard solution at the same time as it is added to the saponified sample(see 5.2.2).
5.1.1. Sitosterol standard chromatographic solution; transfer 1 ml of sitosterol
standard solution (4.8.1) to each of two reaction vials (3.7) and removethe diethyl ether with a stream of nitrogen. Add 1 ml of internal standardsolution (4.3.1.1) and remove the diethyl ether with a stream of nitrogen.
5.1.2. Stigmasterol standard chromatographic solution; transfer 1 ml of stig-
masterol standard solution (4.9.1) to each of two reaction vials (3.7) andremove the diethyl ether with a stream of nitrogen. Add 1 ml of internalstandard solution (4.3.1.2) and remove the diethyl ether with a stream ofnitrogen.
5.2. Preparation of the unsaponifiables.
5.2.1. Melt the butter sample at a temperature not exceeding35 °C, mix the
sample thoroughly by stirring, weigh, to the nearest 1 mg, approximately1 gof butter (W
2) into a 150 ml flask (3.1). Add 50 ml ethanol (4.1) and
10 ml potassium hydroxide solution (4.2). Fit the reflux condenser andheat at approximately 75 °C for 30 minutes. Detach the condenser and
cool the flask to approximately ambient temperature.
5.2.2. Add 1,0 ml of internal standard solution (4.3.1.1) to the flask if sitos-
terol is to be determined, or (4.3.1.2) if stigmasterol is to be determined.Mix thoroughly. Transfer the contents of the flask quantitatively into a500 ml separatingfunnel (3.2) washingthe flask in turn with 50 mlwater and 250 ml diethyl ether (4.4). Shake the separatingfunnel vig or-ously for two minutes and allow the phases to separate. Run of the lower
1994R0086 — EN — 03.02.1999 — 001.001 — 5
▼B
aqueous layer and wash the ether layer by shakingwith four successive
100 ml aliquots of water.
Note 2: To avoid formation of an emulsion, it is essential that the first
two water washes are carried out gently (10 inversions). The third washcan be shaken vigorously for 30 seconds. If an emulsion is formed it canbe destroyed by the addition o f5—1 0m lo f ethanol. If ethanol is
added it is essential to carry out a further two vigorous water washes.
5.2.3. Pass the clear, soap free ether layer through a glass column (3.5)
containing30 ganhydrous sodium sulphate (4.5). Collect the ether in a250 ml flask (3.3). Add one anti-bumpingg ranual and evaporate to neardryness in a water or isomantle, takingcare to collect the waste solvents.
Note 3: If sample extracts are taken to complete dryness at too high a
temperature sterol losses may occur.
5.3. Preparation of trimethyl silyl ethers.
5.3.1. Transfer the ether solution remainingin the flask t oa2m l reaction vial
(3.7) with 2 ml diethyl ether and remove the ether usinga stream ofnitrogen. Wash the flask with two further 2 ml aliquots of diethyl ether,transferringto the vial and removingthe enter with nitrog en each time.
5.3.2. Silylate the sample by addition of 1 ml TRI-SIL (4.6). Close the vial and
shake vigorously to dissolve. If dissolution is incomplete, warm to65—70 °C. Allow to stand for at least five minutes before injectinginto
the gas-chromatograph. Silylate standards in the same way as samples.Silylate the resolution test mixture (4.10) in the same way as samples.
Note 4: Silylation must be affected in a water free environment. Incom-
plete silylation of betulin is indicated by a second peak close to that ofbetulin.
The presence of ethanol at the silylation stage will interfere with silyla-
tion. This may result from inadequate washingat the extraction stag e. Ifthis problem persists a fifth wash, shaken vigorously for 30 seconds,may be introduced at the extraction stage.
5.4. Gas-Chromatographic analysis.
5.4.1. Choice of operatingconditions.
Set up the gas-chromatograph according to the manufacturers instruc-
tions.
The guideline operating conditions are as follows:
— column temperature 265 °C
— injector temperature 280 °C
— detector temperature 300 °C
— carrier gas flow rate 0,6 ml/minute— hydrogen pressure 84 kPa— air pressure 155 kPa— sample split 10:1 to 50:1, the split ratio must be optimized in
accordance with the manufacturers instructions and linearity ofdetector response then validated over the concentration range ofinterest.
Note 5: It is especially important that the injection liner is regularly
cleared.
— amount of substance injected 1 µl of TMSE solution.
Allow the system to equilibrate and obtain a satisfactory stable response
before commencingany analysis.
These conditions must be varied in the light of column and gas-chroma-
tograph characteristics so as to obtain chromatograms which meet thefollowingrequirements:
1994R0086 — EN — 03.02.1999 — 001.001 — 6
▼B
— the sitosterol peak must be adequately resolved from lanosterol.
Figure 1 shows a typical chromatogram which should be obtainedfrom a silylated resolution test mixture (4.10),
— the relative retention times of the followingsterols should be
approximately:
Cholesterol 1,0
Stigmasterol 1,3Sitosterol 1,5Betulin 2,5,
— the retention time for betulin should be approximately 24 minutes.
5.4.2. Analytical procedure.
Inject µl of silylated solution (stigmasterol or sitosterol) and adjust the
integrator calibration parameters.
Inject a further 1 µl of silylated standard solution to determine the
response factors with reference to betulin.
Inject 1 µl of silylated sample solution and measure peak areas. Each
chromatographic run must be bracketed by an injection of standards. Asa guide, six injections of sample should be included in each bracketedrun.
Note 6: Integration of the stigmasterol peak should include any tailing
as defined by points 1, 2 and 3 in Figure 2 b.
Integration of the sitosterol peak should include the area of the 22
dihydro B sitosterol (stigmastanol) peak which elutes immediately aftersitosterol, see Figure 3b, when evaluating total sitosterol.
6. CALCULATION OF RESULTS
6.1. Determine the area of the sterol peaks and betulin peaks in both stan-
dards brackertinga batch, and calculate R
1:
R1=Average peak area of sterol in standard
Average peak area of betulin in standard
Determine the area of the sterol peak (stigmasterol of sitosterol) and
betulin peak in the sample and calculate R2:
R2=Peak area of sterol in sample
Peak area of betulin in sample
W1= sterol content of the standard (mg) contained in 1 ml of standard
solution (4.8.1 or 4.9.1).
W2= weight of sample (g) (5.2.1)
P = purity of standard sterol (4.8 or 4.9)
Sterol content of the sample (mg/kg) =R2
R1×W1
W2×P×1 0 .
7. ACCURACY OF THE METHOD
7.1. Repeatability.
7.1.1. Stigmasterol.
The difference between the results of two determinations carried out
within the shortest feasible time interval, by one operator usingthe sameapparatus on identical test material shall not exceed 19,3 mg/kg.
7.1.2. Sitosterol
The difference between the results of two determinations carried out
within the shortest feasible time interval, by one operator usingthe sameapparatus on identical test material shall not exceed 23,0 mg/kg.
1994R0086 — EN — 03.02.1999 — 001.001 — 7
▼B
7.2. Reproducibility.
7.2.1. Stigmasterol.
The difference between the results of two determinations carried out by
operators in different laboratories, usingdifferent apparatus on identicaltest material shall not exceed 31,9 mg/kg.
7.2.2. Sitosterol.
The difference between the results of two determinations carried out by
operators in different laboratories, usingdifferent apparatus on identicaltest material shall not exceed 8,7 % relative of the mean of the deter-mination.
7.3. Source of precision data.
The precision data were determined from an experiment conducted in
1992 involvingeig ht laboratories and six samples (three blind dupli-cates) for stigmasterol; six samples (three blind duplicates) for sitosterol.
8. TOLERANCE LIMITS
▼
M1
8.1. Three samples must be taken from the traced product in order to check
on the correct tracingof the product.
▼B
8.2. Stigmasterol
8.2.1. The incorporation for stigmasterol is 150 grams of at least 95 % pure
stigmasterol per tonne of butter, i.e. 142,5 mg/kg; or 170 grams of atleast 85 % pure stigmasterol per tonne of butter, i.e. 144,5 mg/kg.
▼
M1
8.2.2. The results of the three samples obtained from the analysis of the
product are used to check the rate and the homogeneity of tracerincorporation and the lowest of these results is compared with thefollowinglimits (Critical Difference for a 95 % probability level(CrD95) taken into consideration):
— 116,0 mg/kg (95 % of the minimum incorporation rate for 95 % pure
stigmasterol),
— 118,0 mg/kg (95 % of the minimum incorporation rate for 85 % pure
stigmasterol),
— 81,0 mg/kg (70 % of the minimum incorporation rate for 95 % pure
stigmasterol),
— 82,0 mg/kg (70 % of the minimum incorporation rate for 85 % pure
stigmasterol).
The tracer concentration of the sample giving the lowest result is used in
conjunction with interpolation respectively between 116,0 mg/kg and81,0 mg/kg or 118,0 mg/kg and 82,0 mg/kg.
▼
B
8.3. Sitosterol
8.3.1. The incorporation rate for sitrosterol is 600 grams of at least 90 % pure
sitosterol per tonne of butter, i.e. 540 mg/kg.
▼M1
8.3.2. The results of the three samples obtained from the analysis of the
product are used to check the rate and the homogeneity of tracerincorporation and the lowest of these results is compared with thefollowinglimits (Critical Difference for a 95 % probability level(CrD95) taken into consideration):
1994R0086 — EN — 03.02.1999 — 001.001 — 8
▼M1
— 486,0 mg/kg (95 % of the minimum incorporation rate for 90 % pure
sitosterol),
— 358,0 mg/kg (70 % of the minimum incorporation rate for 90 % pure
sitosterol).
The tracer concentration of the sample giving the lowest result is used inconjunction with interpolation between 486,0 mg/kg and 358,0 mg/kg.
1994R0086 — EN — 03.02.1999 — 001.001 — 9
▼
M1
1994R0086 — EN — 03.02.1999 — 001.001 — 10
▼
M1
1994R0086 — EN — 03.02.1999 — 001.001 — 11
▼M1