1992R3942 — EN — 01.02.1993 — 001.001 — 1
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►B COMMISSION REGULATION (EEC) No 3942/92
of 22 December 1992
establishing a reference method for the determination of sitosterol and stigmasterol in butteroil
(OJ L 399, 31.12.1992, p. 29)
Amended by:
Official Journal
No page date
►M1Commission Regulation (EEC) No 2539/93 of 15 September 1993 L233 1 16.9.1993
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COMMISSION REGULATION (EEC) No 3942/92
of 22 December 1992
establishing a reference method for the determination of sitosterol
and stigmasterol in butteroil
THE COMMISSION OF THE EUROPEAN COMMUNITIES,
Having regard to the Treaty establishing the European EconomicCommunity,
HavingregardtoCouncilRegulation(EEC)No804/68of27June1968
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 thereof,
Whereas butteroil has to be traced and the traced butteroil has to be
controlled according to Commission Regulation (EEC) No 3143/85(3)
as last amended by Regulation (EEC) No 1264/92(4), and in particular
Articles 5 and 6 thereof;
Whereas butteroil may be traced and the traced products have to be
controlledaccordingtoCommissionRegulation(EEC)No570/88(5)as
last amended by Regulation (EEC) No 124/92(6), and in particular
Articles 3 and 6 thereof;
Whereas butteroil has to be traced and the traced butteroil has to be
controlledaccordingtoCommissionRegulation(EEC)No429/90(7),as
last amended by Regulation (EEC) No 1264/92, and in particularArticles 10 and 11 thereof;
Whereas strict compliance with the conditions relating to the tracing of
butteroil is essential in order to prevent the risk of unauthorized use ofsubsidized butter;
Whereas taking account of the importance of tracing in the proper
functioning of these schemes it is necessary to establish commonmethods for detection of all tracers required by these schemes applic-able in the same way throughout the Community; this would in partic-ular ensure equal treatment of all operators having access to theseschemes and eliminate unequal conidtions of competition which mayresult at present, as a consequence of different national methods ofanalysis.
Whereasitisdifficulttoestablishsuchreferencemethodsforalltracers
simultaneously; whereas establishing a reference method for the deter-minationofstigmasterolandsitosterolinbutteroilconstitutesafirststepin this direction;
WhereasthemeasuresprovidedforinthisRegulationareinaccordance
with the opinion of the Management Committee for Milk and MilkProducts,
HAS ADOPTED THIS REGULATION:
(1) OJ No L 148, 28. 6. 1968, p. 13.
(2) OJ No L 215, 30. 7. 1992, p. 64.
(3) OJ No L 298, 12. 11. 1985, p. 9.
(4) OJ No L 135, 19. 5. 1992, p. 5.
(5) OJ No L 55, 1. 3. 1988, p. 31.
(6) OJ No L 14, 21. 1. 1992, p. 28.
(7) OJ No L 45, 21. 2. 1990, p. 8.
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Article 1
▼M1
The reference method of analysis to determine the stigmasterol contentof butteroil according to Article 6 of Regulation (EEC) No 3143/85,Article 6 of Regulation (EEC) No 570/88 or Article 11 of Regulation(EEC) No 429/90 and the β-sitosterol content of butteroil according to
Article 6 of Regulation (EEC) No 570/88, is specified in the Annex.
▼
B
Butteroil has been traced correctly if the results obtained are in accord-ance 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 February 1993.
ThisRegulationshallbebindinginitsentiretyanddirectlyapplicablein
all Member States.
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ANNEX
DETERMINATION OF SITOSTEROL OR STIGMASTEROL IN
BUTTEROIL BY CAPILLARY-COLUMN GAS CHROMATOGRAPHY
1. SCOPE AND FIELD OF APPLICATION
The method describes a procedure for the quantitative determination of
sitosterolorstigmasterolinbutteroil.SitosterolistakentobethesumofB-sitosterol and 22 dihydro B sitosterol, other sitosterols are assumed tobe insignificant. It is applicable to samples received under Regulations(EEC) No 3143/85, (EEC) No 570/88 and (EEC) No 429/90.
2. PRINCIPLE
The butteroil is saponified with potassium hydroxide in ethanolic solu-
tion 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 150mlsaponificationflaskfittedwitharefluxcondenserhavingground-
glass joints.
3.2 500ml separating funnels.3.3 250ml flasks.3.4 Pressure euqalising funnels, 250ml or similar, to collect waste diethyl
ether.
3.5 Glass column, 350mm×20mm fitted with sintered glass plug.3.6 Waterbath or isomantle.3.7 Reaction vials, 2ml.3.8 Gas chromatograph suitable for use with a capillary column, provided
with a splitting system consisting of:
3.8.1 a thermostatic chamber for columns capable of maintaining the desired
temperature with an accuracy of ±1 °C;
3.8.2 a temperature-adjustable vaporisation 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 Afused-silicacapillarycolumnentirelycoatedwithBP1orequivalentin
a uniform thickness 0,25 µm; the column must be capable of resolving
trimethyl silyl derivatives of lanosterol and sitosterol, a BP1, length12m, internal diameter 0,2mm is suitable.
3.10 A 1 µl gas chromatography microsyringe with hardened needle.
4. REAGENTS
Allreagentsmustbeofrecognizedanalyticalgrade.Thewaterusedmust
be distilled water or water of at least equivalent purity.
4.1 Ethanol, at least 95% pure.4.2 Potassiumhydroxide,60%solution,dissolve600gpotassiumhydroxide
(minimum 85%) in water and make up to one litre with water.
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 1,0mg/ml;
4.3.1.2 Theconcentrationofbetulinsolutionusedforstigmasteroldetermination
should be 0,4mg/ml.
4.4 Diethyl ether, analytical purity (free from peroxides or residue).4.5 Sodium sulphate, anhydrous, granular previously dried at 2 °Cfor two
hours.
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4.6 Silylatingreagent,forexampleTRI-SIL(availablefromPierceChemical
Co, Cat No 49001) or equivalent. (WARNING: TRI-SIL is flammabletoxic,corrosiveandapossiblecarcinogen.Laboratorypersonnelmustbefamiliar with TRI-SIL safety data and take the appropriate precautions.)
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 using the method of normalisation. Assume that all sterolspresentinthesamplearerepresentedonthechromatogramthetotalareaof 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 0,001mg/ml, approximately 0,5mg/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 Stigmasterl standard solution — prepare a solution containing, to the
nearest 0,001mg/m, approximately 0,2mg/ml (W
1) stigmasterol (4.9) in
diethyl ether (4.4).
4.10 Resolutiontestmixture.Prepareasolutioncontaining0,05mg/mllanos-
terol (4.7) and 0,5mg/ml sitosterol (4.8) in diethyl ether (4.4).
5. PROCEDURE
5.1 Preparation of standard solutions for chromatography. The internal
standardsolution(4.3.1)mustbeaddedtotheappropriatesterolstandardsolution at the same time as it is added to the saponified sample (see5.2.2).
5.1.1 Sitosterol standard chromatographic solution; transfer 1ml of sitosterol
standard solution (4.8.1) to each of two reaction vials (3.7) and removethediethyletherwithastreamofnitrogen.Add1mlofinternalstandardsolution(4.3.1.1)andremovethediethyletherwithastreamofnitrogen.
5.1.2 Stigmasterol standard chromatographic solution; transfer 1ml of stig-
maasterolstandardsolution(4.9.1)toeachoftworeactionvials(3.7)andremove the diethyl ether with a stream of nitrogen. Add 1ml of internalstandardsolution(4.3.1.2)andremovethediethyletherwithastreamofnitrogen.
5.2 Preparation of the unsaponifiables.5.2.1 Weigh, to the nearest 1mg, approximately 1g of butteroil (W
2) into a
150ml flask (3.1). Add 50ml ethanol (4.1) and 10ml potassiumhydroxide solution (4.2). Fit the reflux condenser and heat at approxi-mately75 °Cfor30minutes.Detachthecondenserandcooltheflaskto
approximately ambient temperature.
5.2.2 Add1,0mlofinternalstandardsolution(4.3.1.1)totheflaskifsitosterol
is to be determined, or (4.3.1.2) if stigmasterol is to be determined. Mixthoroughly.Transferthecontentsoftheflaskquantitativelyintoa500mlseparating funnel (3.2) washing the flask in turn with 50ml water and250ml diethyl ether (4.4). Shake the separating funnel vigorously fortwo minutes and alow the phases to separate. Run of the lower aqueouslayer and wash the ether layer by shaking with four successive 100mlaliquots 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 washcanbeshakenvigorouslyfor30seconds.Ifanemulsionisformeditcanbedestroyedbytheadditionof5-10mlofethanol.Ifethanolisaddeditis 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)
containing 30g anhydrous sodium sulphate (4.5). Collect the ether in a250ml flask (3.3). Add one anti-bumping granual and evaporate to neardrynessanawaterorisomantle,takingcaretocollectthewastesolvents.
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 remaining in the flask to a 2ml reaction vial
(3.7) with 2ml diethyl ether and remove the ether using a stream of
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nitrogen. Wash the flask with two further 2ml aliquots of diethyl ether,
transferring to the vial and removing the ether with nitrogen each time.
5.3.2 Silylatethesamplebyadditionof1mlTRI-SIL(4.6).Closethevialand
shake vigorously to dissolve. If dissolution is five incomplete, warm to65-70 °C. Allow to stand for at least five minutes before injecting into
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 washing at the extraction stage. Ifthisproblempersistsafifthwash,shakenvigorouslyfor30seconds,maybe introduced at the extraction stage.
5.4 Gas-Chromatographic analysis.5.4.1 Choice of operating conditions.
Set up the gas-chromatograph according to the manufacturers instruc-
tions.
The guideline operating conditions are as follows:
—columntemperature 265 °C
—injectortemperature 280 °C
—detectortemperature 300 °C
—carriergasflowrate 0,6ml/minute—hydrogenpressure 84kPa—airpressure 155kPa—samplesplit10:1to50:1,thesplitratiomustbeoptimisedin
accordancewiththemanufacturersinstructionsandlinearityofdetectorresponsethenvalidatedovertheconcentrationrangeofinterest.
Note 5:It is especially important that the injection liner is regularly
cleared.
— amount of substance injected 1 µl of TMSE solution.
Allowthesystemtoequilibrateandobtainasatisfactorystableresponse
before commencing any analysis.
These conditions may be varied in the light of column and gas-chroma-
tograph characteristics so as to obtain chromatograms which meet thefollowing requirements:
— 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),
— therelativeretentiontimesofthefollowingsterolsshouldbeapprox-
imately:
Cholesterol 1,0
Stigmasterol 1,3Sitosterol 1,5Betulin 2,5,
— the retention time for betulin should be approximately 24minutes.
5.4.2 Analytical procedure.
Inject 1 µl of silylated standard 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 meaure peak areas. Each
chromatographicrunmustebracketedbyaninjectionofstandards.Asaguide,sixinjectionsofsampleshouldbeincludedineachbracketedrun.
Note 6:Integrationofthestigmasterolpeakshouldincludeanytailingas
defined by points 1, 2 and 3 in Figure 2b.
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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 bracketing a 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 1ml 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
withintheshortestfeasibletimeinterval,byoneoperatorusingthesameapparatus on identical test material shall not exceed 10,2mg/kg.
7.1.2 Sitosterol
The difference between the results of two determinations carried out
withintheshortestfeasibletimeinterval,byoneoperatorusingthesameapparatusonidenticaltestmaterialshallnotexceed3,6%relativeofthemean of the determinations.
7.2 Reproducibility.7.2.1 Stigmasterol.
The difference between the results of two determinations carried out by
operators in different laboratories, using different apparatus on identicaltest material shall not exceed 25,3mg/kg.
7.2.2 Sitosterol.
The difference between the results of two determinations carried out by
operators in different laboratories, using different apparatus on identicaltest material shall not exceed 8,9% relative of the mean of the determi-nations.
7.3 Source of precision data.
The precision data were determined from an experiment conducted in
1991involvingninelaboratoriesandsixsamples(threeblindduplicates)for stigmasterol; six samples (three blind duplicates) for sitosterol.
8. TOLERANCE LIMITS
8.1 Three samples must be taken from the traced product in order to check
on homogeneity.
8.2 Stigmasterol8.2.1 Theincorporationrateforstigmasterolis150gramsofatleast95%pure
stigmasterol per tonne of butteroil, ie 142,5mg/kg; or 170grams of atleast 85% pure stigmasterol per tonne of butteroil, ie 144,5mg/kg.
8.2.2 Taking the Critical Difference for a 95% probability level (CrD
95) into
consideration, the mean of the three results shall not be less than
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— in the case of incorporation of 95% pure stigmasterol, 128,3mg/kg,
— in the case of incorporation of 85% pure stigmasterol, 130,3mg/kg.
8.2.3 In addition to the criteria given in 8.2.2 the lowest result obtained from
analysis of the product is used to check homogeneity of tracer distribu-tion. This is done by a comparison with the following limits:
— 120,4mg/kg(95%oftheminimumincorporationratefor95%pure
stigmasterol, single sample CrD
95taken into consideration)
— 122,3mg/kg(95%oftheminimumincorporationratefor85%pure
stigmasterol, single sample CrD95taken into consideration)
— 99,0mg/kg (80% of the minimum incorporation rate for 95% pure
stigmasterol, single sample CrD95taken into consideration)
— 100,6mg/kg(80%oftheminimumincorporationratefor85%pure
stigmasterol, single sample CrD95taken into consideration).
Thetracerconcentrationinthesamplegivingthelowestresultisusedin
confunction with interpolation respectivels between 120,4mg/kg and99,0mg/kg or 122,3mg/kg and 100,6mg/kg.
8.3 Sitosterol8.3.1 The incorporation rate for sitosterol is 600grams of at least 90% pure
sitosterol per tone of butteroil, ie 540mg/kg.
8.3.2 Taking the CrD
95into consideration the mean of the three results shall
not be less than 513,0mg/kg.
8.3.3 Inadditiontothecriteriongivenin8.3.2thelowestresultobtainedfrom
analysis of the product is used to check homogeneity of tracer distribu-tion. This is done by comparison with the following limits:
— 484,4mg/kg(95%oftheminimumincorporationratefor90%pure
sitosterol, single sample CrD
95taken into consideration)
— 403,4mg/kg(80%oftheminimumincorporationratefor90%pure
sitosterol, single sample CrD95taken into consideration).
Thetracerconcentrationinthesamplegivingthelowestresultisusedin
conjunction with interpolation between 484,4mg/kg and 403,4mg/kg.
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Figure 1 Chromatogram of resolution test mixture.
Complete resolution is preferable, ie the peak trace for lanosterol
shouldreturntobaselinebeforeleavingforthesitosterolpeakalthoughincomplete resolution is tolerable.

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Figure 2a
Stigmasterol standard

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Figure 2b
Butteroil sample denatured with stigmasterol
Note: Integration of the stigmasterol peak should include any tailing as defined
by points 1, 2 and 3.
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Figure 3a
Sitosterol standard

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Figure 3b
Butteroil sample denatured with B-sitosterol
Note: B-sitosterol often contains an impurity (identified as stigmastanol) which
elutes immediately after B-sitosterol. The areas of these two peaks should besummed when evaluating the total B-sitosterol present.