Commission Regulation (EC) No 273/2008 (repealed)Show full title

DETERMINING SITOSTEROL OR STIGMASTEROL IN BUTTER OR CONCENTRATED BUTTER BY CAPILLARY-COLUMN GAS CHROMATOGRAPHY

1. SCOPE AND FIELD OF APPLICATION

The method describes a procedure for the quantitative determination of sitosterol or stigmasterol in butter and concentrated butter. Sitosterol is taken to be the sum of β-sitosterol and 22 dihydro-β-sitosterol, other sitosterols are assumed to be insignificant.

2. PRINCIPLE

The butter or concentrated 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 internal standard/betulin.

3. APPARATUS

3.1. 150 ml saponification flask fitted with a reflux condenser having ground-glass joints

3.2. 500 ml separating funnels

3.3. 250 ml flasks

3.4. Pressure equalising funnels, 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 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 ionisation 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 (or any other column of at least equal resolution) in a uniform thickness 0,25 μm; the column must be capable of resolving trimethyl-silyl derivatives of lanosterol and sitosterol. A column, 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 recognised 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 g potassium hydroxide (minimum 85 %) in water and make up to one litre with water

4.3.

Betulin of at least 99 % purity

4.3.1.

Solutions of 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

4.3.1.2. The concentration of betulin solution used for stigmasterol determination 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. Silylating reagent, for example TRI-SIL (available from Pierce Chemical Co, Cat No 49001) or equivalent (Important: TRI-SIL is inflammable, toxic, corrosive and possibly carcinogenic. Laboratory personnel must be familiar 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 sterols present in the sample are represented on the chromatogram, the total area of the peaks represents 100 % of the sterol constituents and that the sterols give the same detector response. Linearity of the system must be validated 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 (W₁) 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 (W₁) stigmasterol (4.9) in diethyl ether (4.4)

4.10. Resolution test mixture. Prepare a solution containing 0,05 mg/ml lanosterol (4.7) and 0,5 mg/ml sitosterol (4.8) in diethyl ether (4.4).

5. METHOD

5.1. Preparation of standard solutions for chromatography

The internal standard solution (4.3.1) must be added to the appropriate sterol standard 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 remove the diethyl ether with a stream of nitrogen. Add 1 ml of internal solution (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 stigmasterol standard solution (4.9.1) to each of two reaction vials (3.7) and remove the diethyl ether with a stream of nitrogen. Add 1 ml of internal standard solution (4.3.1.2) and remove the diethyl ether with a stream of nitrogen

5.2. Preparation of the unsaponifiables

5.2.1. Melt the butter sample at a temperature not exceeding 35 °C, mix the sample thoroughly by stirring

Weigh, to the nearest 1 mg, approximately 1 g of butter (W₂) or concentrated butter (W₂) 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 and heat 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 sitosterol 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 a 500 ml separating funnel (3.2), washing the flask in turn with 50 ml water and 250 ml diethyl ether (4.4). Shake the separating funnel vigorously for two minutes and allow the phases to separate. Run off the lower aqueous layer and wash the ether layer by shaking with 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 wash can be shaken vigorously for 30 seconds. If an emulsion is formed it can be destroyed by the addition of 5-10 ml of 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) containing 30 g anhydrous sodium sulphate (4.5). Collect the ether in a 250 ml flask (3.3). Add one anti-bumping granule and evaporate to near dryness in a waterbath or isomantle, taking care to collect the waste solvents.

Note 3: If sample extracts are taken to complete dryness at too high 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 2 ml reaction vial (3.7) with 2 ml diethyl ether and remove the ether using a stream of nitrogen. Wash the flask with two further 2 ml aliquots of diethyl ether, transferring to the vial and removing the ether with nitrogen 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 to 65-70 °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 effected in a water-free environment. Incomplete silylation of betulin is indicated by a second peak close to that of betulin.

The presence of ethanol at the silylation stage will interfere with silylation. This may result from inadequate washing at the extraction stage. If this problem persists, a fifth wash may be introduced at the extraction stage, shaking vigorously for 30 seconds.

5.4. Gas-chromatographic analysis

5.4.1. Choice of operating conditions

Set up the gas-chromatograph according to the manufacturer’s instructions.

The guideline operating conditions are as follows:

• column temperature: 265 °C

• injector temperature: 265 °C

• detector temperature: 300 °C

• carrier gas flow rate: 0,6 ml/min.

• hydrogen pressure: 84 kPa

• air pressure: 155 kPa

• sample split: 10:1 to 50:1; the split ratio must be optimised in accordance with the manufacturer’s instructions and linearity of detector response, then validated over the concentration range of interest.

  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 commencing any analysis.

These conditions must be varied in the light of column and gas-chromatograph characteristics so as to obtain chromatograms which meet the following requirements:

• the sitosterol peak must be adequately resolved from lanosterol. Figure 1 shows a typical chromatogram which should be obtained from a silylated resolution test mixture (4.10),

• the relative retention times of the following sterols should be approximately:

  • cholesterol: 1,0

  • stigmasterol: 1,3

  • sitosterol: 1,5

  • betulin: 2,5

• the retention time for betulin should be approximately 24 minutes.

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 measure peak areas. Each chromatographic run must be bracketed by an injection of standards.

As a guide, six injections of sample should be included in each bracketed run.

Note 6: Integration of the stigmasterol peak should include any tailing as defined by points 1, 2 and 3 in Figure 2b.

Integration of the sitosterol peak should include the area of the 22 dihydro-β-sitosterol (stigmastanol) peak which elutes immediately after sitosterol (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 standards bracketing a batch, and calculate R₁:

R₁ = (average area of the sterol peak in the standard)/(average area of the betulin peak in the standard)

Determine the area of the sterol peak (stigmasterol and sitosterol) and betulin peak in the sample and calculate R₂:

R₂ = (area of the sterol peak in the sample)/(area of the betulin peak in the 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) = ((R ₂) / (R ₁)) × ((W ₁) / (W ₂)) × P × 10.

7. ACCURACY OF THE METHOD

7.1. Butter

7.1.1. Repeatability

7.1.1.1. Stigmasterol

The difference between the results of two determinations carried out within the shortest feasible time interval, by one operator using the same apparatus on identical test material, may not exceed 19,3 mg/kg.

7.1.1.2. Sitosterol

The difference between the results of two determinations carried out within the shortest feasible time interval, by one operator using the same apparatus on identical test material, may not exceed 23,0 mg/kg.

7.1.2. Reproducibility

7.1.2.1. Stigmasterol

The difference between the results of two determinations carried out by operators in different laboratories, using different apparatus on identical test material, may not exceed 31,9 mg/kg.

7.1.2.2. Sitosterol

The difference between the results of two determinations carried out by operators in different laboratories, using different apparatus on identical test material, may not exceed 8,7 % relative to the mean of the determination.

7.1.3. Source of precision data

The precision data were determined from an experiment conducted in 1992 involving eight laboratories and six samples (three blind duplicates) for stigmasterol and six samples (three blind duplicates) for sitosterol.

7.2. Concentrated butter

7.2.1. Repeatability

7.2.1.1. Stigmasterol

The difference between the results of two determinations carried out within the shortest feasible time interval, by one operator using the same apparatus on identical test material, may not exceed 10,2 mg/kg.

7.2.1.2. Sitosterol

The difference between the results of two determinations carried out within the shortest feasible time interval, by one operator using the same apparatus on identical test material, may not exceed 3,6 % relative to the mean of the determinations.

7.2.2. Reproducibility

7.2.2.1. Stigmasterol

The difference between the results of two determinations carried out by operators in different laboratories, using different apparatus on identical test material, may not exceed 25,3 mg/kg.

7.2.2.2. Sitosterol

The difference between the results of two determinations carried out by operators in different laboratories, using different apparatus on identical test material, may not exceed 8,9 % relative to the mean of the determinations.

7.2.3. Source of precision data

The precision data were determined from an experiment conducted in 1991 involving nine laboratories and six samples (three blind duplicates) for stigmasterol and 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 the correct tracing of the product.

8.2. Butter

8.2.1. Stigmasterol

8.2.1.1. The incorporation rate for stigmasterol is 150 g of at least 95 % pure stigmasterol per tonne of butter, i.e. 142,5 mg/kg, or 170 g of at least 85 % pure stigmasterol per tonne of butter, i.e. 144,5 mg/kg.

8.2.1.2. The results for the three samples obtained from the analysis of the product are used to check the rate and the homogeneity of tracer incorporation and the lowest of these results is compared with the following limits:

• 115,8 mg/kg (95 % of the minimum incorporation rate for 95 % pure stigmasterol),

• 117,7 mg/kg (95 % of the minimum incorporation rate for 85 % pure stigmasterol),

• 80,1 mg/kg (70 % of the minimum incorporation rate for 95 % pure stigmasterol),

• 81,5 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 115,8 mg/kg and 80,1 mg/kg or 117,7 mg/kg and 81,5 mg/kg.

8.2.2. Sitosterol

8.2.2.1. The incorporation rate for sitosterol is 600 g of at least 90 % pure sitosterol per tonne of butter, i.e. 540 mg/kg

8.2.2.2. The results for the three samples obtained from the analysis of the product are used to check the rate and the homogeneity of tracer incorporation and the lowest of these results is compared with the following limits:

• 482,6 mg/kg (95 % of the minimum incorporation rate for 90 % pure sitosterol),

• 347,6 mg/kg (70 % of the minimum incorporation rate for 90 % pure sitosterol).

The tracer concentration of the sample giving the lowest result is used in conjunction with interpolation between 482,6 mg/kg and 347,6 mg/kg.

8.3. Concentrated butter

8.3.1. Stigmasterol

8.3.1.1. The incorporation rate for stigmasterol is 150 g of at least 95 % pure stigmasterol per tonne of concentrated butter, i.e. 142,5 mg/kg; or 170 g of at least 85 % pure stigmasterol per tonne of concentrated butter, i.e. 144,5 mg/kg

8.3.1.2. The results for the three samples obtained from the analysis of the product are used to check the rate and the homogeneity of tracer incorporation and the lowest of these results is compared with the following limits:

• 118,5 mg/kg (95 % of the minimum incorporation rate for 95 % pure stigmasterol),

• 120,4 mg/kg (95 % of the minimum incorporation rate for 85 % pure stigmasterol),

• 82,9 mg/kg (70 % of the minimum incorporation rate for 95 % pure stigmasterol),

• 84,3 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 118,5 mg/kg and 82,9 mg/kg or 120,4 mg/kg and 84,3 mg/kg.

8.3.2. Sitosterol

8.3.2.1. The incorporation rate for sitosterol is 600 g of at least 90 % pure sitosterol per tonne of concentrated butter, i.e. 540 mg/kg

8.3.2.2. The results for the three samples obtained from the analysis of the product are used to check the rate and the homogeneity of tracer incorporation and the lowest of these results is compared with the following limits:

• 480,9 mg/kg (95 % of the minimum incorporation rate for 90 % pure sitosterol),

• 345,9 mg/kg (70 % of the minimum incorporation rate for 90 % pure sitosterol).

The tracer concentration in the sample giving the lowest result is used in conjunction with interpolation between 480,9 mg/kg and 345,9 mg/kg.

Figure 1

Chromatogram of resolution test mixture

Complete resolution is preferable, i.e. the peak trace for lanosterol should return to baseline before leaving for the sitosterol peak although incomplete resolution is tolerable.

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

Note: β-sitosterol often contains an impurity (identified as stigmastanol) which elutes immediately after β-sitosterol. The areas of these two peaks should be summed when evaluating the total β-sitosterol present.
Figure 3a	Figure 3b
Sitosterol standard	Butter sample denatured with β-Sitosterol