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Commission Regulation (EU) No 61/2011 of 24 January 2011 amending Regulation (EEC) No 2568/91 on the characteristics of olive oil and olive-residue oil and on the relevant methods of analysis
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a Total isomers which could (or could not) be separated by capillary column. | ||||||||||||
b Or where the median defect is less than or equal to 3,5 and the fruity median is equal to 0. | ||||||||||||
c Oils with a wax content of between 300 mg/kg and 350 mg/kg are considered to be lampante olive oil if the total aliphatic alcohol content is less than or equal to 350 mg/kg or if the erythrodiol and uvaol content is less than or equal to 3,5 %. | ||||||||||||
d Oils with a wax content of between 300 mg/kg and 350 mg/kg are considered to be crude olive-residue oil if the total aliphatic alcohol content is above 350 mg/kg and if the erythrodiol and uvaol content is greater than 3,5 %. | ||||||||||||
Category | Fatty acid methyl esters (FAMEs) and fatty acid ethyl esters (FAEEs) | Acidity(%)(*) | Peroxide indexmEq O2/kg(*) | Waxesmg/kg(**) | 2-glyceril monopalmitate(%) | Stigmastadienemg/kga | Difference: ECN42 (HPLC) and ECN42(theoretical calculation) | K232 (*) | K270 (*) | Delta-K (*) | Organoleptic evaluationMedian defect (Md) (*) | Organoleptic evaluationFruity median (Mf) (*) |
---|---|---|---|---|---|---|---|---|---|---|---|---|
1.Extra virgin olive oil | Σ FAME + FAEE ≤ 75 mg/kg or 75 mg/kg <Σ FAME + FAEE ≤ 150 mg/kg and (FAEE/FAME) ≤ 1,5 | ≤ 0,8 | ≤ 20 | ≤ 250 | ≤ 0,9 if total palmitic acid % ≤ 14 % | ≤ 0,10 | ≤ 0,2 | ≤ 2,50 | ≤ 0,22 | ≤ 0,01 | Md = 0 | Mf > 0 |
≤ 1,0 if total palmitic acid % > 14 % | ||||||||||||
2.Virgin olive oil | — | ≤ 2,0 | ≤ 20 | ≤ 250 | ≤ 0,9 if total palmitic acid % ≤ 14 % | ≤ 0,10 | ≤ 0,2 | ≤ 2,60 | ≤ 0,25 | ≤ 0,01 | Md ≤ 3,5 | Mf > 0 |
≤ 1,0 if total palmitic acid % > 14 % | ||||||||||||
3.Lampante olive oil | — | > 2,0 | — | ≤ 300c | ≤ 0,9 if total palmitic acid % ≤ 14 % | ≤ 0,50 | ≤ 0,3 | — | — | — | Md > 3,5b | — |
≤ 1,1 if total palmitic acid % > 14 % | ||||||||||||
4.Refined olive oil | — | ≤ 0,3 | ≤ 5 | ≤ 350 | ≤ 0,9 if total palmitic acid % ≤ 14 % | — | ≤ 0,3 | — | ≤ 1,10 | ≤ 0,16 | — | — |
≤ 1,1 if total palmitic acid % > 14 % | ||||||||||||
5.Olive oil composed of refined and virgin olive oils | — | ≤ 1,0 | ≤ 15 | ≤ 350 | ≤ 0,9 if total palmitic acid % ≤ 14 % | — | ≤ 0,3 | — | ≤ 0,90 | ≤ 0,15 | — | — |
≤ 1,0 if total palmitic acid % > 14 % | ||||||||||||
6.Crude olive-residue oil | — | — | — | > 350d | ≤ 1,4 | — | ≤ 0,6 | — | — | — | — | — |
7.Refined olive-residue oil | — | ≤ 0,3 | ≤ 5 | > 350 | ≤ 1,4 | — | ≤ 0,5 | — | ≤ 2,00 | ≤ 0,20 | — | — |
8.Olive-residue oil | — | ≤ 1,0 | ≤ 15 | > 350 | ≤ 1,2 | — | ≤ 0,5 | — | ≤ 1,70 | ≤ 0,18 | — | — |
a Other fatty acids content (%): palmitic: 7,5-20,0; palmitoleic: 0,3-3,5; heptadecanoic: ≤ 0,3; heptadecenoic: ≤ 0,3; stearic: 0,5-5,0; oleic: 55,0-83,0; linoleic: 3,5-21,0. | ||||||||||||||||
b Total: Delta-5,23-stigmastadienol+chlerosterol+beta-sitosterol+sitostanol+delta-5-avenasterol+delta-5,24-stigmastadienol. | ||||||||||||||||
c Oils with a wax content of between 300 mg/kg and 350 mg/kg are considered to be lampante olive oil if the total aliphatic alcohol content is less than or equal to 350 mg/kg or if the erythrodiol and uvaol content is less than or equal to 3,5 %. | ||||||||||||||||
d Oils with a wax content of between 300 mg/kg and 350 mg/kg are considered to be crude olive-residue oil if the total aliphatic alcohol content is above 350 mg/kg and if the erythrodiol and uvaol content is greater than 3,5 %. | ||||||||||||||||
Category | Acid contenta | Total transoleic isomers(%) | Total translinoleic + translinolenic isomers(%) | Sterols composition | Total sterols(mg/kg) | Erythrodiol and uvaol(%) (**) | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Myristic(%) | Linolenic(%) | Arachidic(%) | Eicosenoic(%) | Behenic(%) | Lignoceric(%) | Cholesterol(%) | Brassicasterol(%) | Campesterol(%) | Stigmasterol(%) | Betasitosterol(%)b | Delta-7-stigmastenol(%) | |||||
1.Extra virgin olive oil | ≤ 0,05 | ≤ 1,0 | ≤ 0,6 | ≤ 0,4 | ≤ 0,2 | ≤ 0,2 | ≤ 0,05 | ≤ 0,05 | ≤ 0,5 | ≤ 0,1 | ≤ 4,0 | < Camp. | ≥ 93,0 | ≤ 0,5 | ≥ 1 000 | ≤ 4,5 |
2.Virgin olive oil | ≤ 0,05 | ≤ 1,0 | ≤ 0,6 | ≤ 0,4 | ≤ 0,2 | ≤ 0,2 | ≤ 0,05 | ≤ 0,05 | ≤ 0,5 | ≤ 0,1 | ≤ 4,0 | < Camp. | ≥ 93,0 | ≤ 0,5 | ≥ 1 000 | ≤ 4,5 |
3.Lampante olive oil | ≤ 0,05 | ≤ 1,0 | ≤ 0,6 | ≤ 0,4 | ≤ 0,2 | ≤ 0,2 | ≤ 0,10 | ≤ 0,10 | ≤ 0,5 | ≤ 0,1 | ≤ 4,0 | — | ≥ 93,0 | ≤ 0,5 | ≥ 1 000 | ≤ 4,5c |
4.Refined olive oil | ≤ 0,05 | ≤ 1,0 | ≤ 0,6 | ≤ 0,4 | ≤ 0,2 | ≤ 0,2 | ≤ 0,20 | ≤ 0,30 | ≤ 0,5 | ≤ 0,1 | ≤ 4,0 | < Camp. | ≥ 93,0 | ≤ 0,5 | ≥ 1 000 | ≤ 4,5 |
5.Olive oil composed of refined and virgin olive oils | ≤ 0,05 | ≤ 1,0 | ≤ 0,6 | ≤ 0,4 | ≤ 0,2 | ≤ 0,2 | ≤ 0,20 | ≤ 0,30 | ≤ 0,5 | ≤ 0,1 | ≤ 4,0 | < Camp. | ≥ 93,0 | ≤ 0,5 | ≥ 1 000 | ≤ 4,5 |
6.Crude olive-residue oil | ≤ 0,05 | ≤ 1,0 | ≤ 0,6 | ≤ 0,4 | ≤ 0,3 | ≤ 0,2 | ≤ 0,20 | ≤ 0,10 | ≤ 0,5 | ≤ 0,2 | ≤ 4,0 | — | ≥ 93,0 | ≤ 0,5 | ≥ 2 500 | > 4,5d |
7.Refined olive-residue oil | ≤ 0,05 | ≤ 1,0 | ≤ 0,6 | ≤ 0,4 | ≤ 0,3 | ≤ 0,2 | ≤ 0,40 | ≤ 0,35 | ≤ 0,5 | ≤ 0,2 | ≤ 4,0 | < Camp. | ≥ 93,0 | ≤ 0,5 | ≥ 1 800 | > 4,5 |
8.Olive-residue oil | ≤ 0,05 | ≤ 1,0 | ≤ 0,6 | ≤ 0,4 | ≤ 0,3 | ≤ 0,2 | ≤ 0,40 | ≤ 0,35 | ≤ 0,5 | ≤ 0,2 | ≤ 4,0 | < Camp. | ≥ 93,0 | ≤ 0,5 | ≥ 1 600 | > 4,5 |
The last digit must be increased by one unit if the following digit is greater than 4.
for lampante olive oil, it is possible for both the relevant limits to be different from the stated values at the same time,
for virgin olive oils, if at least one of these limits is different from the stated values, the category of the oil will be changed, although they will still be classified in one of the categories of virgin olive oil.
This method is for the determination of the content of waxes, fatty acid methyl and ethyl esters in olive oils. The individual waxes and alkyl esters are separated according to the number of carbon atoms. The method is recommended as a tool for distinguishing between olive oil and olive-pomace oil and as a quality parameter for extra virgin olive oils enabling the detection of fraudulent mixtures of extra virgin olive oils with lower quality oils whether they are virgin, lampante or some deodorised oils.
Addition of suitable internal standards to the oil and fractionation by chromatography on a hydrated silica gel column. Recovery of the fraction eluted under the test conditions (with a lower polarity than that of the triacylglycerols) and direct analysis by capillary gas chromatography.
WARNING – Fumes may ignite. Keep away from sources of heat, sparks or naked flames. Make sure the bottles are always properly closed. Ensure proper ventilation during usage. Avoid build-up of fumes and remove any possible fire risk, such as heaters or electric apparatus not manufactured from non-inflammable material. Pernicious if inhaled, because it may cause nerve cell damage. Avoid breathing in the fumes. Use a suitable respiratory apparatus if necessary. Avoid contact with eyes and skin.
WARNING – Highly inflammable and moderately toxic. Irritates the skin. Pernicious if inhaled. May cause damage to eyes. Effects may be delayed. It can form explosive peroxides. Fumes may ignite. Keep away from sources of heat, sparks or naked flames. Make sure the bottles are always properly closed. Ensure proper ventilation during usage. Avoid build-up of fumes and remove any possible fire risk, such as heaters or electric apparatus not manufactured from non-inflammable material. Do not evaporate to dryness or near dryness. The addition of water or an appropriate reducing agent can reduce peroxide formation. Do not drink. Avoid breathing in the fumes. Avoid prolonged or repeated contact with skin.
WARNING – Inflammable. Pernicious if inhaled. Keep away from sources of heat, sparks or naked flames. Make sure the bottles are always properly closed. Ensure proper ventilation during usage. Avoid breathing in the fumes. Avoid prolonged or repeated contact with skin.
Hydrogen. Highly inflammable, under pressure. Keep away from sources of heat, sparks, naked flames or electric apparatus not manufactured from non-inflammable material. Make sure the bottle valve is shut when not in use. Always use with a pressure reducer. Release the tension of the reducer spring before opening the bottle valve. Do not stand in front of the bottle outlet when opening the valve. Ensure proper ventilation during usage. Do not transfer hydrogen from one bottle to another. Do not mix gas in the bottle. Make sure the bottles cannot be knocked over. Keep them away from sunlight and sources of heat. Store in a corrosive-free environment. Do not use damaged or unlabelled bottles.
Helium. Compressed gas at high pressure. It reduces the amount of oxygen available for breathing. Keep the bottle shut. Ensure proper ventilation during usage. Do not enter storage areas unless they are properly ventilated. Always use with a pressure reducer. Release the tension of the reducer spring before opening the bottle valve. Do not transfer gas from one bottle to another. Make sure the bottles cannot be knocked over. Do not stand in front of the bottle outlet when opening the valve. Keep them away from sunlight and sources of heat. Store in a corrosive-free environment. Do not use damaged or unlabelled bottles. Do not inhale. Use solely for technical purposes.
Hydrogen, pure, gas chromatography grade.
Air, pure, gas chromatography grade.
Air. Compressed gas at high pressure. Use with caution in the presence of combustible substances as the self-ignition temperature of most of the organic compounds in the air is considerably lower under high pressure. Make sure the bottle valve is shut when not in use. Always use a pressure reducer. Release the tension of the reducer spring before opening the bottle valve. Do not stand in front of the bottle outlet when opening the valve. Do not transfer gas from one bottle to another. Do not mix gas in the bottle. Make sure the bottles cannot be knocked over. Keep them away from sunlight and sources of heat. Store in a corrosive-free environment. Do not use damaged or unlabelled bottles. Air intended for technical purposes must not be used for inhaling or respiratory apparatus.
Suspend 15 g of silica gel (point 4.1) in n-hexane (point 4.2) and introduce into the column (point 3.2). Allow to settle spontaneously. Complete settling with the aid of an electric shaker to make the chromatographic bed more homogeneous. Percolate 30 ml of n-hexane to remove any impurities. Weigh exactly about 500 mg of the sample into the 25-ml flask (point 3.1), using the analytical balance (point 3.8), and add a suitable amount of internal standard (point 4.5), depending on the assumed wax content, e.g. add 0,1 mg of lauryl arachidate in the case of olive oil, 0,25-0,50 mg in the case of olive-pomace oil and 0,05 mg of methyl heptadecanoate for olive oils (point 4.6).
Transfer the prepared sample to the chromatography column with the aid of two 2-ml portions of n-hexane (point 4.2).
Allow the solvent to flow to 1 mm above the upper level of the absorbent. Percolate a further of n-hexane/ethyl ether (99:1) and collect 220 ml at a flow of about 15 drops every 10 seconds. (This fraction contains the methyl and ethyl esters and waxes). (Note 4) (Note 5).
The retention time of the dye lies in between that of the waxes and triacylglycerols. Hence, when the dye reaches the bottom of the chromatography column, elution has to be suspended because all the waxes have been eluted.
Evaporate the resultant fractions in a rotary evaporator until the solvent is almost removed. Remove the last 2 ml under a weak current of nitrogen. Collect the fraction containing the methyl and ethyl esters is diluted with 2-4 ml of n-heptane or iso-octane.
Fit the column to the gas chromatograph (point 3.3), connecting the inlet port to the on-column system and the outlet port to the detector. Check the gas chromatography apparatus (operation of gas loops, efficiency of detector and recorder system, etc.).
If the column is being used for the first time, it is advisable to condition it. Run a light flow of gas through the column, then switch on the gas chromatography apparatus. Gradually heat until a temperature of 350 °C is reached after approximately 4 h.
Maintain this temperature for at least 2 h, then regulate the apparatus to the operating conditions (regulate gas flow, light flame, connect to electronic recorder (point 3.3.4), regulate oven temperature for column, regulate detector, etc.). Record the signal at a sensitivity at least twice as high as that required for the analysis. The base line should be linear, with no peaks of any kind, and must not have any drift.
Negative straight-line drift indicates that the column connections are not correct while positive drift indicates that the column has not been properly conditioned.
The operating conditions are generally as follows:
:
350 °C.
:
1 μl of n-heptane solution (2-4 ml).
:
helium or hydrogen at the optimal linear speed for the gas chosen (see Appendix A).
:
suitable for fulfilling the above conditions.
These conditions may be modified to suit the characteristics of the column and the gas chromatograph in order to separate all the waxes and fatty acid methyl and ethyl esters and to obtain satisfactory peak separation (see Figures 2, 3 and 4) and a retention time of 18 ± 3 minutes for the lauryl arachidate internal standard. The most representative peak of the waxes must be over 60 % of the full-scale value while the methyl heptadecanoate internal standard for the methyl and ethyl esters must reach the full-scale value.
The peak integration parameters should be determined in such a way as to obtain a correct evaluation of the peak areas considered.
Take up 10 μl of the solution with the aid of the 10 μl micro-syringe, drawing back the plunger until the needle is empty. Introduce the needle into the injection system and inject quickly after 1–2 s. After about 5 s, gently extract the needle.
Perform the recording until the waxes or stigmastadienes are completely eluted, depending on the fraction being analysed.
The base line must always meet the required conditions.
Identify the peaks from the retention times by comparing them with mixtures of waxes with known retention times, analysed under the same conditions. The alkyl esters are identified from mixtures of methyl and ethyl esters of the chief fatty acids in olive oils (palmitic and oleic).
Figure 1 provides a chromatogram of the waxes in a virgin olive oil. Figures 2 and 3 show the chromatograms of two retail extra virgin olive oils, one with methyl and ethyl esters and the other without them. Figure 4 gives the chromatograms for a top-quality extra virgin olive oil and the same oil spiked with 20 % deodorised oil.
Determine the area of the peaks corresponding to the lauryl arachidate internal standard and the aliphatic esters from C40 to C46 with the aid of the integrator.
Determine the total waxes content by adding each individual wax, in mg/kg of fat, as follows:
where:
=
area corresponding to the peak for the individual ester, in computer counts
=
area corresponding to the peak for the lauryl arachidate internal standard, in computer counts
=
mass of the lauryl arachidate internal standard added, in milligrams;
=
mass of the sample taken for determination, in grams.
With the aid of the integrator, determine the areas of the peaks corresponding to the methyl heptadecanoate internal standard, the methyl esters of the C16 and C18 fatty acids and the ethyl esters of the C16 and C18 fatty acids.
Determine the content of each alkyl ester, in mg/kg of fat, as follows:
where:
=
area corresponding to the peak for the individual C16 and C18 ester, in computer counts
=
area corresponding to the peak for the methyl heptadecanoate internal standard, in computer counts
=
mass of the methyl heptadecanoate internal standard added, in milligrams;
=
mass of the sample taken for determination, in grams.
Report the sum of the contents of the different waxes from C40 to C46 (Note 7) in milligrams per kilograms of fat.
Report the sum of the contents of the methyl esters and ethyl esters from C16 to C18 and the total of the two.
Results should be expressed to the nearest mg/kg.
Report the ratio between ethyl esters and methyl esters
Figure 4
Part of a chromatogram of an extra virgin olive oil and the same oil spiked with deodorised oil
Inject 1:3 μl of methane (or propane) into the gas chromatograph after adjusting it to the normal operating conditions. Measure the time the gas takes to run through the column from the moment it is injected until the peak emerges (tM).
The linear speed in cm/s is given by L/tM where L is the length of the column, in cm, and tM is the time measured in s.”
After elution of the sterol esters, the chromatogram should not show any significant peaks (triacylglycerols).
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