Commission Regulation (EEC) No 2568/91Dangos y teitl llawn

[F1 ANNEX VII U.K. DETERMINATION OF THE PERCENTAGE OF 2-GLYCERYL MONOPALMITATE

Textual Amendments

F1 Substituted by Commission Regulation (EC) No 702/2007 of 21 June 2007 amending Commission Regulation (EEC) No 2568/91 on the characteristics of olive oil and olive-residue oil and on the relevant methods of analysis.

1. PURPOSE AND SCOPE U.K.

This method describes the analysis procedure for determining the percentage of palmitic acid in position 2 of the triglycerides by evaluating 2-glyceryl monopalmitate.

This method can be applied to liquid vegetable oils at ambient temperature (20 °C).

2. PRINCIPLE U.K.

After preparation the oil sample is subjected to the action of pancreatic lipase: partial and specific hydrolysis in positions 1 and 3 of the triglyceride molecule causes monoglycerides to appear in position 2. The percentage of 2-glyceryl monopalmitate in the monoglyceride fraction is determined after silylation by capillary-column gas chromatography.

3. APPARATUS AND MATERIALS U.K.

3.1. 25 ml Erlenmeyer flask U.K.

3.2. 100, 250 and 300 ml beakers U.K.

3.3. Glass chromatograph column, internal diameter 21-23 mm, length 400 mm, fitted with a sintered glass disc and a stopcock U.K.

3.4. 10, 50, 100 and 200 ml measuring cylinders U.K.

3.5. 100 and 250 ml flasks U.K.

3.6. Rotary evaporator U.K.

3.7. 10 ml conical-bottomed centrifuge tubes with groundglass stopper U.K.

3.8. Centrifuge for 10 and 100 ml tubes U.K.

3.9. Thermostat permitting a stable temperature of 40 ± 0,5 °C U.K.

3.10. 1 and 2 ml graduated pipettes U.K.

3.11. 1 ml hypodermic syringe U.K.

3.12. 100 μl microsyringe U.K.

3.13. 1 000 ml funnel U.K.

3.14. Capillary gas chromatograph with an on-column cold injector for direct injection of the sample into the column and a furnace able to maintain the selected temperature to approximately 1 °C U.K.

3.15. On-column cold injector for direct injection of the sample into the column U.K.

3.16. Flame ionisation detector and electrometer U.K.

3.17. Recorder-integrator adapted to the electrometer with a response rate no greater than 1 sec and a variable paper roll rate U.K.

3.18. Capillary column made of glass or fused silica 8-12 metres long, 0,25-0,32 mm internal diameter, covered with methylpolysiloxane or phenyl methylpolysiloxane 5 %, 0,10-0,30 μm thick, useable at 370 °C U.K.

3.19. 10 μl microsyringe fitted with a hardened needle, at least 7,5 cm long for direct on-column injection. U.K.

4. REAGENTS U.K.

4.1. Silica gel with a grain size of between 0,063 and 0,200 mm (70/280 mesh) prepared as follows: Place the silica gel in a porcelain capsule, dry in an incubator at 160 °C for four hours, then leave to cool at room temperature in a desiccator. Add water equivalent to 5 % of the mass of the silica gel as follows: Weigh 152 g silica gel into an Erlenmeyer flask then add 8 g of distilled water, stopper and shake gently to distribute the water evenly. Leave to stand for at least 12 hours before use. U.K.

[F2 4.2. n-hexane (chromatography grade). Hexane may be replaced by iso-octane (2,2,4- trimethylpentane in chromatography grade), provided that comparable precision values are achieved.] U.K.

Textual Amendments

F2 Substituted by Commission Implementing Regulation (EU) 2019/1604 of 27 September 2019 amending Regulation (EEC) No 2568/91 on the characteristics of olive oil and olive-residue oil and on the relevant methods of analysis.

4.3. Isopropanol U.K.

4.4. Isopropanol, 1/1 (v/v) aqueous solution U.K.

4.5. Pancreatic lipase. It must have an activity of between 2,0 and 10 lipase units per mg. (Pancreatic lipases with an activity of between 2 and 10 units per mg enzyme are commercially available.) U.K.

4.6. Buffer solution of trishydroxymethylaminomethane: 1 M aqueous solution adjusted to pH 8 (potentiometric control) by conc. HCl (1/1 v/v) U.K.

4.7. Enzyme-quality sodium cholate, 0,1 % aqueous solution (this solution must be used within two weeks of its preparation) U.K.

4.8. Calcium chloride, 22 % aqueous solution U.K.

4.9. Diethyl ether for chromatography U.K.

4.10. Developer solvent: mixture of n-hexane/diethyl ether (87:13 v:v) U.K.

4.11. Sodium hydroxide, 12 % by weight solution U.K.

4.12. Phenolphthalein, 1 % solution in ethanol U.K.

4.13. Carrier gas: hydrogen or helium, for gas chromatography U.K.

4.14. Auxiliary gases: hydrogen, 99 % minimum purity, free from moisture and organic substances, and air, for gas chromatography, of the same purity U.K.

4.15. Silanisation reagent: mixture of pyridine/hexamethyldisilazane, trimethylchlorosilane 9/3/1 (v/v/v). (Ready-to-use solutions are commercially available. Other silylation reagents may be used, particularly bis-trimethylsilyl trifluoracetamide + 1 % trimethylchlorosilane, diluted with an identical volume of anhydrous pyridine.) U.K.

4.16. Reference samples: pure monoglycerides or monoglyceride mixtures with a known percentage composition similar to that of the sample. U.K.

5. METHOD U.K.

5.1. Sample preparation U.K.

5.1.1. Oils with a free acidity of less than 3 % do not need to be neutralised before chromatography on a silica gel column. Oils with a free acidity of more than 3 % must be neutralised as per point 5.1.1.1. U.K.

5.1.1.1. Pour 50 g of oil and 200 ml n-hexane into the 1 000 ml funnel (3.13). Add 100 ml of isopropanol and a quantity of 12 % sodium hydroxide solution (4.11) equivalent to the free acidity of the oil plus 5 %. Shake vigorously for one minute. Add 100 ml of distilled water, shake again and leave to stand. U.K.

After decanting, remove the lower layer containing the soaps. Remove any intermediate layers (mucilage and insoluble substances). Wash the hexane solution of the neutralised oil with successive portions of 50-60 ml of the 1/1 (v/v) isopropanol/water solution (4.4) until the pink colouration of the phenolphthalein disappears.

Remove most of the hexane by vacuum distillation (use a rotary evaporator, for example) and transfer the oil into a 100 ml flask (3.5). Dry the oil in vacuum until the solvent is completely removed.

After that procedure is completed, the acidity of the oil should be less than 0,5 %.

5.1.2. Put 1,0 g of the oil prepared as above into a 25 ml Erlenmeyer flask (3.1) and dissolve in 10 ml of developer mixture (4.10). Leave the solution to stand for at least 15 minutes before silica gel column chromatography. U.K.

If the solution is cloudy centrifuge it to ensure optimum conditions for chromatography. (Ready-to-use 500 mg silica gel SPE cartridges can be used).

5.1.3. Preparation of the chromatography column U.K.

Pour about 30 ml of the developer solvent (4.10) into the column (3.3), insert a piece of cotton into the bottom part of the column using a glass rod; press to eliminate the air.

In a beaker prepare a suspension of 25 g of silica gel (4.1) in about 80 ml of developer solvent and pour it into the column using a funnel.

Check that all the silica gel is in the column; wash with developer solvent (4.10), open the stopcock and allow the liquid to reach a level about 2 mm above the level of the silica gel.

5.1.4. Column chromatography U.K.

Weigh accurately 1,0 g of sample prepared as in point 5.1 into a 25 ml Erlenmeyer flask (3.1).

Dissolve the sample in 10 ml of developer solvent (4.10). Pour the solution into the chromatography column prepared as in point 5.1.3. Avoid disturbing the surface of the column.

Open the stopcock and pour the sample solution until it reaches the level of the silica gel. Develop with 150 ml of the developer solvent. Adjust the flow rate to 2 ml/min (so that 150 ml enters the column in about 60-70 minutes).

Recover the eluate in a previously weighed 250 ml flask. Evaporate the solvent under vacuum and remove the final traces of the solvent under a nitrogen current.

Weigh the flask and calculate the recovered extract.

(If ready-to-use silica gel SPE cartridges are used use the following method: Put 1 ml of solution (5.1.2) into the prepared cartridges with 3 ml of n-hexane.

After percolating the solution develop with 4 ml of n-hexane/diethyl ether 9/1 (v/v).

Recover the eluate in a 10 ml tube and evaporate to dry in a nitrogen current.

Expose the dry residue to pancreatic lipase (5.2). (It is essential to check the fatty acid composition before and after crossing the SPE cartridge.)

5.2. Hydrolysis by pancreatic lipase U.K.

5.2.1. Weigh into the centrifuge tube 0.1 g of the oil prepared as in point 5.1. Add 2 ml of buffer solution (4.6), 0,5 ml of the sodium cholate solution (4.7) and 0,2 ml of the calcium chloride solution, stirring well after each addition. Close the tube with the groundglass stopper and place in the thermostat at 40 + 0,5 °C. U.K.

5.2.2. Add 20 mg of lipase, shake carefully (avoid wetting the stopper) and place the tube in the thermostat for exactly two minutes. Then remove it, shake vigorously for exactly 1 minute and leave to cool. U.K.

5.2.3. Add 1 ml of diethyl ether, stopper and shake vigorously, then centrifuge and transfer the ether solution into a clean, dry tube using a microsyringe. U.K.

5.3. Preparation of the silanised derivatives and gas chromatography U.K.

5.3.1. With a microsyringe insert 100 μl of solution (5.2.3) into a 10 ml conical-bottomed tube. U.K.

5.3.2. Remove the solvent under a slight nitrogen current, add 200 μl of silanisation reagent (4.15), stopper the tube and leave to stand for 20 minutes. U.K.

5.3.3. After 20 minutes, add 1 to 5 ml of n-hexane (depending on the chromatography conditions): the resulting solution is ready for gas chromatography. U.K.

5.4. Gas chromatography U.K.

Operating conditions:

• Injector temperature (on-column injector) lower than solvent boiling point (68 °C);

• Detector temperature: 350 °C;

• Column temperature: programming of furnace temperature: 60 °C for 1 minute, increasing by 15 °C per minute up to 180 °C, then by 5 °C per minute up to 340 °C, then 340 °C for 13 minutes;

• Carrier gas: hydrogen or helium, set at a linear velocity sufficient to obtain the resolution reflected in Figure 1. The retention time of the C ₅₄ triglyceride must be 40 ± 5 minutes (see Figure 2). (The operating conditions indicated above are indicative. Operators will have to optimise them to obtain the desired resolution. The peak corresponding to 2-glyceryl monopalmitate must have a minimum height equal to 10 % of the recorder scale.)

• Quantity of substance injected: 0,5-1 μl of the n-hexane solution (5 ml) (5.3.3).

5.4.1. Identification of the peaks U.K.

The individual monoglycerides are identified from their retention times and by comparison with those obtained for standard monoglyceride mixtures under the same conditions.

5.4.2. Quantitative evaluation U.K.

The area of each peak is calculated using an electronic integrator.

6. EXPRESSION OF RESULTS U.K.

The percentage of glyceryl monopalmitate is calculated from the ratio between the area of the corresponding peak and the areas of the peaks of all the monoglycerides (see Figure 2) using the formula:

glyceryl monopalmitate (%):

where:

A _x

=

area of the peak corresponding to glyceryl monopalmitate

ΣA

=

sum of the areas of all the monoglyceride peaks

The result must be to one decimal place.

7. ANALYSIS REPORT U.K.

The analysis report must specify:

• reference to this method,

• all the information needed for a full identification of the sample,

• the analysis result,

• any deviation from the method, whether as the result of a decision by the parties concerned or for another reason,

• details to identify the laboratory, the date of the analysis and the signatures of those responsible for the analysis.

Figure 1

Chromatogram of the products of the silanisation reaction obtained by the action of lipase on a refined olive oil with 20 % esterified oil added (100 %)

Figure 2 U.K.

Chromatogram of :

( A ) unesterified olive oil, after lipase; after silanisation; under these conditions (8-12 m capillary column) the wax fraction is eluted at the same time as the diglyceride fraction or slightly afterwards . U.K.

After lipase, the triglyceride content should not exceed 15 %

Chromatogram of :

( B ) unesterified oil after lipase; after silanisation; under these conditions (8-12 m capillary column) the wax fraction is eluted at the same time as the diglyceride fraction or slightly afterwards . U.K.

After lipase, the triglyceride content should not exceed 15 % .

8. NOTES U.K.

Note 1. PREPARATION OF THE LIPASE U.K.

Lipases with satisfactory activity are commercially available. They can also be prepared in the laboratory in the following manner:

Cool to 0 °C 5 kg of fresh pig’s pancreas. Remove the surrounding solid fat and the connective tissue and grind to a liquid paste in a blender. Stir the paste with 2,5 litres of anhydrous acetone for 4-6 hours, then centrifuge. Extract the residue three more times with the same volume of anhydrous acetone, then twice with an acetone/diethyl ether mixture (1/1 v/v) and twice with diethyl ether.

Vacuum-dry the residue for 48 hours to obtain a stable powder which can be stored for a long time in a refrigerator away from moisture.

Note 2. MONITORING LIPASE ACTIVITY U.K.

Prepare an olive oil emulsion as follows:

In a mixer stir for 10 minutes a mixture of 165 ml of a 100 g/l gum arabic solution, 15 g of crushed ice and 20 ml of a previously neutralised olive oil.

Pour 10 ml of the emulsion into a 50 ml beaker, then 0,3 ml of a 0,2 g/ml sodium cholate solution and then 20 ml of distilled water.

Put the beaker in a thermostat set at 37 °C; introduce the electrodes of the pH meter and the screw agitator.

Using a burette, add a 0,1 N sodium hydroxide solution drop by drop until a pH of 8,3 is obtained.

Add an aliquot of the lipase powder suspension in water (0,1 g/ml of lipase). As soon as the pH meter reads 8,3, start the chronometer and add the sodium hydroxide solution drop by drop at a rate which maintains the pH at 8,3. Note every minute the volume of solution consumed.

Record the data on an x/y graph with the time on the x-axis and millilitres of 0,1 N alkaline solution consumed to keep a constant pH on the y-axis. A linear graph should be obtained.

Lipase activity, expressed in lipase units per mg, is given by the following formula:

where:

A

is activity in lipase units/mg

V

is the number of millilitres of 0,1 N sodium hydroxide solution per minute (calculated on the basis of the graph)

N

is the titre of the sodium hydroxide solution

m

is the mass in mg of the test lipase.

A lipase unit is defined as the quantity of enzyme which releases 10 micro-equivalents of acid per minute.]