3.

The following Annex XVIII is added:



‘ANNEX XVIII DETERMINATION OF TRIACYLGLYCEROLS WITH ECN 42 (DIFFERENCE BETWEEN HPLC DATA AND THEORETICAL CONTENT) 1. Scope Determination of the composition of triacylglycerols (TAGs) in olive oils, in terms of their equivalent carbon number by differences between the analytical results obtained by high performance liquid chromatography (HPLC) and the theoretical content, calculated starting from the fatty acid composition. 2. Field application The standard is applicable to olive oils. The method is applicable to the detection of the presence of small amounts of seed oils (rich in linoleic acid) in every class of olive oils. 3. Principle The content of triacylglycerols with ECN42 determined by HPLC analysis and the theoretical content of triacylglycerols with ECN42 (calculated on the basis of GLC determination of fatty acid composition) correspond within a certain limit for pure oils. A difference larger than the values stated in the Regulation for each type of oil points out that the oil contains seed oils. 4. Method The method for calculation of theoretical content of triacylglycerols with ECN42 and of the difference between the HPLC data and this one essentially is made by the coordination of analytical data obtained by means of other methods: it is possible to distinguish three phases: determination of fatty acid composition by capillary gas chromatography, calculation of theoretical composition of triacylglycerols with ECN42, HPLC determination of ECN42 triacylglycerols 4.1. Apparatus 4.1.1. Round bottom flasks, 250 and 500 ml. 4.1.2. Beakers 100 ml. 4.1.3. Glass chromatographic column, 21 mm internal diameter, 450 mm length, with cock and normalized cone (female) at the top. 4.1.4. Separator funnels, 250 ml, with normalized cone (male) at the bottom, suitable to be connected with the top of the column. 4.1.5. Glass rod, 600 mm length. 4.1.6. Glass funnel, 80 mm diameter. 4.1.7. Volumetric flasks, 50 ml. 4.1.8. Volumetic flasks, 20 ml. 4.1.9. Rotative evaporator. 4.1.10. High performance liquid chromatography, allowing thermostatic control of column temperature. 4.1.11. Injection units for 10 μl delivery. 4.1.12. Detector: differential refractometer. The full scale sensitivity should be at least 10−4 units of refractive index. 4.1.13. Column: stainless steel tube 250 mm length and 4,5 mm internal diameter packed with 5 μm diameter particles of slica with 22 to 23 % carbon in the form of octadecylsilane (note 2). 4.1.14. Recorder and/or integrator. 4.2. Reagents The reagents should be of analytical purity. Elution solvents should be de-gassed, and may be recycled several times without effect on the separations. 4.2.1. Petroleum ether 40 to 60 °C chromatographic grade. 4.2.2. Ethil ether, peroxides free, freshly distilled. 4.2.3. Glass chromatographic elution solvent: mixture petroleum ether/ethil ether 87/13 (v/v). 4.2.4. Silicagel, 70-230 mesh, type Merck 7734, with water content standardized at 5 % (w/w). 4.2.5. Glass wool. 4.2.6. Acetone. 4.2.7. Acetonitrile. 4.2.8. HPLC elution solvent: acetonitrile + acetone (proportions to be adjusted to obtain the desired separation; begin with 50:50 mixture). 4.2.9. Solubilization solvent: acetone. 4.2.10. Reference triglycerides commercial triglycerides tripalmitin, triolein, etc.) may be used and the retention times thence plotted in accordance with the equivalent carbon number, or alternatively reference chromatograms obtained from soya oil, mixture 30:70 soya oil/olive oil and pure olive oil (see notes 3 and 4 and figure 1, 2, 3, 4). 4.3. Sample preparation As a number of interfering substances can rise false positive results, the sample must always be purified according to IUPAC method 2.507, used for determination of polar substances in oxidised oils. 4.3.1. Chromatographic column preparation Fill the column (4.1.3) with about 30 ml of elution solvent (4.2.3), then introduce inside the column some glass wool (4.2.5) pushing it to the bottom of the column by means of the glass rod (4.1.5). In a 100 ml beaker, suspend 25 g of silicagel (4.2.4) in 80 ml of elution mixture (4.2.3), then transfer it inside the column, by means of a glass funnel (4.1.6). To ensure the complete transfer of silicagel inside the column, wash the beaker with the elution mixture and transfer the washing portions inside the column, too. Open the cock and let solvent elute from the column until its level is about 1 cm over the silicagel. 4.3.2. Column chromatography Weigh with the accuracy of 0,001 g, 2,5 ± 0,1 g of oil, previously filtered, homogenized and anhydrified, if necessary, in a 50 ml volumetric flask (4.1.7). Solve it in about 20 ml of elution solvent (4.2.3), if necessary, slightly heat it to make the dissolution easily. Cool at room temperature and adjust the volume with elution solvent. By means of a volumetric pipette, introduce 20 ml of solution inside the column prepared according to 4.3.1, open the cock and let solvent elute to the silicagel layer level. Then elute with 150 ml of elution solvent (4.2.3), adjusting the solvent rate at about 2 ml/min (150 ml will take about 60 to 70 minutes to pass through the column). The eluated is recovered in a 250 ml round bottom flask (4.1.1) previously tared in an oven and exactly weighted. Eliminate solvent at reduce pressure (Rotavapor) and weigh the residue that will be used to prepare the solution for HPLC analysis and for methyl ester preparation. The sample recovery from the column must be 90 % at least for extra virgin, virgin, ordinary refined and olive oil categories, and a minimum of 80 % for lampante and residue olive oils. 4.4. HPLC analysis 4.4.1. Preparation of the samples for chromatographic analysis A 5 % solution of the sample to be analysed is prepared by weighing 0,5 ± 0,001 g of the sample into a 10 ml graduated flask and making up to 10 ml with the solubilization solvent (4.2.9). 4.4.2. Procedure Set up the chromatographic system. Pump elution solvent (4.2.8) at a rate of 1,5 ml/min to purge the entire system. Wait until a stable base line is obtained. Inject 10 μl of the sample prepared as in 4.3. 4.4.3. Calculation and expression of results Use the area normalization method, i.e. assume that the sum of the areas of the peaks corresponding to TAGs from ECN42 up to ECN52 is equal to 100 %. Calculate the relative percentage of each triglyceride using the formula: % triglyceride = area of peak × 100/ sum of peak areas. The results are to be given to within at least two decimal places. Note 1: The elution order can be determined by calculating the equivalent carbon numbers, often defined by the relation ECN = CN-2n, where CN is the carbon number and n is the number of double bounds, it can be calculated more precisely by taking into account the origin of the double bond. If no, nl and nln are the numbers of double bonds attributed to oleic, linoleic and linolenic acids respectively, the equivalent carbon number can be calculated by means of the relation of the formula: ECN = CN − dono − dlnl − dlnnln, where the coefficient do, dl and dln can be calculated by means of the reference triglycerides. Under the conditions specified in this method, the relation obtained will be close in: ECN = CN − (2,60 no) − (2,35 nl) − (2,17 nln) Note 2:



Examples: Lichrosorb (Merck) RP 18 Art 50333 Lichrosphere or equivalent (Merck) 100 CH18 Art 50377. Note 3: With several reference triglycerides, it is also possible to calculate the resolution with respect to triolein: α = RT' / RT triolein by use of the reduced retention time RT' = RT − RT solvent. The graph of log α against f (number of double bonds) enables the retention values to be determined for all the triglycerides of fatty acids contained in the reference triglycerides — see figure 2. Note 4: The efficiency of the column should permit clear separation of the peak of trilinoein from the peaks of the triglycerides with an adjacent RT. The elution is carried out up to ECN52 peak. Note 5: A correct measure of the areas of all peaks of interest for the present determination is ensured if the second peak corresponding to ECN50 is 50 % of full scale of the recorder. 4.5. Calculation of triacylglycerols composition 4.5.1. Determination of fatty acid composition Fatty acid composition is carried out by means of the EEC gas chromatographic method reported in Annex X A of Regulation (EEC) No 2568/91, by means of a capillary column. The methyl esters preparation is carried out according to Annex X B (sodium methylate alcohol solution). 4.5.2. Fatty acids for calculation Glycerides are grouped by their equivalent carbon number (ECN), taking into account the following equivalencies between ECN and fatty acids. Only fatty acids with 16 and 18 carbon atoms were taken in consideration, because only these are important for olive oil.



Fatty acid (FA) Abbreviation Molecular weight (MW) ECN Palmatic acid P 256,4 16 Palmatoleic acid Po 254,4 14 Stearic acid S 284,5 18 Oleic acid O 282,5 16 Linoleic acid L 280,4 14 Linolenic acid Ln 278,4 12 4.5.3. Conversion of area % into moles for all fatty acids

4.5.4. Normalization of fatty acids to 100 %

The result gives the percentage of each fatty acid in moles % in the overall (1,2,3-) position of the TAGs. Then the sum of the saturated fatty acids P and S (SFA) and the unsaturated fatty acids Po, O, L and Ln (UFA) are calculated:

4.5.5. Calculation of the fatty acid composition in 2- and 1,3- positions of TAGs The fatty acids are distributed to three pools as follows: two identical for 1- and 3- positions and one for 2- position, with different coefficients for the saturated (P and S) and unsaturated acids (Po, O, L and Ln). 4.5.5.1. Saturated fatty acids in 2- position [P(2) and S(2)]

4.5.5.2. Unsaturated fatty acids in 2- position [Po(2), O(2), L(2) and Ln(2)]:

4.5.5.3. Fatty acids in 1,3-positions [P(1,3), S(1,3), Po(1,3) O(1,3), L(1,3) and Ln(1,3)]:

4.5.6. Calculation of triacylglycerols 4.5.6.1. TAGs with one fatty acid (AAA, here LLL, PoPoPo)



moles % AAA = moles % A1,3 * moles % A2 * moles % A1,310 000 (7) 4.5.6.2. TAGs with two fatty acids (AAB, here PoPoL, PoLL)

4.5.6.3. TAGs with three different fatty acids (ABC, here OLLn, PLLn, PoOLn, PPoLn)

4.5.6.4. Triacylglycerides with ECN42 The following triglycerides with ECN42 are calculated according equation 7, 8 and 9 in order of expected elution in HPLC (normally only three peaks). LLL PoLL and the positional isomer LPoL OLLn and the positional isomers OLnL and LnOL PoPoL and the positional isomer PoLPo PoOLn and the positional isomers OPoLn and OLnPo PLLn and the positional isomers LLnP and LnPL PoPoPo SLnLn and the positional isomer LnSLn PPoLn and the positional isomers PLnPo and PoPLn The triacylglycerides with ECN42 are given by the sum of the nine triacylglycerols including their positional isomers. The results to be given with at least two decimal places. 5. Evaluation of the results The calculated theoretical content and the content determined by the HPLC analysis are compared. If the difference between HLPC data minus theoretical data is greater than the values states for the appropriate oil category in the Regulation, the sample contains seed oil. Note: Results are given to within one decimal figure. 6. Example (The numbers refer to the sections in the text of the method) 4.5.1. Calculation of moles % fatty acids from GLC data (area %) The following data are obtained for the fatty acid composition by GLC:



FA MW P 256,4 S 284,5 Po 254,4 O 282,5 L 280,4 Ln 278,4 area % 10,0 3,0 1,0 75,0 10,0 1,0 4.5.3. Conversion of area % into moles for all fatty acids



moles P = 10256,4 = 0,03900 moles P See formula (1) moles S = 3284,5 = 0,01054 moles S See formula (1) moles Po = 1254,4 = 0,00393 moles Po See formula (1) moles O = 75282,5 = 0,26549 moles O See formula (1) moles L = 10280,4 = 0,03566 moles L See formula (1) moles Ln = 1278,4 = 0,003594 moles Ln See formula (1)

4.5.4. Normalization of fatty acids to 100 %



moles % P1,2,3 = 0,03900 moles P * 1000,35822 moles = 10,888 % See formula (2) moles % S1,2,3 = 0,01054 moles S * 1000,35822 moles = 2,944 % See formula (2) moles % Po1,2,3 = 0,00393 moles Po * 1000,35822 moles = 1,097 % See formula (2) moles % O1,2,3 = 0,26549 moles O * 1000,35822 moles = 74,113 % See formula (2) moles % L1,2,3 = 0,03566 moles L * 1000,35822 moles = 9,956 % See formula (2) moles % Ln1,2,3 = 0,00359 moles Ln * 1000,35822 moles = 1,003 % See formula (2)

Sum of the saturated and unsaturated fatty acids in the 1,2,3- position of TAGs



moles % SFA = 10,888 % + 2,944 % = 13,831 % See formula (3) moles % UFA = 100,000 % — 13,831 % = 86,169 % See formula (3) 4.5.5. Calculation of the fatty acid composition in 2- and 1,3- positions of the TAGs 4.5.5.1. Saturated fatty acids in 2- position [P(2) and S(2)]



moles % P(2) = 10,888 % * 0,06 = 0,653 moles % See formula (4) moles % S(2) = 2,944 % * 0,06 = 0,177 moles % See formula (4) 4.5.5.2. Unsaturated fatty acids in 1,3-position [Po(1,3), O(1,3), L(1,3) and Ln(1,3)]



moles % Po2 = 1,097 %86,169 %* 100 − − 0,659 − 0,177 = 1,263 moles % See formula (5) moles % O2 = 74,113 %86,169 %* 100 − − 0,659 − 0,177 = 85,295 moles % See formula (5) moles % L2 = 9,956 %86,169 %* 100 − − 0,659 − 0,177 = 11,458 moles % See formula (5) moles % Ln2 = 1,003 %86,169 %* 100 − − 0,659 − 0,177 = 1,154 moles % See formula (5) 4.5.5.3. Fatty acids in 1,3-positions [P(1,3), S(1,3), Po(1,3), O(1,3), L(1,3) and Ln(1,3)]



moles % P1,3 = 10,888 − 0,6592 10,888 = 16,005 moles % See formula (6) moles % S1,3 = 2,944 − 0,1772 2,944 = 4,327 moles % See formula (6) moles % Po1,3 = 1,097 − 1,2632 1,097 = 1,015 moles % See formula (6) moles % O1,3 = 74,113 − 85,2952 74,113 = 68,522 moles % See formula (6) moles % L1,3 = 9,956 − 11,4582 9,956 = 9,205 moles % See formula (6) moles % Ln1,3 = 1,003 − 1,1542 1,003 = 0,927 moles % See formula (6) 4.5.6. Calculation of triacylglycerols From the calculated fatty acid composition in sn-2- and sn-1,3- positions (see above):



FA in 1,3-pos. 2-pos. P 16,005 % 0,653 % S 4,327 % 0,177 % Po 1,015 % 1,263 % O 68,522 % 85,295 % L 9,205 % 11,458 % Ln 0,927 % 1,154 % Sum 100,0 % 100,0 % the following triacylglycerols are calculated: LLL PoPoPo PoLL with 1 positional isomer SLnLn with 1 positional isomer PoPoL with 1 positional isomer PPoLn with 2 positional isomers OLLn with 2 positional isomers PLLn with 2 positional isomers PoOLn with 2 positional isomers. 4.5.6.1. TAGs with one fatty acid (LLL, PoPoPo) See formula (7)



mol % LLL = 9,205 % * 11,458 % * 9,205 %10 000 = 0,09708 mol LLL mol % PoPoPo = 1,015 % * 1,263 % * 1,015 %10 000 = 0,00013 mol PoPoPo 4.5.6.2. TAGs with two fatty acids (PoLL, SLnLn, PoPoL) See formula (8)



mol % PoLL + LLPo = 1,015 % * 11,458 % * 9,205 % * 210 000 = 0,02141 mol % LPoL = 9,205 % * 1,263 % * 9,205 %10 000 = 0,01070 0,03211 mol PoLL mol % SLnLn + LnLnS = 4,327 % * 1,154 % * 0,927 % * 210 000 = 0,00093 mol % LnSLn = 0,927 % * 0,177 % * 0,927 %10 000 = 0,00002 0,00095 mol SLnLn mol % PoPoL + LPoPo = 1,015 % * 1,263 % * 9,205 % * 210 000 = 0,00236 mol % PoLPo = 1,015 % * 11,458 % * 1,015 %10 000 = 0,00118 0,00354 mol PoPoL 4.5.6.3. TAGs with three different fatty acids (PoPLn, OLLn, PLLn, PoOLn) See formula (9)



mol % PPoLn = 16,005 % * 1,263 % * 0,927 % * 210 000 = 0,00375 mol % LnPPo = 0,927 % * 0,653 % * 1,015 % * 210 000 = 0,00012 mol % PoLnP = 1,015 % * 1,154 % * 16,005 % * 210 000 = 0,00375 0,00762 mol PPoLn mol % OLLn = 68,522 % * 11,458 % * 0,927 % * 210 000 = 0,14577 mol % LnOL = 0,927 % * 85,295 % * 9,205 % * 210 000 = 0,14577 mol % LLnO = 9,205 % * 1,154 % * 68,522 % * 210 000 = 0,14577 0,43671 mol OLLn mol % PLLn = 16,005 % * 11,458 % * 0,927 % * 210 000 = 0,03400 mol % LnPL = 0,927 % * 0,653 % * 9,205 % * 210 000 = 0,00111 mol % LLnP = 9,205 % * 1,154 % * 16,005 % * 210 000 = 0,03400 0,06911 mol PLLn mol % PoOLn = 1,015 % * 85,295 % * 0,927 % * 210 000 = 0,01605 mol % LnPoO = 0,927 % * 1,263 % * 68,522 % * 210 000 = 0,01605 mol % OLnPo = 68,522 % * 1,154 % * 1,015 % * 210 000 = 0,01605 0,04815 mol PoOLn ECN42 = 0,69540 mol TAGs’