ROUTINE AND INNOVATIVE CHEMICAL

ANALYSES OF OLIVE OIL

May 2008

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1. Introduction In this report are described the chemicals analyses of extra virgin olive oil. They comprise the traditional analyses (free acidity, peroxide value, ultraviolet spectroscopy, fatty acid composition, sterols composition and olive oil sensory quality), innovative analyses (1H and 13C NMR, IRMS and metals) and analyses of minor compounds (phenolic compounds, volatile compounds, adulteration, chlorophylls, pheophytins, β-carotene, tocopherols and tocotrienols, oxidation stability, wax and aliphatic alcohols). 2. Free acidity Definition Free acidity expresses the percentage content (in weight) of the free fatty acids in the olive oil. Cause The lipolytic action of lipase (specific enzyme, found in the olive fruit) produces free fatty acids which are responsible for the acidity of the oil. Method Free acidity, expressed as percent of oleic acid, was determined by titration of a solution of olive oil dissolved in ethanol/ether 1:1 with ethanolic potassium, according to Reg. EEC n. 2568/91, Annex II. Procedure 10 g of the sample is weighted into a conical flask. It was then dissolved in 50 ml of the solvent mixture (1:1 of 95% (V/V) ethanol and diethyl ether) and titrated, while shaking, with a solution of potassium hydroxide (0.1 N) to the end point of the indicator (pink colour of phenolphthalein persisting for at least 10 sec). Calculation Free acidity, expressed as % of oleic acid, was given to:

V * c * (M / 1000) * (100 / m) = (V * c * m / 10 * m) where: V = volume, in ml, of solution titrated with potassium hydroxide; c = concentration, in mol/l, of solution of potassium hydroxide; M = molar weight, in g/mol, of oleic acid (= 282); m = weight, in g, of oil.

3. Peroxide Value (PV) Definition Peroxide value is the quantity of those substances in the sample, which oxidise potassium iodide. Cause Peroxide value is related to olive oil oxidative status and it depends on storage conditions. The peroxide value is caused by hydroperoxides (primary stage of oxidation). The oxidation may be enzymatic or chemical. Method Peroxide value, expressed in milliequivalents of active oxygen per kilogram of olive oil (meq/kg), was determined as follows: a mixture of olive oil and chloroform/acetic acid was left to react in darkness with saturated potassium iodide solution; the free iodine was then titrated with a sodium thiosulfate solution. The determination of the peroxide value was performed according to Reg. EEC n. 2568/91, Annex III. Procedure The determination was carried out in diffuse daylight or in artificial light. 1-2 g of the sample was weighted in a flask (with ground neck and stopper, of 250 ml of capacity) to the nearest 0.001 g. It was dissolved rapidly by stirring with the addition of 10 ml of chloroform (analytical reagent quality). 15 ml of acetic acid (analytical reagent quality), and then 1 ml of potassium iodide solution (saturated aqueous solution) were added. The stopper was quickly inserted; the sample was shaken for 1 minute and left for exactly 5 minutes in the dark at a temperature from 15 to 25°C. 75 ml of distilled water were added. The liberated iodine was titrated with the sodium thiosulphate solution (0.002 N for expected values less than 12, and 0.01 N for expected values above 12), with vigorous shaking, using starch solution as an indicator (10 g/l aqueous dispersion) from a purple to a slightly yellow or colourless endpoint. Calculation Peroxide Value (P.V.), expressed in milliequivalents of active oxygen per kilogram of olive oil (meq/kg), was given to:

(P.V.) = (V * T / m) * 1000

where: V = ml of solution of titrant (sodium thiosulphate); T = Normality of sodium thiosulphate solution; m = weight of sample of olive oil, in grams. Limits EEC 1989/2003 of peroxide value ≤ 20 (meq O2/kg olive oil).

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4. Ultraviolet Spectrophotometry Definition Spectrophotometric examination in the ultraviolet is the absorption at the wavelengths specified in the method and expressed as specific extinctions (the extinction of 1% solution of olive oil in isooctane (or cyclohexane), in a thickness of 1 cm, 1 cm conventionally indicated by K (also referred to as "extinction coefficient"). Spectrophotometric examination in the ultraviolet can provide information on the quality of olive oil, its state of preservation and changes brought about in it by technological processes. Causes The absorbency at 232 nm is caused by hydroperoxides (primary stage of oxidation) and conjugated dienes (intermediate stage of oxidation). The absorbency at 270 nm is caused by carbonylic compounds (secondary stage of oxidation) and conjugated trienes (technological treatments). The index ΔK is a criterion of discrimination between a bad quality virgin olive oil and a virgin olive oil adulterated with refined olive oil. The degree of oxidation of olive oil is reflected by its specific extinction at 232 nm and 270 nm. In fact, virgin olive oils, of good quality and correctly stored, contain very few products of oxidation; these mainly peroxides in nature, have a maximum absorption at approximately 232 nm. The values of E, at 232 and 270 nm, in such olive oils are below the maximum provided for in the standard. On the other hand, when the olive oil is treated with a decolourising agent (i.e. an absorbent earth) during the refining process, conjugated trienoic compounds are formed. These compounds have a maximum absorption situated at approximately 270 nm; this means that refined olive oils have higher values of E at 270 nm. Method The olive oil is dissolved in the required solvent and the extinction of the solution is then determined at the specified wavelengths with reference to pure solvent. Specific extinctions are calculated from the spectrophotometer readings. It was in according to Reg. EEC n. 2568/91, Annex IX. Procedure Weigh accurately 0.25 g of the sample into a 25 ml graduated flask, make up to the mark with isooctane (or cyclohexane) and homogenise. Fill a cuvette with the solution obtained and measure the extinctions at wavelength between 232 and 270 nm, using isooctane (or cyclohexane) as a reference. The extinction values recorded must lie within the range 0.1 to 0.8. If not, the measurements must be repeated using more concentrated or more dilute solutions as appropriate. Calculation Calculation of Specific Extinction at 232 and 270 nm was given by:

where: Eλ = specific extinction at wavelength λ nm; Aλ = extinction measured at wavelength λ nm; c = concentration of the solution in g/100 ml; l = thickness of the cuvette in cm.

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Determination of the variation of the specific extinction at the wavelength of maximum absorption near 270 nm, which was given by:

where: ΔE = variation of specific extinction at λm; Eλm = specific extinction at the wavelength of maximum adsorption near 270 nm; Eλm-4 and Eλm+4 = specific extinctions at wavelengths λm plus or minus 4 nm.

Limits EEC 1989/2003 K232 ≤ 2.40 K270 ≤ 0.22 ΔK ≤ 0.01

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5. Fatty acid composition Definition The aim of this determination is to establish the percentage composition of fatty acids in olive oil, more commonly known as acidic composition. The fatty acid composition of an olive oil tends to be characteristic and may be used as a means of detecting the presence of undeclared oils. Method The fatty acid composition was determined by means of preparation of the fatty acid methyl esters from the olive oil followed by capillary column gas chromatography with FID according to Reg. EEC n. 2568/91, Annex Xa and Annex Xb, modified by Reg. EEC n. 796/2002. Procedure Preparation of the methyl esters. Weigh 0.2 g of olive oil and dissolve it in 3 ml of hexane; shake vigorously and add 0.4 ml of methanolic potassium 2 N. The methyl esters formed were analysed by gas chromatography on polar column (30 m × 0.25 mm i.d.). Peak identification. The order in which the fatty acid methyl esters appear on the chromatogram is a direct function of their number of carbon atoms. The unsaturated esters are eluted after the corresponding saturated esters and their elution is a direct function of the number of double bonds. The individual methyl esters are then identified by their retention times, which are compared with the retention times of the reference portions. Quantitative analysis. Calculate the areas of each peak using the electronic integrator. The percentage for each fatty acid is calculated from the ratio of the area under the corresponding peak to the sum of the areas under all the peaks using the formula:

where: Ax = peak area for fatty acid x; Σ A = total of all the peak areas.

Limits EEC 1989/2003 (%)

C14:0 (Myristic acid) ≤ 0.05 C16:0 (Palmitic acid) 7.5 - 20.0

C16:1(Palmitoleic acid) 0.3 - 3.5 C17:0 (Heptadecanoic acid) ≤ 0.3 C17:1 (Heptadecenoic acid) ≤ 0.3

C18:0 (Stearic acid) 0.5 - 5.0 C18:1 (Oleic acid) 55.0 - 83.0

C18:2 (Linoleic acid) 3.5 - 21.0 C18:3 (Linolenic acid) ≤ 0.9 C20:0 (Arachidic acid) ≤ 0.6 C20:1 (Eicosenoic acid) ≤ 0.4

C22:0 (Behenic acid) ≤ 0.2 C24:0 (Lignoceric acid) ≤ 0.2

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6. Sterols composition Definition Sterols make up sizeable part of the unsaponifiable matter. The use of individual sterols combined with the total sterols are very effective in the detection of olive oil adulteration with other vegetable oils. 6.1 1st Method The fatty substance, with added a-cholestanol as an internal standard, is saponified with potassium hydroxide in ethanolic solution and the unsaponifiables are then extracted with ethyl ether. The sterol fraction is separated from the unsaponifiable extract by chromatography on a basic silica gel plate. The sterols recovered from the silica gel are transformed into trimethyl-silyl ethers and are analysed by capillary-column gas chromatography in according to Reg. EEC n. 2568/91, Annex V. Procedure Preparation of the unsaponifiables. Using the 500 µl microsyringe introduce into the 250 ml flask a volume of 0.2% a-cholestanol solution in chloroform containing an amount of cholestanol corresponding to approximately 10% of the sterol content of the sample aliquot taken for the determination. Evaporate to dryness in a current of nitrogen and then weigh accurately 5 g of the dry filtered sample into the same flask. Add 50 ml of 2 N ethanolic potassium hydroxide solution, fit the reflux condenser and heat to gentle boiling on a water bath with continuous vigorous stirring until saponification takes place (the solution becomes clear). Continue heating for a further 20 minutes, then add 50 ml of distilled water from the top of the condenser, detach the condenser and cool the flask to approximately 30ºC. Transfer the contents of the flask quantitatively into a 500 ml separating funnel using several rinses of distilled water, amounting in all to about 50 ml. Add approximately 80 ml of ethyl ether, shake vigorously for approximately 30 seconds and allow to settle. Separate off the lower aqueous phase collecting it in a second separating funnel. Perform two further extractions on the water-alcohol phase in the same way using 60 to 70 ml of ethyl ether on each occasion. Pool the ether extracts into a single separating funnel and wash with distilled water (50 ml at a time) until the wash water gives a neutral reaction. When the wash water has been removed, dry with anhydrous sodium sulphate and filter on anhydrous sodium sulphate into a previously weighed 250 ml flask, washing the funnel and filter with small quantities of ethyl ether. Distil the ether down to a few ml, then bring to dryness under a slight vacuum or in a current of nitrogen, completing drying in a stove at l00ºC for approximately a quarter of an hour, and then weigh after cooling in a desiccator. Separation of the sterol fraction. Immerse the silica gel plates completely in the 0.2 N ethanolic potassium hydroxide solution for 10 seconds, then allow to dry in a fume cupboard for two hours and finally place in a stove at 100ºC for one hour. Remove from the stove and keep in a calcium chloride desiccator until required for use (plates treated in this way must be used within 15 days). Place a toluene-acetone mixture in the plate-developing chamber 95:5 (V/V) to a depth of approximately 1 cm. As an alternative a 65:35 (V/V) hexane/ethyl ether mixture may be used. Close the chamber with the appropriate cover and leave thus for at least half an hour so that liquid-vapour equilibrium is established. Strips of filter paper dipping into the eluent may be placed on the internal surfaces of the chamber. This reduces developing time by approximately one-third and brings about more uniform and regular elution of the components. Prepare an approximately 5% solution of the unsaponifiables in chloroform and, using the 100 µl microsyringe, streak a chromatographic plate with 0.3 ml approximately 2 cm from one end in a streak which is as thin and as uniform as possible. In line with the streak place 2 to 3 µl of the sterol reference solution at one end of the plate so that the sterol band can be identified after developing. Place the plate in the developing chamber. The ambient temperature should be maintained between 15 and 20ºC. Immediately close the chamber with the cover and allow to elute until the solvent front reaches approximately 1 cm from the upper edge of the plate. Remove the plate from the developing chamber and evaporate the solvent in a flow of hot

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air or by leaving the plate for a short while under a hood. Spray the plate lightly and uniformly with the 2,7-dichlorofluorescein solution. When the plate is observed under ultraviolet light the sterol band can be identified through being aligned with the stain obtained from the reference solution. Mark the limits of the band along the edges of the fluorescence with a black pencil. Using a metal spatula scrape off the silica gel in the marked area. Place the finely comminuted material removed into the filter funnel. Add 10 ml of hot chloroform, mix carefully with the metal spatula and filter under vacuum, collecting the filtrate in the conical flask attached to the filter funnel. Wash the residue in the flask three times with ethyl ether (approximately 10 ml each time), collecting the filtrate in the same flask attached to the funnel, evaporate the filtrate to a volume of 4 to 5 ml, transfer the residual solution to the previously weighed 10 ml test tube, evaporate to dryness by mild heating in a gentle flow of nitrogen, make up again using a few drops of acetone, evaporate again to dryness, place in a stove at l05ºC for approximately 10 minutes and then allow to cool in a desiccator and weigh. The residue contained in the test tube consists of the sterol fraction. Add the silylation reagent, consisting of a 9:3:1 (V/V/V) mixture of pyridine/hexamethyl disilazane/trimethyl chlorosilane in the ratio of 50 µl for every milligram of sterols to the test tube containing the sterol fraction, avoiding any uptake of moisture. Stopper the test tube, shake carefully (without overturning) until the sterols are completely dissolved. Stand for at least 15 minutes at ambient temperature and then centrifuge for a few minutes. The clear solution is ready for gas chromatographic analysis. Gas chromatographic analysis. The operating conditions of gas chromatographic analysis were:

Column's temperature 260°C ± 5°C Evaporator's temperature 280°C Detector's temperature 290°C

Linear speed of the transport gas: helium 20 + 35 cm/s Linear speed of the transport gas: hydrogen 30 + 50 cm/s

Splitting's relationship from 1:50 to 1:100

Instrumental sensibility rom 4 to 16 times the minimal attenuation

Recording sensibility 1 + 2 mV Speed of the paper 30 + 60 cm/h

Amount of injected substance 0,5 + 1 µl of TMSE solution Peak identification. Identify individual peaks on the basis of retention times and by comparison with mixture of sterol TMSE analysed under the same conditions. The sterols are eluted in the following order: cholesterol, brassicasterol, 24-methylene cholesterol, campesterol, campestanol, stigmasterol, Δ-7-campesterol, Δ-5,23-stigmastadienol, clerosterol, ß-sistosterol, sitostanol, Δ-5-avenasterol, Δ-5,24-stigmastadienol, Δ-7-stigmastenol, Δ-7-avenasterol. Quantitative analysis. Calculate the areas of the a-cholestanol and the sterol peaks using the integrator. Calculate the concentration of each individual sterol in mg/kg of fatty material as follows:

where: Ax = peak area for sterol x, in square millimetres; As = area of the a-cholestanol peak, in square millimetres; ms = mass of a-cholestanol added, in milligrams; m = mass of the sample used for determination, in grams.

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Record individual sterol concentrations as mg/kg of fatty material and their sum as "total sterols". Calculate the percentage of each individual sterol from the ratio of the relevant peak area to the total peak area for sterols.

where: Ax = peak area for x;

ÓA = total peak area for sterols.

Limits EEC 1989/2003 (%) of total sterols

holesterol ≤ 0.5 rassicasterol ≤ 0.1 ampesterol ≤ 4.0 igmasterol ≤ CAMPESTEROL -7 - stigmastenol ≤ 0.5 etasitosterol + �Δ - 5avenaster + 5.23 stigmastadienol + clerosterol + tostanol + Δ 5.24 stigmastadienol

≥ 93.0

otal Sterol content ≥ 1000 (ppm) 6.2 2nd Method Separation and determination of sterols in olive oil by HPLC-MS. [1] The sample were saponified by refluxing with 2 N ethanolic KOH and the unsaponificable fraction was extracted with diethyl ether. This fraction was subjected to thin layer chromatography (TLC) on silica gel plates and then the band of sterols was isolated and extracted with methanol. Sterols were quantified by LC-MS. Procedure 5 g of olive oil sample was saponified by refluxing with 50 ml of ethanolic solution of KOH 2 M for 1 h. After cooling at room temperature, 100 ml of water were added. After phase separation in a separation funnel, the aqueous phase was washed three times with diethyl ether. Finally, the diethyl fractions were collected and washed with water and dried with sodium sulphate anhydrous, filtered and then evaporated to dryness using a rotary evaporator at reduced pressure, and the residue (the unsaponificable material) was dissolved in chloroform (1 ml). The extract (300 μl) was subjected to thin layer chromatography (TLC) on silica gel plates yielding different separated bands corresponding to the different classes of minor components. Then the band of sterols was isolated and extracted with methanol (25 ml) and subsequent liquid chromatographic analysis. Sterols in the unsaponificable fractions of the olive oil sample were analysed by HPLC-MS. The compounds were isolated with a gradient of acetonitrile/water (0.01% acetic acid) at a flow of 0.5 ml/min. The gradient LC conditions were: the mobile phase was water (acetic acid 0.01%) (Phase A) and acetonitrile (Phase B) and the solvent gradient changed according to the following conditions: from 0 to 2 min, 30% (A):70% (B) to 0% (A) :100% (B); this value was maintained for 28 min and then, from 30 to 31 min, 0% (A): 100% (B) to 30% (A): 70% (B) using this percentage during 4 min, and the run was ended. Analytes were detected with an APCI probe in the positive mode.

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7. Olive oil sensory quality Sensory attributes of olive oils are usually evaluated through the so-called panel test. Positive and negative attributes are due to the presence of particular compounds such as polyphenols, aldehydes, terpenes, oxidation products etc. The sensory components of olive oil are: aroma, flavour, pungency, bitterness. Sensory assessment is based on the positive and negative descriptors of olive oil sensory components. Positive descriptors are fruity, green, citrus, spicy, fragrant, tropical, soft, over-ripe, bitter and pungent, and they are determined by the quality of fruit produced on the tree. Negative descriptors are caused by human error and include, fusty, musty, muddy, winey, metallic, rancid, burnt and others. According to the IOC standards, extra virgin olive oil, should not have any sensory defects and should have some fruitiness. Sensory assessment begins with stipulated tasting conditions, then assessment of the aroma by sniffing followed by tasting to determine flavour, pungency and bitterness. Panel because human perceptions of tastes and smells can be personal and subjective, sensory assessment is usually done by a panel of 8–12 people who are trained to IOC standards and led by a panel leader. The panel leader co-ordinates activities and educates members in perception, descriptors, etc.

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8. 1H and 13C NMR Introduction High resolution NMR plays an important role in olive oil analysis, in fact it can be used for the following purposes: authentication purpose, that is detection of the fraudulent addition of hazelnut oil and other non-olive oils to the olive oil; determination of geographical origin of olive oils; study of the effect of climatic condition on the olive oil's composition. The attractive feature of this technique is that qualitative and quantitative information can be obtained on a wide range of chemical species in a single experiment and in a very short time. Free acidity The 1H NMR spectrum of an olive oil does not allow one to obtain the measurement of free acidity directly; however, it is possible to have an indirect measurement of it using the measurable amount of diglycerides. The 1H NMR spectrum shows the resonances of sn 1,2 and sn 1,3- diglycerides. The total glycerides profile and the 1,2 : 1,3 ratio are strongly related to the quality and the freshness of olive oils. [2-3] Fatty acid profile The determination of fatty acid profile, obtained by gas chromatographic analysis, give the full composition of fatty chains without information about the fatty chains distribution on glycerol; the high-resolution 13C NMR spectroscopy is able to provide valuable information about the acyl composition and the sn (strictly numbered) -1,3 and sn-2 acyl positional distribution of glycerol. The common unsaturated fatty acids (oleic, linoleic and linolenic) in an olive oil can be quantified using 1H NMR. This method utilises the area per proton, determined by integration. [4] The high resolution spectroscopy allows the determination of the fatty acid compositions without distinguishing, however, the homologous chains, i.e. C16:0 and C18:0 that appear as a single resonance, and their distribution between the 1(3)- and 2- glycerol positions (no differentiation of the sn-1 and sn-3 positions is achieved). The compositional data for 1(3)- and 2- positions appeared to be correlated to differences in fatty acid compositions of the whole olive oil triglyceride, whereas acyl chain 2-specificity seemed not to be strictly correlated to oleyl and linoleyl chain concentration. [5] Unsaturated fatty acids The degree of unsaturation in olive oils is an important indicator of the development of rancidity. The conventional method used to determine the total degree of unsaturation is the measure of the Iodine Number (IN), that consists in the quantitative reaction of iodine to fatty acids double bonds. The IN of unsaturated fatty acids is not a quantitative measure; it is an empirical number that is useful in defining the total amount of unsaturation. From a 1H NMR spectrum one can obtain a quantitative measure of the unsaturation. All resonances of trans double bonds lie between 5 and 5.2 ppm and are due to two protons; all methyl resonances, three protons, lie between 0.85 and 0.95 ppm; thus the total unsaturation number, in moles, is given by the ratio between the normalised integrals of these two spectral regions. However, by observing the spectral region 120 - 130 ppm in the 13C spectrum, we can obtain the full composition in terms of unsaturated chains as well as their position on the glycerol moiety. [2] Sterols analysis In 1H NMR spectra the signal due to methyl CH3 in position 18 of the sterols resonates between 0.6 and 0.7 ppm. The signal due to methyl 18 of β-sitosterol, resonating at 0.62 ppm, has been clearly identified in virgin olive oils. [6] Signals due to stigmasterol [2] and to cyclo-arthenol [7] have been also detected in olive oils.

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Determination of Mg-depleted chlorophyll Chlorophyll is sometimes added to improve the green colour of olive oils. In this case usually the added product is defective, lacking the co-ordinated magnesium and contains NH groups in the centre of the macro-cycle of the porphyrin structure. In this case, the 1H NMR spectrum of olive oil shows a broad signal at -0.42 ppm indicating adulteration of the product. [2] Phenolic compounds Virgin olive oil is particularly rich in these antioxidants, that contribute in a characteristic way to olive oil sensory qualities, since they are responsible for its bitter and pungent flavour. The bitter taste is particularly related to the presence of oleuropein glucoside and its aglycon. The content of phenolic compounds in olive oils is heavily affected by the variety, origin and maturity degree of olives. 13C NMR spectroscopy cannot be directly used for the analysis of minor compounds due to its low sensitivity. However, 13C-NMR spectroscopy of the polar fraction extracted from virgin olive oils permits to calculate the ratio of free and esterified tyrosol + hydroxytyrosol comparing the intensities of C1 carbon of free tyrosol + hydroxytyrosol with the C1 signal of the corresponding esterified compounds. Aldehydes and volatile compounds The aldehydic compounds are important in the evaluation of olive oil quality for two reasons: first of all they permit to follow the olive oils oxidative processes; secondly, due to their volatility, they play an important role in the sensory attributes of olive oils. The oxidation process occurs in two stages: in the first stage of thermal oxidation, the fatty acids take oxygen forming hydroperoxides. When the hydroperoxide concentration reaches a certain level, and when the thermal process is intensified, second oxidation occurs with the formation of aldehydic compounds that are responsible of the undesirable rancid flavour. These aldehydes can be easily detected in the low-field region of the 1H NMR spectrum [8] and can be used to follow the oxidation process of an olive oil and to evaluate its oxidative stability. The composition of volatiles plays an important role in defining the sensory quality of virgin olive oils. Positive (green-fruit odour, etc.) and negative attributes (bitter tasting, etc.) have been correlated with the presence of aldehydes, alcohols, polyphenols, terpenes and acetic acid. The very low concentration of these compounds in virgin olive oil makes possible their direct determination only by high field (500-600 MHz) 1H NMR. [2] Glycerol Esters The evaluation of the total amount of the diacylglycerols and the ratio between 1,2 and 1,3- diacylglycerols can be useful to detect the freshness of the olive oils and to reveal the presence of partially neutralised oils to olive oils that are given out as "virgin". A quantitative 13C-NMR study carried out on olive oil showed that significant differences in diacylglycerols content occurred among cultivars [9], probably due to its relation with maturation stage. In fact it is observed that lower total diacylglycerol content is characteristic of late maturing cultivars, whereas early maturing fruits produce higher levels of total diacylglycerides. NMR tube preparation The sample is prepared by dissolution of an amount of olive oil in an appropriate solvent, in specific proportions, usually in a 5 mm diameter NMR tube. The solvents most commonly used are deuterated chloroform [10], carbon tetrachloride, deuterated dimethyl sulfoxide or mixtures of the first with one of the two latter or with deuterated methanol. The addition of deuterated dimethyl sulfoxide to dichloromethane has been recommended for a better dissolution of olive oil minor components [6]. The chemical shift of an atom or a group of atoms is measured with respect to a reference compound which for 1H is tetramethylsilane (TMS). This compound is usually added to the sample at a concentration near 0.03%.

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9. IRMS Introduction Isotope ratio mass spectrometers (IRMS) are specialised mass spectrometers that produce precise and accurate measurements of variations in the natural isotopic abundance of light stable isotopes. Due to its chemical composition, olive oil can be characterised by 13C, 18O, and 2H of organic matter. Stable carbon isotope analyses have proven to be a powerful tool for assessing the purity of vegetable products from plants of different photosynthetic pathways. In fact, isotopic discrimination against the heavier carbon isotope (13C) occurs during photosynthesis and is reflected in the isotopic compositions of the plant tissues and products. The isotopic measurements are expressed in the usual (δ) notation as the per thousand (‰) deviations of the isotope ratio of a sample relative to that of a standard:

where X = 13C or 18O and R = 13C/12C or 18O/16O. The standard is the Peedee belemnite limestone (PDB) that has a defined value of 0.0‰ on the δ scale. The international standard for 18O is standard mean ocean water (SMOW). The most important atmospheric carbon dioxide-fixing reactions are the C3 and C4 pathways. The C3 plants use the Calvin cycle (ribulose-1,5-biphosphate carboxylase) for CO2 fixation, and the first metabolite has three carbons. All trees operate with C3 pathway, and their carbon isotope compositions fall into the range -22 to -34‰. The C4 plants use the Hatch-Stack cycle and are isotopically heavier (-6 to -23‰). Food chemists are increasingly using the instrumentation of GC/C/IRMS for carbon isotope analysis of the individual lipids as a tool for assessing adulteration of olive oils [11-12]. The isotopic results enable a distinction between genuine extra virgin olive oil and the other quality olive oils, and also serve as a way of assessing if the olive oil was blended with refined olive oils or other vegetable oils. The 13C/12C of whole olive oils and of the aliphatic alcohols and sterols has been determined [13-15]. The 18O/16O of the whole olive oil by pyrolysis-IRMS [16] have been proposed for the characterisation of olive oils. The combined 18O/16O and 13C/12C isotopic profiles provide information about the geographical origin of samples. [17] Method Bulk samples are combusted with oxygen in an elemental analyser and passed through various oxidation and reduction reactors to remove excess of oxygen and to reduce the nitrous (NOx) products to elemental nitrogen (N2). The gases are then separated in a short GC column and analysed on a continuos flow stable isotope ratio mass spectrometer (IRMS).

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10. Metals Introduction The determination of trace of elements in olive oils is important because of both the metabolic role of metals and possibilities for adulteration detection and olive oil characterisation. The presence of metals in olive oils may be due to: natural contamination and introduction of metals during the refining process and contact with the storage material. Trace levels of metals like Fe, Cu, Ca, Mg, Co, Ni and Mn are known to increase the rate of olive oil oxidation, even at levels of µg/g, while other elements such as Cr, Cd and Pb are very important on account of their toxicity and metabolic role. The oxidation processes cause premature rancidity of the olive oil and may generate peroxides, aldehydes, ketones, acids, epoxides and other compounds. 10.1 1st Method The most commonly used technique for the determination of metals in olive oil samples is based on a simple and rapid method for the simultaneous determination of various metals in olive oil by the formation of emulsions and determination by ICP-MS. Procedure Sample solutions were prepared gravimetrically: portions of around 2 g of olive oil were weighed in a 100 ml flask and mixed with 1.5 ml of Triton X-100 emulsifer. The mixture was stirred mechanically until the solution was homogeneous. An aqueous solution of In was then added so that the final concentration was 100 μg/l and the resulting solution had a final weight of 50 g with distilled water. It was stirred mechanically for 5 min and then placed in an ultrasonic bath for another 5 min. The samples were prepared adding a multielemental solution containing the different metals (Ba, Cd, Co, Cr, Cu, Mn, Ni, Pb and Zn) so that the final concentration were 100 and 200 μg/l. When 1.5 ml of Triton was added after mechanical stirring for 5 min and sonication for a further 5 min, the emulsion was formed and remained stable for at least 24 h. The semiquantitative determination of Ba, Cd, Co, Cr, Cu, Mn, Ni and Pb in olive oil was carried out by ICP-MS with direct introduction of the emulsions into the plasma after optimisation of the instrumental parameters (power, pump flow, nebulizer gas flow, use of auxiliary oxygen flow). [18] Detection limits The detection limits were calculated by 3 times standard deviation of the blank. The measurements of the blank were made in order to calculate the deviation. The blanks were prepared in the same way as the samples, but without adding olive oil. The detection limits are in a range of 0.2-8 ng/g for most of the elements. It is noteworthy that the detection limit for Cr is slightly higher (50 ng/g), due to Cr contamination of Triton X-100. [18]

Metals Detection limits in the determination

of olive oil emulsions by ICP-MS (ng/g)

Cr 50 Mn 3.5 Co 0.75 Ni 6 Cu 7.7 Cd 1 Ba 1.7 Pb 3.2

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10.2 2nd Method Methodology for determination of trace metals in olive oil using ICP-AES. [19] Procedure Samples underwent acid decomposition using HNO3 and H2O2 in a microwave oven followed by determination of the following trace metals (Ag, Al, B, Ba, Bi, Ca, Cd, Co, Cr, Cu, Fe, Ga, Mn, Mg, Ni, Pb, Te and Zn). Data were treated using cluster analysis to determine similarities and differences among them.

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11. Phenolic compounds Introduction Olive fruit contains simple and complex phenolic compounds. Most of these compounds pass into the olive oil, increase its oxidative stability and improve the taste. The three phenolic compounds in highest concentration in olive oil are the glycoside oleuropein, hydroxytyrosol (3,4-dihydroxyphenyl ethanol) and tyrosol. The phenol content and the specific composition of these phenols in olive oil depend on the altitude where the olive trees are grown, on the harvesting time and on the processing conditions. 11.1 1st Method Phenolic compounds analysis in olive oils was carried out by Solid-Phase Extraction, HPLC-DAD and HPLC-MS/MS. [20] Procedure Extraction of phenolic compounds. To clean and concentrate the sample, the polar fraction was obtained from 3 g of olive oil sample using an SPE diol cartridge (Vac RC 500 mg, Waters, Milford, MA). The cartridge was activated with 6 ml of n-hexane, 6 ml of methanol/water mixture (80:20) and 3 ml of acetonitrile. The olive oil was dissolved in 6 ml of n-hexane and percolated into the cartridge. To remove the non polar fraction, the olive oil was washed under vacuum with 10 ml of n-hexane. Afterward, phenolic compounds were eluted with 8 ml of methanol/water mixture and 4 ml of acetonitrile. During the entire process, the vacuum was maintained at less than 30 kPa. The eluent was evaporated to 2 ml by a rotary evaporator, and the temperature was always controlled (<40°C), to avoid the deterioration of phenols. The samples were then filtered through a 13 mm PTFE 0.45 μm membrane filter from Waters. Filters were checked after various retention assays of the phenolic compounds with olive oil extracted samples. After this, 20 μl was injected into the liquid chromatograph. The entire process was performed in conditions of darkness and with brown glass material. HPLC-DAD conditions and quantification. The column was kept at 40°C. The mobile phase consisted of a binary solvent system using water acidified with 0.1% formic acid (solvent A) and 100% acetonitrile (solvent B), kept at a flow rate of 0.5 ml/min. The gradient program started with 90% eluent A and 10% eluent B, which ramped linearly to 25% in 12 min. This percentage was maintained for 7 min, and eluent B was ramped again linearly to 40% at 30 min and to 60% at 40 min. Initial conditions were reached in 5 min, so the total run time was 45 min. Detection and quantification were performed at 280, 240 and 320 nm. Each phenolic compound was expressed with its standard when it was available. Due to the absence of standard for all of the compounds detected, both phenolic alcohols 3,4-dihydroxyphenyl and 4-hydroxyphenyl were expressed as tyrosol, secoiridoids and elenolic acid were expressed as oleuropein, and unknown flavonoids were expressed as luteolin. HPLC-MS conditions. The HPLC conditions were as described for the HPLC-DAD system. All of the analyses used the turbo ion spray source in negative mode with the following settings:

Capillary voltage -3500 V Nebulizer gas (N2) 10 (arbitrary units) Curtain gas (N2) 12 (arbitrary units)

Collision gas (N2) 4 (arbitrary units) Declustering potential (DP) -30 V

Focusing potential -250 V Entrance potential 10 V

Collision energy (CE) -30 V

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Drying gas was heated to 300°C and introduced at a flow-rate of 5000 cm3/min. Full-scan data were acquired by scanning from m/z 100 to 800 in profile mode using a cycle time of 2 s with a step size of 0.1 u and a pause between each scan of 2 ms. In product ion scan experiments, MS/MS product ions were produced by collision-activated dissociation (CAD) of selected precursor ions in the collision cell of the triple quadrupole mass spectrometer and mass-analysed with the second analyser of the instrument. 11.2 2nd Method Phenolic compounds analysis in olive oils was carried out by Coulometric Electrode Array System. [21] Procedure Phenolic extraction. After centrifugation of olive oil with N,N dimethylformamide (DMF), the organic solvent was separated from the lipid phase by using a Pasteur pipet. An internal standard (syringic acid) was added to the pooled DMF extract, and the residual olive oil was eliminated by washing with hexane. Traces of the solvent were removed by flushing nitrogen for 10 min. Finally, the ~2 ml of DMF extract was diluted with 2 ml of methanol and centrifuged, and 20 μl was injected into the chromatograph. HPLC analysis of phenolic compounds using electrochemical detection. The mobile phases were a 30 mM LiClO4 solution (pH adjusted to 3.1 with HClO4) and methanol containing 30 mM LiClO4. The elution profile was the same as used for UV detection. The detector was an ESA coulometric system, which consisted of one cell containing four electrodes in series. 11.3 3rd Method Phenolic compounds analysis in olive oils was carried out by direct injection in High-Performance Liquid Chromatography with Fluorometric Detection. [22] Procedure 2 g of olive oil was dissolved in 10 ml of acetone and then this solution was filtered through a PVDF syringe filter (0.2 μm). The injected sample volume was 20 μl. The C18 columns used were a ChromSep Inertsil ODS-3 250 mm × 4.6 mm column with a particle size of 5 μm with a ChromSep guard column 100 mm × 3.0 mm (Varian Inc., Walnut Creek, CA) and a Spherisorb ODS-1 250 mm × 4.6 mm column with a particle size of 5 μm (Phase Separation Ltd., Deeside, United Kingdom). They were operated with the same mobile phase that was composed of 0.2% acetic acid (pH 3.1) in water (solvent A)/methanol (solvent B) at a flow rate of 1.5 ml/min when the ChromSep Inertsil ODS-3 column was used and 1 ml/min when the Spherisorb ODS-1 was utilised; for both columns, the gradient changed as follows: 95% A/5% B for 2 min, 75% A/25% B in 8 min, 60% A/40% B in 10 min, 50% A/50% B in 16 min, and 0% A/100% B in 14 min; this composition was maintained for 10 min, then returned to initial conditions and equilibration in 13 min; the total running time was 73 min. The FLD was operated at an excitation wavelength set at 280 nm and emissions at 313, 339, 353 and 453 nm. The photomultiplier (PMT) gain of the FLD was set to 13 to increase its sensitivity. The acquisition of chromatographic data was done during 60 min from the beginning of the analysis. 11.4 4th Method Phenolic compounds analysis in olive oils was carried out by enzymatic assay. [23] This enzymatic assay adopts a substrate-recycling procedure that employs tyrosinase together with NADH as a reducing agent. Tyrosinase (polyphenol oxidase, EC 1.14.18.1) is a multifunctional copper-containing enzyme that catalyses both the hydroxylation of phenols and the oxidation of catechols to quinones. In the presence of excess NADH, the resulting quinone is reduced to

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catechol; the rate of NADH oxidation is directly related to the phenol/catechol content, thus allowing the estimation of the total substrate amount. Procedure After extraction of polypenols from olive oils, an aliquot of the extract solution was diluted 1:20 with 7% acetic acid. 40 μl of this diluted solution was treated with 20 EU of tyrosinase and 150 μM NADH in 1 ml of 50 mM phosphate buffer, pH 7.4. Reaction was followed by measuring the rate of NADH oxidation to NAD+ at 340 nm. 11.5 5th Method Determination of total phenol content of olive oil colorimetrically using the Folin Ciocalteau reagent. [24] Procedure The total phenol content of the methanol extracts was evaluated colorimetrically using the Folin Ciocalteau reagent, expressed as gallic acid, was obtained as follows: a diluted extract (0.5 ml of 1:10 v/v) or phenilic standard was mixed with Folin-Ciocalteau reagent (5 ml 1:10 diluted with Nanopure water) and acqueous Na2CO3 (4 ml 1 M). Solutions were maintained at room temperature for 60 min and the total polyphenols were determined colorimetrically at 725 nm. Gallic acid standard solutions were used to calibrate the method. 11.6 6th Method Polyphenols were determined according to the methodology proposed by Kiritsakis. [25] Procedure Olive oil samples were dissolved in n-hexane and extracted, three times, with a water/methanol mixture (60:40 v/v). Total polyphenols were determined by adding the Folin-Ciocalteau reagent and a 20% sodium carbonate solution to a suitable aliquot of the combined extracts. After centrifugation, the absorbance at 725 nm was measured. Results are given as mg/kg of caffeic acid.

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12. Volatile compounds Introduction Flavour and aroma of virgin olive oil are generated by a number of volatile constituents that are present at extremely low concentrations. The volatile fraction of virgin olive oils includes saturated, unsaturated, aromatic and terpenic hydrocarbons, as well as aldehydes, alcohols, ketones, esters, ethers, furans and other compounds. The volatile composition of olive oil depends on the levels and activity of the enzymes involved in the various pathways. Other factors that influence the volatiles are ripening cycle of the fruit. 12.1 1st Method Volatile compounds analysis was carried out by dynamic headspace thermal desorption. [26] Procedure Dynamic headspace thermal desorption. The Carbotrap-300 and Tenax-TA traps of 11.5 cm × 6 mm dimensions (Supelco, Bellefonte, PA) were used, after preconditioning for 8 h at 350ºC and purging for 30 min with nitrogen gas, prior to use. Stripping of volatile compounds from olive oil, placed in 250 ml GWB (Gas wash bottles) with a fritted dispersion glass tube, was performed by the dynamic headspace trapping and desorption technique. Dry nitrogen gas was bubbled into the bottles containing either 80, 160 or 240 g of olive oil. The applied gas flow rates were either 100 or 200 ml/min, while the stripping times were 10, 20, 40, 80 or 100 min. During the stripping process GWB remained in a water bath at 37ºC while the fitted glass traps at the outlet of the bottles were maintained at 23ºC. A Dynatherm 1000 (Dynatherm Analytical Instruments Inc., Kelton, PA) apparatus was used as another means for stripping olive oil volatiles in traps for further analysis with the DHS-TD technique. Helium gas was used as a stripping gas. Olive oil samples of 1, 3 and 6 g were placed in the 9 ml glass-stripping receptacle. Samples were preheated for 5 min at 37ºC and purged for 15 or 30 min applying 100 or 200 ml/min flow rate. Dry helium flowed through the traps for 1 min at 25 ml/min to remove any possible moisture. The desorption of the compounds stripped from the olive oil placed in both GWB and Dynatherm apparatus and retained by the traps, was performed by a Dynatherm desorption unit Model 890. The desorption unit was connected to a gas chromatography by a transfer line. Helium at 7 ml/min and 750 kPa was flowing to desorb the molecules from the trap and convey them into the chromatographic column. During desorption traps were kept at 300ºC for 8 min. All the transfer lines and the valves were maintained at 230ºC to avoid condensation of volatiles. Before using the traps again, they were cleaned by heating them for 30 min at 320ºC while purged with helium. The chromatographic analysis for the DHS-TD technique implemented a Hewlett Packard 5890 Series II GC (Hewlett Packard, Philadelphia, PA) with a 30 m × 0.32 mm ID × 0.25 μm film thickness, fused silica SPB-5, capillary column from Supelco (Bellefonte, PA). A FID detector was used for quantifying the volatile compounds separated by both GWB and Dynatherm apparatus. Integration of chromatographic peaks was performed using a Hewlett Packard HP 3395 integrator (Hewlett Packard, Philadelphia, PA). Conditions used for the analysis were: initial temperature, 35ºC for 1 min, increased to 80ºC at a rate of 3ºC/min, held for 1 min, then increased to 180ºC at 10ºC/min, held for 1 min, and finally up to 260ºC at 4ºC/min where held for 10 min. Carrier gas was Helium at 88 kPa (8 psi) flowed at 1.75 ml/min at 40ºC. Calibration curves were obtained by injecting known amounts of standard compounds in the gas-washing bottle. Selected standard compounds were hexanal, 2-hexenal, nonanal, 2-nonenal, 2-nonanone, acetaldehyde, ethyl butyrate, methylbutanal, hexene, octanal, 2,4-decadienal, butanal, pentanal, 1-pentanol, heptanal, 2-decenal, 2-heptenal, decanal, 2-butylfuran, hexyl acetate and 3-pentanone (Sigma–Aldrich, St. Louis, MO, USA). Ion trap-mass spectrometry (ITMS) was used to identify compounds from the extra virgin olive oil retained in the traps by the DHS-TD procedure. A Varian 2000 mass spectrometer (Varian, TX, USA) was interfaced with the Dynatherm desorption unit operating at the aforementioned

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conditions. Tuning values for the ITMS were 100 using cedrol as tuning standard. Tune sensitivity: 9000. The acquisition parameters (full scan) were:

Scan range 41–300 amu Scan time 1.0 s Threshold 1 count

From 1500 to 2300 V depending on multiplier conditions Multiplier

Transfer line temperature 240ºC Exit nozzle 240ºC Manifold 240ºC

12.2 2nd Method Volatile compounds analysis was carried out by Solid Phase Micro-Extraction – Gas Chromatography/Mass Spectrometry (SPME – GC/MS). [27-28] Two kinds of SPME fibers were used:

1. polydimethylsiloxane (PDMS), 100 μm (non polar compounds); 2. PVB/CAR/PDMS, 50/30 μm (polar and non polar compounds).

12.2.1 1st Procedure SPME procedure was performed using the polydimethylsiloxane (PDMS) 1 cm long–100 μm phase thickness, and the polydimethylsiloxane-divinylbenzene (PDMS-DVB) 1 cm long-65 μm fibers (Supelco, Bellefonte, PA). Fibers were preconditioned at 250ºC for 1 h. During sampling fibers were exposed for 10, 20, 30 and 40 min to the headspace of tightly closed 12 ml glass vials containing 3.5, 7 or 10.5 g of olive oil. Before sampling, the vials were allowed to equilibrate for 2, 4 and 6 h at 23ºC. Sampling was performed at the same temperature. Rubber septum closures with a Teflon liner were used to close the vials. No agitation or stirring were applied to the vials during equilibrium or sampling. All analyses were performed at least in triplicate. Desorption of volatile compounds from the SPME fibers was performed by placing the fibers into the injection port of the GC and exposing them to a helium flow at 250ºC for 2 min. Volatiles were cold trapped by liquid nitrogen at the beginning of the GC column. At the end of the 2 min period the fibers were retracted out of the injection port, the liquid nitrogen was removed, the GC door closed and the chromatographic program was initiated. The gas chromatography-time-of-flight mass spectrometry (GC-TOFMS) was used to separate and identify the compounds isolated by SPME. A Hewlett- Packard HP-6890 gas chromatography (Hewlett Packard, Philadelphia, PA), equipped with a fused silica capillary column HP-5 (Hewlett Packard, Philadelphia, PA) 5.00 m × 0.1 mm ID, and 0.34 mm coating thickness was used. Carrier gas was 99.999% purity helium flowing at a rate of 0.5 ml/min. Temperature program was: 1.5 min at 35ºC, raised to 250ºC at 50ºC/ min and held for 2 min. The GC/MS transfer line was kept at 220°C. The detection and identification of volatile compounds was carried out in a Leco (Leco Inc., St. John, MI, USA) electron impact ionisation. Time of Flight detector model FCD-650. Mass spectra were collected at a rate of 40 spectra/s over a range of m=z 40–300 with ionisation energy of 70 eV. 12.2.2 2nd Procedure [29] The fibre used for the extraction of the volatile components was divinylbenzene/ carboxen/polydimethylsiloxane (DVB/CAR/PDMS) 50/30 μm. Before use, the fibre was conditioned. The olive oil (5 g) was placed in a 20 ml vial closed by PTFE/silicone septum (Interchim). Before extraction, the stabilisation of the headspace in the vial was accomplished by equilibration for 60 min at 25°C. The extraction was carried out at 25°C (room temperature). To

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determine the optimal adsorption time of the fibre with the sample headspace, the fibre DVB/CAR/PDMS was exposed for time periods of 10, 30, 60, 90 and 120 min. After extraction, injections were performed using a SPME autosampler (CTC Analytics, Swiss). The fibre was thermally desorbed into a GC and left in the injection port (equipped with a 0.75 mm i.d. inlet liner) for 4 min. The injector was set at 250°C and operated in the splitless mode for 2 min unless otherwise stated. Before sampling, the fibre was reconditioned for 5 min in a washing port at 250°C. GC–MS analyses. Each olive oil was analysed by GC–MS using an Agilent 6890N/5973N system, with fused-silica capillary columns HP-1 (50 m × 0.20 mm; film thickness: 0.5 μm). Oven conditions were: carrier gas helium; injector temperature, 250°C, the ion source and the transfer line temperatures were 170°C and 280°C, respectively; splitless mode. GC–MS analyses on a polar column were performed on a Hewlett-Packard 5890/5970A system with HP20M capillary column (50 m × 0.20 × 0.1 μm). Injector and transfer line temperature: 220°C. Retention indices were determined with C5–C26 alkane standards as references. The mass spectra were performed at 70 eV over a mass range of 35–350 amu. The identification of the constituents was based on comparison of the retention times with those of authentic samples, on computer matching against commercial (NIST 1998, Wiley 6N, MassFinder 2.1 Library 2001) libraries built up from pure substances and MS literature data and then, confirmed by comparison of retention indices with published index data.

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13. Adulteration The following techniques are based on the vibrational transitions occurring in the ground electronic state of the molecules. 13.1 1st Method The detection and quantification of adulteration in olive oil was carried out by Raman Spectroscopy. [30] Procedure Raman spectra were excited with a near-infrared 780 nm laser with the power at the sampling point typically at 20 mW; backscattering radiation was collected by a Renishaw System 100 instrument (Renishaw plc, New Mills, Wotton-under-Edge, Gloucestershire GL12 8JR, U.K.). Each olive oil was pipetted into a 4 ml Supelco vial (Supelco Park, Bellfonte, PA). The vial was placed into a pre-fixed sample holder such that the laser was focused into the center of the vial (12 mm from the collection lens). Spectra were collected for 2 min over the spectral range 1000-3000 wavenumbers (cm-1) at a resolution of about 6 cm-1. Calibration was periodically checked by recording the position of known Raman lines of a silicon wafer (520 cm-1) and the wavenumber accuracy was estimated to be 1 cm-1. 13.2 2nd Method The detection and quantification of adulteration in olive oil was carried out by Fourier Transform Raman Spectroscopy. [31] Procedure The FT-Raman spectra were acquired on a Nicolet 910 FT-Raman spectrometer (Nicolet Analytical Instrument, Madison, WI). FT-Raman spectra were obtained by placing each sample in front of the laser and focusing the Nd: YAG laser beam onto the sample. Spectra were produced over the Raman shift 3250–0 cm-1. Typically, 200 interferograms were co-added at 4 cm-1 resolution with a sampling time of 4 min. 13.3 3rd Method The detection and quantification of adulteration in olive oil was carried out by Near Infrared Spectroscopy. [32] Procedure The NIR measurements were made using a transmittance technique in the region of 12000-4000 cm-

1 by using a Bruker Vector 22/N FT-NIR spectrometer equipped with a Ge diode detector. A total of 32 scans were collected on each sample with a spectral resolution of 4 cm-1. The quartz cell was thermostatted at 25ºC during the measurements.

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14. Chlorophylls, pheophytins, β-carotene, tocopherols and tocotrienols Introduction The colour of olive oil is mainly related to the presence of chlorophyll and carotenoids. The presence of these constituents depend on several factors, such as cultivar, soil and climate, and fruit maturation as well as applied conditions during olive fruit processing. Chlorophyll is one of the main pigments in olive oil. The chlorophyll content decreases as the fruit matures so olives picked green produce a greener oil with a "grassy" flavour. According to Apostolos Kiritsakis, fresh olive oil contains between 1 to 10 parts per million. The olive cultivar, weather, pressing method, etc. also determine chlorophyll content. Olives are invariably pressed with some leaves still present so some of the chlorophyll comes from that source. Some producers have been know to deliberately allow leaves in the mill to increase the "grassiness" of the olive oil. In the light, chlorophyll will promote formation of oxygen radicals and speed up oxidation but in the dark chlorophyll acts as an antioxidant. Chlorophylls, pheophytins, β-carotene and other carotenoids, tocopherols and tocotrienols are naturally occurring constituents found in olive oils. Chlorophyll a and b and their primary degradation products, pheophytins a and b, are green pigments that are destrable in some oils such as olive oil, but are typically removed, along with β-carotene, from other vegetable oils during processing. [33] The tocopherol homologues (α-, β-, γ- and δ- tocopherol) and the tocotrienol homologues ((α-, β-, γ- and δ-tocotrienol) are natural antioxidants that contribute to the oxidative stability of oils. The presence of these compounds is important in relation to oil stability and nutritional labeling. 14.1 1st Method High-performance liquid chromatography (HPLC) is widely used for determining chlorophyll pigments, carotenoids, tocopherols and tocotrienols in vegetatable oils. [34-35] A high-performance liquid chromatographic method was used for the simultaneous measurement of chlorophylls, pheophytins, β-carotene, tocopherols, and tocotrienols in olive oils using 2 spectrophotometers in series and one normal-phase silica column. Olive oil samples (0.5 g) were diluted in the mobile phase, isopropanol-hexane (1.5:98.5 vol/vol) in a 10 ml volumetric flask, and injected directly into the column. A programmable UV/Vis spectrophotometer was used to measure chlorophylls a and b, pheophytins a and b, and β-carotene at their appropriate absorption maxim (430, 452, 409, 433, and 452 nm, respectively) programming the detector to change the wavelength during elution; instead of α-, γ- and δ-tocopherols and α-, γ- and δ-tocotrienols were measured by fluorescence at 295 nm excitation and 330 nm emission. The minimum detection was determined to be 0.01 μg for β-carotene, 0.01 μg for chlorophyll a and b, 0.02 μg for pheophytin a and b and 0.01 μg per injection for α-tocopherol. 14.2 2nd Method [36] 7.5 g of olive oil were accurately weighed and dissolved in cyclohexane up to a final volume of 25 ml. Chlorophyll and carotenoid contents were calculated from the absorption spectra of the olive oils. The absorption at 670 nm is usually considered to be related to the chlorophylls fraction, pheophytin ‘‘a’’ being its major component, the dominant pigment in the carotenoids fraction is lutein, and the absorption is measured at 470 nm. Thus, chlorophyll and carotenoid contents were expressed as mg of pheophytin ‘‘a’’ and lutein per kg of olive oil, respectively.

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15. Oxidation stability Introduction The resulting induction time that was determined characterises the resistance of olive oils to oxidation. The Rancimat method is also called the automated Swift test or accelerated oxidation test and is, in general, used today as an automated version of the previously used and extremely complicated Active Oxygen Method (AOM). During the measurement a stream of air is passed through the oil sample contained in a sealed and heated reaction vessel. This treatment results in oxidation of the olive oil molecules in the sample, with peroxides initially being formed as the primary oxidation products. After some time the fatty acids are completely destroyed; the secondary oxidation products formed include low-molecular organic acids in addition to other volatile organic compounds. These are transported in the stream of air to a second vessel containing distilled water. The conductivity in this vessel is recorded continuously. The organic acids can be detected by the increase in conductivity. The time that elapses until these secondary reaction products appear is known as the induction time, induction period or Oil Stability Index (OSI). Method The determination of the oxidation stability of olive oils is the classical application for the Rancimat method. [37-38] Procedure Stability was expressed as the oxidation induction time (hours), measured with the Rancimat apparatus, using an olive oil sample of 3.5 g warmed to 100°C, and an air flow of 10 l/h. With this well-established methodology, the volatile oxidation products were stripped from the olive oil and dissolved in cold water, whose conductivity increased progressively. The time taken to reach a fixed level of conductivity was measured.

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16. Wax

Introduction The individual waxes are separated according to the number of carbon atoms. The method is recommended for distinguishing between olive oil obtained by pressing and that obtained from olive pomace (olive-pomace oil). Method The determination of wax content was carried out by capillary column gas chromatography according to Reg. EEC n. 183/93. Addition of a suitable internal standard to the olive oil, then fractionation by chromatography on a hydrated silica gel column. Recovery of the fraction eluted under the test conditions (which has a lower polarity than the triglycerides), then direct analysis by capillary column gas chromatography. Procedure Capillary column gas chromatography analysis. Suspend 15 g of silica gel in n-hexane and introduce into the column. Allow to settle spontaneously. Complete settling with the aid of an electric shaker to make the chromatographic band 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 and add a suitable amount of internal standard depending on the assumed wax content, e.g. add 0.1 mg of lauryl arachidate in the case of olive oil. Transfer the prepared sample to the chromatographic column with the aid of 2 ml portions of n-hexane. Allow the solvent to flow to 1 mm above the upper level of the absorbent. Percolate a further 70 ml of n-hexane to remove any n-alkanes naturally present. Then start chromatographic elution by collecting 180 ml of the n-hexane/ethyl ether mixture at 99:1, at a flow of about 15 drops every 10 seconds. Evaporate the resultant fraction in a rotary evaporator until the solvent is almost removed. Remove the last 2 ml of solvent under a weak current of nitrogen, then add 2-4 ml n-heptane. Fit the column to the gas chromatograph, 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, detector and recorder efficiency, etc.). Choice of operating conditions. The operating conditions are generally as follows: column temperature: column temperature 80°C at first 1 min, then increased to 120ºC at a rate of 30ºC/min; then increased to 340ºC at a rate of 5ºC/min; detector temperature: 350ºC; amount injected: 0.5-1 µl of n-heptane solution (2-4 ml); carrier gas: helium or hydrogen at the optimal linear speed: 20-35 cm/s; instrument sensitivity: from 4 to 16 times minimum attenuation; recording sensitivity: 1-2 mV f.s.; paper rate: 30 cm/h. Performance of the analysis. Take up 1 µl of the solution with the aid of the 10 µl micro-syringe; draw back the plunger until the needle is empty. Introduce the needle in the injection system and inject quickly after 1-2 seconds. After about 5 seconds, gently extract the needle. Perform the recording until the waxes are completely eluted. The base line must always satisfy the required conditions. Peak identification. Identify the peaks from the retention times by comparing them with mixtures of waxes with known retention times, analysed under the same conditions. Quantitative analysis. Determine the areas of the peaks corresponding to the internal standard and the aliphatic esters from C40 to C46 with the aid of the integrator. Determine the wax content of each ester, in mg/kg of fat, according to the formula:

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where: Ax = area corresponding to the peak for each single ester in square millimetres; As = area corresponding to the internal standard peak, in square millimetres; ms = mass of the internal standard added, in milligrams; m = mass of the sample taken for determination, in grams. Expression of the results. Report the sum of the contents of the different waxes from C40 to C46 in mg/kg of fat (ppm).

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17. Aliphatic alcohols Method The determination of aliphatic alcohols content was carried out by capillary gas chromatography according to Reg. EEC n. 183/93. The fatty substance, with 1-eicosanol added as internal standard, is saponified with ethanolic potassium hydroxide and then the unsaponifiable matter extracted with ethyl ether. The alcoholic fraction is separated from the unsaponifiable matter by chromatography on a basic silica gel plate; the alcohols recovered from the silica gel are transformed into trimethylsilyl ethers and analysed by capillary gas chromatography. Procedure Preparation of the unsaponifiables. Using a 500 µl microsyringe place, into a 250 ml round-bottom flask, a volume of 0.1% 1-eicosanol solution in chloroform containing a quantity of 1-eicosanol approximately equal to 10% of the aliphatic alcohols content in that portion of sample to be taken for analysis. For example, to 5 g of olive oil sample add 250 µl of the 0.1% 1-eicosanol solution. Evaporate to dryness in current of nitrogen and then weigh accurately 5 g of the dry filtered sample into the same flask. Add 50 ml of 2 N potassium hydroxide ethanolic solution, fit the reflux condenser and heat the apparatus to slight boiling on a steam bath, stirring continuously throughout the heating process until saponification has taken place (the solution becomes clear). Continue heating for a further 20 minutes and then add 50 ml of distilled water through the condenser. The condenser is then disconnected and the flask cooled to approximately 30°C. The contents of the flask are quantitatively transferred to a separating funnel of 500 ml capacity by adding distilled water several times, using a total of around 50 ml distilled water. Add approximately 80 ml of ethyl ether, shake vigorously for approximately 30 seconds and allow to settle. Separate off the lower aqueous phase collecting it in a second separating funnel. Two further extractions are effected on the aqueous phase, in the same manner, using each time 60 to 70 ml ethyl ether. Emulsions may be eliminated by adding, using as a spray, small quantities of ethyl alcohol or methyl alcohol. The ethyl ether extracts are combined in a separating funnel and washed with distilled water (50 ml at a time) until the washing water gives a neutral reaction. Discard the washing water, dry with anhydrous sodium sulphate and filter, into a flask of 250 ml capacity which has been weighed beforehand, the funnel and filter being washed with small quantities of ethyl ether which are added to the total. Distil the ether down to a few ml, then bring to dryness under a slight vacuum or in a current of nitrogen, completing drying in an oven at 100°C for approximately a quarter of an hour, and then weigh after cooling in a desiccator. Separation of alcoholic fractions. Preparation of basic TLC plates: the silica gel plates are immersed completely, in 0.2 N potassium hydroxide solution for 10 seconds, and then left to dry for two hours under an extractor hood and finally placed in an oven at 100°C for one hour. Remove from the oven and keep in a calcium chloride desiccator until required for use (plates treated in this way must be used within 15 days). When basic silica gel plates are used to separate the alcoholic fraction there is no need to treat the unsaponifiables with alumina. It follows that all acid compounds (fatty acids and others) are retained at the origin thereby obtaining both aliphatic alcohol and terpenic alcohol bands which are both separated distinctly from the sterol band. Place a 95:5 by volume benzene/acetone eluent mixture in the plate-developing chamber to a depth of approximately 1 cm or it can be used a 65/35 by volume hexane/ethyl ether mixture. Close the chamber using an appropriate cover and leave for half an hour to allow equilibration between vapour and liquid. Strips of filter paper dipping into the eluent may be affixed to the inside surfaces of the tank to reduce the development time by approximately one-third and obtain more uniform, regular elution of the components. An approximately 5% solution of unsaponifiable matter in chloroform is prepared and 0.3 ml of the solution is streaked as a uniform strip of minimum thickness, using the 100 µl microsyringe, on a TLC plate at approximately 2 cm from the bottom of

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the TLC plate. Aligned with the origin, 2 to 3 µl of the aliphatic alcohols reference solution are spotted for the identification of the aliphatic alcohols band after development has been completed. Place the plate inside the development tank. The ambient temperature should be maintained between 15 and 20°C. Immediately close the chamber with the cover and allow to elute until the solvent front reaches approximately 1 cm from the upper edge of the plate. The plate is then removed from the development chamber and the solvent evaporated under a hot air current or the plate is left for a while under the extractor hood. The plate is sprayed lightly and evenly with the solution of 2,7-dichlorofluorescein when the plate is observed under ultra violet light. The aliphatic alcohols band can be identified through being aligned with the stain obtained from the reference solution: mark the limits of the band with a black pencil; outlining the band of aliphatic alcohols and the band immediately above that, which is the terpenic alcohols band, together. The aliphatic alcohols band and the terpenic alcohols band are to be grouped together in view of the possible migration of some aliphatic alcohols into the triterpenic alcohols band. Using a metal spatula scrape off the silica gel in the marked area. Place the finely comminuted material removed into the filter funnel. Add 10 ml of hot chloroform, mix carefully with the metal spatula and filter under vacuum, collecting the filtrate in the conical flask attached to the filter funnel. Wash the pomace in the flask three times with ethyl ether (approximately 10 ml each time) collecting the filtrate in the same flask attached to the funnel. Evaporate the filtrate to a volume of 4-5 ml, transfer the residual solution to the previously weighed 10 ml test tube, evaporate to dryness by mild heating in a gentle flow of nitrogen, make up again using a few drops of acetone, evaporate again to dryness, place in an oven at 105°C for approximately 10 minutes and then allow to cool in a desiccator and weigh. The pomace inside the test tube is composed of the alcoholic fraction. Preparation of the trimethylsilyl ethers. The reagent for silylation, consisting of a mixture of 9:3:1 by volume of pyridine-hexamethyldisilazane-trimethylchlorosilane in the proportion of 50 µl for each milligram of aliphatic alcohols, is added to the test tube containing the alcoholic fraction, avoiding all absorption of moisture. Stopper the test tube, shake carefully (without overturning) until the aliphatic alcohols are completely dissolved. Stand for at least 15 minutes at ambient temperature and then centrifuge for a few minutes. The clear solution is ready for gas chromatographic analysis. Gas chromatography analysis. Preliminary operations, column packing. Fit the column in the gas chromatograph, attaching the inlet end to the injector connected to the splitting system and the outlet end to the detector. Carry out a general check of the gas chromatography assembly (tightness of gas fittings, efficiency of the detector, efficiency of the splitting system and of the recording system, etc.). Choice of operating conditions. The guideline operating conditions for a chromatographic system with a split injection unit are as follows: – column temperature: the initial isotherm is set at 180°C for 8 minutes and then programmed at

5°C/minute to 260°C and a further 15 minutes at 260°C; – temperature of injector: 280°C; – temperature of detector: 290°C; – linear velocity of carrier gas: helium 20-35 cm/s, hydrogen 30-50 cm/s; – splitting ratio: 1:50 to 1:100; – sensitivity of instrument: 4 to 16 times the minimum attenuation; – sensitivity of recording: 1 to 2 mV fs; – paper speed: 30 to 60 cm/h; – quantity of substance injected: 0.5 to 1 µl of TMSE solution. The above conditions may be modified according to the characteristics of the column and of the gas chromatograph to obtain chromatograms satisfying the following conditions: – alcohol C26 retention time shall be 18 ± 5 minutes; – the alcohol C22 peak shall be 80 ± 20% of the full scale value for olive oil. The above requirements are checked by repeated injection of the standard TMSE mixture of alcohols and the

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operating conditions are adjusted to yield the best possible results. The parameters for the integration of peaks shall be set so that a correct appraisal of the areas of the peaks considered is obtained. Analytical procedure. Using the microsyringe of 10 µl capacity draw in 1 µl of hexane followed by 0.5 µl of air and subsequently 0.5 to 1 µl of the sample solution; raise the plunger of the syringe further so the needle is emptied. Push the needle through the membrane of the injection unit and after 1-2 seconds inject rapidly, then slowly remove the needle after some 5 seconds. Recording is effected until the TMSE of the aliphatic alcohols present have been eluted completely. Peak identification. The identification of individual peaks is effected according to the retention times and by comparison with the standard TMSE mixture, analysed under the same conditions. Quantitative evaluation. The peak areas of 1-eicosanol and of the aliphatic alcohols C22, C24, C26 and C28 are calculated by electronic integration. The contents of each aliphatic alcohol, expressed in mg/1000 g fatty substance, are calculated as follows:

where: Ax = area of the alcohol peak x; As = area of 1-eicosanol; ms =. mass of 1-eicosanol in milligrams; m = mass of sample drawn for determination, in grams. Expression of the results. The contents of the individual aliphatic alcohols in mg/1000 g of fatty substance and the sum of the ‘total aliphatic alcohols’ are reported.

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18. 31P NMR Introduction The amount of information contained in an NMR spectrum obtained fairly rapidly, combined with easy sample preparation, renders this spectroscopic technique very attractive for the determination of the composition of olive oil. 1H NMR spectroscopy has provided valuable information [39-41] about lipid classes, fatty acid composition, unsaturation levels and several minor compounds (sterols, squalene, terpenes, volatile compounds, etc.), whereas 13C NMR, among others, has given unique information about the positional distribution of fatty acids on the glycerol moiety and the stereochemistry of unsaturation. An alternative nuclear magnetic resonance method that supplements 1H NMR and 13C NMR spectroscopy, especially in cases in which strong signal overlap and dynamic range problems in 1H NMR spectra and/or long relaxation times of the insensitive 13C nuclei render the analysis of olive oil a difficult task. This method [42] is based on the derivatization of the labile hydrogens of functional groups, such as hydroxyl and carboxyl groups, of olive oil constituents with the phosphorus reagent 2-chloro-4,4,5,5-tetramethyldioxaphospholane (1) according to the reaction scheme illustrated in Fig. 1, and the use of the 31P chemical shifts to identify the labile centers (compound 2).

Figure 1. Reaction of hydroxyl and carboxyl groups of olive oil constituents with the phosphorus reagent 2-chloro-4,4,5,5-tetramethyldioxaphospholane (1). 1H and 13C NMR spectroscopies are used in the analysis of extra-virgin olive oil. Although these magnetic resonance methods are quantitative and do not require any sample pre-treatment, they are not so effective under certain circumstances. For instance, the unambiguous identification and quantification of certain minor constituents of olive oil, such as mono- and diacylglycerols, demand 1H NMR spectrometers operating at rather high magnetic field strengths (≥14.1 T or 600 MHz in terms of the proton Larmor frequency). At lower magnetic field strengths, the diacylglycerol resonances are overlapped by the strong resonances of the triacylglycerols, [39] questioning therefore the ability of this magnetic resonance method for reliable quantitative determination of these minor components at lower magnetic field strengths. [43] On the other hand, the large range of chemical shifts (~1000 ppm) reported for the 31P nucleus [44] ensures a good separation of the diacylglycerol signals. The excellent resolution between the 31P chemical shifts allows a reliable detection of the phosphitylated mono- and diacylglycerols, even at a magnetic field strength of 9.4 T or 162.0 MHz. The study of diacylglycerols (but not monoacylglycerols due to their low concentration in olive oil, ≤ 0.3%) in olive oil by 31P NMR spectroscopy was feasible at even lower magnetic field strengths. Another advantage of the 31P NMR method is the introduction of an IS (Internal Standard) of known amount (usually cyclohexanol) in the reaction mixture, which allows the determination of the absolute concentration of the phosphitylated product 2, avoiding thereby normalisation conditions. Limitations of the present methodology may be the preparation of the phosphorus reagent and the phosphitylation reaction preceding the acquisition of the 31P NMR spectrum, lengthening the duration of the analysis relative to 1H NMR spectroscopy. Although this method is less simple and requires more time than the 1H NMR method, it is much faster than the corresponding classical methods of titration and gas chromatography (GC) because it determines several constituents (e.g. monoacylglycerols, diacylglycerols, total free sterols and free acidity) in a single spectrum. The

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quantification of phenolic compounds and, in particular, total tyrosol and total hydroxytyrosol that contribute to the stability of extra virgin olive oil against oxidation [45] involves one more step, i.e. extraction, which lengthens further the duration of this NMR method. NMR instrumentation For the detection and quantification of the diacylglycerols, total free sterols and acidity, which give well-separated signals, relatively low magnetic field strengths from 7.05 to 9.4 T (Larmor frequencies 121.5 and 162.0 MHz for the 31P nucleus) are adequate. [42,43] However, the detection of phenolic compounds in the polar fraction of olive oil may require higher magnetic field strengths up to 11.74 T (202.3 MHz) to improve the sensitivity and resolution of the experiment. [46] Sample preparation A stock solution was prepared by dissolving 0.6 mg of chromium acetylacetonate [Cr(acac)3, (0.165 µM)] and 13.5 mg cyclohexanol (13.47 mM) in 10 ml of a mixture of pyridine and CDCl3 solvents (1.6:1.0 volume ratio) and protected from moisture with 5 Å molecular sieves. Each model compound (0.1–3.0 mg depending on its molecular weight and the number of hydroxyl groups) and 100 to 150 mg of olive oil were dissolved in 0.4 ml of the stock solution in a 5-mm NMR tube. For the determination of phenolic compounds, the polar extract obtained from 35 g of olive oil was dissolved in 0.4 ml stock solution. Predetermined amounts of reagent 1 (5–30 µl for model compound and olive oil, depending on the number of the functional groups, and 50 µl for the olive oil extract) were added. The mixture was left to react for about 15 min at room temperature. Upon completion of the reaction, the solution was used to obtain the 31P NMR spectra. Pyridine was selected as the second component of the solvent mixture because it captures immediately the hydrogen chloride gas liberated during the phosphitylation reaction forming the pyridine hydrochloride salt. This drives the overall phosphitylation reaction to total conversion. Pyridine solvent should be in excess relative to the phosphorus reagent, since the latter is still a strong derivatizing reagent and the HCl liberated in further derivatization is capable of inducing decomposition of the derivatized compounds. [47] The role of the chloroform solvent is twofold: first it ensures the dissolution of olive oil, and second it does not allow the precipitation of the pyridine- HCl salt. Quantitative one-dimensional 31P NMR spectra To obtain reliable quantitative data by using one dimensional 31P NMR spectra, several criteria must be considered: (i) Complete derivatization with the tagging reagent 1 must be achieved; (ii) Thermal equilibrium must be reached by the phosphorus nuclei before the pulse sequence repetition. This was guaranteed by using repetition time at least 5 times greater than the longest spin–lattice relaxation time (31P-T1). The measured 31P spin–lattice relaxation times for several phosphitylated model compounds in the mixture of pyridine and chloroform solvents, including the phosphitylated IS (cyclohexanol) were very long (5–10 s), [46, 48, 49] lengthening considerably the duration of the experiment; (iii) Addition of paramagnetic Cr(acac)3 lowers the spin-lattice relaxation times of the phosphorus nuclei, shortening thus the duration of the measurements significantly. For instance, the largest 31P spin–lattice relaxation time (4.6 s) measured in the presence of Cr(acac)3 was found to be that of the phosphitylated IS cyclohexanol. [46] Therefore a repetition time of 25 s is considered the most appropriate for the present quantitative 31P NMR experiments; (iv) To eliminate NOE effects, the inverse gated decoupling technique was used; (v) The S/N ratio of the 31P NMR experiments depends on the concentration of the constituent in extra virgin olive oil, the number of the functional groups in the molecule to be derivatized and the chemical nature of the functional group and its environment. A reasonable S/N ratio was achieved by optimising the number of scans to 16 for diacylglycerols and model compounds and to 32 scans for the derivatized phenolic compounds in extra virgin olive oil extracts. Spectroscopic acquisition is carried out at room temperature, mainly because decomposition of the phosphitylated compounds

30

was observed at high temperatures. At any rate, no sensitivity or resolution enhancement was observed when recording spectra at higher temperatures. Two-dimensional 31P NMR spectra Apart from coupled and/or decoupled 1D 31P NMR spectra, two-dimensional (2D) gradient selected homonuclear 31P–31P (gradient 31P–31P homonuclear spectroscopy, g-PP-COSY) and heteronuclear 1H–31P experiments provide the experimentalist with additional means for clarifying assignments in 1D 31P NMR spectra. Heteronuclear 1H–31P spectra were obtained either with magnetisation transfer from protons to phosphorus-31 nuclei through chemical bonding (gradient 1H–31P heteronuclear multiple quantum coherence, g-H-P-HMQC), or through-space (1H–31P heteronuclear NOE, H-P-HOESY). Phenolic compounds in olive oil The strong signals at õ 138.19 and õ 139.20 reflect the total amount of tyrosol and hydroxytyrosol contained in olive oil, respectively, since the phosphitylated aromatic hydroxyl groups of these compounds in their free and esterified forms are expected to show about the same chemical shifts. The esterified hydroxytyrosol and tyrosol involve their acetate derivatives and the hydrolysis products of oleuropein and ligstroside (the dialdehydic form of elenolic acid linked to hydroxytyrosol, the dialdehydic form of elenolic acid linked to tyrosol, the oleuropein aglycon and the ligstroside aglycon), respectively, formed during olive oil extraction from olive fruits and/or during storage. [50] Free and esterified hydroxytyrosol constitute an important class of phenolic compounds that contributes to the stability of extra virgin olive oil against oxidation. Quality control of extra virgin olive oil Evaluation of extra virgin olive oil quality can be made by employing 31P NMR spectroscopy and using the information it provides on minor compounds. In particular, the minor constituents 1,2-diacylglycerols (1,2-DGs) and 1,3-diacylglycerols (1,3-DGs) accompanying the major triacylglycerol components have been proven to be useful indices to assess the quality of extra virgin olive oil. [43] There are two sources generating 1,2-DGs: incomplete biosynthesis of triacylglycerols, [51] and the limited hydrolysis of triacylglycerols during extra virgin olive oil storage. [52] On the other hand, 1,3-DGs are considered to be secondary products resulting from the isomerization of 1,2-DGs during the extraction process and continued during the storage of the olive oil. [52-54] Therefore, freshly made extra virgin olive oil from healthy olive fruits is expected to contain almost solely 1,2-DGs, the concentration of which decreases during storage, while the 1,3-DG content and the total DG concentration increase. In this respect, the concentration levels of both 1,2-DGs and 1,3-DGs are indicative of extra virgin olive oil freshness. From these facts, the ratio D of 1,2-diglycerides to the total amount of diacylglycerols [D = 1,2-DGs/(1,2-DGs + 1,3-DGs)] has been considered as a potential index to assess the freshness of EVOO. [43] Detection of extra virgin olive oil fraud Contrary to the natural content of total diacylglycerols in fresh extra virgin olive oils, which does not exceed 1–3%, olive oils of lower quality contain higher levels of total diacylglycerols. For instance, refined olive oil contains 4–5% of total diacylglycerols (mainly 1,3-DGs), which go up to 15–20% in olive-pomace oils. [55] The isomerization of 1,2-DGs to 1,3-DGs in refined olive oils reaches very rapidly its equilibrium state, where the ratio D assumes its equilibrium value, Deq ~ 0.33. Therefore, diacylglycerols can be used to detect extra virgin olive oil adulteration with olive oils of lower quality, such as lampante olive oils and refined olive oil.

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19. Triglycerides Method The determination of triglycerides is carried out by High Performance Liquid Chromatography (HPLC). The High performance liquid chromatograph must be these characteristic: − Column: stainless steel tube 250 mm length x 4.5 mm internal diameter packed with 5 μm

diameter particles of silica with 22 to 23% carbon in the form of octadecylsilane; − Thermostatic control of column temperature; − Recorder and/or integrator. − Detector: differential refractometer. The full scale sensitivity should be at least 10-4 units of

refractive index. Procedure 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 solubilisation solvent (acetone). Set up the chromatographic system. Pump elution solvent (acetonitrile + acetone (proportions to be adjusted to obtain the desired separation; begin with 50:50 mixture)) at a rate of 0.7 ml/min to purge the entire system. Wait until a stable base line is obtained. Inject 20 μl of the sample prepared. Calculation and expression of results. Use the area normalisation method, i.e. assume that the sum of the areas of the peaks corresponding to TAGs from ECN 42 up to ECN 52 is equal to 100%. Calculate the relative percentage of each triglyceride using the formula:

% triglyceride = area of peak x 100/ sum of peak areas. The results to be given with at least two decimal places. Reference triglycerides Commercial triglycerides (tripalmitin, triolein, etc.) may be used and the retention times then 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 Lichrosorb (Merck) RP 18 Art 50333, Lichrosphere (Merck) 100 CH18 Art 50377 or equivalent.

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Index 1. Introduction 2. Free acidity 3. Peroxide Value (PV) 4. Ultraviolet spectrophotometry 5. Fatty acid composition 6. Sterols composition

6.1 1st Method 6.2 2nd Method

7. Olive oil sensory quality 8. 1H and 13C NMR 9. IRMS 10. Metals

10.1 1st Method 10.2 2nd Method

11. Phenolic compounds M 11.1 1st Method

11.2 2nd Method 11.3 3rd Method 11.4 4th Method 11.5 5th Method 11.6 6th Method

12. Volatile compounds 12.1 1st Method 12.2 2nd Method

12.2.1 1st Procedure 12.2.2 2nd Procedure

13. Adulteration 13.1 1st Method 13.2 2nd Method 13.3 3rd Method

14. Chlorophylls, pheophytins, β-carotene, tocopherols and tocotrienols 14.1 1st Method 14.2 2nd Method

15. Oxidation stability 16. Wax 17. Aliphatic compounds 18. 31P NMR 19. Triglycerides References

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