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Fisheries Research Services Report No 02/01 ADULTERATION OF OLIVE OIL WITH HAZELNUT OIL: TO ENABLE DETECTION OF UNREFINED AND REFINED HAZELNUT OIL IN VIRGIN AND REFINED OLIVE OIL

L Webster, P Simpson and A M Shanks

June 2001 Fisheries Research Services Marine Laboratory Victoria Road Aberdeen AB11 9DB

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ADULTERATION OF OLIVE OIL WITH HAZELNUT OIL: TO ENABLE DETECTION OF UNREFINED AND REFINED HAZELNUT

OIL IN VIRGIN AND REFINED OLIVE OIL

Lynda Webster, Pamela Simpson and Aileen M Shanks

FRS Marine Laboratory, Victoria Road, Aberdeen, AB11 9DB, UK

SUMMARY 1. Samples of crude hazelnut oil and refined hazelnut oil were analysed for n-alkanes by

gas chromatography with flame ionisation detection to determine if the pattern and composition was oil specific and, therefore, if the hydrocarbon patterns could be used as determinants for assessing adulteration of virgin and refined olive oil.

2. A total of 13 crude hazelnut oils were analysed. The n-alkane concentrations ranged

from 1.62 to 62.96 mg kg-1. Unlike other edible oils, the n-alkane profile of crude hazelnut oil showed more than one type of profile. However, from the GC-FID chromatograms, these could broadly be divided into two types; ‘Type 1' with an unresolved complex mixture, ‘Type 2' without.

3. Four refined hazelnut oils were analysed for n-alkanes and sterenes. The n-alkane

concentrations ranged from 1.65 to 6.68 mg kg-1. The total sterene concentration ranged from 5.18 to 30.69 mg kg-1. The sterene composition was different to that of olive oil with 24-methylcholesta-3,5-diene accounting for 8.17% of the total sterene concentration of refined hazelnut oil; this sterene was not detected in olive oil.

4. The crude hazelnut oil n-alkane data was added to an existing database that included

rapeseed, safflower, sunflower, corn, palm, palm kernel, coconut, groundnut and soyabean oils and analysed by principal component analysis (PCA). Hazelnut oil could clearly be differentiated from olive oil.

5. Using principal component analysis (PCA) adulteration of extra virgin olive oil with crude

hazelnut oil could be detected at levels as low as 5.29% w/w for the ‘Type 1' oil and 0.76% w/w for the ‘Type 2' oil. Adulteration of refined olive oil with refined hazelnut oil could be detected at levels as low as 0.84% w/w using PCA.

INTRODUCTION Consumption of olive oil has increased considerably over the last decade due to its stability, delicate flavour and reported health benefits. Olive oil consumption represents 15.2% of the total oil consumption in the European Union, compared with 18.2% for sunflower, 17.7% for soya, 17.3% for rape and 16.0% for palm oils. It is produced mainly in Mediterranean countries and can be consumed directly without purification. It is expensive and has always been the

Adulteration of olive oil with hazelnut oil

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subject of fraud by mixing with less expensive vegetable oils or else traded as a higher grade product.1,2 In 1993/94 1,829,000 tonnes of olive oil were consumed world wide. In 1995 Turkish hazelnut oil was illegally introduced into the European union in order to adulterate olive oil. The loss to European consumers was estimated at 40 million ECUs due to hazelnut oil being three times as cheap as olive oil. In 1997 the European Commissions= anti-fraud body, Unit for the Coordination of Fraud Prevention (UCLAF), estimated that more than 20,680 tonnes of hazelnut oil was used for producing 103,400 tonnes of adulterated olive oil.3 The hydrocarbon composition of edible oils has been studied to a limited extent and the odd carbon numbered, long chain predominance is well documentated.4, 5, 6, 7,8 These n-alkanes are endogenous to a plant and are thought to be the result of decarboxylation of long chain fatty acids.9, 10 Previous published work indicated that it was possible, using the n-alkane pattern and composition and the sum of the individual n-alkanes between pentadecane (nC15) and tritriacontane (nC33), to distinguish between both crude and refined oils of different plant origin.8,11 By using principal component analysis (PCA) and discriminant analysis, the specific patterns have been shown to be significantly different, especially for crude oils.8 In a previous MAFF project authentic extra virgin olive oil was adulterated with various amounts of either crude sunflower or crude rapeseed oil, at levels between 0.5 and 11% w/w. Using the carbon number profiles alone it was possible to determine adulteration of the extra virgin olive oil with as little as 2.6% crude rapeseed oil or crude sunflower oil. Analysis of the n-alkane pattern by PCA made it possible to identify adulterants at levels as low as 0.5% w/w.8 The similar fatty acid and triglyceride composition of olive oil and hazelnut oil means adulteration of olive oil with hazelnut oil is difficult to detect at levels less than 20%. Fatty acid composition is often used to establish the authenticity of vegetable oils. However, olive oil is difficult to distinguish on this basis from hazelnut oil and sunflower oil as all these oils are high in oleic acid. In the case of sunflower oil, sterol composition may be used to distinguish it from olive oil but the same is not true for hazelnut oil. Other criteria for the detection of seed oils also includes determining the maximum difference between real and theoretical ECIV42 triglyceride content, however the pattern of major triglycerides in hazelnut oil is similar to that of olive oil; there are no triglycerides present in hazelnut oil that are not in olive oil. The fatty acid, triglyceride and sterol composition do not differ sufficiently to distinguish the two oils, even before mixing. Minor components, such as tocopherols do differ but the small proportions make distinguishing the two oils difficult. A number of alternative techniques for assessing both quality and authenticity have been reported. These include Nuclear Magnetic Resonance (NMR)12,13, stable isotope mass spectrometry14, CO2 laser infrared optothermal spectroscopy15, and near infrared spectroscopy.16 The latest method proposed for detecting unrefined hazelnut oil in virgin olive oil is based on the analysis of the flavouring components of hazelnut oil, in particular 5-methylhepta-2-en-4-one (filbertone).17,18 However, during deodorisation hazelnut oil will lose flavour marker compounds such as filbertone. Therefore adulteration of olive oil with deodorised hazelnut oil may not be detected using this method. The determination of authenticity on the basis of n-alkane composition could provide an alternative to the current methods employed and the more recent alternative methods. Detection of refined hazelnut oil in refined olive oil has proven to be even more difficult than the detection of crude hazelnut in virgin olive oil. Flavour components such as filbertone are lost during the refining process, but sterenes are produced during the refining process. They are derived from sterols and are thought to be oil specific.19 24-Ethylcholesta-3,5-diene is used as a determinant for detecting the adulteration of virgin olive oil with refined olive oil. In 1994 the European Union introduced a regulation limiting the content of 24-ethylcholesta-3,5-diene in


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olive oil to 0.15 mg kg-1 with a total sterene content not exceeding 0.3 mg kg-1. In the previous study a method was developed for the co-joint analysis of n-alkanes and sterenes in edible oils.20 Groundnut, safflower and olive oil were analysed for sterenes, aswell as n-alkanes. The sterene concentration and composition was found to be oil specific. As the n-alkane profile is also oil specific it may be possible, using a combination of the n-alkane profiles and steroidal hydrocarbon composition, to detect the adulteration of refined olive oil with refined hazelnut oil. Experimental Reagents Analytical reagent grade iso-hexane, dichloromethane (DCM), methanol, acetone and water were supplied by Rathburn Chemicals Ltd (Walkerburn, UK). Squalane (Sq) was obtained from Kodak Eastman Fine Chemicals (New York, USA). The internal standard 2,2,4,4,6,8,8-heptamethylnonane (HMN) was obtained from Sigma-Aldrich Chemical Company Ltd (Dorset, UK) as were undecane (nC11), dodecane (nC12), tridecane (nC13), pentadecane (nC15), heptadecane (nC17), 2,6,10,14-tetramethylpentadecane (pristane), heneicosane (nC21), docosane (nC22), pentacosane (nC25), heptacosane (nC27), nonacosane (nC29), triacontane (nC30) and tritriacontane (nC33). Tetradecane (nC14), hexadecane (nC16), octadecane (nC18), eicosane (nC20), tricosane (nC23), tetracosane (nC24), hexacosane (nC26), octacosane (nC28) and dotriacontane (nC32) were purchased from Eastman Chemical Company (New York, USA). Hentriacontane (nC31) and 2,6,10,14-tetramethylhexadecane (phytane) were obtained from Restek Corporation (Bellefonte, USA). Silicic acid (500 g, 100 mesh) was obtained from Promochem Ltd (Hertfordshire, UK) and treated as described below. Edible Oils Authentic extra virgin olive oil and refined olive oil were obtained through The International Olive Oil Council (Madrid, Spain. Crude and refined hazelnut oils were supplied by Anglia Oils Ltd. (Hull, UK) and Liberty Vegetable Oil Company (Santa Fe Springs, USA). Crude hazelnut oils were also obtained from the Central Science Laboratory (York, UK). Samples of authentic extra virgin olive oil, authentic refined olive oil and crude and refined hazelnut oil were stored in the glass bottles in which they were received in the dark at 20ΕC ± 2ΕC. Preventative Measures For Reducing Background Contamination Hydrocarbons are ubiquitous to the environment and great care must be taken to avoid adventitious contamination of samples. Therefore all analytical glassware was soaked in Decon 90 (Decon Laboratories Ltd, Hove, UK) before being thoroughly scrubbed and then rinsed with water. The glassware was then dried in an oven at 85ΕC. After cooling, and just prior to use, the glassware was rinsed with DCM and then with iso-hexane, the latter being allowed to evaporate before proceeding. The columns used for the silicic acid column chromatography were soaked, at three monthly intervals, in concentrated nitric acid to clean the frits. The columns were then washed with copious amounts of water before being washed as previously described.


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Individual batches of all solvents were checked for n-alkanes by removal of an aliquot (100 ml) to which was added a two component internal standard (100 µl), containing HMN and Sq in iso-hexane. This mixture was then concentrated, by rotary evaporation, to ~300 µl. The solvent was transferred, with washings, to a tapered vial where it was further concentrated to 25 µl under a stream of scrubbed nitrogen. An aliquot (1 µl) was analysed by gas chromatography with flame ionisation detection (GC-FID). GC-FID chromatograms were qualitatively and quantitatively assessed. If there were unexpected peaks, or individual hydrocarbons at levels higher than 100 ng the batch of solvent was rejected. Preparation of Silicic Acid Silicic acid, 100 mesh, was heated at 550ΕC for 18 ± 2 hours. The silicic acid was then cooled prior to deactivation with HPLC grade water (1% w/v). Isolation of n-alkanes

Hazelnut oil (800 mg) was accurately weighed and the internal standard added (100 µl containing 1,000-1,500 ng each of squalane and heptamethylnonane in iso-hexane). In the case of refined hazelnut oils, cholesta-3,5-diene (200 µl containing 1,300 ng) was also added as an internal standard for the determination of steroidal hydrocarbons. To the oil containing internal standard was added iso-hexane (5 ml). The iso-hexane/oil solution was applied to a 1% deactivated silicic acid column (11 cm x 2 cm) which had been pre-washed with iso-hexane (100 ml). The analytes were then eluted with iso-hexane (130 ml), the column eluent being collected in a 250 ml round bottomed flask. The solution was then concentrated by rotary evaporation to approximately 1 ml and then further concentrated to 300 µl under a stream of scrubbed nitrogen, following transfer to a 2 dram vial. The n-alkanes were isolated from any aromatic components by high performance liquid chromatography using a previously calibrated Lichrosorb Si-60, 5 µm column (25 x 0.46 cm; Jones Chromatography, Mid Glamorgan, UK) with a flow rate of 2 ml min-1 iso-hexane. The aliphatic fraction was collected over the first 2:30-3:00 minutes; the time was dependent on the columns predetermined split of the aliphatic hydrocarbons from the aromatic hydrocarbons. The split time was determined by injecting 150 µl from a mixture comprising 200 µl of the concentrated aliphatic standard and 200 µl of a concentrated aromatic standard. Fractions were collected, concentrated and analysed by GC-FID to determine the fraction where all the aliphatic components have been eluted and no aromatic components are present. The aliphatic fraction was then concentrated, using rotary evaporation, to ~500 µl, then transferred with washings to a tapered vial insert where it was further concentrated, under a stream of scrubbed nitrogen, to 25 µl. An aliquot (1 µl) was analysed by GC-FID. The extraction and analyses were each performed in duplicate with a procedural blank being undertaken with each batch of six duplicate analyses. Preparation of Adulterated Olive Oil Authentic extra virgin olive oil or refined olive oil was accurately weighed into a conical flask and to this was accurately weighed either crude hazelnut oil or refined hazelnut oil, resulting in adulteration levels between 0.75 and 11.16% w/w. The prepared mixtures were ultrasonicated for 15 minutes to ensure thorough mixing of the oils. Aliquots (500 mg) were accurately weighed and extracted as detailed above. The extraction and analyses were each performed in triplicate with a procedural blank being undertaken with each batch of 12 analyses.


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Analysis by GC-FID n-Alkanes and sterenes were analysed by GC-FID using an HP 5890 Series II GC (Hewlett Packard, Berkshire, UK) fitted with an HP 7673 automatic injector and flame ionisation detector (300oC) using a fused silica capilliary column (25 m x 0.25 mm id) coated with 0.33 µm film of Ultra 1 (Hewlett Packard, Berkshire, UK). On-column injections were made at 60oC and after three minutes the temperature was elevated at 10oC min-1 up to 240oC and then 0.5oC min-1 up to 252oC prior to a final elevation at 25oC min-1 up to 280oC where it was held for 20 minutes. Nitrogen (17 psi) was used as the carrier gas. The data was collected via a PE Nelson 610 link box and processed using Perkin-Elmer Turbochrom version 6 software (Perkin-Elmer Ltd, Cheshire, UK). This temperature programme was also used for the analysis of sterenes by gas chromatography with mass selective detection (GC-MSD). Analysis of Sterenes by Gas Chromatography with Mass Selective Detection (GC-MSD) The presence of stetoidal hydrocarbons was confirmed by GC-MSD using an HP6890 Series gas chromatograph interfaced with an HP5973 MSD and fitted with a cool on-column injector. A non-polar methylsilicone column was used for the analyses (HP 5, 30 m x 0.25 mm with 0.25 µm film thickness; Hewlett-Packard Ltd, Stockport, England). The carrier gas was helium set at a constant flow of 0.7 ml/min. The temperature programme was equivalent to that of the GC-FID analysis of n-alkanes detailed above. The MSD was set with a peak threshold of 1000 and a sample number of 4, resulting in 0.95 scans per second in the mass range 500 to 50 Da.

Determination of Steranes and Triterpanes by GC-MSD The sterane and triterpane composition was determined by GC-MSD using an HP6890 Series gas chromatograph interfaced with an HP5973 MSD and fitted with a cool on-column injector. A non-polar methylsilicone column was used for the analyses (HP 5, 30 m x 0.25 mm; 0.25 µm film thickness: Hewlett-Packard Ltd, Stockport, England). The carrier gas was helium set at a constant flow of 0.7 ml/min. Injections were made at 50 oC and the oven temperature held at this for three minutes. Thereafter the temperature was raised at 20oC min-1 up to 100oC. This was followed by a slower ramp of 4oC min-1 up to a final temperature of 270oC. The MSD was set for selective ion monitoring (SIM) with a dwell time of 50 ms. Triterpanes were monitored using m/z 177 and 191 and steranes monitored using m/z 217 and 218 with a dwell time of 80 msec and a delay of 10 msec. Fatty Acid Esterification Hazelnut oil or rapeseed oil (10 mg) was accurately weighed into a screw-topped test tube and dissolved in distilled toluene (1 ml). Esterification of the fatty acids was carried out by the addition of sulphuric acid in methanol (1%, 2 ml) together with one crystal of butylated hydroxytoluene (BHT), and heating overnight at 50ºC. Water (5 ml), containing sodium chloride (5%), was then added and the methyl esters were extracted with iso-hexane (2 x 5 ml). The organic layers were combined, washed with water (4 ml) containing potassium bicarbonate (2%), and dried over anhydrous sodium sulphate. The iso-hexane extract was concentrated, prior to analysis by GC-FID.


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GC-FID Analysis of Fatty Acid Methyl Esters Samples were analysed on a Hewlett Packard 5890 gas chromatograph (Hewlett Packard, Berkshire, UK) equipped with an HP 7673 on-column injector and fitted with a J & W Scientific DB-23 fused silica capillary column (30 m x 02 mm id) coated with a 0.25 µm film of 50% cyanopropyl (Crawford Scientific, Strathaven, UK). Injections were made at 60ºC and the temperature ramped at 25ºC min-1 up to 150ºC. This was followed by a ramp of 1ºC min-1 up to 200ºC, were it was held at this temperature for two minutes. The temperature was then elevated at 10ºC min-1 to 210ºC and held for two minutes at this temperature. ECD grade nitrogen (13 psi) was used as the carrier gas. The data was collected via the Perkin Elmer Turbochrom navigator data system (Perkin Elmer, Beconsfield, UK).

RESULTS AND DISCUSSION

Quality Control

Hydrocarbons are ubiquitous in the environment and thus particular care has to be taken when carrying out trace analysis of n-alkanes in food matrices to avoid background contamination of sample isolates. Quality control, through regular analysis of a procedural blank, is thus essential. Furthermore, each sample was analysed in duplicate to ensure any spurious peaks could be identified. Similarly, it was important that the peak identification was assessed. This was achieved by regularly running a standard mixture containing the internal standards, pristane, phytane, and the n-alkanes undecane to tritriacontane. Where appropriate peak identification was confirmed by GC-mass spectroscopy.

Calibration curves were prepared for the n-alkanes nC11 - nC33 and for HMN, pristane, phytane and Sq using standard solutions covering the concentration range ~0.1 ng µl-1 - ~50 ng µl-1. Quantification was on the basis of the added internal standard, squalane, since this compound had a retention time approximating to the major determinants in olive oil. The detector response for each of the n-alkanes, pristane, phytane, HMN and Sq was linear; a correlation coefficient of 0.9991, 0.9998, 0.9998, 0.9997 and 0.9998 was obtained for nC11, nC16, nC23 squalane and nC31 respectively. To ensure the detector response was maintained a standard mixture containing, nC12, nC20, nC25, nC33, pristane, HMN and Sq was analysed at the beginning of each batch of six samples and the resulting data was monitored using Shewhart charts.

The limit of detection of n-alkanes for the FID, when using automated integration, was 25 pg on-column. However, it was possible, by manual integration of the peaks, to determine n-alkane levels as low as 5 pg on-column, with a signal to noise ratio of 7:1 in the region of octadecane (nC18). The limit of detection for the procedure was determined to be 2 ng g-1 with the term >trace= being used for concentrations between 2 and 16 ng g-1. These figures were determined by taking a standard nC11-nC33 mixture, containing ~2 ng of each component, through the analytical procedure, resulting in an on-column concentration equivalent to ~50 pg. The recoveries of the n-alkanes were determined by extracting a known amount of a standard mixture, containing tridecane (nC13), heneicosane (nC21), nonacosane (nC29) and squalane, using the same procedure as for edible oils, on six separate occasions. Mean recoveries of at least 86% (SD 11.2) were obtained for nC13, this figure rising to in excess of 100% for the remaining components.


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Quantification of the steroidal hydrocarbons was based on cholesta-3,5-diene. There was limited availability of standard sterenes. It was, therefore, assumed that the response factor for the steroidal hydrocarbons was equivalent to that of cholesta-3,5-diene. The amount, calculated using the product of the peak area for the sterene of interest and the quotient of the peak area for cholesta-3,5-diene and the amount of cholesta-3,5-diene added, was calculated manually. The limited availability of standard sterenes resulted in recoveries for these compounds being based on that of cholesta-3,5-diene. The recoveries were determined by extracting a known amount of a standard mixture containing HMN, Sq and cholesta-3,5-diene using the same procedure as for the edible oils. Average recoveries for the three compounds were in excess of 70%. Hazelnut Oil Sources of hazelnut oil, in particular refined hazelnut oil, were limited. In the UK, Anglia Oils Ltd was the only producer of crude and refined hazelnut oil. Hazelnut oil supplied by Anglia Oils Ltd were of Turkish and French origin. Other sources, such as Leon Frenkle, only produced a blend of crude and refined, which was not suitable for our purpose. The Central Science Laboratory (CSL) in York supplied seven crude oils. These oils were cold-pressed from nuts purchased from retailers and wholesalers by CSL. The hazelnuts used to produce these oil came from Turkey, USA and Italy. Liberty Vegetable Oil Company in the USA supplied one sample of crude and one of refined hazelnut oil. The hazelnuts were of Turkish origin and had not been roasted. Crude Hazelnut Oil Crude Hazelnut Oil A total of thirteen crude hazelnut oils were analysed in duplicate. The mean total n-alkane (nC12-nC33) concentrations for the crude hazelnut oils ranged from 1.62 mg kg-1 - 62.96 mg kg-1

(mean = 14.02 mg kg-1, n=12, SD = 18.82; Table 1). The highest concentrations were found in the American oils and the lowest in the Turkish oils. The n-alkane distribution of some of the crude hazelnut oils was typical of other edible oils, showing a long chain, odd-carbon predominance (Table 1), with nC29 having the highest concentration of the long chain n-alkanes. However a number of oils also contained a significant proportion of shorter chain n-alkanes and in some cases an unresolved complex mixture (UCM), covering the boiling range nC12 to nC21, was observed (Fig. 1). The n-alkane profiles could broadly be divided into two groups; ‘Type 1' containing a UCM and ‘Type 2' without a UCM (Table 2). Koprivnjak et al. also reported two types of hazelnut oil profile; hazelnut oil I containing a large UCM and hazelnut oil II containing a very small UCM.21 Seven of the crude hazelnut oils analysed could be classed as ‘Type 1'. Four were supplied by Anglia Oils Ltd and were of Turkish, French or unknown origin, one was from Liberty Vegetable Oil Company and was of Turkish origin and two were from CSL, one of Turkish origin the other Italian. ‘Type 1' profiles contained low concentrations of the long chain n-alkanes, nC27, nC29 and nC31 (Fig. 2). The highest concentration of the long chain n-alkanes was due to nC29 in all samples except 2559/00, where nC30 had the higher concentration. The proportion of nC12-nC25, in relation to the total n-alkane concentration the ‘Type 1' oils was high ranging from 19% to 97%, with a median value of 89%. In addition a UCM was present, however the size and position of this UCM varied from sample to sample. In samples 2571/00 and 2559/00 the UCMs were small. A UCM comprises of mainly saturated and unsaturated alicyclic hydrocarbons in the region of nC12 to nC25. UCMs are normally associated with petrogenic contamination, however in previous studies a number of edible oils (groundnut oil, corn oil and soyabean oil) were found to contain small UCMs.8, 20 Geochemical biomarker


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(triterpanes and steranes) analysis is often used for fingerprinting crude oils. Biomarker analysis carried out on the hazelnut oils containing UCMs showed no evidence of crude oil contamination. However it is still not clear if the UCMs in these hazelnut oils were a result of petrogenic contamination or a natural phenomenon. ‘Type 2' oils showed a long chain, odd carbon predominance from nC27, with nC29 having the highest concentration (Fig. 3). In comparison to the ‘Type 1' oils, the ‘Type 2' oils contained a low proportion of shorter chain n-alkanes (<29%), and no UCM. However within this group the concentrations of the longer chain odd carbon n-alkanes varied greatly; the concentration of nC29 ranged from 1.17 mg kg-1 in a Turkish oil (2562/00) to 50.39 mg kg-1 in an American oil (2564/00). Five hazelnut oils could be classed as ‘Type 2', all were supplied by CSL. Two of these oils were American, two Turkish and one Italian. The total n-alkane concentrations in the American oils, were 62.96 mg kg-1 and 24.55 mg kg-1. The percentage concentration of the total n-alkanes represented by nC12-nC25 was low with values of 1% and 2% in samples 2564/00 and 2565/00 respectively (Figure 4). This was mainly due to the high concentration of nC29 which was accounted for 80% (2564/00) and 81% (2565/00) of the total n-alkane concentration. One of the two Italian oils also had a high concentration of nC29 (78% of the total n-alkanes) with n-alkanes in the region nC12-nC25 accounting for 3% of the total n-alkane concentration. The total n-alkane concentration in this oil was 42.35 mg kg-1. These profiles were similar to that of rapeseed oil determined during a previous study. The n-alkane concentration in rapeseed oil was found to range from 65.35 mg kg-1 to 91.25 mg kg-1 (mean = 83.89 mg kg-1, SD = 6.97, n=15) with nC29 comprising 80.8 - 90.6% of the total n-alkane concentration.22 In order to prove these hazelnut oils were not in fact rapeseed oils, fatty acid analysis was carried out. Hazelnut oils (Type 1 and Type 2) and rapeseed oil were analysed, in duplicate, for fatty acids. The fatty acids were converted to the methyl ester, prior to analysis by GC-FID. The fatty acid profile of both types of hazelnut oil were similar to each other (Table 3) but were different to that of rapeseed. The dominant fatty acid in both rapeseed and hazelnut oils was oleic acid [18:1(n-9)]. However there was a higher proportion of oleic acid in both types of hazelnut oil with 76.00% (Type 2) and 77.98% (Type 1). This compared to 58.66% in rapeseed oil (Fig. 5). In addition, rapeseed contained a higher proportion of linoleic acid [18:2 (n-6)] (18.49%) and α-linolenic acid [18:3 n-3] (7.83%). This compared to 13.70% (Type 2) and 11.39% (Type 1), for 18:2 (n-6), and 0.14% (Type 2) and 0.13% (Type 1) for 18:3 (n-3). Two other CSL oils gave a Type 2 n-alkane profile, both of these were of Turkish origin. The n-alkane concentrations in these oils were considerably lower with values of 2.27 mg kg-1 and 2.40 mg kg-1 for nC12-nC33. The percentage concentration of the total n-alkanes represented by nC12-nC25 was 29% and 14%, with nC29 accounting for 52% and 29% of the total. Oil (911/00) of Turkish origin, supplied by Anglia Oils Ltd, did not contain a UCM but did contain a significant proportion of lower chain n-alkanes, with an even carbon predominance. The total n-alkane concentration in this oil was 5.30 mg kg-1 with n-alkanes in the range nC12-nC25 accounting for 90% of the total n-alkanes. In addition there was evidence for the presence of sterenes, which was confirmed by mass spectroscopy, indicating this oil was refined. During a previous MAFF project one commercial hazelnut oil was analysed in duplicate.22 The total n-alkane (nC12-nC33) concentration of this oil was 18.7 mg kg-1 and would be classed as a ‘Type 2' oil, containing no UCM. However the proportion of shorter chain n-alkanes was high with 34.9% of the total being accounted for by nC12-nC25. Refined Hazelnut Oils A method for the co-joint analysis of n-alkanes and sterenes was developed for a previous MAFF project on the authentication of olive oil.20 This method was employed for the analysis of


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n-alkanes and sterenes in refined hazelnut oil. Four hazelnut oils were analysed, three from Anglia Oils Ltd and one from Liberty Vegetable Oil Company (Table 4). The total n-alkane (nC12-nC33) concentration ranged from 1.65 mg kg-1 to 6.68 mg kg-1 in refined hazelnut oils (Table 5). Compared to the crude hazelnut oils there was a reduction in the concentration of the lower chain n-alkanes. The UCM present in the crude hazelnut oil from Liberty Vegetable Oil Company was no longer present (Fig. 6). The UCMs in oils 2568/00 and 2569/00 were greatly reduced in size in comparison to the corresponding crude oils (2570/00 and 2571/00). The French refined hazelnut oil from Anglia Oil Ltd (912/00) still showed a high proportion of short chain n-alkanes with an even carbon predominance, similar to the corresponding crude oil (911/00). During a previous MAFF project five authentic refined olive oils were analysed in duplicate for steroidal hydrocarbons by GC-FID and confirmed by GC-MSD. The four major sterenes present in olive oil were 24-ethylcholesta-4,6-diene, 24-ethylcholesta-3,5,22-triene, 24-ethylcholesta-2,5-diene and 24-ethylcholesta-3,5-diene, with the total sterene concentration ranging from 0.92 mg kg-1 to 5.74 mg kg-1. The dominant sterene was 24-ethylcholesta-3,5-diene. Refined groundnut and safflower oils were also analysed for sterenes.22 The variation found in the sterene composition of the different oils suggested the sterene composition of edible oils may be oil specific. Therefore the refined hazelnut oils were analysed in duplicate for steroidal hydrocarbons using a GC-FID method, for quantification, and a GC-MS method for confirmation. The total sterene concentration of the refined hazelnut oils ranged from 5.18 to 30.69 mg kg-1 (mean 11.51 mg kg-1, n = 4, SD = 10.08; Table 6). Similar to olive oil, the dominant sterene was 24-ethylcholesta-3,5-diene, however the concentration was higher than that found in olive oil, with the concentration ranging from 4.56 to 26.91 mg kg-1 in refined hazelnut oil (Fig. 7) and between 0.52 and 4.52 mg kg-1 in refined olive oil. In addition, 24-methylcholesta-3,5-diene accounted for 8.17% of the total sterene composition of refined hazelnut oil but was not present in refined olive oil (Table 7). Adulteration of Extra Virgin Olive Oil with Crude Hazelnut Oil An authentic extra virgin olive oil was adulterated with a ‘Type 1' crude hazelnut oil, at levels of 0.75, 1.18, 3.24, 5.29 and 11.12% w/w, and a ‘Type 2' hazelnut oil at levels of 0.76, 1.07, 2.61, 5.37 and 11.16%. The extra virgin olive oil/crude hazelnut oil mixtures were analysed in triplicate to determine the n-alkane concentrations (nC12-nC33) and carbon number profiles.Despite the adulteration of the extra virgin olive oil with various concentrations of crude hazelnut oil (Type 1) there was not any significant effect on the total n-alkane concentration. The n-alkane concentrations (nC12-nC33) in the extra virgin olive oil was 51.74 mg kg-1 and the concentration in the adulterated olive oil mixtures ranged from 44.27 mg kg-1 in the 11.12% mixture to 53.19 mg kg-1 in the 1.18% mixture (mean = 49.19 mg kg-1, SD = 3.58; Table 8). The low concentrations of the long chain n-alkanes also meant that there was little difference in the concentration in the dominant n-alkanes found in olive oil (nC25, nC27, nC29, nC31). However the UCM present in the crude hazelnut oil could be seen in the adulterated mixtures at levels as low as 0.75%. Adulteration of the same olive oil with a ‘Type 2' crude hazelnut oil (Table 9), with the lowest concentration of nC29, again made little difference to the total n-alkane concentration. The n-alkane concentrations (nC12-nC33) in the adulterated olive oil mixtures ranged from 57.70 mg kg-1 in the 11.62% mixture to 61.60 mg kg-1 in the 5.37% mixture (Table 9). Although the concentration of the dominant hazelnut oil n-alkane, nC29, was higher than found in the Type 1


10

hazelnut oil it was still lower than the concentration found in olive oil. Therefore there was not a significant effect on the n-alkane profile of the olive oil. Adulteration of olive oil with rapeseed oil can be detected at levels as low as 2.56% on the basis of n-alkane profiles alone.20 Hazelnut oils 2563/00, 2564/00 and 2565/00 had similar n-alkane concentrations and profiles to rapeseed oil. Therefore if these ‘Type 2' oils were used for adulteration of olive oil it would be expected that a difference in the concentration of nC29 would be detected in the adulterated mixtures at the 3.24% level from their n-alkane profiles. Adulteration of Refined Olive Oil with Refined Hazelnut Oil Refined olive oil was adulterated with refined hazelnut oil (10868/00) at levels of 11.11, 5.38, 2.59, 1.10 and 0.84%. The refined olive oil/refined hazelnut oil mixtures were analysed in triplicate to determine the n-alkane (nC12-nC33) concentrations and carbon number profiles and sterene composition. The n-alkane (nC12-nC33) concentrations in the refined olive oil was 12.72 mg kg-1 (Table 10) and the concentration in the adulterated olive oil mixtures ranged from 9.22 mg kg-1 (0.84% mixture) to 10.78 mg kg-1 (5.38% mixture; Table 10). Again the refined olive oil adulterated with refined hazelnut oils showed little difference in the total n-alkane concentration or in the concentration of the dominant olive oil n-alkanes. The low concentrations of n-alkanes in the hazelnut oil also meant that there was little difference in the n-alkane pattern. However the sterene pattern of refined olive oil and olive oil adulterated with hazelnut oil did show differences (Table 11). 24-Methylcholesta-3,5-diene was present in all refined olive oil/refined hazelnut oil mixtures but not in the refined olive oil (Fig. 8). Statistical Analysis Crude Hazelnut Oils In order to display any major differences between duplicate determinations, a principal component analysis was carried out for the twenty six samples using nC12 to nC33. In a small number of cases the concentration of nC28 or nC30 was ‘masked’ by the presence of an unknown component therefore these were treated as missing values and excluded from the PCA. The first five principal components accounted for over 92% of the variability. Considerable variation between oils was found, but not between duplicate analyses of the same oil. Therefore the mean n-alkane concentration for each oil was calculated to represent an overall profile for each oil. Principal component analysis (PCA) was repeated using the means for nC12 to nC33, excluding nC28 and nC30. The first five principal components accounted for 92% of the variability. A plot of the second principal component against the first (Fig. 9) showed a number of differences among the oils, with oil 2559/00 (Type 1) of unknown origin (oil 10 on PCA plot) being different from the others. This oil has particularly high concentrations of even carbon n-alkanes from nC12 to nC24. Oils 2563/00 and 2564/00, both ‘Type 2' oils supplied by CSL (oils 7 and 8), appear to be similar, although they originate on different continents, and are quite different from the other oils. These oils both have a high concentration of nC29 and have among the highest concentrations of nC27 to nC33. Oils 2562/00 (Type 2), 2566/00 (Type 2), 2558/00 (Type 1) and 2571/00 (Type 1) (oils 4, 5, 9 and 12) appear to be similar with oil 2565/00 (oil 2) reasonably closely associated with these four oils. Oils 2562/00, 2566/00, 2558/00 all originate in Turkey


11

while oil 2571/00 is described as extra virgin hazelnut oil from France. Oil 2565/00 (Type 2) originated from the USA and gave high concentrations of nC27, nC29 and nC31, although not as pronounced as oil 2564/00. Oils 911/00 and 2570/00 (oils 6 and 11) were similar in that the concentrations of nC27, nC29 and nC31 were low. However oil 911/00 did not contain a UCM and was thought to be refined, due to the presence of sterenes. Oil 2570/00 was a ‘Type 1' oil. Oil 2567/00 (oil 3), ‘Type 1' of Turkish origin, was not particularly like any other oil and oil 2557/00 (oil 1), ‘Type 1' originating in Italy, was different from any other oil. Oil 10867/00 (oil 13), ‘Type 1' originating in Turkey, was not noticeably like any other oil, particularly other Turkish oils, but was most like oil 2557/00. This oil gave highest concentrations for nC14 to nC18. Finally the hazelnut oils were combined with the dataset of all crude samples of edible oils which was examined in 1998.8 During the 1998 study PCA was carried out using nC25 to nC33 as these components accounted for the greatest variance. Therefore, for this study, PCA was carried out using nC25 to nC33, excluding nC28 and nC30 (due to the masking problems). The first three principal components accounted for 97% of the variability. Figure 10 shows the second principal component plotted against the first. The hazelnut oils were similar to a number of the other oils including coconut, groundnut, palm, palm kernel, rapeseed and soyabean. Oils 2563/00 and 2564/00, which had the highest concentration of nC29, appear to be most like rapeseed or soyabean oils. However all crude hazelnut oils were well separated from the virgin olive oil. Refined Hazelnut Oils The n-alkane profiles of the refined hazelnut oils were analysed by PCA using nC12 to nC33. PCA confirmed that the duplicate analyses of the oils produced similar profiles. Therefore PCA was repeated using the mean values for nC12 to nC33. The four refined oils were found to be different in character (Fig. 11). Extra Virgin Olive Oil Adulterated with Crude Hazelnut Oil Extra virgin olive oil was adulterated with 11.12%, 5.29%, 3.24%, 1.18% and 0.75% crude hazelnut oil (Table 8). The hazelnut oil (2570/00) was from Turkey and contained a UCM (Type 1). Each adulterated oil was analysed in triplicate. From PCA the replicates were found to have similar profiles. Therefore PCA was repeated using the mean n-alkane concentrations for nC12 to nC33, excluding nC28 and nC30, on the five adulterated oils and the two original oils. The first three components accounted for over 98% of the variability. When the second principal component is plotted against the first, the crude hazelnut oil is completely separate from all the other oils, as might be expected. The extra virgin olive oil and the three adulterated oils with the lowest percentages of hazelnut oil were clustered fairly closely but the adulterated oils with 5.29% (EVOA1) and 11.12% (EVOA2) were removed some distance from the cluster (Fig. 12). The same extra virgin olive oil was also adulterated with crude hazelnut oil 2562/00 (Type 2 from Turkey), at levels of 11.16%, 5.37%, 2.61%, 1.07% and 0.76%. PCA was carried out, using the mean nC12 to nC33 concentrations, on the five adulterated oils and the two original oils. The first three components accounted for over 98% of the variability. When the second component was plotted against the first, the crude hazelnut oil was completely separated from all other oils as might be expected. The extra virgin olive oil was also separated from all the adulterated oils and the crude hazelnut oil. Thus adulteration with this type of hazelnut oil can be detected at levels as low as 0.76% (Fig. 13).


12

Refined Olive Oil Adulterated with Refined Hazelnut Oil A refined olive oil was adulterated with 11.11%, 5.38%, 2.59%, 1.10% and 0.84% refined hazelnut oil. The refined hazelnut oil (10868/00), was of Turkish origin. Triplicate analyses were carried out on each adulterated oil. PCA confirmed that replicate analyses of the oils produced consistent profiles for each of the adulterations. Therefore PCA of the five adulterated oils and the two original oils was repeated using the mean concentration for nC12 to nC33, excluding nC28 and nC30. The first three components accounted for over 96% of the variability on the PCA. A plot of the second component against the first shows that the adulterations, at all five levels, clustered together with the original oils well separated from the cluster and each other (Fig. 14). Thus adulteration of refined olive oil with refined hazelnut oil can be detected at levels as low as 0.84%.

CONCLUSIONS 1. n-Alkane concentrations and profiles of crude hazelnut oils varied widely, but could

broadly be divided into two groups; ‘Type 1' containing a UCM and ‘Type 2' with no UCM.

2. When compared to other crude edible oils using PCA, crude hazelnut oil was similar to

coconut, groundnut, palm, palm kernel, rapeseed and soyabean oils, and was well separated from olive oil.

3. Extra virgin olive oil was adulterated with crude hazelnut oil at varying levels. Due to the

low concentrations of n-alkanes in most crude hazelnut oils, detection of adulteration of extra virgin olive oil by crude hazelnut oil could not be achieved on the basis of their n-alkane profiles alone. However a UCM could just be seen at all levels of adulteration with the ‘Type 1' hazelnut oil.

4. Using PCA it was possible to detect adulteration of extra virgin olive oil with crude

hazelnut oil at levels as low as 5.29% for the ‘Type 1' hazelnut oil and as low as 0.76% with the ‘Type 2' oil.

5. Only four refined hazelnut oils were analysed for n-alkanes and sterenes due to the

limited availability of this oil. The oils showed a reduction in the concentration of the lower carbon numbered n-alkanes and in one case the UCM was absent.

6. Again the detection of adulteration of refined olive oil with refined hazelnut oil was

impossible by looking at the carbon number profiles alone due to the low concentration of n-alkanes. However, using PCA, adulteration could be detected at levels as low as 0.84%. In addition refined hazelnut oil contained the sterene 24-methylcholesta-3,5-diene which was not found in olive oil.


13

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olive oil on the basis of hydrocarbon concentration and composition. Analyst, 125, 97-104.

9. Kolattukudy, P.E., Buckner, J.S. and Brown, L. 1972. Direct evidence for a

decarboxylation mechanism in the biosynthesis of alkanes in B. oleracea. Biochem. Biophys. Res. Commun., 47, 1306-1313.

10. Khan A. and Kolattukudy, P.E. 1974. Decarboxylation of long chain fatty acids to

alkanes by cell free preparations of pea leaves (Pisum sativum). Biochem. Biophys. Res. Commun., 61, 1379-1386.

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and concentration of n-alkanes in retail samples of edible oils . J. Sci. Food Agric., 61, 357-362.

12. Sacco, A., Brescia, M.A., Liuzzi, V., Reniero, C.G., Ghelli, S. and Meer, P. van der.

2000. Characterisation of Italian olive oils based on Analytical and nuclear magnetic resonance determinations. J. Amer. Oil Chem. Soc., 77, 619-625.

13. Mavromoustakos, T., Zervou, M., Bonas, G., Kolocouris, A. and Petrakis, P. 2000. A

novel analytical method to detect adulteration of virgin olive oils by other oils. J. Amer. Oil Chem. Soc., 77, 405- 411.


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14. Angerosa, F., Camera, L., Cumitini, S., Gleixner, G. and Reneiro, F. 1997. Carbon stable isotopes and olive oil adulteration with pomace oil. J. Agric. Food Chem., 45, 3044-3048.

15. Favier, J.P., Bicanic, D., Cozijnsen, J., B. van and Helander, P. 1998. CO2 laser

infrared optothermal spectroscopy for quantitative adulteration studies in binary mixtures of extra virgin olive oil. J. Amer. Oil Chem. Soc., 75, 359-382.

16. Wesley, I.J., Pacheco, F. and McGill, A.E.J. 1996. Identification of adulterants in olive

oil. J. Amer. Oil Chem. Soc., 73, 515-518. 17. Blanch, G.P., Caja, M., Ruiz del Castillo, M.L., and Herraiz, M. 1998. Comparison of

different methods for the evaluation of the authenticity of olive oil. J. Agric. Food Chem., 46, 3153-3157.

18. Ruiz del Castillo, M.L., Caja, M. del mar, Herraiz, M. and Blanch, G.P. 1998. Rapid

recognition of olive oil adulterated with hazelnut oil by diret analysis of the enantiomeric composition of filbertone. J. Agric. Food Chem., 46, 5128-5131.

19. Mennie, D, Moffat, C.F. and McGill, A.S. 1994. Identification of sterene compounds

produced during processing of edible oils. J High Res. Chromatogr., 17, 831 - 838. 20. Simpson, P., Webster, L., Shanks, A.M. and Moffat, C.F. 1999. The authentication of

olive oil on the basis of hydrocarbon concentration and composition. FRS Report No 3/99.

21. Koprivnjak, O., Procida, G. and Favretto, L. 1997. Determination of endegenous

aliphatic hydrocarbons of virgin olive oils of four autochthonous cultivars from Krk Island (Croatia). Food Technol. Biotechnol., 35, 125 - 131.

22. Moffat C.F., Cruickshank P., Brown, N.A., Mennie, D., Anderson D.A and McGill, A.S.

1995. The concentration and composition of n-alkanes in edible oils. Report submitted to Food Science Division, MAFF.

TABLE 1

n-Alkane composition of crude hazelnut oil together with the mean and standard deviation (SD) for the individual n-alkanes and the sum of nC12-nC33.

Carbon No *911/00

Turkey 2557/00

Italy 2558/00Turkey

2559/00 Unknown

2562/00 Turkey

2563/00 Italy

2564/00 USA

2565/00 USA

2566/00 Turkey

2567/00 Turkey

2570/00 Turkey

2571\00France

10867/00 Turkey Mean SD

nC12 1.42 ND 0.02 2.16 0.25 0.02 0.02 0.02 ND 0.01 Tr Tr 0.04 0.21 0.59 nC13 0.05 0.03 0.03 0.06 0.02 ND Tr 0.02 ND 0.03 0.01 Tr 0.19 0.03 0.05 nC14 1.29 0.02 0.07 2.74 0.03 0.12 Tr 0.01 ND 0.09 ND Tr 0.96 0.34 0.77 nC15 0.12 0.38 0.09 0.14 0.05 0.20 0.05 0.04 Tr 0.41 0.19 0.06 2.27 0.32 0.60 nC16 0.89 1.80 0.19 1.84 0.03 0.01 0.06 0.04 Tr 0.98 0.10 Tr 2.11 0.60 0.84 nC17 0.09 2.04 0.11 0.16 0.03 0.08 0.06 0.03 0.03 1.45 0.59 0.07 1.40 0.50 0.68 nC18 0.53 1.65 0.08 0.93 0.02 0.11 0.05 0.03 0.04 1.41 0.66 0.06 0.89 0.49 0.57 nC19 0.05 1.20 Tr 0.01 0.03 0.03 0.01 0.01 0.04 0.94 0.08 Tr 0.63 0.25 0.41 nC20 0.30 0.42 0.11 0.62 0.01 0.10 0.03 ND 0.02 0.48 0.56 0.03 0.43 0.23 0.23 nC21 0.02 0.17 0.04 0.05 0.02 0.10 0.03 ND 0.02 0.02 0.35 0.04 0.27 0.09 0.11 nC22 ND 0.04 0.02 0.40 0.02 0.09 0.03 ND Tr 0.12 0.18 0.04 0.12 0.09 0.11 nC23 0.02 0.04 0.04 0.16 0.03 0.14 0.14 0.06 0.04 0.26 0.18 0.04 0.08 0.10 0.07 nC24 0.10 Tr 0.04 0.30 0.03 0.09 0.07 0.02 0.02 0.09 0.02 0.03 0.04 0.06 0.08 nC25 0.07 ND 0.03 0.48 0.08 0.36 0.63 ND 0.14 0.69 0.18 0.04 0.08 0.25 0.23 nC26 0.05 0.01 0.06 0.20 0.03 0.13 0.06 0.02 Tr 0.05 0.22 0.08 0.06 0.08 0.07 nC27 0.03 Tr 0.09 0.12 0.11 1.20 2.76 1.23 0.14 0.10 0.04 0.25 0.05 0.51 0.80 nC28 ND 0.02 0.08 0.05 0.05 0.69 1.19 0.57 ND 0.05 M ND 0.02 0.22 0.37 nC29 0.24 0.09 0.18 0.25 1.17 32.94 50.39 19.82 1.65 0.19 0.12 0.77 0.17 8.98 15.95nC30 M 0.07 0.15 0.28 0.05 1.19 1.48 0.57 0.09 0.04 ND ND ND 0.33 0.48 nC31 ND 0.02 0.09 0.11 0.22 4.28 5.71 2.04 0.15 0.10 0.05 0.20 ND 1.08 1.85 nC32 0.02 Tr 0.05 0.12 0.01 0.17 0.09 0.01 ND ND 0.01 ND ND 0.04 0.06 nC33 0.03 0.01 0.05 0.06 0.01 0.30 0.10 0.01 0.01 0.03 0.04 0.09 ND 0.06 0.08

Pristane 0.03 1.16 0.17 0.17 3.75 0.05 0.05 ND 0.01 0.70 0.38 0.04 0.74 0.60 1.01 Phytane 0.05 1.62 0.07 0.09 0.08 0.07 0.05 ND 0.05 0.68 0.50 0.15 0.58 0.33 0.45

Sum nC12-nC33 5.3 8.01 1.62 11.24 2.27 42.35 62.96 24.55 2.40 7.54 3.68 1.80 9.81 14.02 18.82

15

Sum nC12-nC25 4.9 7.79 0.87 10.05 0.65 1.45 1.18 0.28 0.35 6.98 3.1 0.35 9.51 3.55 3.7

Tr - Trace = 2 - 16 ng g-1; ND - Not Detected = < 2 ng g-1; M - Masked


16

TABLE 2 Supplier, origin and n-alkane profile type for crude hazelnut oils.

Sample Supplier Origin Alkane profile

2558/00 CSL Turkey Type 1



2557/00 CSL Italy Type 1

2563/00 CSL Italy Type 2 2564/00 CSL USA Type 2

2565/00 CSL USA Type 2

911/00 Anglia Oils Ltd Turkey Sterenes present, possibly refined

2567/00 Anglia Oils Ltd Turkey Type 1

2570/00 Anglia Oils Ltd Turkey Type 1

10867/00 Liberty Vegetable Oil Company Turkey Type 1

2559/00 Anglia Oils Ltd Unknown Type 1*

2571/00 Anglia Oils Ltd France Type 1* Type 1, low concentrations of nC27, nC29 and nC31 and a UCM *very small UCM; Type 2 no UCM


17

TABLE 3 Mean Percentage composition of fatty acids in rapeseed, and Type 1 and Type 2 hazelnut oils. The fatty acids were converted to the methyl esters before analysis by GC-FID. Type 1 and Type 2 hazelnut oils have a similar fatty acid composition and is different to that of rapeseed.

Normalised area % Crude Hazelnut Crude Hazelnut Fatty Acid

2564/00 Type 2

2567/00 Type 1

Crude Rapeseed

14:0 0.03 ND ND 15:0 0.03 0.03 0.04 16:0 4.91 5.37 4.89 16:1 0.20 0.19 0.30 16:2 0.08 0.27 0.07 16:3 0.05 0.06 0.06 16:4 ND ND ND 18:0 1.88 2.37 1.80

18:1 a 76.00 77.98 58.66 18:1 b 2.61 1.80 5.70

18:2 (n-6) 13.70 11.39 18.49 18:3 (n-6) ND ND ND 18:3 (n-3) 0.14 0.13 7.83 18:4 (n-3) ND ND ND

20:1 a 0.17 0.21 1.24 20:1 b 0.20 0.23 0.22

20:4 (n-6) ND ND ND 20:4 (n-3) 0.02 ND 0.01

20:5 ND ND ND 22:1 ND 0.01 0.37 21:5 ND ND ND 22:5 ND ND ND

22:6 (n-3) ND ND 0.18 24:1 ND ND 0.19 Total 100.00 100.00 100.00


18

TABLE 4 Supplier, origin and n-alkane profile type for refined hazelnut oils.

Sample Supplier Origin n-alkane profile 2568/00

Anglia Oils Ltd

Turkey

Type 1*

2569/00

Anglia Oils Ltd

France

Type1*

912/00

Anglia Oils Ltd

Turkey

Type 2

10868/00

Liberty Vegetable Oil Company

Turkey

Type 2

Type 1, low concentrations of nC27, nC29 and nC31 and a UCM *very small UCM; Type 2 no UCM


19

TABLE 5

n-Alkane composition of refined hazelnut oil together with the mean and standard deviation (SD) for the individual n-alkanes, sum of nC12-nC25 and the sum of nC12-nC33.

Mean n-alkane concentration (mg kg-1) from the duplicate analysis Carbon No

2568/00 2569/00 912/00 10868/00 nC12 Tr Tr 1.5 ND nC13 Tr Tr 0.07 ND nC14 Tr Tr 1.32 Tr nC15 0.02 0.04 0.07 0.04 nC16 0.07 0.17 0.80 Tr nC17 0.15 0.30 0.15 ND nC18 0.19 0.30 0.53 ND nC19 0.19 0.28 0.39 ND nC20 0.14 0.18 0.36 ND nC21 0.10 0.11 0.11 ND nC22 0.05 0.06 0.19 ND nC23 0.14 0.06 0.07 ND nC24 0.02 ND 0.09 ND nC25 0.27 0.05 0.10 ND nC26 ND ND 0.07 ND nC27 0.32 ND ND 0.39 nC28 0.09 ND M 0.05 nC29 0.50 0.08 0.09 1.68 nC30 0.22 ND 0.77 ND nC31 0.27 0.02 ND 0.80 nC32 ND ND ND ND nC33 0.07 ND ND 0.08

Pristane (Pr) 0.04 0.17 ND ND Phytane (Ph) 0.12 0.2 ND ND

Sum of n-Alkanes nC12-nC33 2.81 1.65 6.68 3.04 Sum of n-Alkanes nC12-nC25 1.3 1.55 5.75 0.04

Tr - Trace = 2 - 16 ng g-1 ND - Not Detected = <2 ng g-1 M - Masked


20

TABLE 6

Sterene composition of refined hazelnut oil together with the mean and standard deviation (SD), along with the percentage composition of individual sterenes relative to the total sterene concentration.

Mean sterene concentration (mg kg-1) of the duplicate analysis Sterene

912/00 10868/00 2568/00 2569/00 Mean SD Percentage composition

24-ethylcholesta-4,6-diene 0.51 0.08 2.51 0.13 0.81 1.00 5.86

24-methylcholesta-3,5-diene 1.94 0.59 1.68 0.31 1.13 0.70 8.17




21

TABLE 7

Comparison of the sterene composition of refined hazelnut oil with refined oils analysed previously.19 The sterene concentration (mg kg-1) of groundnut, hazelnut, safflower and olive refined oils is the mean of oil samples analysed in duplicate. Standard deviations are shown in brackets.

Mean sterene concentration (mg kg-1) Sterene

Groudnut Hazelnut Safflower Olive

24-ethylcholesta-4,6-diene 0.31(0.17) 0.81(1.00) ND 0.46 (0.70)

24-ethylcholesta-3,5,22-triene 0.20 (0.13) ND 0.84 (0.09) 0.36 (0.20)

24-ethylcholesta-2,5-diene 0.52 (0.25) 0.78 (0.41) 0.26 (0.06) 0.28 (0.16)

24-ethylcholesta-3,5-diene 4.37 (2.4) 11.11(9.17) 0.83 (0.19) 2.90 (1.52)

24-methylcholesta-3,5-diene 1.31(0.78) 1.13 (0.69) 0.21(0.04) ND

24-methylcholesta-3,5,22-triene 0.02 ND ND ND


22

TABLE 8

n-Alkane concentrations (mg kg-1) of an authentic extra virgin olive oil (EXOO15)8 which had been adulterated with various amounts of ‘Type 1' crude hazelnut oil.

Sample No Description

EXOO15

Extra Virgin Olive

2570/00

Crude Hazel (Type 1)

11198/00 11.12%

11199/00

5.29%

11200/00

3.24%

11201/00

1.18%

11202/00

0.75%

nC12 ND Tr Tr 0.02 Tr Tr Tr

nC13 ND 0.02 Tr Tr Tr Tr Tr

nC14 0.09 ND 0.06 0.07 0.07 0.07 0.07

nC15 0.11 0.20 0.08 0.09 0.10 0.10 0.08

nC16 0.09 0.10 0.10 0.09 0.09 0.09 0.08

nC17 0.07 0.59 0.11 0.08 0.07 0.05 0.05

nC18 0.09 0.66 0.13 0.11 0.09 0.08 0.08

nC19 0.10 0.08 0.16 0.12 0.10 0.09 0.09

nC20 0.26 0.56 0.13 0.11 0.10 0.09 0.09

nC21 0.21 0.35 0.21 0.21 0.20 0.20 0.20

nC22 0.28 0.18 0.23 0.24 0.24 0.25 0.26

nC23 2.86 0.18 2.34 2.52 2.67 2.80 2.83

nC24 2.78 0.02 2.20 2.38 2.58 2.72 2.73

nC25 15.83 0.18 12.87 13.44 14.89 15.73 15.84

nC26 2.81 0.22 2.42 2.54 2.95 3.13 3.09

nC27 12.64 0.04 10.34 10.87 12.07 12.88 12.77

nC28 1.32 M 1.36 1.43 1.25 1.31 1.28

nC29 6.60 0.12 5.97 6.17 6.51 7.02 6.87

nC30 0.53 ND 0.67 0.70 0.50 0.55 0.53

nC31 3.28 0.05 2.98 3.06 3.44 3.77 3.63

nC32 0.37 0.03 0.32 0.33 0.37 0.42 0.38

nC33 1.42 0.04 1.46 1.46 1.65 1.84 1.70

Pristane (Pr) ND 0.38 0.05 0.03 Tr ND Tr

Phytane (Ph) ND 0.5 0.08 0.06 0.02 ND 0.03

Sum nC12-nC33 51.74 3.62 44.14 46.04 49.94 53.19 52.65 Sum nC12-nC25 22.77 3.12 18.62 19.48 21.2 22.27 22.4

Tr, Trace = 2 - 16 ng g-1; ND, Not Detected = < 2 ng g-1; M, Masked


23

TABLE 9 n-Alkane concentrations (mg kg-1) of an authentic extra virgin olive oil which had been adulterated with various amounts of ‘Type 2' crude hazelnut oil.


EXO015 Extra Virgin

Olive

2562/00 Crude hazelnut

(Type 2)

1549/01 11.16%

1550/01 5.37%

1551/01 2.61%

1552/01 1.07 %

1553/01 0.76%

nC12 ND 0.25 0.05 0.04 0.03 0.02 0.03

nC13 ND 0.02 0.02 0.02 0.02 0.02 0.02

nC14 0.09 0.03 0.08 0.10 0.09 0.09 0.09

nC15 0.11 0.05 0.06 0.08 0.10 0.08 0.11

nC16 0.09 0.03 0.08 0.09 0.10 0.1 0.09

nC17 0.07 0.03 0.05 0.06 0.07 0.06 0.06

nC18 0.09 0.02 0.07 0.08 0.08 0.08 0.08

nC19 0.10 0.03 0.08 0.08 0.09 0.09 0.08

nC20 0.26 0.01 0.08 0.08 0.08 0.08 0.08

nC21 0.21 0.02 0.18 0.19 0.20 0.19 0.19

nC22 0.28 0.02 0.24 0.26 0.27 0.26 0.26

nC23 2.86 0.03 3.17 3.42 3.47 3.45 3.43

nC24 2.78 0.03 2.45 2.64 2.66 2.66 2.64

nC25 15.83 0.08 16.30 17.60 17.67 17.63 17.55

nC26 2.81 0.03 3.36 3.61 3.62 3.60 3.58

nC27 12.64 0.11 13.80 14.89 14.93 14.77 14.74

nC28 1.32 0.05 1.58 1.64 1.59 1.91 1.61

nC29 6.60 1.17 8.21 8.73 8.64 8.51 8.49

nC30 0.53 0.05 0.78 0.77 0.72 0.81 0.70

nC31 3.28 0.22 3.92 4.20 4.14 4.05 4.05

nC32 0.37 0.01 0.5 0.50 0.47 0.50 0.47

nC33 1.42 0.01 2.38 2.56 2.51 2.45 2.46

Pristane (Pr) ND 3.75 0.4 0.22 0.06 0.06 0.04

Phytane (Ph) ND 0.08 ND ND 0.06 0.04 0.05

Sum nC12-nC33 51.74 2.27 57.44 61.64 61.55 61.41 60.81

Sum nC12-nC25 22.77 0.65 22.91 24.74 24.93 24.81 24.71

ND - Not Detected = < 2 ng g-1


24

TABLE 10

n-Alkane concentrations (mg kg-1) of an authentic refined olive oil which had been adulterated with various amounts of refined hazelnut oil.


RFOO1 Refined

Olive

10868/00 Refined Hazel

11837/00 11.11%

11838/00 5.38%

11839/00 2.59%

11840/00 1.10%

11841/00 0.84%

nC12 0.04 ND 0.03 0.03 0.04 0.04 0.03

nC13 Tr 0.04 Tr ND ND ND ND

nC14 0.06 Tr 0.05 0.05 0.05 0.05 0.04

nC15 0.02 ND 0.02 0.02 0.02 0.02 0.02

nC16 0.03 ND 0.02 0.02 0.02 Tr Tr

nC17 0.02 ND Tr Tr Tr Tr Tr

nC18 0.02 ND ND Tr Tr 0.02 ND

nC19 0.02 ND ND ND Tr Tr ND

nC20 0.03 ND Tr ND Tr Tr Tr

nC21 Tr ND Tr Tr Tr Tr Tr

nC22 0.04 ND Tr Tr Tr Tr ND

nC23 0.10 ND 0.08 0.09 0.09 0.09 0.08

nC24 0.09 ND 0.03 0.05 0.04 0.04 Tr

nC25 0.90 ND 0.85 0.91 0.87 0.94 0.80

nC26 ND ND 0.21 0.21 0.20 0.18 0.12

nC27 2.04 0.38 1.92 1.93 1.79 2.03 1.66

nC28 0.40 0.05 0.40 0.34 0.33 0.33 0.16

nC29 2.32 1.66 2.28 2.32 2.06 2.36 2.10

nC30 1.18 ND 0.03 0.08 Tr 0.08 0.17

nC31 2.77 0.80 2.71 2.61 2.35 2.83 2.47

nC32 0.30 Tr 0.30 0.29 0.27 0.30 0.23

nC33 1.44 0.08 1.34 1.33 1.12 1.53 1.34

Pristane (Pr) ND ND Tr Tr Tr Tr Tr

Phytane (Ph) ND Tr ND Tr 0.02 0.02 ND

Sum nC12-nC33 12.72 3.01 10.29 10.28 9.25 10.66 9.22

Sum nC12-nC25 1.37 0.04 1.08 1.17 1.13 1.18 0.97

Tr - Trace = 2-16 ng g-1 (ie 0.002-0.016 mg kg-1); ND - Not Detected = < 2 ng g-1


25

TABLE 11

Sterene concentrations of an authentic refined (R) olive oil, an authentic refined hazelnut oil and refined olive oil adulterated with various amounts of refined hazelnut oil.

Mean sterene concentration (mg kg-1)

R Olive R Hazel 11.11% 5.38% 2.59% 1.10% 0.84%


24-methylcholesta-3,5-diene ND 0.59 0.28 0.28 0.26 0.30 0.29



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