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MALDI-TOF mass spectrometry detection of extra-virgin olive oil adulteration with hazelnut oil by analysis of phospholipids using an ionic liquid as matrix and extraction solvent
Food Chemistry 134 (2012) 1192–1198
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Analytical Methods
MALDI-TOF mass spectrometry detection of extra-virgin olive oil adulteration with hazelnut oil by analysis of phospholipids using an ionic liquid as matrix and extraction solvent Cosima D. Calvano ⇑, Cristina De Ceglie, Lucia D’Accolti, Carlo G. Zambonin Università degli Studi di Bari Aldo Moro, Dipartimento di Chimica, Via Orabona, 4, 70126-BARI, Italy Centro Interdipartimentale di Spettrometria di Massa Analitica per Ricerche Tecnologiche (SMART), Italy
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Article history: Received 24 May 2011 Received in revised form 26 October 2011 Accepted 26 February 2012 Available online 10 March 2012 Keywords: Ionic liquids TBA-CHCA Extra-virgin olive oil Hazelnut oil MALDI-TOF-MS Phospholipids
a b s t r a c t The adulteration of extra virgin olive oil (EVOO) with hazelnut oil (HO) is frequent and constitutes a serious concern both for oil suppliers and consumers. The high degree of similarity between the two oils as regards triacylglycerol, total sterol and fatty acid profile, complicates the detection of low percentages of HO in EVOO. However, phospholipids (PLs) are usually present in seed oils at a concentration range of 10– 20 g/kg, while the amounts of PLs in VOOs are 300–400 times lower. Thus, in this work a sample pretreatment procedure focused towards the selective PLs extraction was developed; the Bligh–Dyer extraction procedure was modified introducing the ionic liquid resulting from the combination of TBA (tributylamine) and CHCA (a-Cyano-4-hydroxycinnamic acid) as extraction solvent. The selective extraction and enrichment of phospholipids from EVOO and HO samples was then achieved. The relevant extracts were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDITOF-MS) using the same ionic liquid TBA-CHCA as MALDI matrix, that was found to be very suitable for PLs analysis. In fact, a remarkable increase of the phospholipids signals, with a simultaneous decrease of those relevant to triacylglycerols and diacylglycerols, was observed in the relevant mass spectra. The applicability of the whole method to the individuation of the presence of HO in EVOO was demonstrated by the analysis of EVOO samples progressively adulterated with variable quantities of HO, that was still detectable at a 1% contamination level. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Olive oil (OO), obtained from the fruit of olive trees (Olea europaea L.), is a fundamental component of the Mediterranean diet (Serra-Majem, de la Cruz, Ribas, & Tur, 2003). Extra virgin olive oil (EVOO) is the highest quality and the most expensive product among OOs as it is obtained from olive fruits using only mechanical processing steps or other physical means under conditions that do not lead to oil alteration (European Union Commission Regulation, 1513/2001). This leads to the preservation of its characteristic properties responsible for its pleasant flavor and for nutritional and health benefits, which have been mainly related to the optimal balance between saturated, monounsaturated, and polyunsaturated fatty acids, as well as to minor components such as chlorophyll, squalene (Zambonin, Calvano, D’Accolti, & Palmisano, 2006), polyphenols, tocopherols (Lazzez, Perri, Caravita, Khlif, & Cossentini, 2008) and sterols. It appears to be an example of a func⇑ Corresponding author. E-mail address: [email protected] (C.D. Calvano). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.02.154
tional food, with a components variety that may contribute to its overall therapeutic characteristics including reduction of risk factors for coronary heart disease, prevention of several forms of cancer, and modification of immune and inflammatory responses (Stark & Madar, 2002). EVOO is of enormous economic importance, especially for Mediterranean countries. During the 2008–2009 season, more than 2.8 106 tons of extra virgin olive oil was produced in the world. The European Community is responsible for more than 75% of this amount (Torrecilla, Rojo, Domínguez, & Rodríguez, 2010). This means that a guarantee of the quality of EVOO as high as possible is required, and any fraudulent activity from the harvest of olives to the manufacture of EVOO must be avoided. However, the adulteration of extra virgin olive oil with small amounts of other edible oils of lower commercial value is frequent (Calvano, Palmisano, & Zambonin, 2005); with extra-virgin olive oil in high demand with concomitant high prices, adulterated olive oil has become the biggest source of agricultural fraud problems in the European Union. Besides the economic fraud, this may sometimes have severe health implications for consumers, such as hap-
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pened in the case of the Spanish toxic oil syndrome (Ruiz-Mendez et al., 2001). Those oils normally added to EVOO can be either olive oils of lower quality (e.g. olive-pomace oil or virgin olive oil obtained by second centrifugation of the olives) or seed oils (e.g. corn, hazelnut, soybean, palm or sunflower oil, among others). In particular, adulteration of extra virgin olive oil with hazelnut oil (HO) is a serious concern since it cannot be easily detected by well-established techniques because of their similarities in the triacylglycerol, sterol and fatty acid compositions. In addition adulteration of EVOO with HO may introduce hazelnut-derived allergens (Arlorio et al., 2010; Bremer, Smits, & Haasnoot, 2009). Several methods have been proposed in order to detect such adulteration principally based on chromatographic analyses (Blanch, Caja, Leon, & Herraiz, 2000; Garcia-Gonzalez, Viera-Macias, Aparicio-Ruiz, Morale, & Aparicio, 2007; Zabaras & Gordon, 2004), differential scanning calorimetry (Chiavaro, Vittadini, Rodriguez-Estrada, Cerretani, & Bendini, 2008), nuclear magnetic resonance (Agiomyrgianaki, Petrakis, & Dais, 2010; Mannina et al., 2009; Vlahov, 2009) and mass spectrometry (Arlorio et al., 2010; Calvano, Aresta, & Zambonin, 2010; Pena, Cardenas, Gallego, & Valcarcel, 2005). The simplest methods which can easily be implemented routinely in laboratories, are those based upon the analysis of a particular marker compound. These methods are useful for the qualitative detection of a particular type of adulterant, such as the determination of filbertone (Blanch et al., 2000; Flores, del Castillo, Herraiz, & Blanch, 2006). Another option is the direct analysis of the sample without pretreatment but with dilution, even if the high amount of data in these fingerprints cannot be processed without complicate chemometric tools (Agiomyrgianaki et al., 2010). Other works have been devoted to the analysis of particular classes of compounds such as sterols (Azadmard-Damirch, 2010), triacylglycerols (Garcia-Gonzalez et al., 2007), a specific fraction of the oil sample such as the polar fraction (Zabaras & Gordon, 2004) or, more specifically, the phospholipid fraction (Calvano et al., 2010). This approach seems to be very promising, since phospholipids (PLs) are usually present in seed oils at a concentration range of 10–20 g/kg (Bernardini, 1983), while their amount in VOOs is 300–400 times lower than in seed oils (Koidis & Boskou, 2006). For instance, a new micro-solid phase extraction (l-SPE) procedure based on hydrophilic liquid chromatography (HILIC) micro-columns (Calvano et al., 2010) was developed for the extraction and enrichment of lysophosphatidylcholine from HO and from EVOO adulterated with HO before matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis, using 2,5-dihydroxybenzoic acid (2,5–DHB) as matrix; the method was capable to detect the adulteration down to a level of 5%. On the basis of the above considerations, it can be hypothesized that the development of a sample pretreatment procedure focused towards the selective PLs extraction, together with the use of a MALDI matrix more suitable for PLs analysis, could be very useful to individuate adulteration of extra virgin olive oil with hazelnut oil. A possible approach can consider the use of ionic liquids (ILs), a class of ionic compounds with melting points below 100 °C (Liu, Jonsson, & Jiang, 2005), that have shown for the PLs classes very good solubilizing properties and excellent performances as MALDI matrices (Li & Gross, 2005). In the present work, a new selective extraction procedure involving the use of the ionic liquid arising from the combination of TBA (tributylamine) and CHCA (a-cyano-4-hydroxycinnamic acid) was developed for the extraction and enrichment of the PLs fraction of EVOO and HO. The resulting extracts were analyzed by MALDI-TOF-MS using TBA-CHCA as matrix. The whole procedure was optimized in order to obtain a PLs fingerprint for each oil that can be used to detect potential markers for the presence of HO in EVOO.
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2. Materials and methods 2.1. Materials 2,5-Dihydroxybenzoic acid (DHB), a-cyano-4-hydroxycinnamic acid (CHCA), tributylamine (TBA) were obtained from Sigma–Aldrich (St. Louis, MO, USA). Lipids (trilaurin, trimyristin, tripalmitin, and tristearin) were obtained from Supelco. Water, acetonitrile (ACN), methanol (MeOH), chloroform (CHCl3) (Sigma Aldrich, St. Louis, MO, USA) were HPLC grade and were used without further purification. Extra-virgin olive oil samples were purchased from local supermarket. Raw pressed hazelnut oil samples were purchased from retail outlets in Germany while refined hazelnut oil samples were obtained from local Agency Customs. 2.2. Synthesis of the ionic liquid matrix TBA-CHCA The ionic liquid matrix was prepared as already described (Armstrong, Zhang, He, & Gross, 2001). Briefly, organic salt of CHCA was prepared by dissolving 0.5 g of CHCA in 15 mL of methanol to yield a 176 mM solution. An equimolar amount of tributylamine was added and the mixture sonicated for 5 min and, subsequently, filtered. The solvent was then evaporated using a vacuum evaporator and the TBA-CHCA allowed to dry at room temperature under vacuum for 12 h to remove the last traces of the solvent. Then, the viscous ionic liquid was dissolved in 7.5 mL of MeOH and the resulting stock solution was used for further experiments. 2.3. Oil sample pretreatment The Bligh–Dyer (BD) (Bligh & Dyer, 1959) method was initially used for oil sample pretreatment. Briefly, 1.87 mL of CHCl3/MeOH (1:2) were added to 0.5 mL of oil followed by vigorous vortex-mixing. Then, 0.62 mL of CHCl3 were added and the solution vortexed again. Afterwards, 0.62 mL of H2O were added and the solution vortexed again. Finally, the solution was centrifuged (15 min at 2000 g) and the organic layer was collected in a new tube and dried under a stream of nitrogen. The residue was reconstituted in 100 lL of a CHCl3/MeOH (1:1) solution and mixed (1:1 v/v) with DHB matrix (20 mg/mL in MeOH:ACN (1:1, v:v)) or ionic liquid matrix TBA-CHCA; 1 lL of the sample/matrix solution was spotted, allowed to dry and analyzed by MALDI-TOF-MS. In the case of the modified Bligh–Dyer (BD) method, the same procedure described above was repeated using the ionic liquid TBA-CHCA dissolved in MeOH (at the concentration of 0.035 M), instead of MeOH as such. The organic layer was directly collected, diluted 1:10 in a CHCl3/ MeOH (1:1) solution and spotted on the target plate. 2.4. Maldi-tof-ms MS experiments were performed using a Micromass M@LDI™ – LR time-of-flight mass spectrometer (Waters MS Technologies, Manchester, UK) equipped with a nitrogen UV laser (337 nm wavelength), a precision flat target plate sample introduction system bearing a micro-titer target plate, reflectron optics with effective path length of 2.3 m, a fast dual micro-channel plate (MCP) detector, and a high magnification (70) camera system. Positive ion spectra were acquired in reflectron mode. The following voltages were applied: pulse, 2610 V; source, 15,000 V; reflectron, 2000 V; MCP, 1900 V. The laser firing rate was 5 Hz, and, unless otherwise specified, 80 laser shots were used for each well. The 80 resulting spectra were averaged, background subtracted, and smoothed by a Savitzky–Golay algorithm. External mass calibration (reflectron mode) was performed using a lipid
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mixture composed of trilaurin, trimyristin, tripalmitin, and tristearin.
3. Results and discussion As stated in the introduction, a promising approach for the individuation of adulteration of extra virgin olive oil with hazelnut oil could be based on a sample pre-treatment focused towards the extraction of phospholipids, usually present at higher concentration in seed oils. Thus, in the present work, the Bligh–Dyer (BD) method was used as starting point since it proved to extract PLs more efficiently than non-polar lipids (Cabrini, Landi, Stefanelli, Barzanti, & Sechi, 1992). Furthermore, since most MALDI analyses of PLs have employed 2,5-dihydroxybenzoic acid (DHB) as matrix because its use causes little fragmentation (Harvey, 1995), it was initially adopted. Fig. 1, A and B reports the MALDI-TOF-MS (DHB matrix) spectra relevant to the Bligh–Dyer extracts of (A) an extra-virgin olive oil sample and (B) an hazelnut oil sample, while Table 1 reports the list of the main m/z ions observed in those spectra and their probable attribution. At a first sight, one can immediately notice that the two oils produced very similar profiles, even regarding the relative abundance of the m/z ions, clearly underlining the difficulties in the determination of a potential adulteration.
Table 1 Attribution of the main ions observable in the spectra of Fig. 1A and B. Observed m/z
Probable attribution
577.40 601.41 603.42 615.42 617.43 641.41 643.43 659.39 771.50 797.54 855.65 877.64 879.66 881.69 883.70 895.70 897.69 899.70 905.70 907.70 909.71 923.68
[PO]+ [OL]+ [OO]+ [dg(PL) + Na]+* [dg(PO) + Na]+* [dg(OL) + Na]+* [dg(OO) + Na]+* [dg(OO) + K]+* [PG(C34:1) + Na]+, [GPA(C40:6) + Na]+ [PG(C36:2) + Na]+, [GPA(C42:7) + Na]+ [POP + Na]+, [PPO + Na]+ [PLL + Na]+, [POLn + Na]+ [PLO + Na]+, [POL + Na]+, [OPL + Na]+ [POO + Na]+, [OPO + Na]+ [POS + Na]+, [SPO + Na]+, [PSO + Na]+ [PLO + K]+, [POL + K]+, [OPL + K]+ [POO + K]+, [OPO + K]+ [POS + K]+, [SPO + K]+, [PSO + K]+ [OLO + Na]+, [OOL + Na]+ [OOO + Na]+, [SOL + Na]+, [SLO + Na]+, [LSO + Na]+ [SOO + Na]+ [OOO + K]+, [SOL + K]+, [SLO + K]+, [LSO + K]+
P = palmitic acid; O = oleic acid; S = stearic acid; L = linoleic acid; Ln = linolenic acid; PG = phosphatidylglycerol; GPA = glycerophosphatidic acid. * dg = diacylglycerol
Fig. 1. MALDI-TOF-MS spectra relevant to the Bligh–Dyer extracts of an extra-virgin olive oil sample (A) and an hazelnut oil sample (B) obtained using DHB as matrix. Zoom of the m/z range 730–850 for olive oil (C) and hazelnut oil (D).
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As expected, the spectra are dominated by sodium adducts of the main EVOO and HO triacylglycerols (TAGs), that constitute the major components, and by peaks attributable to the fragmentation of
Table 2 Attribution of the main ions observable in the spectra of Fig. 1C, D. Observed m/z
Probable attribution
758.50 760.45 771.48 780.46 782.48 784.51 786.51 788.50 795.50 797.50 806.49 808.50 811.50 813.50
[PC(C16:0/C18:2) + H]+ [PC(C16:0/C18:1) + H]+ [PG(C34:1) + Na]+, [GPA(C40:6) + Na]+ [PC(C16:0/C18:2) + Na]+ [PC(C16:0/C18:1) + Na]+, [PC(C18:2/C18:2) + H]+ [PC(C18:1/C18:2) + H]+ [PC(C18:1/C18:1) + H]+ [PC(C18:1/C18:0) + H]+ [PG(C36:3) + Na]+, [GPA(C42:8) + Na]+ [PG(C36:2) + Na]+, [GPA(C42:7) + Na]+ [PC(C18:2/C18:1) + Na]+ [PC(C18:1/C18:1) + Na]+, [PC(C18:0/C18:2) + Na]+ [PG(C36:3) + K]+, [GPA(C42:8) + K]+ [PG(C36:2) + K]+, [GPA(C42:7) + K]+
PC = phosphatidylcholine; glycerol.
GPA = glycerophosphatidic
acid;
PG = phosphatidyl-
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the most abundant TAGs (high mass region), corresponding to the loss of RCOO-Na+ molecules, with the consequent formation of the shorter positively charged diacylglycerols (low-mass region). However, from a more careful inspection of the intermediate mass range, some additional signals relevant to minor components are clearly present. Fig. 1, C and D, shows the expanded views of the m/z range 730–850 of the spectra reported in Fig. 1A (olive) and 1B (hazelnut), respectively. The list of the main m/z ions observed in those spectra and their hypothetical attribution is shown in Table 2. As apparent, the profiles relevant to this region showed some differences; in particular, the m/z ions 758.50, 760.45, 780.46, 782.48, 784.51, 786.51, 806.49 and 808.50 were observed only in the HO spectrum, while the m/z ions 771.50, 795.50, 797.50, 811.50 and 813.50 were present in both spectra. On the basis of literature data and with the help of database LIPID MAPS (The LIPID MAPS–Nature Lipidomics Gateway), the peaks observed only in HO samples were attributed to different classes of phospholipids (see Table 2), mainly phosphatidylcholines. However, in order to use some of these compounds as potential markers for the presence of HO in EVOO at low levels, the detection limit has to be improved. It is likely that the low S/N ratios observed for PLs are due mainly to a very low concentration of phospholipids in the analyzed sample compared to TAGs (that are the major components in oils) and,
Fig. 2. MALDI-TOF-MS spectra relevant to the Bligh–Dyer extracts of an extra-virgin olive oil sample (A) and an hazelnut oil sample (B) obtained using TBA-CHCA as matrix. Zoom of the m/z range 730–850 for olive oil (C) and hazelnut oil (D). M indicates matrix related peaks.
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probably to a not sufficient ionization efficiency (a modest competition for cation ion can involve TAGs and PLs). Furthermore, there could be some issues regarding the acidic conditions experienced with DHB. Indeed, an aqueous DHB sample solution at a typical concentration for MALDI analysis (20 mg/mL) exhibits a pH value < 2.5 that can cause a degradation of labile molecules such as lipids and phospholipids by hydrolysis. Thus, two different paths can be simultaneously followed at this point, i.e. the increase of both the ionization and extraction efficiency of phospholipids. As far as the ionization is concerned, the attention should be focused towards the MALDI matrix. Issues that affect the use of traditional matrices like DHB are well known: solvents used to dissolve the matrix also solubilize PLs causing the spreading of the sample spots; in a classical dried droplet approach, all ‘‘crystalline matrices’’ co-crystallize with the analyte, producing a heterogeneous sample, with the consequent lack of shot-to-shot and sample-tosample reproducibility. As previously reported (Liu et al., 2005), these problems can be avoided using ionic liquids as matrix, which may also be crystalline solids at room temperature, but their crystal size is smaller, producing homogeneous samples and better shot-to-shot, spot-to-spot reproducibility. Moreover, they do not strongly acidify the matrix-sample mixture maintaining relatively
moderate pH values. Furthermore, they have excellent solubilizing properties and vacuum stability, and, in many cases, higher ion peak intensity and equivalent or lower detection limits than currently used solid matrixes. Thus, the ionic liquid resulting from the combination of tributylamine and a-Cyano-4-hydroxycinnamic acid (TBA-CHCA) was selected, since it showed good performances in the ionization of PLs (Liu et al., 2005). Fig. 2, A and B reports the MALDI-TOF-MS (TBA-CHCA matrix) spectra relevant to the Bligh–Dyer extracts of (A) an extra-virgin olive oil sample and (B) an hazelnut oil sample. The differences between these spectra and those obtained with the DHB matrix are noticeable. In particular, the comparison between the spectra obtained from the analysis of hazelnut oil with the two matrices (Fig. 1B and Fig. 2B, respectively) showed that the use of TBA-CHCA brings to a terrific increase of the phospholipids signals, with a simultaneous decrease of those relevant to triacylglycerols and diacylglycerols, clearly showing the potential of this ionic liquid as suitable matrix for a selective PLs ionization in oil samples extracts. Moreover, while the MS spectra of EVOO and HO obtained with the DHB matrix were almost equal, very different profiles were obtained in this case, thus confirming that PLs could be useful markers for the detection of HO in EVOO. This aspect can be high-
Fig. 3. MALDI-TOF-MS spectra relevant to the modified Bligh–Dyer extracts of an extra-virgin olive oil sample (A) and an hazelnut oil sample (B) obtained using TBA-CHCA as extraction solvent and MALDI matrix. Zoom of the m/z range 730–850 for olive oil (C) and hazelnut oil (D).
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lighted observing Fig. 2, C and D, that shows the expanded views of the m/z range 730–850 of the spectra reported in Fig. 2A (olive) and 2B (hazelnut), respectively. As it is well known (Van Rantwijk & Sheldon, 2007) that ionic liquids can preferably dissolve polar compounds, it is reasonable to assume that PLs, in particular phosphatidylcholines, can be better extracted by using the ionic liquid TBA-CHCA as matrix. In addition, due to the steric effects of the bulky trialkyl ammonium cation, the positive charge is shielded, forcing far away the CHCA counterion; as a consequence, the smaller choline’s head, with a more exposed positive charge, could be strongly retained by the CHCA anion, increasing the solubility of PCs in this medium. Then, a further increase to the S/N ratio of PLs could be given by the improvement of their extraction efficiency from oil samples. Thus, the Bligh–Dyer method was modified (see ‘‘material and methods’’) by the addition of TBA-CHCA, that has shown for the PLs classes very good solubilizing properties (Li & Gross, 2005). Furthermore, very recently the properties of ionic liquids as good extraction media have been deeply reviewed (Zhao & Anderson, 2011). The relevant results are shown in Fig. 3, A–D that reports the MALDI-TOF-MS (TBA-CHCA matrix) spectra relevant to (A) an extra-virgin olive oil extract and (B) an hazelnut oil extract obtained using the modified Bligh–Dyer method; Fig. 3, C and D, shows the expanded views of the m/z range 730–850 of the spectra reported in Fig. 3A (olive) and 3B (hazelnut), respectively. As apparent, the new extraction approach produced very good results; the m/z ions relevant to triacylglycerols and diacylglycerols were almost absent from both spectra while in the HO spectrum all peaks previously attributed to PLs (see Table 2) were present at higher S/N ratios, while in the EVOO spectrum even the m/z ion 788.56, attributable to [PC(C18:0/C18:1) + H]+, was now clearly observable. These data confirmed a strong increase in selectivity and
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extraction efficiency towards PLs. In fact, even lysoPLs and the relevant dimers were observed in the spectra in the m/z ranges 490– 540 and 990–1100, respectively. Then, in order to exclude the possible existence of a variability between oil samples of different origins or producers, and to confirm that the marker compounds observed above are peculiar of hazelnut oil, different EVOO and HO samples were analyzed. No significant differences, with the exception of very slight intensity variations for some peaks, were found between the MALDI profiles relevant to the analysis of EVOO samples; the same results were obtained also in the case of HO samples, indicating that the method does not seem to be affected by natural variations, at least in the case of the investigated samples.
Fig. 5. Plot of the ratios between the intensities of two diagnostic peaks of HO (the m/z ions 760.50, 786.51) and EVOO (the m/z ion 788.51) versus the percentage composition of HO in the hazelnut oil/extra-virgin olive oil mixture.
Fig. 4. MALDI-TOF-MS spectra relevant to the modified Bligh–Dyer extracts of an extra-virgin olive oil sample adulterated with (A) 1% (B) 5% (C) 10% and (D) 20% of hazelnut oil.
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The present approach permitted to obtain different profiles for EVOO and HO and appears to be sensitive enough to discover adulteration of extra-virgin olive oil with hazelnut oil at low amounts. In order to demonstrate this hypothesis, an EVOO sample was progressively adulterated with variable quantities of hazelnut oil (from 1% to 20% in volume), and analyzed with the optimized procedure. Fig. 4, A–D, reports the MALDI-TOF-MS spectra relevant to the extracts of EVOO adulterated with 1%, 5%, 10% and 20% of HO, respectively. As apparent, the presence of the marker PLs was already observable at the lowest adulteration level, demonstrating the applicability of the method. In Fig. 5, the average ratios (three replicates) between the intensities of two diagnostic peaks of HO (the m/z ions 760.50, 786.51) and EVOO (the m/z ion 788.51) versus the percentage composition of HO in the hazelnut oil/extra-virgin olive oil mixture were plotted. Again, the presence of the marker compounds is already observable at the lowest adulteration level (i.e. 1%), furthermore, the value of each selected ratio increases with the percentage increase of hazelnut oil, demonstrating the sensitivity of the method. 4. Conclusions The ionic liquid resulting from the combination of tributylamine and a-Cyano-4-hydroxycinnamic acid (TBA-CHCA) was employed as extraction solvent in order to develop a fast and simple sample pretreatment procedure focused towards the selective extraction and enrichment of phospholipids from EVOO and HO samples. The approach, together with the use of the same ionic liquid as MALDI matrix, permitted a sensitive and selective detection of the PLs fraction of the analyzed samples. The whole procedure was found to be very useful to individuate adulteration of extra virgin olive oil with hazelnut oil down to a level of 1%. Acknowledgment We gratefully acknowledge Dr. Antonio Monopoli for the matrix synthesis and for helpful discussion. S. Giacummo is gratefully acknowledged for his skilled technical help. The University of Bari is acknowledged for financial support. References Agiomyrgianaki, A., Petrakis, P. V., & Dais, P. (2010). Detection of refined olive oil adulteration with refined hazelnut oil by employing NMR spectroscopy and multivariate statistical analysis. Talanta, 5, 2165–2171. Arlorio, M., Coisson, J. D., Bordiga, M., Travaglia, F., Garino, C., Zuidmeer, L., et al. (2010). Olive oil adulterated with hazelnut oils: simulation to identify possible risks to allergic consumers. Food Add. Contam. Part A, 27, 11–18. Armstrong, D. W., Zhang, L. K., He, L., & Gross, M. L. (2001). Ionic liquids as matrixes for matrix-assisted laser desorption/ionization mass spectrometry. Anal. Chem., 73, 3679–3686. Azadmard-Damirchi, S. (2010). Review of the use of phytosterols as a detection tool for adulteration of olive oil with hazelnut oil. Food Add. Contam. Part A, 27, 1–10. Bernardini, E. (1983). Oilseeds, oils and fats (Vol. I, B.E.). Rome: Oil Publishing House, pp. 173–178. Blanch, G. P., Caja, M. M., Leon, M., & Herraiz, M. (2000). Determination of (E)-5methylhept-2-en-4-one in deodorised hazelnut oil. Application to the detection of adulterated olive oils. J. Sci. Food Agr., 80, 140–144.
Bligh, E. G., & Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol., 37, 911–917. Bremer, M. G. E. G., Smits, N. G. E., & Haasnoot, W. (2009). Biosensor immunoassay for traces of hazelnut protein in olive oil. Anal. Bioanal. Chem., 395, 119–126. Cabrini, L., Landi, L., Stefanelli, C., Barzanti, V., & Sechi, A. M. (1992). Extraction of lipids and lipophilic antioxidants from fish tissues – a comparison among different methods. Comp. Biochem. Physiol., 101B, 383–386. Calvano, C. D., Aresta, A., & Zambonin, C. G. (2010). Detection of hazelnut oil in extra-virgin olive oil by analysis of polar components by micro-solid phase extraction based on hydrophilic liquid chromatography (HILIC l-SPE) and MALDI-TOF mass spectrometry. J. Mass Spectrom., 45, 981–988. Calvano, C. D., Palmisano, F., & Zambonin, C. G. (2005). Laser desorption/ionization time-of-flight mass spectrometry of triacylglycerols in oils. Rapid Commun. Mass Spectrom., 19, 1315–1320. Chiavaro, E., Vittadini, E., Rodriguez-Estrada, M. T., Cerretani, L., & Bendini, A. (2008). Differential scanning calorimeter application to the detection of refined hazelnut oil in extra virgin olive oil. Food Chem., 110, 248–256. European Union Commission Regulation 1513/2001 of 23 July 2001 amending Regulations No. 136/66/EEC. J. Eur. Union, L201. Flores, G., del Castillo, M. L. R., Herraiz, M., & Blanch, G. P. (2006). Study of the adulteration of olive oil with hazelnut oil by on-line coupled high performance liquid chromatographic and gas chromatographic analysis of filbertone. Food Chem., 97, 742–749. Garcia-Gonzalez, D. L., Viera-Macias, M., Aparicio-Ruiz, R., Morale, M. T., & Aparicio, R. (2007). Validation of a method based on triglycerides for the detection of low percentages of hazelnut oil in olive oil by column liquid chromatography. J. AOAC Int., 90, 1346–1353. Harvey, D. J. (1995). Matrix-assisted laser desorption/ionization mass spectrometry of phospholipids. J. Mass Spectrom., 30, 1333–1346. Koidis, A., & Boskou, D. (2006). The contents of proteins and phospholipids in cloudy (veiled) virgin olive oils. Europ. J. Lip. Sci. Technol., 108, 323–328. Lazzez, A., Perri, E., Caravita, M. A., Khlif, M., & Cossentini, M. (2008). Influence of olive maturity stage and geographical origin on some minor components in virgin olive oil of the Chemlali Variety. J. Agric. Food Chem., 56, 982–988. Li, Y. L., & Gross, M. L. (2005). Ionic-liquid matrices for improved analysis of phospholipids by MALDI-TOF mass spectrometry. J Am Soc Mass Spectrom., 16, 679–682. Liu, J., Jonsson, J. A., & Jiang, G. (2005). Application of ionic liquids in analytical chemistry. Trends Anal. Chem., 24, 20–27. Mannina, L., D’Imperio, M., Capitani, D., Rezzi, S., Guillou, C., Mavromoustakos, T., et al. (2009). H-1 NMR-based protocol for the detection of adulterations of refined olive oil with refined hazelnut oil. J. Agr. Food Chem., 57, 11550–11556. Pena, F., Cardenas, S., Gallego, M., & Valcarcel, M. (2005). Direct olive oil authentication: Detection of adulteration of olive oil with hazelnut oil by direct coupling of headspace and mass spectrometry, and multivariate regression techniques. J. Chrom. A, 1074, 215–221. Ruiz-Mendez, M. V., Posada de la Paz, M., Abian, J., Calaf, R. E., Blount, B., CastroMolero, N., et al. (tul=0?>Ruiz-Mendez). Storage time and deodorization temperature influence the formation of aniline-derived compounds in denatured rapeseed oils. Food Chem. Toxicol., 39, 91–96. Serra-Majem, L., de la Cruz, J., Ribas, L., & Tur, J. A. (2003). Olive oil and the Mediterranean diet: beyond the rhetoric. Eur. J. Clin. Nutr., 57, S2–S7. Stark, A. H., & Madar, Z. (2002). Olive oil as a functional food: Epidemiology and nutritional approaches. Nutr. Rev., 60, 170–176. The LIPID MAPS–Nature Lipidomics Gateway, http://www.lipidmaps.org/. Torrecilla, J. S., Rojo, E., Domínguez, J. C., & Rodríguez, F. (2010). A novel method to quantify the adulteration of extra virgin olive oil with low grade olive oils by UV–vis. J. Agr. Food Chem., 58, 1679–1684. Van Rantwijk, F., & Sheldon, R. A. (2007). Biocatalysis in Ionic Liquids. Chem. Rev., 107, 2757–2785. Vlahov, G. (2009). C-13 Nuclear magnetic resonance spectroscopic detection of the adulteration of extra virgin olive oils extracted from different cultivars with cold-pressed hazelnut oil. J. AOAC Int., 92, 1747–1754. Zabaras, D., & Gordon, M. H. (2004). Detection of pressed hazelnut oil in virgin olive oil by analysis of polar components: improvement and validation of the method. Food Chem., 84, 475–483. Zambonin, C. G., Calvano, C. D., D’Accolti, L., & Palmisano, F. (2006). Laser desorption/ionization time-of-flight mass spectrometry of squalene in oil samples. Rapid Commun. Mass Spectrom., 20, 325–327. Zhao, Q., & Anderson, J. L. (2011). Task-specific microextractions using ionic liquids. Anal Bioanal Chem., 400, 1613–1618.
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Analytical Methods
MALDI-TOF mass spectrometry detection of extra-virgin olive oil adulteration with hazelnut oil by analysis of phospholipids using an ionic liquid as matrix and extraction solvent Cosima D. Calvano ⇑, Cristina De Ceglie, Lucia D’Accolti, Carlo G. Zambonin Università degli Studi di Bari Aldo Moro, Dipartimento di Chimica, Via Orabona, 4, 70126-BARI, Italy Centro Interdipartimentale di Spettrometria di Massa Analitica per Ricerche Tecnologiche (SMART), Italy
a r t i c l e
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Article history: Received 24 May 2011 Received in revised form 26 October 2011 Accepted 26 February 2012 Available online 10 March 2012 Keywords: Ionic liquids TBA-CHCA Extra-virgin olive oil Hazelnut oil MALDI-TOF-MS Phospholipids
a b s t r a c t The adulteration of extra virgin olive oil (EVOO) with hazelnut oil (HO) is frequent and constitutes a serious concern both for oil suppliers and consumers. The high degree of similarity between the two oils as regards triacylglycerol, total sterol and fatty acid profile, complicates the detection of low percentages of HO in EVOO. However, phospholipids (PLs) are usually present in seed oils at a concentration range of 10– 20 g/kg, while the amounts of PLs in VOOs are 300–400 times lower. Thus, in this work a sample pretreatment procedure focused towards the selective PLs extraction was developed; the Bligh–Dyer extraction procedure was modified introducing the ionic liquid resulting from the combination of TBA (tributylamine) and CHCA (a-Cyano-4-hydroxycinnamic acid) as extraction solvent. The selective extraction and enrichment of phospholipids from EVOO and HO samples was then achieved. The relevant extracts were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDITOF-MS) using the same ionic liquid TBA-CHCA as MALDI matrix, that was found to be very suitable for PLs analysis. In fact, a remarkable increase of the phospholipids signals, with a simultaneous decrease of those relevant to triacylglycerols and diacylglycerols, was observed in the relevant mass spectra. The applicability of the whole method to the individuation of the presence of HO in EVOO was demonstrated by the analysis of EVOO samples progressively adulterated with variable quantities of HO, that was still detectable at a 1% contamination level. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Olive oil (OO), obtained from the fruit of olive trees (Olea europaea L.), is a fundamental component of the Mediterranean diet (Serra-Majem, de la Cruz, Ribas, & Tur, 2003). Extra virgin olive oil (EVOO) is the highest quality and the most expensive product among OOs as it is obtained from olive fruits using only mechanical processing steps or other physical means under conditions that do not lead to oil alteration (European Union Commission Regulation, 1513/2001). This leads to the preservation of its characteristic properties responsible for its pleasant flavor and for nutritional and health benefits, which have been mainly related to the optimal balance between saturated, monounsaturated, and polyunsaturated fatty acids, as well as to minor components such as chlorophyll, squalene (Zambonin, Calvano, D’Accolti, & Palmisano, 2006), polyphenols, tocopherols (Lazzez, Perri, Caravita, Khlif, & Cossentini, 2008) and sterols. It appears to be an example of a func⇑ Corresponding author. E-mail address: [email protected] (C.D. Calvano). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.02.154
tional food, with a components variety that may contribute to its overall therapeutic characteristics including reduction of risk factors for coronary heart disease, prevention of several forms of cancer, and modification of immune and inflammatory responses (Stark & Madar, 2002). EVOO is of enormous economic importance, especially for Mediterranean countries. During the 2008–2009 season, more than 2.8 106 tons of extra virgin olive oil was produced in the world. The European Community is responsible for more than 75% of this amount (Torrecilla, Rojo, Domínguez, & Rodríguez, 2010). This means that a guarantee of the quality of EVOO as high as possible is required, and any fraudulent activity from the harvest of olives to the manufacture of EVOO must be avoided. However, the adulteration of extra virgin olive oil with small amounts of other edible oils of lower commercial value is frequent (Calvano, Palmisano, & Zambonin, 2005); with extra-virgin olive oil in high demand with concomitant high prices, adulterated olive oil has become the biggest source of agricultural fraud problems in the European Union. Besides the economic fraud, this may sometimes have severe health implications for consumers, such as hap-
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pened in the case of the Spanish toxic oil syndrome (Ruiz-Mendez et al., 2001). Those oils normally added to EVOO can be either olive oils of lower quality (e.g. olive-pomace oil or virgin olive oil obtained by second centrifugation of the olives) or seed oils (e.g. corn, hazelnut, soybean, palm or sunflower oil, among others). In particular, adulteration of extra virgin olive oil with hazelnut oil (HO) is a serious concern since it cannot be easily detected by well-established techniques because of their similarities in the triacylglycerol, sterol and fatty acid compositions. In addition adulteration of EVOO with HO may introduce hazelnut-derived allergens (Arlorio et al., 2010; Bremer, Smits, & Haasnoot, 2009). Several methods have been proposed in order to detect such adulteration principally based on chromatographic analyses (Blanch, Caja, Leon, & Herraiz, 2000; Garcia-Gonzalez, Viera-Macias, Aparicio-Ruiz, Morale, & Aparicio, 2007; Zabaras & Gordon, 2004), differential scanning calorimetry (Chiavaro, Vittadini, Rodriguez-Estrada, Cerretani, & Bendini, 2008), nuclear magnetic resonance (Agiomyrgianaki, Petrakis, & Dais, 2010; Mannina et al., 2009; Vlahov, 2009) and mass spectrometry (Arlorio et al., 2010; Calvano, Aresta, & Zambonin, 2010; Pena, Cardenas, Gallego, & Valcarcel, 2005). The simplest methods which can easily be implemented routinely in laboratories, are those based upon the analysis of a particular marker compound. These methods are useful for the qualitative detection of a particular type of adulterant, such as the determination of filbertone (Blanch et al., 2000; Flores, del Castillo, Herraiz, & Blanch, 2006). Another option is the direct analysis of the sample without pretreatment but with dilution, even if the high amount of data in these fingerprints cannot be processed without complicate chemometric tools (Agiomyrgianaki et al., 2010). Other works have been devoted to the analysis of particular classes of compounds such as sterols (Azadmard-Damirch, 2010), triacylglycerols (Garcia-Gonzalez et al., 2007), a specific fraction of the oil sample such as the polar fraction (Zabaras & Gordon, 2004) or, more specifically, the phospholipid fraction (Calvano et al., 2010). This approach seems to be very promising, since phospholipids (PLs) are usually present in seed oils at a concentration range of 10–20 g/kg (Bernardini, 1983), while their amount in VOOs is 300–400 times lower than in seed oils (Koidis & Boskou, 2006). For instance, a new micro-solid phase extraction (l-SPE) procedure based on hydrophilic liquid chromatography (HILIC) micro-columns (Calvano et al., 2010) was developed for the extraction and enrichment of lysophosphatidylcholine from HO and from EVOO adulterated with HO before matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis, using 2,5-dihydroxybenzoic acid (2,5–DHB) as matrix; the method was capable to detect the adulteration down to a level of 5%. On the basis of the above considerations, it can be hypothesized that the development of a sample pretreatment procedure focused towards the selective PLs extraction, together with the use of a MALDI matrix more suitable for PLs analysis, could be very useful to individuate adulteration of extra virgin olive oil with hazelnut oil. A possible approach can consider the use of ionic liquids (ILs), a class of ionic compounds with melting points below 100 °C (Liu, Jonsson, & Jiang, 2005), that have shown for the PLs classes very good solubilizing properties and excellent performances as MALDI matrices (Li & Gross, 2005). In the present work, a new selective extraction procedure involving the use of the ionic liquid arising from the combination of TBA (tributylamine) and CHCA (a-cyano-4-hydroxycinnamic acid) was developed for the extraction and enrichment of the PLs fraction of EVOO and HO. The resulting extracts were analyzed by MALDI-TOF-MS using TBA-CHCA as matrix. The whole procedure was optimized in order to obtain a PLs fingerprint for each oil that can be used to detect potential markers for the presence of HO in EVOO.
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2. Materials and methods 2.1. Materials 2,5-Dihydroxybenzoic acid (DHB), a-cyano-4-hydroxycinnamic acid (CHCA), tributylamine (TBA) were obtained from Sigma–Aldrich (St. Louis, MO, USA). Lipids (trilaurin, trimyristin, tripalmitin, and tristearin) were obtained from Supelco. Water, acetonitrile (ACN), methanol (MeOH), chloroform (CHCl3) (Sigma Aldrich, St. Louis, MO, USA) were HPLC grade and were used without further purification. Extra-virgin olive oil samples were purchased from local supermarket. Raw pressed hazelnut oil samples were purchased from retail outlets in Germany while refined hazelnut oil samples were obtained from local Agency Customs. 2.2. Synthesis of the ionic liquid matrix TBA-CHCA The ionic liquid matrix was prepared as already described (Armstrong, Zhang, He, & Gross, 2001). Briefly, organic salt of CHCA was prepared by dissolving 0.5 g of CHCA in 15 mL of methanol to yield a 176 mM solution. An equimolar amount of tributylamine was added and the mixture sonicated for 5 min and, subsequently, filtered. The solvent was then evaporated using a vacuum evaporator and the TBA-CHCA allowed to dry at room temperature under vacuum for 12 h to remove the last traces of the solvent. Then, the viscous ionic liquid was dissolved in 7.5 mL of MeOH and the resulting stock solution was used for further experiments. 2.3. Oil sample pretreatment The Bligh–Dyer (BD) (Bligh & Dyer, 1959) method was initially used for oil sample pretreatment. Briefly, 1.87 mL of CHCl3/MeOH (1:2) were added to 0.5 mL of oil followed by vigorous vortex-mixing. Then, 0.62 mL of CHCl3 were added and the solution vortexed again. Afterwards, 0.62 mL of H2O were added and the solution vortexed again. Finally, the solution was centrifuged (15 min at 2000 g) and the organic layer was collected in a new tube and dried under a stream of nitrogen. The residue was reconstituted in 100 lL of a CHCl3/MeOH (1:1) solution and mixed (1:1 v/v) with DHB matrix (20 mg/mL in MeOH:ACN (1:1, v:v)) or ionic liquid matrix TBA-CHCA; 1 lL of the sample/matrix solution was spotted, allowed to dry and analyzed by MALDI-TOF-MS. In the case of the modified Bligh–Dyer (BD) method, the same procedure described above was repeated using the ionic liquid TBA-CHCA dissolved in MeOH (at the concentration of 0.035 M), instead of MeOH as such. The organic layer was directly collected, diluted 1:10 in a CHCl3/ MeOH (1:1) solution and spotted on the target plate. 2.4. Maldi-tof-ms MS experiments were performed using a Micromass M@LDI™ – LR time-of-flight mass spectrometer (Waters MS Technologies, Manchester, UK) equipped with a nitrogen UV laser (337 nm wavelength), a precision flat target plate sample introduction system bearing a micro-titer target plate, reflectron optics with effective path length of 2.3 m, a fast dual micro-channel plate (MCP) detector, and a high magnification (70) camera system. Positive ion spectra were acquired in reflectron mode. The following voltages were applied: pulse, 2610 V; source, 15,000 V; reflectron, 2000 V; MCP, 1900 V. The laser firing rate was 5 Hz, and, unless otherwise specified, 80 laser shots were used for each well. The 80 resulting spectra were averaged, background subtracted, and smoothed by a Savitzky–Golay algorithm. External mass calibration (reflectron mode) was performed using a lipid
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mixture composed of trilaurin, trimyristin, tripalmitin, and tristearin.
3. Results and discussion As stated in the introduction, a promising approach for the individuation of adulteration of extra virgin olive oil with hazelnut oil could be based on a sample pre-treatment focused towards the extraction of phospholipids, usually present at higher concentration in seed oils. Thus, in the present work, the Bligh–Dyer (BD) method was used as starting point since it proved to extract PLs more efficiently than non-polar lipids (Cabrini, Landi, Stefanelli, Barzanti, & Sechi, 1992). Furthermore, since most MALDI analyses of PLs have employed 2,5-dihydroxybenzoic acid (DHB) as matrix because its use causes little fragmentation (Harvey, 1995), it was initially adopted. Fig. 1, A and B reports the MALDI-TOF-MS (DHB matrix) spectra relevant to the Bligh–Dyer extracts of (A) an extra-virgin olive oil sample and (B) an hazelnut oil sample, while Table 1 reports the list of the main m/z ions observed in those spectra and their probable attribution. At a first sight, one can immediately notice that the two oils produced very similar profiles, even regarding the relative abundance of the m/z ions, clearly underlining the difficulties in the determination of a potential adulteration.
Table 1 Attribution of the main ions observable in the spectra of Fig. 1A and B. Observed m/z
Probable attribution
577.40 601.41 603.42 615.42 617.43 641.41 643.43 659.39 771.50 797.54 855.65 877.64 879.66 881.69 883.70 895.70 897.69 899.70 905.70 907.70 909.71 923.68
[PO]+ [OL]+ [OO]+ [dg(PL) + Na]+* [dg(PO) + Na]+* [dg(OL) + Na]+* [dg(OO) + Na]+* [dg(OO) + K]+* [PG(C34:1) + Na]+, [GPA(C40:6) + Na]+ [PG(C36:2) + Na]+, [GPA(C42:7) + Na]+ [POP + Na]+, [PPO + Na]+ [PLL + Na]+, [POLn + Na]+ [PLO + Na]+, [POL + Na]+, [OPL + Na]+ [POO + Na]+, [OPO + Na]+ [POS + Na]+, [SPO + Na]+, [PSO + Na]+ [PLO + K]+, [POL + K]+, [OPL + K]+ [POO + K]+, [OPO + K]+ [POS + K]+, [SPO + K]+, [PSO + K]+ [OLO + Na]+, [OOL + Na]+ [OOO + Na]+, [SOL + Na]+, [SLO + Na]+, [LSO + Na]+ [SOO + Na]+ [OOO + K]+, [SOL + K]+, [SLO + K]+, [LSO + K]+
P = palmitic acid; O = oleic acid; S = stearic acid; L = linoleic acid; Ln = linolenic acid; PG = phosphatidylglycerol; GPA = glycerophosphatidic acid. * dg = diacylglycerol
Fig. 1. MALDI-TOF-MS spectra relevant to the Bligh–Dyer extracts of an extra-virgin olive oil sample (A) and an hazelnut oil sample (B) obtained using DHB as matrix. Zoom of the m/z range 730–850 for olive oil (C) and hazelnut oil (D).
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As expected, the spectra are dominated by sodium adducts of the main EVOO and HO triacylglycerols (TAGs), that constitute the major components, and by peaks attributable to the fragmentation of
Table 2 Attribution of the main ions observable in the spectra of Fig. 1C, D. Observed m/z
Probable attribution
758.50 760.45 771.48 780.46 782.48 784.51 786.51 788.50 795.50 797.50 806.49 808.50 811.50 813.50
[PC(C16:0/C18:2) + H]+ [PC(C16:0/C18:1) + H]+ [PG(C34:1) + Na]+, [GPA(C40:6) + Na]+ [PC(C16:0/C18:2) + Na]+ [PC(C16:0/C18:1) + Na]+, [PC(C18:2/C18:2) + H]+ [PC(C18:1/C18:2) + H]+ [PC(C18:1/C18:1) + H]+ [PC(C18:1/C18:0) + H]+ [PG(C36:3) + Na]+, [GPA(C42:8) + Na]+ [PG(C36:2) + Na]+, [GPA(C42:7) + Na]+ [PC(C18:2/C18:1) + Na]+ [PC(C18:1/C18:1) + Na]+, [PC(C18:0/C18:2) + Na]+ [PG(C36:3) + K]+, [GPA(C42:8) + K]+ [PG(C36:2) + K]+, [GPA(C42:7) + K]+
PC = phosphatidylcholine; glycerol.
GPA = glycerophosphatidic
acid;
PG = phosphatidyl-
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the most abundant TAGs (high mass region), corresponding to the loss of RCOO-Na+ molecules, with the consequent formation of the shorter positively charged diacylglycerols (low-mass region). However, from a more careful inspection of the intermediate mass range, some additional signals relevant to minor components are clearly present. Fig. 1, C and D, shows the expanded views of the m/z range 730–850 of the spectra reported in Fig. 1A (olive) and 1B (hazelnut), respectively. The list of the main m/z ions observed in those spectra and their hypothetical attribution is shown in Table 2. As apparent, the profiles relevant to this region showed some differences; in particular, the m/z ions 758.50, 760.45, 780.46, 782.48, 784.51, 786.51, 806.49 and 808.50 were observed only in the HO spectrum, while the m/z ions 771.50, 795.50, 797.50, 811.50 and 813.50 were present in both spectra. On the basis of literature data and with the help of database LIPID MAPS (The LIPID MAPS–Nature Lipidomics Gateway), the peaks observed only in HO samples were attributed to different classes of phospholipids (see Table 2), mainly phosphatidylcholines. However, in order to use some of these compounds as potential markers for the presence of HO in EVOO at low levels, the detection limit has to be improved. It is likely that the low S/N ratios observed for PLs are due mainly to a very low concentration of phospholipids in the analyzed sample compared to TAGs (that are the major components in oils) and,
Fig. 2. MALDI-TOF-MS spectra relevant to the Bligh–Dyer extracts of an extra-virgin olive oil sample (A) and an hazelnut oil sample (B) obtained using TBA-CHCA as matrix. Zoom of the m/z range 730–850 for olive oil (C) and hazelnut oil (D). M indicates matrix related peaks.
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probably to a not sufficient ionization efficiency (a modest competition for cation ion can involve TAGs and PLs). Furthermore, there could be some issues regarding the acidic conditions experienced with DHB. Indeed, an aqueous DHB sample solution at a typical concentration for MALDI analysis (20 mg/mL) exhibits a pH value < 2.5 that can cause a degradation of labile molecules such as lipids and phospholipids by hydrolysis. Thus, two different paths can be simultaneously followed at this point, i.e. the increase of both the ionization and extraction efficiency of phospholipids. As far as the ionization is concerned, the attention should be focused towards the MALDI matrix. Issues that affect the use of traditional matrices like DHB are well known: solvents used to dissolve the matrix also solubilize PLs causing the spreading of the sample spots; in a classical dried droplet approach, all ‘‘crystalline matrices’’ co-crystallize with the analyte, producing a heterogeneous sample, with the consequent lack of shot-to-shot and sample-tosample reproducibility. As previously reported (Liu et al., 2005), these problems can be avoided using ionic liquids as matrix, which may also be crystalline solids at room temperature, but their crystal size is smaller, producing homogeneous samples and better shot-to-shot, spot-to-spot reproducibility. Moreover, they do not strongly acidify the matrix-sample mixture maintaining relatively
moderate pH values. Furthermore, they have excellent solubilizing properties and vacuum stability, and, in many cases, higher ion peak intensity and equivalent or lower detection limits than currently used solid matrixes. Thus, the ionic liquid resulting from the combination of tributylamine and a-Cyano-4-hydroxycinnamic acid (TBA-CHCA) was selected, since it showed good performances in the ionization of PLs (Liu et al., 2005). Fig. 2, A and B reports the MALDI-TOF-MS (TBA-CHCA matrix) spectra relevant to the Bligh–Dyer extracts of (A) an extra-virgin olive oil sample and (B) an hazelnut oil sample. The differences between these spectra and those obtained with the DHB matrix are noticeable. In particular, the comparison between the spectra obtained from the analysis of hazelnut oil with the two matrices (Fig. 1B and Fig. 2B, respectively) showed that the use of TBA-CHCA brings to a terrific increase of the phospholipids signals, with a simultaneous decrease of those relevant to triacylglycerols and diacylglycerols, clearly showing the potential of this ionic liquid as suitable matrix for a selective PLs ionization in oil samples extracts. Moreover, while the MS spectra of EVOO and HO obtained with the DHB matrix were almost equal, very different profiles were obtained in this case, thus confirming that PLs could be useful markers for the detection of HO in EVOO. This aspect can be high-
Fig. 3. MALDI-TOF-MS spectra relevant to the modified Bligh–Dyer extracts of an extra-virgin olive oil sample (A) and an hazelnut oil sample (B) obtained using TBA-CHCA as extraction solvent and MALDI matrix. Zoom of the m/z range 730–850 for olive oil (C) and hazelnut oil (D).
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lighted observing Fig. 2, C and D, that shows the expanded views of the m/z range 730–850 of the spectra reported in Fig. 2A (olive) and 2B (hazelnut), respectively. As it is well known (Van Rantwijk & Sheldon, 2007) that ionic liquids can preferably dissolve polar compounds, it is reasonable to assume that PLs, in particular phosphatidylcholines, can be better extracted by using the ionic liquid TBA-CHCA as matrix. In addition, due to the steric effects of the bulky trialkyl ammonium cation, the positive charge is shielded, forcing far away the CHCA counterion; as a consequence, the smaller choline’s head, with a more exposed positive charge, could be strongly retained by the CHCA anion, increasing the solubility of PCs in this medium. Then, a further increase to the S/N ratio of PLs could be given by the improvement of their extraction efficiency from oil samples. Thus, the Bligh–Dyer method was modified (see ‘‘material and methods’’) by the addition of TBA-CHCA, that has shown for the PLs classes very good solubilizing properties (Li & Gross, 2005). Furthermore, very recently the properties of ionic liquids as good extraction media have been deeply reviewed (Zhao & Anderson, 2011). The relevant results are shown in Fig. 3, A–D that reports the MALDI-TOF-MS (TBA-CHCA matrix) spectra relevant to (A) an extra-virgin olive oil extract and (B) an hazelnut oil extract obtained using the modified Bligh–Dyer method; Fig. 3, C and D, shows the expanded views of the m/z range 730–850 of the spectra reported in Fig. 3A (olive) and 3B (hazelnut), respectively. As apparent, the new extraction approach produced very good results; the m/z ions relevant to triacylglycerols and diacylglycerols were almost absent from both spectra while in the HO spectrum all peaks previously attributed to PLs (see Table 2) were present at higher S/N ratios, while in the EVOO spectrum even the m/z ion 788.56, attributable to [PC(C18:0/C18:1) + H]+, was now clearly observable. These data confirmed a strong increase in selectivity and
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extraction efficiency towards PLs. In fact, even lysoPLs and the relevant dimers were observed in the spectra in the m/z ranges 490– 540 and 990–1100, respectively. Then, in order to exclude the possible existence of a variability between oil samples of different origins or producers, and to confirm that the marker compounds observed above are peculiar of hazelnut oil, different EVOO and HO samples were analyzed. No significant differences, with the exception of very slight intensity variations for some peaks, were found between the MALDI profiles relevant to the analysis of EVOO samples; the same results were obtained also in the case of HO samples, indicating that the method does not seem to be affected by natural variations, at least in the case of the investigated samples.
Fig. 5. Plot of the ratios between the intensities of two diagnostic peaks of HO (the m/z ions 760.50, 786.51) and EVOO (the m/z ion 788.51) versus the percentage composition of HO in the hazelnut oil/extra-virgin olive oil mixture.
Fig. 4. MALDI-TOF-MS spectra relevant to the modified Bligh–Dyer extracts of an extra-virgin olive oil sample adulterated with (A) 1% (B) 5% (C) 10% and (D) 20% of hazelnut oil.
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The present approach permitted to obtain different profiles for EVOO and HO and appears to be sensitive enough to discover adulteration of extra-virgin olive oil with hazelnut oil at low amounts. In order to demonstrate this hypothesis, an EVOO sample was progressively adulterated with variable quantities of hazelnut oil (from 1% to 20% in volume), and analyzed with the optimized procedure. Fig. 4, A–D, reports the MALDI-TOF-MS spectra relevant to the extracts of EVOO adulterated with 1%, 5%, 10% and 20% of HO, respectively. As apparent, the presence of the marker PLs was already observable at the lowest adulteration level, demonstrating the applicability of the method. In Fig. 5, the average ratios (three replicates) between the intensities of two diagnostic peaks of HO (the m/z ions 760.50, 786.51) and EVOO (the m/z ion 788.51) versus the percentage composition of HO in the hazelnut oil/extra-virgin olive oil mixture were plotted. Again, the presence of the marker compounds is already observable at the lowest adulteration level (i.e. 1%), furthermore, the value of each selected ratio increases with the percentage increase of hazelnut oil, demonstrating the sensitivity of the method. 4. Conclusions The ionic liquid resulting from the combination of tributylamine and a-Cyano-4-hydroxycinnamic acid (TBA-CHCA) was employed as extraction solvent in order to develop a fast and simple sample pretreatment procedure focused towards the selective extraction and enrichment of phospholipids from EVOO and HO samples. The approach, together with the use of the same ionic liquid as MALDI matrix, permitted a sensitive and selective detection of the PLs fraction of the analyzed samples. The whole procedure was found to be very useful to individuate adulteration of extra virgin olive oil with hazelnut oil down to a level of 1%. Acknowledgment We gratefully acknowledge Dr. Antonio Monopoli for the matrix synthesis and for helpful discussion. S. Giacummo is gratefully acknowledged for his skilled technical help. The University of Bari is acknowledged for financial support. References Agiomyrgianaki, A., Petrakis, P. V., & Dais, P. (2010). Detection of refined olive oil adulteration with refined hazelnut oil by employing NMR spectroscopy and multivariate statistical analysis. Talanta, 5, 2165–2171. Arlorio, M., Coisson, J. D., Bordiga, M., Travaglia, F., Garino, C., Zuidmeer, L., et al. (2010). Olive oil adulterated with hazelnut oils: simulation to identify possible risks to allergic consumers. Food Add. Contam. Part A, 27, 11–18. Armstrong, D. W., Zhang, L. K., He, L., & Gross, M. L. (2001). Ionic liquids as matrixes for matrix-assisted laser desorption/ionization mass spectrometry. Anal. Chem., 73, 3679–3686. Azadmard-Damirchi, S. (2010). Review of the use of phytosterols as a detection tool for adulteration of olive oil with hazelnut oil. Food Add. Contam. Part A, 27, 1–10. Bernardini, E. (1983). Oilseeds, oils and fats (Vol. I, B.E.). Rome: Oil Publishing House, pp. 173–178. Blanch, G. P., Caja, M. M., Leon, M., & Herraiz, M. (2000). Determination of (E)-5methylhept-2-en-4-one in deodorised hazelnut oil. Application to the detection of adulterated olive oils. J. Sci. Food Agr., 80, 140–144.
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