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Extraction Techniques for the Analysis of Virgin Olive Oil Aroma
Chapter 66
Extraction Techniques for the Analysis of Virgin Olive Oil Aroma Stefania Vichi Departament de Nutrició i Bromatologia, Facultat de Farmàcia, Universitat de Barcelona, Spain
66.1 Introduction Sensory characteristics are used to define virgin olive oil (VOO) quality. This oil has a characteristic flavor that distinguishes it from other edible vegetable oils. After its extraction from the fruit of Olea europaea, VOO can be consumed without refining and it preserves its typical aroma. In recent years, the need for analytical procedures to evaluate the quality of VOO has led to several studies addressing its volatile fraction. Various analytical methods have been developed to examine these volatile compounds. In this way, a large number of components that contribute to the aroma of olive oil have been identified.
The volatile profile of VOO closely depends upon the method of extraction used (Cavalli et al., 2003; Vichi et al., 2007).
66.2 Features of virgin olive oil aroma The characteristic aroma of VOO, and in particular, the green and fruity attributes, depend on many volatile compounds derived from the degradation of polyunsaturated fatty acids through a chain of enzymatic reactions known as the lipoxygenase (LOX) pathway taking place during the oil extraction process (Figure 66.1).
Linoleic acid (LA)
Linolenic acid (LNA)
LOX
LOX
13-LA Hydroperoxide
13-LNA Hydroperoxide
HPL Hexanal
HPL 13-Alcoxy radical
(Z)-3-Hexenal
ADH Hexanol
Isomerase ADH
Pentene radical
(E)-2-Hexenal
AAT Hexyl acetate
ADH Pentene dimers
(Z)-3-Hexenol
(E)-2-Hexenol
AAT 2-Penten-1-ol 1-Penten-3-ol 2-Pentenal 1-Penten-3-one
(Z)-3-Hexenyl acetate
Figure 66.1 VOO volatile compounds derived from the LOX action on LA and LNA. The figure resumes the pathways of formation of C6 and C5 volatile compounds of virgin olive oil, following the attack of LOX on polyunsaturated fatty acids. LA: linoleic acid; LNA: linoleic acid; LOX: lipoxygenase; HPL: hydroperoxide lyase; ADH: alcohol dehydrogenase; AAT: alcohol acyl transferase. Olives and Olive Oil in Health and Disease Prevention. ISBN: 978-0-12-374420-3
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Variable amounts of hexanal, hexanol, and hexyl acetate derive from the degradation of linoleic acid, whereas (Z)-3-hexenal, (E)-2-hexenal, (E)-2-hexenol, (Z)-3-hexenol, and (Z)-3-hexenyl acetate result from the enzymatic degradation of linolenic acid (Olías et al., 1993). Moreover, pentene dimers, pentenols, and C5 carbonyl compounds are thought to originate from -scission of alkoxy radicals formed from 13-hydroperoxides by a LOX-mediated mechanism (Angerosa et al., 1998). Other linear alcohols, acids, esters and ketones, together with mono- and sesquiterpenes, deriving from the fruit metabolism, have also been found as constituents of VOO volatile fraction (Cavalli et al., 2003; Vichi et al., 2003a, 2006; Zunin et al., 2004). The volatile profiles of VOOs are believed to be influenced by factors such as the cultivar of the olives, climate, soil quality, degree of ripeness of the fruit, and oil extraction process (Montedoro et al., 1978; Angerosa, 2002). The preservation of olive fruits or olive oil under inadequate conditions, as well as unsuitable harvesting methods or technological processes, may decrease the sensory quality of olive oil by altering its typical volatile fraction composition. Basically fermentations, exogenous enzymatic processes and oxidation reactions give rise to undesirable volatile compounds affecting the VOO aromatic profile (Angerosa, 2002).
66.3 Headspace extraction techniques 66.3.1 Static Headspace (SHS) Headspace extraction techniques are selective techniques exploiting the volatility of some compounds with respect to the sample matrix, and their capacity to move from the sample to the headspace of a sealed container, from where they are collected. Static headspace (SHS) sampling is the simplest of these techniques, and it consists in removing a volume of the sample headspace after the equilibrium has been reached and introducing it into the analytical instrument. The concentration of the analyte in the headspace depends on its amount in the sample, its volatility and solubility in the sample matrix, the volume of the headspace and the temperature. Therefore, the sample can be heated to increase the concentration of analytes in the headspace. Because of its limited sensitivity, SHS is rarely employed for olive oil analysis. This extraction technique was inefficient for the analysis of VOO volatiles when used prior to gas chromatographic techniques (Cavalli et al., 2003). However, SHS directly coupled to a mass spectrometer (SHS-MS) has been recently applied for the characterization of olive oil (Cerrato Oliveros et al., 2005), for the quantification of VOO sensory attributes (López-Feria et al., 2007), and for the detection of adulterants (Lorenzo et al., 2002) and contaminants (Peña et al., 2004) in olive oil. In these works, the
Section | I Natural Components
total ion current signal was considered a spectral fingerprint of the oil sample, and statistical analyses were applied to relate the independent variables with the dependent variables represented by each m/z ratio obtained by the MS.
66.3.2 Dynamic Headspace (DHS) In order to overcome some of the sensitivity limitations of static headspace sampling, a dynamic headspace (DHS) technique, also known as purge and trap sampling, has been introduced. DHS involves the passing of carrier gas through a liquid sample, sweeping out the volatiles, and their continuous transferring through a trap which retains them. Heating the sample can increase the concentration of the compounds in the headspace and so increase the amount of sample on the trap. Nevertheless, in the case of VOO, the oxidative degradation must be taken into account when fixing the extraction temperature. In addition to temperature, also the flow rate, the stripping time and the volume of sample have to be optimized to obtain the highest uptakes and to avoid losses of analytes through the trap. One of the critical points of DHS is the loading capacity of traps. Over a certain volume of gaseous phase, the trap can be overloaded, with the resulting loss of analytes. This breakthrough can be prevented by carefully choosing the most adequate trapping material and working conditions. The use of several different trapping materials has been reported in the literature: solid or liquid sorbents, cold-traps or solvents (Pillonel et al., 2002). Carbon-based trapping materials and Tenax are the most widely used trapping media for the analysis of olive oil aroma. Tenax is characterized by a high thermal stability, a low bleed, and a good adsorption capacity for medium and high boiling compounds, in particular aldehydes, esters and hydrocarbons. Carbon-based traps show a strong adsorbent capacity for both low and medium molecular weight and in particular for alcohols and ketones (Angerosa, 2002; Pillonel et al., 2002). The high affinity of these traps against water is not a relevant inconvenience for olive oil analysis because of the low amount of water of this matrix. A comparative study showed that carbon-based traps are more efficient than Tenax for the analysis of VOO volatiles, independently from the applied conditions (Kanavouras et al., 2005). Desorption of thermally stable analytes from Tenax is generally performed by heating the trap and transferring the released volatiles to the gas chromatographic system by a carrier gas, while carbon traps require the elution at room temperature with a suitable solvent. Table 66.1 reports some applications of DHS in the analysis of VOO volatile composition, using Tenax and carbon-based traps.
66.3.2.1 Closed-Loop Stripping Apparatus (CLSA) A variation on dynamic headspace sampling is the closedloop stripping apparatus (CLSA). In this configuration the
Chapter | 66 Extraction Techniques for the Analysis of Virgin Olive Oil Aroma
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Table 66.1 Applications of principal sorbents used in DHS for the analysis of VOO aroma. Adsorbent
Application
Reference
Tenax
Identification of volatile compounds in VOO
Montedoro et al., 1978; Dobarganes-García et al., 1980; Morales et al., 1994a
Analysis of volatile compounds in relation with olive ripeness
Morales et al., 1996
Analysis of volatile compounds related with VOO sensory attributes
Morales et al., 1994b; Aparicio et al., 1996
Analysis of volatiles related with off-flavors
Morales et al., 1997, 2005
Identification and evaluation of VOO biogenic compounds
Olías et al., 1993; Angerosa et al., 1998, 1999
Volatile determination for cultivar characterization
Dhifi et al., 2005
Analysis of volatile compounds related with VOO sensory attributes
Servili et al., 1995; Angerosa et al., 2000
Analysis of volatiles related with off-flavors
Solinas et al., 1987; Angerosa et al., 1992, 1996
Determination of monoaromatic hydrocarbons as possible products of olive metabolism
Biedermann et al., 1995
Charcoal
Tenax and charcoal are the principal sorbents used in DHS for the analysis of VOO aroma. As can be seen in the table, both of them have been largely used for similar types of applications.
gaseous phase flows through the sample and the trap in a closed circuit. Volatiles are purged from the sample and concentrated in the trap, and since this is a cyclic system, compounds eluting from the end of the trap will not be lost but will be carried back through the system until reaching equilibrium. CLSA has been applied for the analysis of VOO aroma and compared with other extraction techniques (Vichi et al., 2007). CLSA showed a great efficiency in extracting esters and hydrocarbons, including sesquiterpene compounds. Esters principally consisted of hexyl acetate and hexenyl acetate, while hydrocarbons mainly included compounds such as pentene dimers and alkylated benzenes. The latter are environmental pollutants previously documented in VOO volatile fraction (Biedermann et al., 1995; Vichi et al., 2005).
66.3.3 Solid-Phase Microextraction (SPME) Solid-phase microextraction (SPME) technique has been introduced as an alternative to the dynamic headspace technique as a sample preconcentration method prior to chromatographic analysis. SPME is a rapid, sensitive and solvent-free sampling technique first developed by Pawliszyn and co-workers (Arthur and Pawliszyn, 1990) for the analysis of pollutants in water. In recent years,
SPME has extended its applications to numerous other fields, in particular food flavor analysis. SPME is based on the partitioning of organic components between a liquid sample or its vapor phase and a thin sorbent phase coated onto fused silica fibers. In general, the trapped compounds are thermally desorbed by introducing the fiber into the injection port of a gas chromatograph. The amount of analytes adsorbed or absorbed by the fiber is regulated by their concentration in the sample and their partition coefficient. The partition of analytes between the sample and the fiber is influenced by the type of sorbent and the extraction conditions. The uptake of the analytes and therefore the sensitivity of the method can be improved by optimizing parameters such as time and temperature of extraction, stirring, volume of sample and headspace, and in the case of aqueous samples, by regulating the pH and the ionic strength (Pawliszyn, 1999). Due to the nature and composition of olive oil, only headspace-SPME (HSSPME) has been used for the analysis of its volatile fraction. The principal difficulty of the SPME analysis of lipid samples is the matrix effect, which causes the decrease in SPME efficiency. Indeed, the lipid sample participates in the distribution equilibrium of volatiles as well as fiber coatings, having a high affinity with organic compounds. In order to optimize the efficiency and sensitivity of the
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Section | I Natural Components
Table 66.2 Commercial SPME fibers commonly used for the analysis of VOO aroma, and characteristics of sorbent phases. Fibers
Type of phase
Principal sorption process
Polarity
Length
References
PDMS
Polymeric
AB
Apolar
1 cm
Vichi et al., 2003a; Cavalli et al., 2003; Flamini et al., 2003; Tura et al., 2004; Kanavouras et al., 2005; Jimenez et al., 2006; Temime et al., 2006
CAR-PDMS
Porous particles/polymeric
AD
Semipolar
1 cm
Vichi et al., 2003a; Cavalli et al., 2003
PDMS-DVB
Porous particles/polymeric
AD
Semipolar
1 cm
Vichi et al., 2003a; Kanavouras et al., 2005; Jimenez et al., 2006
DVB-CAR-PDMS
Porous particles/polymeric
AD
Semipolar
2 cm
Vichi et al., 2003a
1 cm
Cavalli et al., 2003
2 cm
Runcio et al., 2008
2 cm
Manai et al., 2008
1 cm
Cavalli et al., 2003; Benincasa et al., 2003; Servili et al., 2003; Jimenez et al., 2006
CW-DVB
Porous particles/polymeric
AD
Polar
Mostly porous particles phases imbedded into polymeric phases have been used for the analysis of VOO aroma. AB: absorption; AD: adsorption.
method it is necessary to identify the most suitable SPME conditions. Basically, the type of fiber coating, temperature and time of extraction are the parameters to be taken into account in the case of olive oil since, in the case of lipids, the amount of sample does not affect the mass of analyte absorbed by the SPME coating (Page and Lacroix, 2000). Several sorbent phases for SPME with different characteristics are commercially available and have been used for the analysis of VOO aroma (Table 66.2). The efficiency of SPME for the qualitative and quantitative analysis of VOO volatile compounds was evaluated for the first time by comparing the behavior of four fiber coatings and by identifying the most suitable SPME sampling conditions (Vichi et al., 2003a). Polydimethylsiloxane (PDMS), carboxen-polydimethylsiloxane (CAR-PDMS), polydimethylsiloxane-divinylbenzene (PDMS-DVB) and divinylbenzene-carboxen-polydimethylsiloxane (DVB-CARPDMS) were compared for sensitivity, repeatability and linearity of response, and DVB-CAR-PDMS coating was found to be the most suitable for the analysis of VOO volatiles. Later, this result was also confirmed by comparing PDMS, CAR-PDMS, carbowax-DVB (CW-DVB) and DVB-CAR-PDMS (Cavalli et al., 2003). Sampling at 40 °C during 30 min allowed detection and identification of more than 100 compounds, some of them had not previously been reported in virgin olive oil (Vichi et al.,
2003a). Figure 66.2 reports the chromatographic profile of the same VOO extracted by HS-SPME and CLSA.
66.3.3.1 Specific Applications of SPME in the Analysis of VOO Aroma Among other applications, SPME allowed the characterization of VOOs from different olive varieties and geographical areas (Benincasa et al., 2003; Vichi et al., 2003b; Temime et al., 2006; Manai et al., 2008; Runcio et al., 2008) and the evaluation of processing and storage effects (Servili et al., 2003; Vichi et al., 2003c; Tura et al., 2004; Jiménez et al., 2006). SPME was also applied for the determination of semivolatile compounds of VOO by optimizing the extraction conditions, in particular testing several extraction temperatures (Vichi et al., 2005, 2006). The uptake of less volatile compounds increased at high temperatures because of the improvement of the mass-transfer process from the sample to the headspace. On the other hand, it has to be taken into account that the adsorption of analytes by the fiber coating is an exothermic process, and the partition coefficient decreases by increasing temperature, negatively affecting the adsorption of more volatile analytes. Optimized extraction conditions, with DVB-CAR-PDMS as sorbent during 60 min at 100 °C, allowed the simultaneous determination in VOOs of volatile and semivolatile aromatic hydrocarbons.
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Chapter | 66 Extraction Techniques for the Analysis of Virgin Olive Oil Aroma
2.0 E6
(E)-2-hexenol (Z)-3-hexenol (Z)-3-hexenyl acetate (E)-2-hexenal
SPME
1-hexanol
(E,E)-α-farnesene
2.6 E6 (Z)-3-hexenyl acetate
(E,E)-α-farnesene DHS (CLSA)
hexyl acetate (E)-2-hexenal
8.00
12.00
1-hexanol (Z)-3-hexenol (E)-2-hexenol 16.00
20.00
24.00
Figure 66.2 Chromatograms of VOO volatile profile obtained by SPME (DVB-CAR-PDMS, 40 °C) and DHS (CLSA mode). The figure shows that SPME sampling allows a higher uptake of LOX-derived alcohols to be obtained while DHS led to higher amounts of esters and semivolatile hydrocarbons.
These environmental contaminants can be especially abundant in oils and fats due to their lipophilic nature, especially in VOOs which lack refining processes (Vichi et al., 2005). Also, an SPME method was developed for the determination of semivolatile sesquiterpene hydrocarbons in VOOs, useful to distinguish samples from different cultivar and geographical origin (Vichi et al., 2006). The 30 sesquiterpenes extracted by DVB-CAR-PDMS at 70 °C during 60 min comprised hydrocarbons not previously documented as present in VOO (Figure 66.3).
66.3.4 Headspace Sorptive Extraction (HSSE) The main limit of SPME is the relatively low capacity given by the small amount of sorbent present on the fiber (0.5– 1 L). To overcome this problem, Baltussen et al. (1999) introduced the stir bar sorptive extraction (SBSE), a magnetic stir bar coated with a thick film of PDMS the volume of which ranges from 25 to 250 L. As an extension of SBSE, headspace sorptive extraction (HSSE) was introduced for the extraction of headspace components, and performed by suspending the PDMS stir bar in the vapor phase, in equilibrium or not with the matrix (Bicchi et al., 2000). Only HSSE configuration was applied for the analysis of VOO. After sampling, the stir bar is placed in a glass tube and transferred to a thermo-desorption system from where the analytes are conveyed to the gas chromatographic system. The principles of HSSE are similar to those
of SPME, but with an increased capacity due to the higher amount of sorbent phase. So, the extraction conditions have to be optimized in the same way to allow the highest uptakes of compounds of interest. HSSE was applied for the analysis of VOOs, and compared with SPME, SHS and direct thermal desorption (DTD) (Cavalli et al., 2003). HSSE allowed the determination of all the compounds detected by SPME using a DVB-CAR-PDMS fiber, and a larger number of sesquiterpenes, showing a higher concentration capacity than SPME. Nevertheless, as the introduction of the stir bar into the vial breaks the equilibrium, the best performances are obtained after more than one hour. The optimum extraction time was fixed at 120 min.
66.4 Direct thermal desorption (DTD) Thermal desorption systems were initially developed for the extraction of volatiles from SBSE stir-bars, and later proposed for direct thermal desorption (DTD). In this technique, the sample is directly placed in the thermal desorption unit without the presence of any adsorbent. The analytes are volatilized by increasing the temperature and stripped by the carrier gas into a cold trap placed in the injector. Differently from the techniques based on the use of sorbents, the recovery of the analytes in DTD is only based on their volatility, avoiding their partition between the sample and the sorbent.
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Section | I Natural Components
1 α-pinene 2 β-pinene 3 sabinene 4 δ3-carene 5 myrcene 6 α-terpinene 7 dl-limonene 8 p-mentha-1,5,8-triene 9 (Z)-b-ocimene 10 γ-terpinene 11 (E)-b-ocimene 12 p-cymene 13 4,8-dimethyl-1,3,7-nonatriene 14 (Z)-alloocimene 15 (E)-alloocimene 16 α-cubebene 17 cyclosativene 18 α-copaene 19 sativene 20 α-cedrene 21 β-cubebene 22 (E)-α-bergamotene 23 β-gurjunene (calarene) 24 β-caryophyllene 25 (Z)-β-farnesene 26 (E)-β-farnesene 27 γ-gurjunene 28 γ-muurolene 29 γ-curcumene 30 β-acoradiene 31 α-selinene 32 eremophyllene 33 α-zingiberene 34 α-muurolene 35 δ-guaiene 36 (E,E)-α-farnesene 37 δ-cadinene 38 β-sesquiphellandrene 39 ar-curcumene 40 n.i. sesquiterpene 41 calamenene 42 n.i. m/z 93, 107, 135, 204 43 n.i. m/z 93, 107, 135, 204 44 β-calacorene 45 n.i. m/z 93, 107, 135, 204 Figure 66.3 Extracted ion chromatogram of mono- and sesquiterpene hydrocarbons in VOO, by HS-SPME coupled to gas chromatography/mass spectrometry. Reprinted from Vichi S., et al. J. Chromatogr. A 2006; 1125:117–123, with permission. The figure allows appreciating the sensibility of the SPME method coupled to gas chromatography/mass spectrometry (single ion monitoring) for the analysis of terpene and sesquiterpene hydrocarbons, which are present at trace levels in VOO.
DTD is a rapid, solvent-free technique requiring a very small amount of sample, usually a few microliters, which does not need any manipulation prior to the analysis. DTD has been applied for the characterization of French, Spanish and Italian VOOs (Cavalli et al., 2003; Zunin et al., 2004). Desorption conditions evaluated to develop the analytical methods were related both to the extraction step and
the injection step. They were: final temperature, temperature ramp rate, time, cooling temperature and carrier gas flow. Desorptions at 40 °C and 80 °C during 20 min were chosen for the analysis of VOO volatiles, allowing the detection of a large number of compounds characteristic of VOO (Cavalli et al., 2003; Zunin et al., 2004). As expected, the number and amount of semivolatile sesquiterpenes were proportional
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Chapter | 66 Extraction Techniques for the Analysis of Virgin Olive Oil Aroma
50 SPME
SDE
CLSA
Percentage of areas
40
30
20
10
0 Alcohols
Aldehydes
Ketones
Hydrocarbons
Esters
Terpenoids
Figure 66.4 Percentages of VOO’s major families of volatiles extracted by SPME, SDE and CLSA. Reprinted from Vichi S., et al. Food Chem. 1007; 105: 1171–1178, with permission. Better affinities for alcohols and aldehydes are shown by SPME and SDE, respectively, while CLSA gives higher uptakes of esters and hydrocarbons. The best recoveries of terpenoids are obtained by SDE and DHS (CLSA).
to the temperature applied in these studies. A further study was conducted for the determination of these semivolatile terpenoid hydrocarbons in VOO, and the subsequent discrimination of the samples according to the geographical origin (Zunin et al., 2005). When compared with SPME, DTD was more efficient in the extraction of semivolatile sesquiterpenes and esters of fatty acids, while no relevant differences were observed in the recovery of other volatiles (Cavalli et al., 2003). Anyway, it must be taken into account that the extraction temperatures applied in this study were not the same for both methods. In fact, SPME was performed at 25°C, while DTD was performed at 80°C, which is likely to facilitate the volatilization of semivolatile compounds. Recently, a multi-step direct thermal desorption method coupled to comprehensive gas chromatography-time-offlight-mass spectrometry was developed and applied for the characterization of fresh and aged olive oils at typical frying temperatures (de Koning et al., 2008). Different temperatures of desorption were tested to evaluate the compounds present in native VOOs (70°C), in olive oil during frying or baking (175 and 250°C), evidencing differences between fresh and aged olive oil.
66.5 Distillation and fluid-based extraction techniques 66.5.1 Simultaneous Distillation/Extraction (SDE) and Hydrodistillation Distillation methods have traditionally been applied in the analysis of plant materials. Hydrodistillation and simultaneous distillation/extraction (SDE) were used for this purpose, and SDE appeared to afford the most favorable uptake for mono- and sesquiterpenes, as well as their oxygenated
analogues (Marriott et al., 2001). Hydrodistillation has been applied for the analysis of leaf, fruit and virgin oil volatiles of an Italian olive cultivar (Flamini et al., 2003). The uptake of olive oil volatiles was comparable to that obtained by SPME using a PDMS fiber. The most remarkable difference was the higher amount of -farnesene obtained by hydrodistillation. However, with hydrodistillation the volatiles in the steam distillate are heavily diluted by water when collected in cold traps. This is overcome in SDE via solvent extraction of the distillate. SDE was evaluated for the analysis of VOO aroma using a modified Likens and Nickerson apparatus, and using pentane and dichloromethane as solvents (Vichi et al., 2007). In comparison with the extraction of the same oil by SPME and CLSA, SDE gave higher percentages of aldehydes correlated with the oxidative degradation of VOO, indicating that the extraction conditions induced the thermal alteration of the oil sample (Figure 66.4). Regarding the extraction of semivolatile compounds, -zingiberene and -farnesene were the only compounds detected at a higher percentage by SDE, while other sesquiterpenes were more abundant by CLSA extraction.
66.5.2 Supercritical Fluid Extraction (SFE) Supercritical fluid extraction (SFE) has been widely used for extracting flavor and fragrance compounds from complex matrices without the use of organic solvents. This method is based on the use of gases at determined conditions of pressure and temperature, known as supercritical region. At these conditions gases can acquire solvent properties that are superior to those of liquid solvents. Supercritical CO2 is among the most used fluids due to its good proven extractive capacity and safety, and it was also tested for the extraction of volatile and semivolatile compounds from VOO (Morales et al., 1998). After SFE, a concentration of volatiles on a Tenax trap was required
622
prior to the chromatographic analysis in order to enhance the sensitivity of the method. Different profiles of volatile and semivolatile compounds were found in the SFE extracts, depending on experimental parameters. All the major VOO volatiles were identified in the extract. Soft extraction conditions allowed higher uptakes of the most volatile compounds to be obtained, while drastic conditions led to a higher extraction of semivolatile compounds and induced the oxidation of the sample.
Summary points VOO has a unique flavor that distinguishes it from other vegetable oils. l Various analytical methods have been developed to examine these volatile compounds. l The volatile profile of VOO closely depends upon the method of extraction used. l Headspace extraction techniques are the most widely used for the analysis of olive oil aroma, and SHS, DHS, SPME, HSSE and DTD have been applied to VOO, depending on the kind of determination required. l Among distillation and fluid-based extraction techniques, only hydrodistillation, SDE and SFE have been tentatively applied for the analysis of VOO aroma. l
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Section | I Natural Components
Benincasa, C., De Nino, A., Lombardo, N., Perri, E., Sindona, G., Tagarelli, A., 2003. Assay of aroma active components of virgin olive oils from southern Italian regions by SPME-GC/ion trap mass spectrometry. J. Agric. Food Chem. 51, 733–741. Bicchi, C., D’Amato, A., David, F., Sandra, P., 2000. Headspace sorptive extraction (HSSE) in the headspace analysis of aromatic and medicinal plants. J. High Res. Chromatogr. 23, 539–546. Biedermann, M., Grob, K., Morchio, G., 1995. On the origin of benzene, toluene, ethylbenzene and xylene in extra virgin olive oil. Z. Lebensm. Unters. Forsch. 200, 266–272. Cavalli, J.F., Fernandez, X., Lizzani-Cuvelier, L., Loiseau, A.M., 2003. Comparative study of different extraction techniques for the analysis of virgin olive oil aroma. J. Agric. Food Chem. 51, 7709–7716. Cerrato Oliveros, C., Boggia, R., Casale, M., Armanino, C., Forina, M., 2005. Optimisation of a new headspace mass spectrometry instrument. Discrimination of different geographical origin olive oils. J. Chromatogr. A 1076, 7–15. de Koning, S., Kaal, E., Janssen, H.G., van Platerink, C., Brinkman, U.A.T., 2008. Characterization of olive oil volatiles by multistep direct thermal desorption-comprehensive gas chromatography-time-of-flightmass spectrometry using a programmed temperature vaporizing injector. J. Chromatogr. A 1186, 228–235. Dhifi, W., Angerosa, F., Serraiocco, A., Oumar, I., Hamrouni, I., Marzouk, B., 2005. Virgin olive oil aroma: characterization of some Tunisian cultivars. Food Chem. 93, 697–701. Dobarganes-García, M.C., Olías, J.M., González-Quijano, R.G., 1980. Componentes volátiles en el aroma del aceite de oliva virgen. III. Reproducibilidad del método utilizado para su aislamiento, concentración y separación. Grasas Aceites 31, 317–321. Flamini, G., Cioni, P.L., Morelli, I., 2003. Volatiles from leaves, fruits, and virgin oil from Olea europaea Cv. Olivastra Seggianese from Italy. J. Agric. Food Chem. 51, 1382–1386. Jimenez, A., Aguilera, M.P., Beltran, G., Uceda, M., 2006. Application of solid-phase microextraction to virgin olive oil quality control. J. Chromatogr. A 1121, 140–144. Kanavouras, A., Kiritsakis, A., Hernandez, R.J., 2005. Comparative study on volatile analysis of extra virgin olive oil by dynamic headspace and solid phase microextraction. Food Chem. 90, 69–79. López-Feria, S., Cárdenas, S., García-Mesa, J.A., Fernández-Hernández, A., Valcárcel, M., 2007. Quantification of the intensity of virgin olive oil sensory attributes by direct coupling headspace-mass spectrometry and multivariate calibration techniques. J. Chromatogr. A 1147, 144–152. Lorenzo, M.I., Pavón, J.L.P., Laespada, M.E.F., Pinto, C.G., Cordero, B.M., 2002. Detection of adulterants in olive oil by headspace-mass spectrometry. J. Chromatogr. A 945, 221–230. Manai, H., Mahjoub-Haddada, F., Oueslati, I., Daoud, D., Zarrouk, M., 2008. Characterization of monovarietal virgin olive oils from six crossing varieties. Sci. Hortic. 115, 252–260. Marriott, P.J., Shellie, R., Cornwell, C., 2001. Gas chromatographic technologies for the analysis of essential oils. J. Chromatogr. A 936, 1–22. Montedoro, G.F., Bertuccioli, M., Anichini, F., 1978. Aroma analysis of virgin olive oil by head space (volatiles) and extraction (polyphenols) techniques. In: Charalambous, G., Inglett, G. (Eds.), Flavor of Foods and Beverages. Academic Press, New York, pp. 247–281. Morales, M.T., Aparicio, R., Rios, J.J., 1994a. Dynamic headspace gas chromatographic method for determining volatiles in virgin olive oil. J. Chromatogr. A 668, 455–462.
Chapter | 66 Extraction Techniques for the Analysis of Virgin Olive Oil Aroma
Morales, M.T., Alonso, M.V., Rios, J.J., Aparicio, R., 1994b. Virgin olive oil aroma: relationship between volatile compounds and sensory attributes by chemometrics. J. Agric. Food Chem. 43, 2925–2931. Morales, M.T., Aparicio, R., Calvente, J.J., 1996. Influence of olive ripeness on the concentration of aroma compounds in virgin olive oil. Flavour Fragrance J. 11, 171–178. Morales, M.T., Rios, J.J., Aparicio, R., 1997. Changes in the volatile composition of virgin olive oil during oxidation: flavours and off-flavours. J. Agric. Food Chem. 45, 2666–2673. Morales, M.T., Berry, A.J., McIntyre, P.S., Aparicio, R., 1998. Tentative analysis of virgin olive oil aroma by supercritical fluid extraction–high-resolution gas chromatography–mass spectrometry. J. Chromatogr. A 819, 267–275. Morales, M.T., Luna, G., Aparicio, R., 2005. Comparative study of virgin olive oil sensory defects. Food Chem. 91, 293–301. Olías, J.M., Pérez, A.G., Ríos, J.J., Sanz, L.C., 1993. Aroma of virgin olive oil: biogenesis of the “green” odor notes. J. Agric. Food Chem. 41, 2368–2373. Pawliszyn, J., 1999. Quantitative aspects of SPME. In: Pawliszyn, J. (Ed.), Applications of Solid Phase Microextraction. Royal Society of Chemistry, Cambridge (UK), pp. 3–21. Page, B.D., Lacroix, G., 2000. Analysis of volatile contaminants in vegetable oils by headspace solid-phase microextraction with carboxenbased fibres. J. Chromatogr. A 873, 79–94. Peña, F., Cárdenas, S., Gallego, M., Valcárcel, M., 2004. Direct screening of olive oil samples for residual benzene hydrocarbon compounds by headspace-mass spectrometry. Anal. Chim. Acta 526, 77–82. Pillonel, L., Bosset, J.O., Tabacchi, R., 2002. Rapid preconcentration and enrichment techniques for the analysis of food volatile. A review. LWT-Lebensm.-Wiss. Technol. 35, 1–14. Runcio, A., Sorgona, L., Mincione, A., Santacaterina, S., Poiana, M., 2008. Volatile compounds of virgin olive oil obtained from Italian cultivars grown in Calabria. Food Chem. 106, 735–740. Servili, M., Conner, J.M., Piggott, J.R., Withers, S.J., Paterson, A., 1995. Sensory characterisation of virgin olive oil and relationship with headspace composition. J. Sci. Food Agric. 67, 61–70. Servili, M., Selvaggini, R., Taticchi, A., Esposto, S., Montedoro, G., 2003. Volatile compounds and phenolic composition of virgin olive oil: optimization of temperature and time of exposure of olive pastes to air contact during the mechanical extraction process. J. Agric. Food Chem. 51, 7980–7988. Solinas, M., Angerosa, F., Cucurachi, A., 1987. Connessione tra i prodotti di neoformazione ossidativa delle sostanze grasse e insorgenza del
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difetto di rancidità all’esame organolettico. Nota II. Determinazione quantitativa. Riv. Ital. Sost. Grasse. 64, 137–145. Temime, S.B., Campeol, E., Cioni, P.L., Daoud, D., Zarrouk, M., 2006. Volatile compounds from Chétoui olive oil and variations induced by growing area. Food Chem. 99, 315–325. Tura, D., Prenzler, P.D., Bedgood, D.R., Antolovich, M., Robards, K., 2004. Varietal and processing effects on the volatile profile of Australian olive oils. Food Chem. 84, 341–349. Vichi, S., Castellote, A.I., Pizzale, L., Conte, L.S., Buxaderas, S., LopezTamames, E., 2003a. Analysis of virgin olive oil volatile compounds by headspace solid-phase microextraction coupled to gas chromatography with mass spectrometric and flame ionization detection. J. Chromatogr. A 983, 19–23. Vichi, S., Pizzale, L., Conte, L.S., Buxaderas, S., Lopez-Tamames, E., 2003b. Solid-phase microextraction in the analysis of virgin olive oil volatile fraction: characterization of virgin olive oils from two distinct geographical areas of northern Italy. J. Agric. Food Chem. 51, 6572–6577. Vichi, S., Pizzale, L., Conte, L.S., Buxaderas, S., Lopez-Tamames, E., 2003c. Solid-phase microextraction in the analysis of virgin olive oil volatile fraction: modifications induced by oxidation and suitable markers of oxidative status. J. Agric. Food Chem. 51, 6564–6571. Vichi, S., Pizzale, L., Conte, L.S., Buxaderas, S., Lopez-Tamames, E., 2005. Simultaneous determination of volatile and semi-volatile aromatic hydrocarbons in virgin olive oil by headspace solid-phase microextraction coupled to gas chromatography/mass spectrometry. J. Chromatogr. A 1090, 146–154. Vichi, S., Guadayol, J.M., Caixach, J., López-Tamames, E., Buxaderas, S., 2006. Monoterpene and sesquiterpene hydrocarbons of virgin olive oil by headspace solid-phase microextraction coupled to gas chromato graphy/mass spectrometry. J. Chromatogr. A 1125, 117–123. Vichi, S., Guadayol, J.M., Caixach, J., López-Tamames, E., Buxaderas, S., 2007. Comparative study of different extraction techniques for the analysis of virgin olive oil aroma. Food Chem. 105, 1171–1178. Zunin, P., Boggia, R., Lanteri, S., Leardi, R., De Andreis, R., Evangelisti, F., 2004. Direct thermal extraction and gas chromatographic-mass spectrometric determination of volatile compounds of extra-virgin olive oils. J. Chromatogr. A 1023, 271–276. Zunin, P., Boggia, R., Salvadeo, P., Evangelisti, F., 2005. Geographical traceability of West Liguria extravirgin olive oils by the analysis of volatile terpenoid hydrocarbons. J. Chromatogr. A 1089, 243–249.
Extraction Techniques for the Analysis of Virgin Olive Oil Aroma Stefania Vichi Departament de Nutrició i Bromatologia, Facultat de Farmàcia, Universitat de Barcelona, Spain
66.1 Introduction Sensory characteristics are used to define virgin olive oil (VOO) quality. This oil has a characteristic flavor that distinguishes it from other edible vegetable oils. After its extraction from the fruit of Olea europaea, VOO can be consumed without refining and it preserves its typical aroma. In recent years, the need for analytical procedures to evaluate the quality of VOO has led to several studies addressing its volatile fraction. Various analytical methods have been developed to examine these volatile compounds. In this way, a large number of components that contribute to the aroma of olive oil have been identified.
The volatile profile of VOO closely depends upon the method of extraction used (Cavalli et al., 2003; Vichi et al., 2007).
66.2 Features of virgin olive oil aroma The characteristic aroma of VOO, and in particular, the green and fruity attributes, depend on many volatile compounds derived from the degradation of polyunsaturated fatty acids through a chain of enzymatic reactions known as the lipoxygenase (LOX) pathway taking place during the oil extraction process (Figure 66.1).
Linoleic acid (LA)
Linolenic acid (LNA)
LOX
LOX
13-LA Hydroperoxide
13-LNA Hydroperoxide
HPL Hexanal
HPL 13-Alcoxy radical
(Z)-3-Hexenal
ADH Hexanol
Isomerase ADH
Pentene radical
(E)-2-Hexenal
AAT Hexyl acetate
ADH Pentene dimers
(Z)-3-Hexenol
(E)-2-Hexenol
AAT 2-Penten-1-ol 1-Penten-3-ol 2-Pentenal 1-Penten-3-one
(Z)-3-Hexenyl acetate
Figure 66.1 VOO volatile compounds derived from the LOX action on LA and LNA. The figure resumes the pathways of formation of C6 and C5 volatile compounds of virgin olive oil, following the attack of LOX on polyunsaturated fatty acids. LA: linoleic acid; LNA: linoleic acid; LOX: lipoxygenase; HPL: hydroperoxide lyase; ADH: alcohol dehydrogenase; AAT: alcohol acyl transferase. Olives and Olive Oil in Health and Disease Prevention. ISBN: 978-0-12-374420-3
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Copyright © 2010 Elsevier Inc. All rights of reproduction in any form reserved.
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Variable amounts of hexanal, hexanol, and hexyl acetate derive from the degradation of linoleic acid, whereas (Z)-3-hexenal, (E)-2-hexenal, (E)-2-hexenol, (Z)-3-hexenol, and (Z)-3-hexenyl acetate result from the enzymatic degradation of linolenic acid (Olías et al., 1993). Moreover, pentene dimers, pentenols, and C5 carbonyl compounds are thought to originate from -scission of alkoxy radicals formed from 13-hydroperoxides by a LOX-mediated mechanism (Angerosa et al., 1998). Other linear alcohols, acids, esters and ketones, together with mono- and sesquiterpenes, deriving from the fruit metabolism, have also been found as constituents of VOO volatile fraction (Cavalli et al., 2003; Vichi et al., 2003a, 2006; Zunin et al., 2004). The volatile profiles of VOOs are believed to be influenced by factors such as the cultivar of the olives, climate, soil quality, degree of ripeness of the fruit, and oil extraction process (Montedoro et al., 1978; Angerosa, 2002). The preservation of olive fruits or olive oil under inadequate conditions, as well as unsuitable harvesting methods or technological processes, may decrease the sensory quality of olive oil by altering its typical volatile fraction composition. Basically fermentations, exogenous enzymatic processes and oxidation reactions give rise to undesirable volatile compounds affecting the VOO aromatic profile (Angerosa, 2002).
66.3 Headspace extraction techniques 66.3.1 Static Headspace (SHS) Headspace extraction techniques are selective techniques exploiting the volatility of some compounds with respect to the sample matrix, and their capacity to move from the sample to the headspace of a sealed container, from where they are collected. Static headspace (SHS) sampling is the simplest of these techniques, and it consists in removing a volume of the sample headspace after the equilibrium has been reached and introducing it into the analytical instrument. The concentration of the analyte in the headspace depends on its amount in the sample, its volatility and solubility in the sample matrix, the volume of the headspace and the temperature. Therefore, the sample can be heated to increase the concentration of analytes in the headspace. Because of its limited sensitivity, SHS is rarely employed for olive oil analysis. This extraction technique was inefficient for the analysis of VOO volatiles when used prior to gas chromatographic techniques (Cavalli et al., 2003). However, SHS directly coupled to a mass spectrometer (SHS-MS) has been recently applied for the characterization of olive oil (Cerrato Oliveros et al., 2005), for the quantification of VOO sensory attributes (López-Feria et al., 2007), and for the detection of adulterants (Lorenzo et al., 2002) and contaminants (Peña et al., 2004) in olive oil. In these works, the
Section | I Natural Components
total ion current signal was considered a spectral fingerprint of the oil sample, and statistical analyses were applied to relate the independent variables with the dependent variables represented by each m/z ratio obtained by the MS.
66.3.2 Dynamic Headspace (DHS) In order to overcome some of the sensitivity limitations of static headspace sampling, a dynamic headspace (DHS) technique, also known as purge and trap sampling, has been introduced. DHS involves the passing of carrier gas through a liquid sample, sweeping out the volatiles, and their continuous transferring through a trap which retains them. Heating the sample can increase the concentration of the compounds in the headspace and so increase the amount of sample on the trap. Nevertheless, in the case of VOO, the oxidative degradation must be taken into account when fixing the extraction temperature. In addition to temperature, also the flow rate, the stripping time and the volume of sample have to be optimized to obtain the highest uptakes and to avoid losses of analytes through the trap. One of the critical points of DHS is the loading capacity of traps. Over a certain volume of gaseous phase, the trap can be overloaded, with the resulting loss of analytes. This breakthrough can be prevented by carefully choosing the most adequate trapping material and working conditions. The use of several different trapping materials has been reported in the literature: solid or liquid sorbents, cold-traps or solvents (Pillonel et al., 2002). Carbon-based trapping materials and Tenax are the most widely used trapping media for the analysis of olive oil aroma. Tenax is characterized by a high thermal stability, a low bleed, and a good adsorption capacity for medium and high boiling compounds, in particular aldehydes, esters and hydrocarbons. Carbon-based traps show a strong adsorbent capacity for both low and medium molecular weight and in particular for alcohols and ketones (Angerosa, 2002; Pillonel et al., 2002). The high affinity of these traps against water is not a relevant inconvenience for olive oil analysis because of the low amount of water of this matrix. A comparative study showed that carbon-based traps are more efficient than Tenax for the analysis of VOO volatiles, independently from the applied conditions (Kanavouras et al., 2005). Desorption of thermally stable analytes from Tenax is generally performed by heating the trap and transferring the released volatiles to the gas chromatographic system by a carrier gas, while carbon traps require the elution at room temperature with a suitable solvent. Table 66.1 reports some applications of DHS in the analysis of VOO volatile composition, using Tenax and carbon-based traps.
66.3.2.1 Closed-Loop Stripping Apparatus (CLSA) A variation on dynamic headspace sampling is the closedloop stripping apparatus (CLSA). In this configuration the
Chapter | 66 Extraction Techniques for the Analysis of Virgin Olive Oil Aroma
617
Table 66.1 Applications of principal sorbents used in DHS for the analysis of VOO aroma. Adsorbent
Application
Reference
Tenax
Identification of volatile compounds in VOO
Montedoro et al., 1978; Dobarganes-García et al., 1980; Morales et al., 1994a
Analysis of volatile compounds in relation with olive ripeness
Morales et al., 1996
Analysis of volatile compounds related with VOO sensory attributes
Morales et al., 1994b; Aparicio et al., 1996
Analysis of volatiles related with off-flavors
Morales et al., 1997, 2005
Identification and evaluation of VOO biogenic compounds
Olías et al., 1993; Angerosa et al., 1998, 1999
Volatile determination for cultivar characterization
Dhifi et al., 2005
Analysis of volatile compounds related with VOO sensory attributes
Servili et al., 1995; Angerosa et al., 2000
Analysis of volatiles related with off-flavors
Solinas et al., 1987; Angerosa et al., 1992, 1996
Determination of monoaromatic hydrocarbons as possible products of olive metabolism
Biedermann et al., 1995
Charcoal
Tenax and charcoal are the principal sorbents used in DHS for the analysis of VOO aroma. As can be seen in the table, both of them have been largely used for similar types of applications.
gaseous phase flows through the sample and the trap in a closed circuit. Volatiles are purged from the sample and concentrated in the trap, and since this is a cyclic system, compounds eluting from the end of the trap will not be lost but will be carried back through the system until reaching equilibrium. CLSA has been applied for the analysis of VOO aroma and compared with other extraction techniques (Vichi et al., 2007). CLSA showed a great efficiency in extracting esters and hydrocarbons, including sesquiterpene compounds. Esters principally consisted of hexyl acetate and hexenyl acetate, while hydrocarbons mainly included compounds such as pentene dimers and alkylated benzenes. The latter are environmental pollutants previously documented in VOO volatile fraction (Biedermann et al., 1995; Vichi et al., 2005).
66.3.3 Solid-Phase Microextraction (SPME) Solid-phase microextraction (SPME) technique has been introduced as an alternative to the dynamic headspace technique as a sample preconcentration method prior to chromatographic analysis. SPME is a rapid, sensitive and solvent-free sampling technique first developed by Pawliszyn and co-workers (Arthur and Pawliszyn, 1990) for the analysis of pollutants in water. In recent years,
SPME has extended its applications to numerous other fields, in particular food flavor analysis. SPME is based on the partitioning of organic components between a liquid sample or its vapor phase and a thin sorbent phase coated onto fused silica fibers. In general, the trapped compounds are thermally desorbed by introducing the fiber into the injection port of a gas chromatograph. The amount of analytes adsorbed or absorbed by the fiber is regulated by their concentration in the sample and their partition coefficient. The partition of analytes between the sample and the fiber is influenced by the type of sorbent and the extraction conditions. The uptake of the analytes and therefore the sensitivity of the method can be improved by optimizing parameters such as time and temperature of extraction, stirring, volume of sample and headspace, and in the case of aqueous samples, by regulating the pH and the ionic strength (Pawliszyn, 1999). Due to the nature and composition of olive oil, only headspace-SPME (HSSPME) has been used for the analysis of its volatile fraction. The principal difficulty of the SPME analysis of lipid samples is the matrix effect, which causes the decrease in SPME efficiency. Indeed, the lipid sample participates in the distribution equilibrium of volatiles as well as fiber coatings, having a high affinity with organic compounds. In order to optimize the efficiency and sensitivity of the
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Section | I Natural Components
Table 66.2 Commercial SPME fibers commonly used for the analysis of VOO aroma, and characteristics of sorbent phases. Fibers
Type of phase
Principal sorption process
Polarity
Length
References
PDMS
Polymeric
AB
Apolar
1 cm
Vichi et al., 2003a; Cavalli et al., 2003; Flamini et al., 2003; Tura et al., 2004; Kanavouras et al., 2005; Jimenez et al., 2006; Temime et al., 2006
CAR-PDMS
Porous particles/polymeric
AD
Semipolar
1 cm
Vichi et al., 2003a; Cavalli et al., 2003
PDMS-DVB
Porous particles/polymeric
AD
Semipolar
1 cm
Vichi et al., 2003a; Kanavouras et al., 2005; Jimenez et al., 2006
DVB-CAR-PDMS
Porous particles/polymeric
AD
Semipolar
2 cm
Vichi et al., 2003a
1 cm
Cavalli et al., 2003
2 cm
Runcio et al., 2008
2 cm
Manai et al., 2008
1 cm
Cavalli et al., 2003; Benincasa et al., 2003; Servili et al., 2003; Jimenez et al., 2006
CW-DVB
Porous particles/polymeric
AD
Polar
Mostly porous particles phases imbedded into polymeric phases have been used for the analysis of VOO aroma. AB: absorption; AD: adsorption.
method it is necessary to identify the most suitable SPME conditions. Basically, the type of fiber coating, temperature and time of extraction are the parameters to be taken into account in the case of olive oil since, in the case of lipids, the amount of sample does not affect the mass of analyte absorbed by the SPME coating (Page and Lacroix, 2000). Several sorbent phases for SPME with different characteristics are commercially available and have been used for the analysis of VOO aroma (Table 66.2). The efficiency of SPME for the qualitative and quantitative analysis of VOO volatile compounds was evaluated for the first time by comparing the behavior of four fiber coatings and by identifying the most suitable SPME sampling conditions (Vichi et al., 2003a). Polydimethylsiloxane (PDMS), carboxen-polydimethylsiloxane (CAR-PDMS), polydimethylsiloxane-divinylbenzene (PDMS-DVB) and divinylbenzene-carboxen-polydimethylsiloxane (DVB-CARPDMS) were compared for sensitivity, repeatability and linearity of response, and DVB-CAR-PDMS coating was found to be the most suitable for the analysis of VOO volatiles. Later, this result was also confirmed by comparing PDMS, CAR-PDMS, carbowax-DVB (CW-DVB) and DVB-CAR-PDMS (Cavalli et al., 2003). Sampling at 40 °C during 30 min allowed detection and identification of more than 100 compounds, some of them had not previously been reported in virgin olive oil (Vichi et al.,
2003a). Figure 66.2 reports the chromatographic profile of the same VOO extracted by HS-SPME and CLSA.
66.3.3.1 Specific Applications of SPME in the Analysis of VOO Aroma Among other applications, SPME allowed the characterization of VOOs from different olive varieties and geographical areas (Benincasa et al., 2003; Vichi et al., 2003b; Temime et al., 2006; Manai et al., 2008; Runcio et al., 2008) and the evaluation of processing and storage effects (Servili et al., 2003; Vichi et al., 2003c; Tura et al., 2004; Jiménez et al., 2006). SPME was also applied for the determination of semivolatile compounds of VOO by optimizing the extraction conditions, in particular testing several extraction temperatures (Vichi et al., 2005, 2006). The uptake of less volatile compounds increased at high temperatures because of the improvement of the mass-transfer process from the sample to the headspace. On the other hand, it has to be taken into account that the adsorption of analytes by the fiber coating is an exothermic process, and the partition coefficient decreases by increasing temperature, negatively affecting the adsorption of more volatile analytes. Optimized extraction conditions, with DVB-CAR-PDMS as sorbent during 60 min at 100 °C, allowed the simultaneous determination in VOOs of volatile and semivolatile aromatic hydrocarbons.
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Chapter | 66 Extraction Techniques for the Analysis of Virgin Olive Oil Aroma
2.0 E6
(E)-2-hexenol (Z)-3-hexenol (Z)-3-hexenyl acetate (E)-2-hexenal
SPME
1-hexanol
(E,E)-α-farnesene
2.6 E6 (Z)-3-hexenyl acetate
(E,E)-α-farnesene DHS (CLSA)
hexyl acetate (E)-2-hexenal
8.00
12.00
1-hexanol (Z)-3-hexenol (E)-2-hexenol 16.00
20.00
24.00
Figure 66.2 Chromatograms of VOO volatile profile obtained by SPME (DVB-CAR-PDMS, 40 °C) and DHS (CLSA mode). The figure shows that SPME sampling allows a higher uptake of LOX-derived alcohols to be obtained while DHS led to higher amounts of esters and semivolatile hydrocarbons.
These environmental contaminants can be especially abundant in oils and fats due to their lipophilic nature, especially in VOOs which lack refining processes (Vichi et al., 2005). Also, an SPME method was developed for the determination of semivolatile sesquiterpene hydrocarbons in VOOs, useful to distinguish samples from different cultivar and geographical origin (Vichi et al., 2006). The 30 sesquiterpenes extracted by DVB-CAR-PDMS at 70 °C during 60 min comprised hydrocarbons not previously documented as present in VOO (Figure 66.3).
66.3.4 Headspace Sorptive Extraction (HSSE) The main limit of SPME is the relatively low capacity given by the small amount of sorbent present on the fiber (0.5– 1 L). To overcome this problem, Baltussen et al. (1999) introduced the stir bar sorptive extraction (SBSE), a magnetic stir bar coated with a thick film of PDMS the volume of which ranges from 25 to 250 L. As an extension of SBSE, headspace sorptive extraction (HSSE) was introduced for the extraction of headspace components, and performed by suspending the PDMS stir bar in the vapor phase, in equilibrium or not with the matrix (Bicchi et al., 2000). Only HSSE configuration was applied for the analysis of VOO. After sampling, the stir bar is placed in a glass tube and transferred to a thermo-desorption system from where the analytes are conveyed to the gas chromatographic system. The principles of HSSE are similar to those
of SPME, but with an increased capacity due to the higher amount of sorbent phase. So, the extraction conditions have to be optimized in the same way to allow the highest uptakes of compounds of interest. HSSE was applied for the analysis of VOOs, and compared with SPME, SHS and direct thermal desorption (DTD) (Cavalli et al., 2003). HSSE allowed the determination of all the compounds detected by SPME using a DVB-CAR-PDMS fiber, and a larger number of sesquiterpenes, showing a higher concentration capacity than SPME. Nevertheless, as the introduction of the stir bar into the vial breaks the equilibrium, the best performances are obtained after more than one hour. The optimum extraction time was fixed at 120 min.
66.4 Direct thermal desorption (DTD) Thermal desorption systems were initially developed for the extraction of volatiles from SBSE stir-bars, and later proposed for direct thermal desorption (DTD). In this technique, the sample is directly placed in the thermal desorption unit without the presence of any adsorbent. The analytes are volatilized by increasing the temperature and stripped by the carrier gas into a cold trap placed in the injector. Differently from the techniques based on the use of sorbents, the recovery of the analytes in DTD is only based on their volatility, avoiding their partition between the sample and the sorbent.
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Section | I Natural Components
1 α-pinene 2 β-pinene 3 sabinene 4 δ3-carene 5 myrcene 6 α-terpinene 7 dl-limonene 8 p-mentha-1,5,8-triene 9 (Z)-b-ocimene 10 γ-terpinene 11 (E)-b-ocimene 12 p-cymene 13 4,8-dimethyl-1,3,7-nonatriene 14 (Z)-alloocimene 15 (E)-alloocimene 16 α-cubebene 17 cyclosativene 18 α-copaene 19 sativene 20 α-cedrene 21 β-cubebene 22 (E)-α-bergamotene 23 β-gurjunene (calarene) 24 β-caryophyllene 25 (Z)-β-farnesene 26 (E)-β-farnesene 27 γ-gurjunene 28 γ-muurolene 29 γ-curcumene 30 β-acoradiene 31 α-selinene 32 eremophyllene 33 α-zingiberene 34 α-muurolene 35 δ-guaiene 36 (E,E)-α-farnesene 37 δ-cadinene 38 β-sesquiphellandrene 39 ar-curcumene 40 n.i. sesquiterpene 41 calamenene 42 n.i. m/z 93, 107, 135, 204 43 n.i. m/z 93, 107, 135, 204 44 β-calacorene 45 n.i. m/z 93, 107, 135, 204 Figure 66.3 Extracted ion chromatogram of mono- and sesquiterpene hydrocarbons in VOO, by HS-SPME coupled to gas chromatography/mass spectrometry. Reprinted from Vichi S., et al. J. Chromatogr. A 2006; 1125:117–123, with permission. The figure allows appreciating the sensibility of the SPME method coupled to gas chromatography/mass spectrometry (single ion monitoring) for the analysis of terpene and sesquiterpene hydrocarbons, which are present at trace levels in VOO.
DTD is a rapid, solvent-free technique requiring a very small amount of sample, usually a few microliters, which does not need any manipulation prior to the analysis. DTD has been applied for the characterization of French, Spanish and Italian VOOs (Cavalli et al., 2003; Zunin et al., 2004). Desorption conditions evaluated to develop the analytical methods were related both to the extraction step and
the injection step. They were: final temperature, temperature ramp rate, time, cooling temperature and carrier gas flow. Desorptions at 40 °C and 80 °C during 20 min were chosen for the analysis of VOO volatiles, allowing the detection of a large number of compounds characteristic of VOO (Cavalli et al., 2003; Zunin et al., 2004). As expected, the number and amount of semivolatile sesquiterpenes were proportional
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Chapter | 66 Extraction Techniques for the Analysis of Virgin Olive Oil Aroma
50 SPME
SDE
CLSA
Percentage of areas
40
30
20
10
0 Alcohols
Aldehydes
Ketones
Hydrocarbons
Esters
Terpenoids
Figure 66.4 Percentages of VOO’s major families of volatiles extracted by SPME, SDE and CLSA. Reprinted from Vichi S., et al. Food Chem. 1007; 105: 1171–1178, with permission. Better affinities for alcohols and aldehydes are shown by SPME and SDE, respectively, while CLSA gives higher uptakes of esters and hydrocarbons. The best recoveries of terpenoids are obtained by SDE and DHS (CLSA).
to the temperature applied in these studies. A further study was conducted for the determination of these semivolatile terpenoid hydrocarbons in VOO, and the subsequent discrimination of the samples according to the geographical origin (Zunin et al., 2005). When compared with SPME, DTD was more efficient in the extraction of semivolatile sesquiterpenes and esters of fatty acids, while no relevant differences were observed in the recovery of other volatiles (Cavalli et al., 2003). Anyway, it must be taken into account that the extraction temperatures applied in this study were not the same for both methods. In fact, SPME was performed at 25°C, while DTD was performed at 80°C, which is likely to facilitate the volatilization of semivolatile compounds. Recently, a multi-step direct thermal desorption method coupled to comprehensive gas chromatography-time-offlight-mass spectrometry was developed and applied for the characterization of fresh and aged olive oils at typical frying temperatures (de Koning et al., 2008). Different temperatures of desorption were tested to evaluate the compounds present in native VOOs (70°C), in olive oil during frying or baking (175 and 250°C), evidencing differences between fresh and aged olive oil.
66.5 Distillation and fluid-based extraction techniques 66.5.1 Simultaneous Distillation/Extraction (SDE) and Hydrodistillation Distillation methods have traditionally been applied in the analysis of plant materials. Hydrodistillation and simultaneous distillation/extraction (SDE) were used for this purpose, and SDE appeared to afford the most favorable uptake for mono- and sesquiterpenes, as well as their oxygenated
analogues (Marriott et al., 2001). Hydrodistillation has been applied for the analysis of leaf, fruit and virgin oil volatiles of an Italian olive cultivar (Flamini et al., 2003). The uptake of olive oil volatiles was comparable to that obtained by SPME using a PDMS fiber. The most remarkable difference was the higher amount of -farnesene obtained by hydrodistillation. However, with hydrodistillation the volatiles in the steam distillate are heavily diluted by water when collected in cold traps. This is overcome in SDE via solvent extraction of the distillate. SDE was evaluated for the analysis of VOO aroma using a modified Likens and Nickerson apparatus, and using pentane and dichloromethane as solvents (Vichi et al., 2007). In comparison with the extraction of the same oil by SPME and CLSA, SDE gave higher percentages of aldehydes correlated with the oxidative degradation of VOO, indicating that the extraction conditions induced the thermal alteration of the oil sample (Figure 66.4). Regarding the extraction of semivolatile compounds, -zingiberene and -farnesene were the only compounds detected at a higher percentage by SDE, while other sesquiterpenes were more abundant by CLSA extraction.
66.5.2 Supercritical Fluid Extraction (SFE) Supercritical fluid extraction (SFE) has been widely used for extracting flavor and fragrance compounds from complex matrices without the use of organic solvents. This method is based on the use of gases at determined conditions of pressure and temperature, known as supercritical region. At these conditions gases can acquire solvent properties that are superior to those of liquid solvents. Supercritical CO2 is among the most used fluids due to its good proven extractive capacity and safety, and it was also tested for the extraction of volatile and semivolatile compounds from VOO (Morales et al., 1998). After SFE, a concentration of volatiles on a Tenax trap was required
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prior to the chromatographic analysis in order to enhance the sensitivity of the method. Different profiles of volatile and semivolatile compounds were found in the SFE extracts, depending on experimental parameters. All the major VOO volatiles were identified in the extract. Soft extraction conditions allowed higher uptakes of the most volatile compounds to be obtained, while drastic conditions led to a higher extraction of semivolatile compounds and induced the oxidation of the sample.
Summary points VOO has a unique flavor that distinguishes it from other vegetable oils. l Various analytical methods have been developed to examine these volatile compounds. l The volatile profile of VOO closely depends upon the method of extraction used. l Headspace extraction techniques are the most widely used for the analysis of olive oil aroma, and SHS, DHS, SPME, HSSE and DTD have been applied to VOO, depending on the kind of determination required. l Among distillation and fluid-based extraction techniques, only hydrodistillation, SDE and SFE have been tentatively applied for the analysis of VOO aroma. l
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Section | I Natural Components
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Chapter | 66 Extraction Techniques for the Analysis of Virgin Olive Oil Aroma
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