Off-Flavors)

4.06 Sample Preparation for Food Flavor Analysis (Flavors/Off-Flavors) , M Majcher and M Dziadas, University of Life Sciences, Pozna HH Jelen n, P...

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4.06

Sample Preparation for Food Flavor Analysis (Flavors/Off-Flavors)

, M Majcher and M Dziadas, University of Life Sciences, Pozna HH Jelen n, Poland Ó 2012 Elsevier Inc. All rights reserved.

4.06.1 Speciﬁcity of Food Flavor/Off-Flavor Analysis 4.06.1.1 Volatile Compounds, Flavor Compounds, Key Odorants – An Introduction 4.06.1.2 Odor Thresholds vs. Limits of Detection 4.06.1.3 Instability of Flavor Compounds 4.06.1.4 Inﬂuence of the Matrix on Flavor Compounds 4.06.2 Strategies for Flavor Compound Analysis 4.06.3 Methods for Isolation and Analysis of Free Flavor Compounds 4.06.3.1 Solvent Extraction Methods 4.06.3.2 Distillation Methods 4.06.3.3 Headspace Methods 4.06.3.4 Sorption Methods 4.06.4 Methods for Isolation of Volatile Compounds for Gas Chromatography–Olfactometry 4.06.5 Methods for Isolation of Volatile Compounds for Electronic Noses 4.06.6 Methods for Isolation and Analysis of Bound Flavor Compounds 4.06.7 Conclusions References Relevant Websites

4.06.1

Specificity of Food Flavor/Off-Flavor Analysis

4.06.1.1

Volatile Compounds, Flavor Compounds, Key Odorants – An Introduction

119 119 120 121 122 122 123 123 125 129 131 138 139 140 144 144 145

Flavor is a key attribute in selection of a particular food product by consumers and, together with texture and color, forms the main features that are crucial for consumer acceptance. Although the term ﬂavor combines aroma and taste, the majority of research performed has been related to odorants not the tastants. The ﬂavor of food is dependent on an array of volatile compounds – their number, character, and quantities. However, because ﬂavor is related to perception of odorants by our olfactory system, unique features of volatile compounds have to be considered as well: their odor threshold being the most important and features that inﬂuence odor thresholds and aroma perception: chirality, concentration, synergistic effects and a type of matrix (food) from which the compounds are released. In ﬂavor analysis, several areas of research exist and intertwine: determination of key odorants (compounds that indeed inﬂuence food aroma), analysis of ﬂavor compounds that are restricted and should be monitored in foods, and analysis of taints and off-ﬂavors. Taints and off-ﬂavors are not synonymous – taints are deﬁned as odors that migrate to food from outer sources, whereas off-ﬂavors are often deﬁned as ﬂavors that develop in food in a result of deteriorative changes caused by enzymatic, chemical, or microbial processes. Very often, compounds present in characteristic product concentration do not impair its sensory quality; however, when present in higher concentrations they inﬂuence negatively its sensory properties. Food volatile ﬂavor compounds are usually volatiles of MW not exceeding 300 Da and represent various chemical classes. Because of their character and molecule size, volatile ﬂavor compounds have been analyzed using gas chromatography, although there are a limited number of publications where HPLC is used for the analysis of some odorants, mainly aldehydes (E-2-nonenal in beer). Inventions in gas chromatography and mass spectrometry have signiﬁcantly accelerated aroma analysis. Development of the capillary column was a milestone and allowed the identiﬁcation of hundreds of new volatiles. The next milestone, which inﬂuenced similarly the number of identiﬁable volatiles, was the invention of two-dimensional (2D) gas chromatography in 1959. Although two-dimensional chromatography using the ‘heart-cut’ technique allowed the use of different separation mechanisms in a single chromatographical run, and improved the separation capacities of GC systems (an especially valuable tool for ﬂavor research as hyphenation of polar or nonpolar with chiral columns), real progress was noted with the invention of comprehensive gas chromatography (GC GC), especially with time-of-ﬂight mass spectrometers as detectors. With peak capacities of 10 000, mass spectral identiﬁcation and deconvolution algorithms, researchers received a powerful tool, which enabled detection of thousands of peaks, where previously tens or hundreds were detectable using single-dimensional chromatography. From one point of view, it provides additional data; from the other it causes a data overﬂow in some cases, especially when not target analysis but proﬁling volatile or ﬂavor compounds is considered. The important distinction has to be made here that analysis of volatile ﬂavor compounds in food is not synonymous with analysis of volatile compounds. Of about 8000 various volatile compounds found in food products, not all contribute signiﬁcantly to the ﬂavor of food. It is estimated that only 5% of food volatiles indeed inﬂuence the aroma of a particular food.1,2 Such aromaactive compounds can be selected from nonactive volatiles by combining the human olfactory system response with instrumental

Comprehensive Sampling and Sample Preparation, Volume 4

doi:10.1016/B978-0-12-381373-2.10130-9

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analysis. The hyphenation is known as gas chromatography–olfactometry (GC–O) and is based on snifﬁng efﬂuent from a chromatographical column. The speciﬁcity of GC–O is discussed in the next part of this chapter. In the next step, evaluation of whether the volatile compound belongs to the key odorants of the food product is based on its odor threshold and concentration and it has been proposed to express it as odor activity value (OAV) or aroma value (AV) – the ratio of the compound concentration to its odor threshold value. According to this approach, compounds present in foods in the concentration above their sensory threshold contribute to overall aroma. The ﬁnest examples of key odorant identiﬁcation can be revealed by the foods with very complex aromas such as coffee, tea, beer, or wine. There have been over 800 volatile compounds identiﬁed in wine or coffee and over 600 in beer or tea. However, the more recent research data substantiated that indeed no more than 20–30 compounds actually belong to potent odorants involved in their aroma formation.3–7 The task of identifying key odorants in foods is very complex and difﬁcult to achieve due to several problems such as poor sensitivity of detectors used in gas chromatography for some aroma compounds as compared to the human olfactory system, instability of ﬂavor compounds during sample preparation or analysis and ﬁnally, inﬂuence of the matrix effect on ﬂavor binding and release. Compounds that are key odorants represent virtually all chemical classes because of the broad spectrum of aromas that are encountered in nature. Therefore, compounds important for the aroma of a particular food can be as well relatively simple molecules, such as dimethylsulﬁde (beer), simple aldehydes, such as acetaldehyde (fermented milk products), and also long chain aldehydes, such as 2,4-decadienals (fried products), monoterpenes (sinensal in oranges), sesquiterpenes (caryophyllene in hops), a large group of heterocyclic compounds (2-acetyl-1-pyroline in basmati rice), to provide only few examples. Often, these compounds have low odor thresholds and are present in low concentration, which makes their analysis challenging. Taints and offﬂavors also represent various chemical classes, mainly haloanisoles, chlorophenols, volatile phenols, sulfur-containing compounds, carbonyls, amines, and fatty acids. They are usually formed as a result of microbial activity, migration from packaging materials, cleaning agents, and disinfectants, or are formed as a result of chemical or enzymatic changes in food. Similar to key odorants, compounds that cause taints and off-ﬂavors often have odor thresholds in ng l1 concentrations.

4.06.1.2

Odor Thresholds vs. Limits of Detection

Odor threshold is deﬁned as the concentration of a compound in a speciﬁed medium that is detectable by 50% of the speciﬁed population. Similar to the limits of detection (LOD) and limits of quantitation (LOQ) concept, two types of odor thresholds are sometimes distinguished: detection threshold – deﬁned as the lowest physical intensity at which a stimulus is perceptible, and the recognition threshold, which is the lowest intensity in which the stimulus could be correctly deﬁned/identiﬁed. As mentioned before, the odor-active compounds are distinguished based on their concentration and odor threshold value – a compound becomes potent odorant when its concentration exceeds its odor threshold. This is achieved either through high concentration in the sample or very low sensory threshold. In case of highly concentrated compounds, identiﬁcation and quantiﬁcation are quite easy as opposed to second group compounds, which are frequently present in food samples in trace concentration, very often lower than instrumental detection limits. In Table 1, some examples of aroma compounds with very low odor threshold values are present. This indicates that compounds such as 2-methyl-3-furanthiol or 2-isobutyl-3-methoxy-pyrazine can inﬂuence the aroma of a food product when present in concentrations as low as 0.05 ppb. Analyzing black tea, Schuh and Schieberle4 found that among 18 identiﬁed potent odorants there are also compounds in concentrations as low as 0.15 mg l1, such as (E)-b-damascenone. Similar ﬁndings7 for 2-furfurylthiol identiﬁed it as the most powerful aroma compound in coffee brew only when present at concentrations of 1.7 mg kg1. Another example that illustrates well the challenges in the analysis of aroma is the determination of polyfunctional mercaptans. This group of compounds including 2-furfurylthiol, 4-mercapto-4-metyl-2-pentanone, 2-methyl-3-furanthiol, and 3-mercaptohexanol, inﬂuence the sensory characteristics of many tropical fruit such as passion fruit, and mango, and also coffee, cooked meat, and some wines. Odor thresholds of all these compounds are in ng l1 levels. This creates a challenge for instrumental analysis, as used methods should be characterized by limits of detection comparable to that achieved by the human nose.8 Detection limits of the method can be improved at several stages of analysis and will depend on: 1. the amount of sample that enters the chromatographical column and detector, which can be inﬂuenced by extraction and injection techniques, Table 1

Odor thresholds and odor descriptions of some potent food odorants8

Compound name

Odor threshold in water [mg l1]

Odor description

methional b-damascenone methylthiol 2-acetyl-1-pyrroline (Z)-1,5-octadiene-3-one 2-methyl-3-furanthiol 2-furfurylthiol 2-isobutyl-3-methoxy-pyrazine

0.2 0.002 0.02 0.1 0.0012 0.007 0.012 0.002

boiled potato fruity, sweet sulfur, garlic, gasoline popcorn geranium-like boiled meat-like roasted, coffee-like hot red pepper

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2. separation process, and, 3. kind of detector used and the analyte treatment that improves its detection (derivatization). The main potential in lowering detection limits in analysis of food ﬂavor compounds is the appropriate choice of extraction and preconcentration methods. The assortment of available extraction techniques covers all those used for volatile compound analysis and the most important of them will be discussed further in this chapter. Introduction of large quantities of analyte done using large volume injections did not gain as much attention as in environmental analysis, as ﬂavor compounds in food comprise also very volatile analytes that elute close to the solvent; therefore, the need for high differences between volatility of analyte and solvent, which is a prerequisite for satisfactory separation and recovery in solvent venting, cannot be met. However, for some high boiling ﬂavor compounds it is an attractive method of improving their detection limits. Regarding chromatographical columns, improvement of detection limits for ﬂavor compounds can be achieved by using comprehensive two-dimensional gas chromatography (GC GC). In this technique, with respect to classical two-dimensional (heart-cut) chromatography (2D) not only a certain fraction but also the entire sample is subjected to two different separation mechanisms performed on two capillaries with opposite polarity connected with a transfer device – modulator. The revolutionary aspects of GC GC are improvements of separation due to the much higher peak capacity, enhancement of signal-to-noise ratio as a result of trapping and refocusing process occurring during modulation and ﬁnally, better compound identiﬁcation on the basis of two retention times instead of one. Adahchour et al. have used GC GC for detection of garlic ﬂavor volatiles and reported a 10–50-fold increase in sensitivity as compared to GC analysis.9 Detection limits can also be improved using a particular detection system. This process is instrument dependent and also sample dependent. For the analysis of volatile ﬂavor compounds, universal GC detectors are used (mainly FID; TCD is not used due to its poor sensitivity) as well as selective ones. Of selective detectors, the electron capture detector is the one offering the highest usefulness. It offers low limits of detection for aliphatic ketones, especially vicinal diketones, and much lower for halo-compounds. Although there are not many ﬂavor compounds containing chlorine, bromine, or iodine atoms (the most representative group would be haloanisoles), derivatization using reagents providing ﬂuorine atoms is the technique of crucial importance in the analysis of aldehydes and there are numerous publications describing analysis of derivatized aldehydes mainly using PFBOA as a derivatization agent.10 In fact, many aroma compounds possessing mainly hydroxyl groups can be derivatized and TFFA, PFPA, and HFBA derivatives provide low limits of detection using ECD detector. Apart from the electron capture detector, other selective detectors are used in ﬂavor research for detection of nitrogen-containing compounds (NPD) or sulfur-containing compounds (FPD). However, not counting the ECD detector, the role of other selective detectors in aroma research decreases as they are replaced by mass spectrometers. Hyphenation of gas chromatography with mass spectrometry is the most powerful, most versatile, and in fact a standard instrument conﬁguration nowadays for volatile ﬂavor compound analysis, providing characterization of analyzed compounds by their mass spectra and can serve as both a universal and a selective detector. There are also two important features of mass spectrometers, which make them a useful tool for analysis of aroma compounds. Firstly, peak coelutions and overlapping in the analytical column can be, in certain instances, improved by mass spectrometry, either by employing deconvolution algorithms or/and by utilizing quantitation of compounds by their unique ions, which often enables quantitation of peaks completely overlapped by solvent or other more abundant peaks. A prerequisite for such an approach is the absence of target ion in the co-eluting compound. The other advantage of mass spectrometers as detectors is the ability to perform SIDA (Stable Isotope Dilution Analysis), unable to be executed using other detectors. This technique is based on the use of isotopomers of analyzed compounds and provides the best results in quantitation of odorants, as the properties of the analyzed compound and the isotopomer are the same, so their relation with matrix and recoveries in all steps of the analytical procedure are similar. The main drawback of using these types of standards is related to their cost and limited availability, especially for rare odorants. It has to be remembered that of 13C- and 2H-substituted compounds, the latter group, although usually easier to synthesize and more available, is sometimes prone to deuterium exchange; thus, their stability should be for some compounds and solvents controlled during their storage. Mass spectrometers as detectors offer also improvement in limits of detection using chemical ionization, both positive and negative. High-resolution mass spectrometers offer also a selectivity based on a precise mass measurement. To summarize, the whole analytical process inﬂuences limits of detection and the suitability of the method in certain types of food ﬂavor analysis. In food aroma research, limits of detection achieved by analytical instruments must be comparable to odor thresholds of particular compounds, as this is required for the proper characterization of odoriferous compounds in foods. As comprehensive gas chromatography and high-resolution mass spectrometry are deﬁnitely not routine tools for ﬂavor research nowadays, the highest potential for improvements in method limits of detection, is on the sample preparation side. Proper sample preparation procedures, on one side, offer a preconcentration of analyte to a level that enables their characterization and quantitation, and offers also a selective enrichment, which minimizes the inﬂuence of other matrix (food) constituents that could interfere with analyzed volatile compounds.

4.06.1.3

Instability of Flavor Compounds

Analysis and isolation of ﬂavor compounds can be difﬁcult also due to the fact that ﬂavor compounds represent various classes of chemical compounds: alcohols, aldehydes, acids, ketones, carbonyls, sulfur and nitrogen, amines, aromatics, compounds with high and low volatility, etc. This means that these compounds are susceptible to various chemical changes such as: oxidation of aldehydes, rearrangement and isomerization of terpenes in presence of acids, hydrolysis of esters, reaction of thiols, amines, and aldehydes in the aroma concentrate, and polymerization of unsaturated compounds or photo-oxidation due to light exposure. The fact that those

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compounds may be present in foods in very low quantity makes it even more difﬁcult to achieve. In general, sample preparation for ﬂavor analysis should be performed in the manner that will allow obtaining a representative extract of the original food aroma. It should allow for extracting all compounds that contribute to the ﬂavor of the food product, though not alter the proﬁle of characteristic volatiles, especially concerning very unstable compounds that may generate artifacts during isolation of ﬂavors. One of the examples of ﬂavor compounds difﬁcult to analyze is sotolone (3-hydroxy-4,5-dimethyl-2(5H)-furanone). This very powerful aroma compound, characterized by a spicy-curry note, has been found to be a main odorant of many food products, e.g., coffee,11 rye bread crust,12 fenugreek seeds,13 or fortiﬁed Madeira wines.14 Unfortunately, with its very low olfaction thresholds of 15 mg l1 sotolone causes also oxidation–spoiling off-ﬂavor of white wines. Therefore, a great deal of research has been done in developing extraction methods that would be capable of detecting sotolone in the mg l1 range. The main problem in quantiﬁcation analysis of sotolone is due to the poor extraction yields and its instability15 and proper quantiﬁcation can be obtained only by the use of labeled internal standards, which compensates almost totally for losses of analyzed compound during sample preparation.16 The analysis of odor-active sulfur compounds, which have very strong odor potential as ﬂavor and off-ﬂavor contributors, is also a challenging task not only because of the low concentrations at which these compounds have to be quantiﬁed (down to ng l1 levels) but also because of their high reactivity and poor spectrometric properties. For the most part, their mass spectra lack characteristic ions of high m/z and their chromatographic properties are reduced because of the adsorptive characteristics of the functional groups, e.g., thiols, which causes intense peak tailing. A second problem is their instability. These compounds are elusive and can react with oxygen and other oxidants and, they form complexes and precipitates with metal ions.17 Because of this, many procedures for their quantitative determination make use of derivatization or selective isolation methods have been developed. The most commonly used strategies for the analysis of these compounds make use of the complexing properties of the thiol group, particularly to certain forms of organic mercury. The most recent application of Mateo-Vivarcho and colleagues proposes the use of headspace solid-phase microextraction with on-ﬁber derivatization as the extraction method followed by identiﬁcation by gas chromatography–negative chemical ionization mass spectrometry.18 They have reached limits of detection as low as 0.1 ng l1 for compounds, such as 2-furanmethanethiol and 3-mercaptohexyl acetate, with RSD <20%. The same authors have developed a procedure for the selective preconcentration and puriﬁcation of mercaptanes, such as 2-methyl-3-furanthiol, 2-furfurylthiol, 4-mercapto-4-methyl-2-pentanone, 3-mercaptohexyl acetate, or 3-mercaptohexanol, using a 20-mg SPE cartridge containing p-hydroxymercurybenzoate.19

4.06.1.4

Inﬂuence of the Matrix on Flavor Compounds

The study of the inﬂuence of a food matrix on the aroma isolation and concentration process is hindered by the complexity of food products. It is mostly due to the reaction of, very often, trace quantities of ﬂavor compounds with main food constituents, such as lipids, proteins, carbohydrates, and water. During sample preparation, it is important to remember that those interactions affect the retention of volatiles within the food and, hence, their levels in the gaseous phase. Selected examples cited herein address the importance of matrix inﬂuence on the partition of volatiles and their interactions with matrix constituents. Afoakwa has studied the inﬂuence of matrix particle size distribution (PSD) (18, 25, 35, and 50 lm) and fat content (25%, 30%, and 35%) on the release of ﬂavor of dark chocolate volatiles. In case of different fat content, the authors noticed an inverse relationship with volatiles such as: 2-phenylethanol, furfuryl alcohol, methylpyrazine, phenylacetaldehyde, 2,3,5-trimethyl-6-ethylpyrazine, and 2-carboxaldehyde-1H-pyrrole, probably due to the lipophilic matrix–ﬂavor interaction. On the other hand, there was a direct relationship of fat content with 3-methylbutanal and also branched pyrazines (trimethypyrazine, 2,3-dimethylpyrazine, 2,5-dimethylpyrazine, tetramethylpyrazine, and 2,3,5-triethyl-5-methylpyrazine). This decreased matrix retention, which could be related to differences in (micro) structure as inter-particle ﬂocculation and aggregates are reduced with higher fat contents.20 Another very recent research paper on the impact of fat reduction on Cheddar cheese ﬂavor21 has reported a large concentration difference between full-fat and low-fat cheeses for key odorants (furaneol, homofuraneol, sotolon, phenylethanal, 1-octen-3-one, free fatty acids, and lactones); however, the same compounds were detected in all cheeses. The authors conﬁrm with this that sensory ﬂavor differences in full-fat and reduced-fat cheeses are due to differences in the matrix that inﬂuence compound release and also to distinct differences in ripening biochemistry. Boland has veriﬁed the release of 11 ﬂavor compounds (diacetyl, 2-butanone, ethyl acetate, 1-butanol, 3-methyl-1-butanol, ethyl butyrate, hexanal, 2-heptanone, heptanal, 2-octanone, and 2-decanone) from three commonly used hydrocolloids in food products: gelatin, starch, and pectin.22 Using static headspace for the compounds’ partition coefﬁcient determination, it was shown that partition coefﬁcients for analyzed ﬂavor compounds varied depending on a matrix, where the higher values were reached for pectin gel, while the gelatin and starch gels were not signiﬁcantly different from each other. Overall, the hydrophilic compounds had the signiﬁcantly highest partition coefﬁcient in the gelatin gel, while the hydrophobic compounds had the lowest, which indicates an effect of the rigidity of the gelatin gel on the thermodynamic component of release. The effect of hydrocolloids on ﬂavor release may be due to physical entrapment of ﬂavor molecules within the food matrix or interaction with gel components. The importance of matrix in ﬂavor compound binding and release is of great importance for the food industry and has been a subject of intensive research.

4.06.2

Strategies for Flavor Compound Analysis

Analysis of ﬂavor compounds can be divided into two areas. The ﬁrst one is target analysis of known compounds, usually those that are limited or banned in foods, or analysis of known odorants or off-odorants. The other is analysis of key odorants in a product,

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123

where usually these compounds’ identity is unknown. Target analysis of ﬂavor compounds relies usually on gas chromatography with either universal (FID) or selective (ECD and NPD) detectors, or more frequently, coupled with mass spectrometry. Depending on the nature of analyzed compounds and their concentrations, extraction and preconcentration techniques used in the analysis of volatiles are used. Key odorant analysis takes into consideration two important features of aroma molecules – odor character of a particular compound and its odor threshold. For the analysis of compounds responsible for the ﬂavor of a particular fruit, beverage, plant, or any food product, identiﬁcation of compounds done with detectors used in chromatography has to be coordinated with their identiﬁcation using the human olfactory system. For the analysis of key odorants, gas chromatography–olfactometry techniques are used. Basically, they are all based on snifﬁng efﬂuent from a chromatographical column, which is usually split between the olfactory port and, generally, mass spectrometer. This setup provides simultaneous detection of compounds. Although the setup of such instruments is usually similar, the differences are in the methods of quantiﬁcation of odoriferous molecules.23 Among the various methods of odor impression quantiﬁcation, analyses based on snifﬁng the extract of ﬂavor compounds, which is subjected to serial dilutions, AEDA is the most frequently used.1 Serially diluted extracts (1:4, 1:8, 1:16, 1:32, etc.) are injected into a GC–O system and sniffed. Compounds that are detectable by the human nose in the highest diluted extracts (having the highest FD (ﬂavor dilution)) play usually a crucial role as key odorants for a given product. Another measure for the relevance of a given compound in the overall ﬂavor of a product is the ratio of its concentration to odor threshold of a given compound (Odor Activity Value). To detect ﬂavorsigniﬁcant compounds from a certain product, the GC–O method applied is only a part of an analysis scheme. As proposed by Grosch, Schieberle, and Hoffmann, the analysis of key odorants (and also tastants) includes a multistep procedure that in fact requires several experiments to be performed: (1) The extract (liquid) of volatiles should be resolved using gas chromatography and potent odorants should be localized by one of the dilution techniques (AEDA or Charm); (2) highly volatile odorants should be detected using GC–O with sample introduction via a static headspace; (3) odorants should be preconcentrated and identiﬁed using various methods (extraction, preparative GC, fractionation, GC/MS, enantioselective GC, and NMR) – potent odorants should be quantiﬁed and their odor activity values should be calculated; (4) a synthetic blend of key odorants should be prepared based on the results of previous experiments and aroma compared with the original aroma of a product; and (5) omission experiments should be carried out based on testing of models lacking one or more identiﬁed compounds to assess their inﬂuence on overall ﬂavor of the product.1,24 Identiﬁcation and quantitation of unknown taints and off-ﬂavors in foods are based on a procedure similar to the sensory guided determination of key odorants. It involves several steps, which are equally important for the analysis results: (1) gathering all available information about the sample, its origin, composition, and operations involved in technological processes; (2) sensory analysis of the sample using proﬁle analysis – descriptors of the characteristic off-odor should be elucidated; (3) isolation of volatile compounds for subsequent analysis by GC-MS and GC–O – the extract obtained should exhibit the same off-odor as the food sample; (4) analysis compounds responsible for the off-ﬂavor using gas chromatography – olfactometry; (5) identiﬁcation of odoriferous compounds detected in GC–O using gas chromatography – mass spectrometry and other required techniques; (6) quantitation of compounds responsible for the off-odor; (7) spiking the food product with identiﬁed off-odorant to verify its role in the formation of a particular taint or off-ﬂavor followed by GC–O analysis and sensory proﬁle analysis of the spiked product; and (8) determination of the origin of off-odor checking possible contamination sources in the technological process, packaging, or storage. For both key aroma odorants, as well as for the analysis of food off-ﬂavors and taints, obtaining the extract of ﬂavor compounds representative of the sample is a crucial step in GC–O analyses. Considering the proﬁle of this chapter, techniques used for the isolation of food aroma compounds will be discussed in the following sections.

4.06.3

Methods for Isolation and Analysis of Free Flavor Compounds

Majority of methods for the analysis of volatile/ﬂavor compounds refer to determination of free ﬂavor compounds, i.e., present in free form in a matrix (though they can be entrapped by matrix macroconsituents, they are bound only by weak physical forces, not using covalent bonds in the form of glycosides). Methods for extraction of ﬂavor compounds in their free form comprise solvent extraction, distillation methods (mainly steam distillation and simultaneous distillation/extraction methods), and headspace analysis, including static headspace, dynamic headspace (purge and trap), and methods based on sorption mechanisms. Analysis of volatile precursors involves mainly sorption-based techniques used with the conjunction of methods used for free aglycones’ analysis.

4.06.3.1

Solvent Extraction Methods

Solvent extraction methods for the analysis of ﬂavor compounds are highly dependent on the type of matrix from which the volatiles are extracted. As majority of ﬂavor compounds are nonpolar, the use of nonpolar solvents is a logical approach. However, for foods rich in lipids these solvents will coextract lipids and lipid-soluble compounds as well. After such extraction, separation of volatile compounds from the lipid fraction requires application of other methods. Other food components soluble in polar solvents will interfere with ﬂavor compounds extracted with polar solvents. Therefore, extraction with solvents is rarely used without additional separation/fractionation techniques in case of solid matrices, and simultaneous distillation extraction techniques are preferred for such types of foods. For the analysis of liquid food, liquid/liquid extraction is used relatively often.

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Solvent extraction is a routine element in the determination of taste compounds in food; however, in the analysis of aroma compounds its use is limited. Usually, the procedure is multistep, as can be seen for the investigation of compounds responsible for buckwheat groats’ aroma.25 Milled buckwheat groats were macerated with water, extracted with petroleum ether, concentrated, extracted with sodium hydrogen carbonate solution, extracted with petroleum ether, and the aqueous layer acidiﬁed to pH 2 to convert aroma compounds from the ionized to the nonionized form, which were extracted subsequently with the organic solvent. Preparative TLC was used to clean and fractionate the extract. In all stages, samples and intermediate compounds were monitored by sensory analysis. By this procedure, salicylaldehyde was identiﬁed as a volatile responsible for the aroma of buckwheat groats. Another example of compounds isolated using solvent extraction can be short-chain fatty acids. Free fatty acids (C2–C6) are a group of compounds important from the sensory point of view in the formation of the characteristic aroma of many cheeses. As a result of milk fat lipolysis, free fatty acids are released in the cheese and, due to the volatility of those having less than 8 carbon atoms, they contribute to the characteristic aroma of cheese. The determination of free volatile fatty acids (FVFA) in cheese described in literature is based on steam distillation of an aqueous, acidiﬁed cheese suspension. Then, the distillate is alkalized and concentrated, and free fatty acids released from corresponding salts by adding a strong acid are solvent extracted for GC analysis. A method of free fatty acid determination in cheese based on solvent extraction was proposed by Innocente. In this method, water containing an internal standard was added to grated cheese, and the mixture was homogenized and centrifuged to eliminate triglycerides and proteins. The extract was acidiﬁed to pH 3–4 to free volatile fatty acids present in the form of salts. Fatty acids were extracted from an acidiﬁed aqueous solution using diethyl ether and determined by GC. To avoid losses of the most volatile fatty acids, homogenization can be performed in a basic environment, with the addition of sodium hydroxide; however, it was observed that no signiﬁcant losses were observed in case of free fatty acids.26 Solvent extraction proves to be a rapid and simple method of isolation of even the most labile compounds, such as the lachrymatory factor in onion. The lachrymatory factor in onion – (Z,E)-propanethial S-oxide – is a very labile volatile compound experienced by a consumer when one cuts an onion. It is formed in a process of tissue disruption in a result of the activity of allinase (EC 4.4.1.4) on (þ)-S-(E)-1-propenylcysteine S-oxide. It is very unstable and tends to decompose rapidly. In the procedure proposed by Schmidt, onion bulbs are crushed, and the juice collected and allowed to react (allinase with ﬂavor precursors) at room temperature. The juice is then extracted with methylene chloride and concentrated under a stream of nitrogen and injected into GC.27 Solvent extraction is a good approach for liquid samples, where the inﬂuence of matrix constituents is less pronounced. The complexity of solvent extraction can be well illustrated using determination of polyfunctional thiols in wine.8 As the odor thresholds of these thiols are in ng l1 concentration, the method for their determination in wine requires an effective extraction separation procedure providing limits of detection comparable to that of the human olfactory system. Solvents used for extraction of ﬂavor compounds also include supercritical ﬂuids. Carbon dioxide is most explored as a solvent for isolation of plant constituents. The liquid-like solvating properties and gas-like transport properties make supercritical ﬂuids an attractive extraction method for diffusion-controlled matrices, such as plant tissues. The best-known industrial processes are decaffeination of coffee and tea or the obtaining of hop extracts. Besides this, there are many publications dealing with extraction of ﬂavors, oils, spices, and essential oils. Supercritical ﬂuid extraction was used for lemon oil, citrus oil, basil, bergamot, borage, eucalyptus, primrose, thymus, Coriandrum, and juniper to name only some of the applications in the ﬁeld of ﬂavor and the extraction of essential oils is one of the most widely discussed applications in supercritical ﬂuid literature.28 Apart from industrial installation for supercritical CO2 extraction, there are commercially available micro-SFE devices designed for analytical purposes, able to work in batch or semibatch modes, and which can be used for extraction of various food components. The main advantage of SFE is the low temperature of the extraction process, which prevents the degradation of the chemical compounds of the extract due to heat, as sometimes observed in a steam distillation process. The extracted compounds can be recovered from the extracting ﬂuid by decreasing the pressure/increasing temperature. The solvating properties of a supercritical carbon dioxide phase can be varied by changing its pressure and temperature, or by addition of small amounts of modiﬁers. These liquid co-solvents are used to enhance the afﬁnity of the solvent mixture toward the polar compounds, which have a very low solubility in supercritical CO2. As modiﬁers, usually methanol or acetone is used. Acetone enhanced the solubility of cis-jasmonate, n-anthranilate, and benzylacetate from jasmine ﬂowers or furaneol from strawberries; methanol increased the recovery of irones and iridals from iris rhizomes.28 Supercritical ﬂuid extraction has been tested as an alternative to hydrodistillation. Its advantages are shorter extraction time (usually up to 30 min compared to up to two hours for hydrodistillation methods), lower temperatures used in the extraction process, and no solvent residues. The disadvantage is the different (to a certain extent) composition of extract obtained by SFE compared to hydrodistillation. Another issue that has to be considered is a nonselective extraction in many cases, where other plant components of similar solubility are extracted (lipophilic cuticular waxes from leaves can be an example). As compounds usually coextracted with essential oils, fatty acids were also detected, along with fatty acid methyl esters and some high molecular weight hydrocarbons. At extraction temperatures between 40 and 50 C and extraction pressures lower than 100 bar, higher molecular weight compounds are usually not coextracted with essential oils.28 The inconvenience of coextraction of waxes can be solved by further sample treatments such as fractional separation. However, this requires a set of separators at different pressures and temperatures to precipitate the compounds selectively. A complete separation of parafﬁns from essential oil terpenes can be achieved. For the studies performed on black cumin, hydrodistillation allowed the detection of 22 compounds compared to 16 detected by SFE; moreover, their proportions were different: no a-thujone, sabinene, p-cymene, terpinen-4-ol, a-terpineol, cumin alcohol, b-caryophylene, g-elemene, b-bisabolene, or myristicin was detected using SFE and the yields of the remaining compounds varied.29 In the comparison of SFE with steam distillation, there are often differences noted in the proportion of compounds. It was

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Figure 1 (a) Evolution of sage oil composition with extraction time.30 Percentages of the various compound families at different extraction times; B – terpenes; , – sesquiterpenes; ; – oxygenated terpenes; and - – oxygenated sesquiterpenes. (b) Evolution of sage oil composition with extraction time. Cumulative quantities expressed as grams of the extracted compound families at various extraction times.

noted that steam distilled oil of rosemary contained higher percentages of terpene hydrocarbons; in contrast, SFE contained a higher percentage of oxygenated compounds, SFE extraction did not produce thermal degradation of cis-sabinene hydrates and acetates in marjoram fragrance, nor was thermal degradation observed in chamomile, whereas steam distillation resulted in conversion of matricine into chamazulene.28 As is the case for other extraction methods, SFE requires optimization of pressure, temperature, particle size, particle size distribution, and solvent ﬂow rate on the yield and composition of the volatile oil obtained. Extraction curves show yield of essential oils in time. It has to be kept in mind that for different groups of compounds their extraction curves may look different and some compounds can be extracted at the beginning, whereas some higher molecular weight groups can be extracted later on. It is especially important in case of essential oils containing such distinctive groups as monoterpene or sesquiterpene hydrocarbons, oxygenated terpenes, fatty acids, and other nonvolatile compounds. Extraction time can inﬂuence the composition of the extract or essential oil. This can be observed in Figure 1 with sage essential oil as an example.30 If properly optimized, SFE yields extracts with no artifacts that are valuable in the determination of the real ﬂavor or fragrance of a product.

4.06.3.2

Distillation Methods

Usefulness of distillation methods for ﬂavor compound analysis depends on the matrix character and the stability of compounds to be isolated. Usually, for the isolation of volatile ﬂavor compounds, with distillation (SD) and simultaneous distillation extraction (SDE) are performed. Steam distillation is usually performed in a Clavenger-type apparatus (Figure 2.) and used usually for the isolation of essential oils and is a well-established method. As a consequence of steam/water boiling, addition points of the compounds are decreased, which makes their evaporation at lower temperatures possible. Distillation at atmospheric pressure used for isolation of ﬂavor compounds uses high temperatures. For the heat-sensitive compounds, vacuum needs to be used to lower the boiling points of extracted compounds.

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Figure 2

Clavenger-type apparatus for the determination of essential oils.31

Figure 3 Schematic process of steam distillation–extraction. (1) Steam distillation–extraction apparatus; (2) stirrer/heater plate; (3) cooling water; (4) sample ﬂask; (5) solvent ﬂask; and (6) mineral oil.32

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For the extraction of ﬂavor compounds from food products, more often steam distillation simultaneous distillation extraction (SDE) is used. For this purpose, the Likens–Nickerson apparatus developed (1964) for the isolation of hop oil volatiles is one of the most widely used devices (Figure 3.). Water containing the sample is boiled in one ﬂask while the extracting solvent is boiled in another one. Vapors from both ﬂasks mix in a central chamber of the apparatus. The condensates form immiscible layers, which are returned by glass tubes to the appropriate ﬂasks. Volatiles extracted from the water vapors are transferred to the organic solvent. The relatively small volumes of solvents are used for SDE. It is a suitable method for the isolation of compounds that are nonsensitive to heat or form products that involve similar temperature treatment (isolation of volatile compounds from tea brew or cooked meat). This technique can be performed in the microdistillation units, which have a different build, depending on the solvent used for extraction (heavier or lighter than water). SDE is usually performed for a relatively long time ranging usually between 60 and 120 min. Extraction time inﬂuences recoveries of the analyzed compounds. To improve the recoveries of basic or acidic compounds, pH adjustments are required to convert acids or bases into their free forms and improve volatility. In addition, the thermal stability of the analyzed compounds should be assessed if unknown. The main drawback of the technique is possible artifact formation due to prolonged heating and the presence of air. As an example, the composition of volatile compounds extracted from Penicillium roqueforti cultures using solid-phase microextraction (SPME) and simultaneous distillation extraction (SDE) can be shown. Prolonged distillation causes oxygenation of terpenes, which are the main volatiles produced in this fungus and as a result of SDE, the fraction of oxygenated sesquiterpenes reaches 50% (Figure 4). Simultaneous distillation and extraction (SDE) is the technique that has been used for isolation of ﬂavor compounds for several decades. The main disadvantage of SDE is the high temperature used for compounds’ isolation and as result, reactions of lipid oxidation, sugar degradation, or the Maillard reaction occur, which change the proﬁle of ﬂavor compounds. Due to the thermal liability of many ﬂavor compounds, high vacuum distillation provides better results, not altering the initial sample composition. Operation of SDE under vacuum requires changing organic solvents into less volatile ones. The matrix composition inﬂuences the volatiles’ release and their partition. In the extraction of ﬂavor compounds, all macromolecules (proteins, lipids, and carbohydrates) inﬂuence the release of volatile compounds. Extraction of ﬂavor compounds from matrices containing high levels of lipids is of special importance. As the partition coefﬁcients of ﬂavor compounds between lipids and extraction solvents are close to 1, lipids have to be removed from ﬂavor compounds prior to further analysis. For the distillation of matrices rich in fat, a modiﬁed high vacuum transfer (HVT) apparatus was designed by Berger and coworkers.34 The liquid sample is introduced in the form of small droplets into the high vacuum zone and the coil of the condenser is thermostated with circulating water at 45 C. High vacuum is required for the apparatus to work effectively (5 103 Pa), under which the sample dosed from the sample reservoir is sprayed over the coil as a thin ﬁlm and subsequently moves downward (Figure 5). This setup was used by Krings et al. for the model mixture of aroma compounds of different character dissolved in oil obtaining satisfactory results for different classes of volatile compounds with the exception of lactones for which the recoveries were poor (d-dodecalactone – 5.1%; g-decalactone – 28.2%). Another modiﬁcation of the SD apparatus is the addition of single drop microextraction (SDME) as a part of the SD process. In SDME techniques, a microdrop of solvent is suspended from the tip of a microsyringe needle and can be immersed either in the liquid matrix or in the headspace. This technique was used together with continuous hydrodistillation (a classical technique used for the analysis of essential oils) for the isolation of essential oil constituents from Lavandula angustifolia Mill35 (Figure 6). Very often, for the full characteristics of ﬂavor/volatile compounds in food, more than one method of extraction is required. A good example is the extraction of volatile compounds from cheese. Ewe’s semi-hard and hard cheeses with a PDO (protected

Figure 4 Main fractions of volatile compounds (percentage of total peak areas) isolated from Penicillum roqueforti using SPME (PDMS ﬁber) and SDE methods.33

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Figure 5

Apparatus used for thin-layer high-vacuum distillation (TLHVD) of lipids containing volatile ﬂavor samples.34

designation origin) were used to compare two methods of extraction to obtain a full proﬁle of volatile compounds.36 Volatile compounds were isolated using a purge-and-trap technique (DHS) and a classical simultaneous distillation extraction (SDE) method. As the cheeses are solid material, two approaches to the analysis of volatiles using dynamic headspace were proposed: sampling volatiles from dry cheese (dry method), and sampling volatiles from solid cheese dispersed in water using a homogenizer (suspension method). Volatiles were trapped using a mixture of Carbosieve SIII and Carbopack B using the purge and trap system. Simultaneous distillation extraction was performed in a microversion of an SDE unit conﬁgured for a solvent lighter than water. All

Figure 6 Apparatus for hydrodistillation–headspace solvent microextraction (a) syringe; (b) needle; (c) microdrop; (d) condenser; (e) round-bottom ﬂask; and (f) Claisen distillation head.35

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the main groups of cheese volatile compounds were compared: hydrocarbons, alcohols, ketones, aldehydes, esters, fatty acids, phenols, and some other compounds including terpenes. Using the dynamic headspace technique, more highly volatile compounds were isolated compared to SDE. Applying the dynamic headspace technique to dry cheese provided more compounds and generally larger quantities of compounds extracted compared to the suspension technique. This can be attributed to solubility of polar compounds in aqueous phase. The SDE technique was more efﬁcient in the extraction of phenols, free fatty acids, lactones, heavier aldehydes, ketone alcohols, and esters. Chromatograms of cheese samples extracted using SDE and DHS using dry and suspension methods are shown in Figure 7.

4.06.3.3

Headspace Methods

Headspace analysis comprises a number of solventless techniques used for the isolation of volatile compounds from the matrix using a gas phase. Gas phase analysis can be achieved using the techniques of static headspace, or dynamic headspace. Supercritical ﬂuid extraction is sometimes also included in gas extraction techniques. The development of headspace analysis can be dated as far as back to 1939, when Harger indicated the possibilities of determination of ethanol in headspace above the solution. Probably, the ﬁrst application of static headspace in the ﬁeld of food ﬂavors was the determination of lipid oxidation products in potato chips by Etre in 1959. Since that time, static headspace developed into a mature technique, which, using automated autosamplers, can offer high reproducibility, ease of operation, and sensitivity in the high mg–mg l1 range. It found applications in many areas of ﬂavor analysis: determination of volatiles from liquid or solid matrices, control of the production processes, measurement of ﬂavor release, etc. The next breakthrough in headspace analysis appeared in the early 70s, when Teranishi and Zlatkis invented the dynamic headspace technique, where gas is used for purging analyte from the matrix, with subsequent trapping of volatiles on various kinds of sorbents. The last major development in headspace analysis was the invention of SPME, developed by Pawliszyn in 1990. Although SPME can be used for direct extraction, due to the nature of food as a matrix, mainly headspace–solid-phase microextraction is used in ﬂavor analysis. SPME will be discussed in a separate paragraph of this chapter. Static headspace is the simplest form of headspace analysis, in which the sample is kept at constant temperature, so equilibrium is reached, and a certain volume of the headspace is transferred to the gas chromatograph to be analyzed. Static headspace is based on Dalton, Rault, and Henry laws. The latter especially provides the basis for headspace analysis. In static headspace, a partition constant describes the distribution of the analyte between liquid and gas phase (K ¼ CL/CG) phases, where CL and CG are the concentration of analyte in liquid and gaseous phases, respectively. The distribution constant will be dependent on the solubility of the analyte in liquid phase, and as a consequence compounds well soluble in liquid phase will have much higher concentrations in the condensed phase compared to the headspace (CL [ CG). For the compounds of weak solubility in condensed phase, their concentration CL will be comparable to CG and as a consequence the K value will be low. The equation describing concentration of analyte in the headspace vial can be summarized as COVL ¼ CLVL þ CGVG; the sensitivity of the headspace analysis will depend on the concentration of analyte in the gas phase and can be expressed as CG ¼ CO/(K þ b), where K ¼ CL/CG and b ¼ VG/VL. This introduction helps us to understand the basic factors that inﬂuence the sensitivity of analysis using the static headspace method. In the analysis of volatile ﬂavor compounds, there are usually mixtures of compounds, which are polar and nonpolar, and characterized by different solubility in the matrix. The majority of applications of static headspace in ﬂavor analysis refer to liquid matrices, although there are numerous examples of using headspace for solid matrices. To facilitate uniform standard addition and improve extraction, solid matrices are often homogenized into slurries or liquids. Vapor pressure depends on the temperature, so increase of temperature decreases the partition constant; therefore, temperature increase can improve the headspace sensitivity method. This is true for compounds for which K >>> b. If K < b, sensitivity of headspace analysis will depend mainly on b. As b is not temperature dependent, as a consequence temperature increase will have little or no effect on increase of headspace sensitivity for these compounds. This can be observed for polar and nonpolar compounds extracted using static headspace from aqueous phase. As polar compounds such as alcohols will be characterized by high K values (well soluble in water), temperature change will inﬂuence their partition to a much higher extent than for nonpolar terpene hydrocarbons, which are characterized by low K values. Static headspace analysis is a method well suited for the analysis of very volatile compounds; in this respect, it is used for GC–O extractions and for characterization of low molecular weight compounds due to the relation between vapor pressure for a compound and the number of carbon atoms in the carbon chain of homologous series ðlog poi ¼ a Cn þ b; where a and b are constantsÞ. In consequence, much lower concentrations of low boiling compounds can be detected by headspace than by direct injection. When performing analysis using headspace, the temperature chosen for the sample equilibrium should be the lowest, guaranteeing sufﬁcient sensitivity. The temperature is restricted mainly by the sample properties. It is especially important for food samples, where often they are thermally labile and prone to oxidation and other changes. An example may be the equilibration of samples of reconstituted milk from milk powder carried out for 30 min at 50 C – performed in the authors’ laboratory. Samples were equilibrated with and without the addition of BHT (5 mg ml1) acting as an antioxidant. When selected, aldehydes in such samples were compared in samples where no antioxidant was added; peak areas of pentanal, hexanal, heptanal, and 2-heptanal were 1.23; 1.28; 1.23; and 2.27 times higher compared to samples with BHT added, indicating an ongoing process of lipid oxidation during sample heating. Static headspace is a very attractive technique in terms of simplicity in sample preparation – only weighing or measuring a certain volume of the sample is required when transferred to the headspace vial as is addition of internal standard (if such calibration is carried out). However, it has to be stressed that heating the sample for sometimes a relatively long time usually in the presence of air in the vial can lead to sample changes. For plant samples (tissues), an additional factor has to be considered – the activity of enzymes that are involved in the formation of ﬂavor compounds.

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Figure 7 Gas chromatograms of volatile compounds extracted from Fiore Sardo cheese using different extraction methods.36 From top to bottom: simultaneous distillation/extraction method (SDE); dynamic headspace method using dry method (grated cheese); dynamic headspace method using suspension method (cheese homogenized with water).

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Usually, lipoxygenases, hydroperoxide lyases, and other enzymes taking part in formation of aroma compounds are inactivated by addition of CaCl2, if extraction performed at relatively low temperatures. Another factor inﬂuencing headspace sensitivity is the phase ratio, which inﬂuences the static headspace method sensitivity only for compounds having low K values. In addition, factors that inﬂuence analyte solubility will inﬂuence the sensitivity of the method inﬂuencing partition of the compounds. Similar to the relation of K value with temperature, compounds that have high K values (i.e., polar compounds in aqueous matrices) will be prone to solubility changes after such operations as salting out. For nonpolar compounds in aqueous solutions, salting out will not provide satisfactory results. Method sensitivity in static headspace will depend also on the pH of the matrix, which is related to the neutral or ionic form of analyzed compounds (therefore, weak acids should be extracted from a well-acidiﬁed matrix (pH 2); the opposite is true for weak bases). Sample mixing is recommended to shortening the time required for reaching equilibrium (improves extraction kinetics). The amount of sample injected into the gas chromatograph, which inﬂuences method sensitivity, is usually restricted by the injection system. A heated headspace syringe is more versatile in this respect; systems that transfer the sample via a loop system usually use 1-ml or sometimes 3-ml loops, so change in the volume injected is performed by exchanging loops. The main drawback of static headspace is its relatively low sensitivity compared to methods of dynamic headspace or other methods based on sorption phenomena. Static headspace is a technique where sample is not preconcentrated, as only a certain volume of gaseous phase is transferred to the gas chromatograph. Therefore, compared to other headspace techniques, static headspace is less suitable for food odorant analysis, especially in the ﬁeld of off-ﬂavor and taints analysis, where compounds are present in very low concentrations.

4.06.3.4

Sorption Methods

Concentration of food aroma compounds using sorptive methods became probably the most widely used group of methods in the past two decades. Methods based on extraction on sorbents comprise purge-and-trap techniques (P&T), solid-phase extraction (SPE), solid-phase microextraction (SPME), or stir bar sorptive extraction (SBSE) to name the main trends. Since its invention 20 years ago, SPME became the most frequently used of sorbent-based method for the analysis of ﬂavors and off-ﬂavors in food. In the adsorption process, the analyte is attached to the inner or external surface of a solid. Adsorbents have a porous structure and, as a result, a large surface area often exceeding 100 m2 g1. The size of pores varies from 2 nm (micropores) to 50 nm (mesopores). The adsorption mechanism in volatile compound’s isolation is based on physical sorption, where weak forces (van der Waals and electrostatic forces) enable the reverse process of desorption. As listed by Nongonierma,37 ﬁve groups of sorbent materials are used: (1) silica gels (polar due to their hydroxyl groups) efﬁcient in removal of polar compounds; (2) activated aluminas (polar) that can adsorb water; (3) activated carbon (apolar), which due to the wide range of pores can be used for extraction of a wide range of analytes; (4) zeolites (porous crystalline aluminosilicate), and (5) polymers – the most frequently used are polystyrene, polyacrylic esters, and phenolic resins. Some (PDMS or PA) are in liquid state at extraction temperature so an absorption mechanism prevails in them and they behave like solvents. For the analysis of aroma compounds, sorbents belonging to the last group are used most frequently; also, activated carbons are utilized for this purpose. They are used for the extraction of aroma compounds in their free form, whereas sorbents belonging to the ﬁrst group (silica gels) are often used for the analysis of bound volatile compounds. Adsorbents used for the extraction of volatiles are usually used in a form of granules employed for ﬁlling traps or can be used as a coating in SPME devices, SBSE or in-tube extraction devices. Among the used sorption techniques, SPME is the most often employed for the extraction of volatile compounds from foods. Twenty years after its development, it found applications in screening volatiles of a product, determination of key aroma compounds, and extraction of compounds responsible for taints and off-ﬂavors. Although the majority of applications of SPME refer to environmental studies, the second group of applications is that related to food and ﬂavor. Among SPME papers related to ﬂavor analysis, the majority refer to beverages, wine, and other alcoholic beverages, followed by fruit and vegetables, dairy products, and plant oils. The popularity of solid-phase microextraction can be attributed to its simplicity, robustness, and low cost of the technique with very good performance parameters – its high sensitivity is usually comparable to that of dynamic headspace and it has good reproducibility. The advantage of SPME compared to other techniques used in ﬂavor compound isolation is the short extraction time, absence of solvent, selectivity of extraction based on K (distribution constant) values, and the wide range of ﬁbers that can be used for extraction and inﬂuence the selectivity. It allows extraction of volatiles at low temperatures. The only stage that can be a potential source of artifact formation is the desorption process in the injection port of GC, where usually temperatures of 250–270 C are applied. Solid-phase microextraction gained its popularity for the analysis of volatile compounds in foods – in proﬁling volatiles as well as in target analysis of selected compounds. It has been used for the analysis of compounds which are present in trace concentrations (ng l1 or ng kg1) as well as for compounds that are present in food in much higher concentrations, up to mg l1. SPME is the single extraction technique that has been, since its invention, the main tool used for food volatile compound characterization. This requires more detailed explanation. Table 2. comprises selected applications of SPME in food volatile characterization that were published in the last 10 years. The applications comprise various food products ranging from fruit, milk, coffee, honey, and meat to wine. When particular foods are concerned, there are relatively many applications on alcoholic beverages, mainly wine, which reﬂect trends in application of SPME for food volatiles, where alcoholic beverages also dominate. There are two types of application of SPME in characterization of food volatiles. The majority of papers included in the table, which refer to aroma compound analysis, provide the characteristics of the volatile compounds of foods using SPME. In this approach, rarely all, or even

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Table 2

SPME ﬁber coatings used in extraction of aroma compounds from various food matrices

Fiber coating

Matrix: analyte: extraction mode/temperature [ C]/time [min.]/detector type; reference

DVB/CAR/PDMS

Apple juice: off-ﬂavors: HS/60/10/GC-MS; Anal. Chim. Acta. 2004, 520, 3-11 Banana: aroma compounds: HS/40/10/GC-MS; LWT, 2009, 42, 1847-1853 Bread: aroma compounds: HS/35/60/GC-MS: Food Res. Intl. 2007, 40, 1170-1184 Boiled potatoes: aroma: HS/37/30/GC-MS; Food Chem. 2010, 118, 283–290 Cheese: aroma compounds: /HS/room temp/20/GC-MS; Food Chem. 2009, 113, 506–512 Cheese: volatile fatty acids: HS/45/60/GC-MS; Int. Dairy. J. 2006, 16, 8860894 Coffee: off-ﬂavors: HS/70/40/GC-MS; Food Chem. 2008, 106, 787-796 Corn ﬂakes: lipid oxidation products: HS/40/30/GC-MS; Food Chem, 2009, 113, 543-549 Cork: chloroanisoles: HS/65/90/GC-ECD; J. Chromatogr. A, 2006, 1107, 240–247 Custard dessert: aroma compounds: HS/30/30/GC-MS; Food Chem. 2008,108, 1183-1191 Dry-fermented sausages: aroma compounds: HS/30/90/GC-MS; Food Chem. 2004, 84, 633–641 French beans: aroma compounds: HS/40/60/GC-MS; Food Chem., 2007, 101, 1279–1284 Honey: aroma compounds: HS/40/20/GC GC-TOFMS; J. Sep. Sci. 2007, 30, 534 – 546 Italian sausage: aroma compounds: HS/40/30/GC-MS; Meat Science, 2009, 81, 77–85 Jackfruit: aldehydes, esters, alcohols: HS/30/10/GC-MS; J. Food Comp. and Anal. 2008, 21, 416– 422 Milk: off-ﬂavors: HS/45/45/GC-MS, GC-FID; Int. Dairy Journal, 2005, 15, 1203–1215 Milk: oxidation product: HS/45/30/GC-MS; International Dairy Journal, 2007, 17, 746–752 Olive oils: volatile phenols: HS/60/30/GC-MS; J. Chromatogr. A, 2008, 1211, 1–7 Olive oil: lipid oxidation product: HS/40/30/GC-MS; J. Agric. Food Chem. 2003, 51, 6564-6571 Pasta and semolina: aroma compounds: HS/30/24h/GC-MS; J. Cereal Sci., 2009, 49, 301–309 Pepper: aroma compounds/HS/ns/240/GC-MS, GC-FID, GC-O; J. Chromatogr. A, 2002, 976, 265–275 Potato crisps: aroma compounds: HS/70/5/GC-MS; J. Chromatogr. A, 2004, 1046, 75–81 Potato crisps: aroma compounds: HS/70/20/GC-MS; J. Chromatogr. A. 2005, 1064, 239-245 Rice: aroma: HS/80/18/GC-MS, GC-O; Food Chem. 2011, 124, 501–513 Sparkling wines: aroma compounds: HS/35/30/GC-MS; Food Chem. 2007, 105, 428–435 Wine: 2-aminoacetophenone: DI/ 30/30/GC-MS; Food Chem. 2007 105, 1144-1150 Wine: aroma compounds: DI/25/30/GC-MS; J. Chromatogr. A, 2003, 985, 233–246 Wine: aroma compounds: HS/20/20/GC-MS/ Anal. Chim. Acta, 2010, 677, 43–49 Wine: carbonyls: HS/40/45/GC-MS: J. Agric. Food Chem, 2010, 58, 12976-12985 Wine: haloanisoles, halophenols: HS(der)/70/60/; J. Chromatogr. A, 2007, 1143, 26–35 Wine: heavy sulfur compounds: HS/35/120/GC-FPD: J. Chromatogr. A, 2002, 945, 211–219 Wine: off-ﬂavors: HS/45/45/HS/GC-MS; J. Chromatogr. A, 2007, 1141, 1–9 Wine: pyrazines: HS/40/30/GC-MS; J. Agric. Food Chem. 2004, 52, 5431-5435 Vinegar: aroma compounds: HS/50/40/GC-MS J. Chromatogr. A, 2003, 1017, 141–149 Vinegar: aroma compounds: HS/70/60/GC-FID; J. Chromatogr. A, 2002, 967, 261–267 Andean blackberry: free aroma compounds: HS/60/20/GC-MS; Food Res. Intl. 2011, 44, 54-60 Cork: chloroanisoles: HS/30/30/GC GC-ToF-MS; J. Chromatogr. A, 2007, 1138, 10–17 Cheese: lipid oxidation product: 60/30/HS/GC-TOFMS; J. Agric. Food Chem. 2003, 51, 1405-1409 Musts: 3-alkyl-2-methoxypyrazines: HS/30/240/GC-NPD: J. Chromatogr. A. 2000, 880, 93-99 Wine: polyfunctional thiols: HS(der)/55/10/GC-NCI-MS; J. Chromatogr. A. 2006, 1121, 1-9 Apricots: aroma compounds: HS/40/20/GC-MS, GC-O; Food Chem. 2006, 96, 147-155 Beef tallow: aroma compounds: HS/55/30/GC-MS; Food Chem., 2011, 124, 203–209 Beer: aroma compounds: HS/20/30/GC-MS; J. Chromatogr. A. 2006, 1121, 145-153 Butter: aroma compounds: HS/40/20/GC GC-ToF-MS; J. Chromatogr. A. 2005, 1086, 99-106 Cheese: aroma: HS/20/30/GC-MS; Food Chem., 2009, 112, 1053-1059 Dry-fermented sausages: aroma: HS/30/90/GC-MS; Food Chem. 2004, 84, 633–641 Dry sausage: aroma: HS/30/180/HS/GC-MS; Meat Science, 2006, 73, 660–673 Fermented sausage: aroma compounds: HS/30/90/GC-MS; Food Chem. 2009, 115, 1464–1472 Honey: aroma compounds: HS/40/30/GC-MS; Food Chem. 2008, 111, 988–997 Meat proteins: carbonyls: HS/30/30/GC-FID; Food Chem. 2008, 108, 1226-1233 Mushroom: aroma compounds: HS/40/30/GC-MS, GC-FID Food Chem. 2010, 123, 983–992 Sausage: aroma compounds: HS/30/60/GC-MS; Food Chem. 2010, 121, 319–325 Tea: aroma compounds: HS/50/30/GC-MS; Food Chem., 2008, 109, 196–206 Whisky: aroma compounds: HS/40/60/GC-MS; J. Chromatogr. A, 2007, 1150, 198–207 Wine: heavy sulfur compounds: DI/10/240/GC-MS; J. Chromatogr. A, 2000, 881, 583–590 Wine: sulphides, disulphides, thiols: HS(der)/25/30/GC-FPD J. Chromatogr. A, 1999, 849, 293–297 Wine: thiols, sulphides: HS/25/30/GC-FPD; J. Chromatogr. A. 1999, 849, 293-297 Wine: sulfur compounds: HS/30/15/GC-PFPD; J. Chromatogr. A. 2005, 1080, 177-185 Wine: volatiles phenols: HS/60/50/ GC-FID; J. Chromatogr. A, 2003, 995, 11–20

DVB/PDMS

Carboxene/PDMS

Sample Preparation for Food Flavor Analysis (Flavors/Off-Flavors)

Table 2

133

SPME ﬁber coatings used in extraction of aroma compounds from various food matricesdcont'd

Fiber coating

Matrix: analyte: extraction mode/temperature [ C]/time [min.]/detector type; reference

PDMS

Coffee: aroma compounds: HS/30/30/GC-MS; J. Sci. Food Agric. 2003, 84, 43–51 Corn, soybean oil: aldehydes: HS/60/60/GC-MS/ J. Food Sci. 2002, 67, 1, 71-76 Grape juice fermentation: esters: HS/18/60/GC-MS; J. Agric. Food Chem, 2001, 49, 589-595 Hops: aroma compounds: HS/50/240/GC-FID; J. Agric. Food Chem. 1996, 44, 1768-1772 Olive oil: aldehydes: HS/45/15; J. Chromatogr. A, 2004, 1028, 321–324 Pepper: aroma compounds: HS/64/44/GC-MS; Microchem. J. 2006, 82, 142-149 Strawberry: aroma compounds: HS/20/45/GC GC-FID; J. Chromatogr. B, 2005, 817, 97–107 Sunﬂower oil: off-ﬂavors: HS/40/30/GC-MS; J. Agric. Food Chem. 2000, 48, 5981-5985 Wine: aroma compounds: HS/25/30/GC-MS; J. Chromatogr. A, 2003, 985, 233–246 Wine: aroma compounds: HS/40/10/GC-MS; J. Chromatogr. A 2001, 922, 267–275 Wine: esters: HS/45/40/GC-MS; Food Chem., 2010, 121, 1236–1245 Wine: off-ﬂavors: HS/50/50/GC-MS-MS; J. Chromatogr. A, 2006, 1112, 133–140 Wine: sulphides, disulphides: HS(der)/30/15/GC-FPD; J. Chromatogr. A, 1998, 808, 211–218 Wine: chlorophenols: HS(der)/45/65/GC-ECD; J. Chromatogr. A. 2004, 1048, 141-151 Wines and grapes: aroma compounds: HS/30/30/GC-MS; J. Chromatogr. A, 2009, 1216, 3012–3022 Wine (Madera): aroma compounds: HS/20/15/GC-MS; Anal. Chim. Acta, 2005, 546, 11–21 Butter: aroma: HS/40/60/GC GC-ToF-MS: J. Chromatogr. A. 2005, 1086, 99-106 Kiwi fruit: aroma compounds: HS/25/30/GC-MS; Food Res. Int. 1999, 32, 175-183 Olive oil: lipid oxidation product: HS/40/20/GC-MS; J. Agric. Food Chem., 2003, 51, 733-741 Wine: phenols: HS/50/30/GC-FID; J. Chromatogr. A. 2003, 995, 11-20 Wine: aroma compounds: HS/20./20/GC-FID; J. of Chromatogr. A, 2003, 991, 13–22

CW-DVB

HS – extraction from headspace; DI – direct extraction; (der) – derivatization; ns – not speciﬁed.

the main compounds are quantiﬁed; usually, area percent of peaks is provided, or some normalization is performed. In this approach, a proﬁle of volatile compound for a given product is provided. However, it has to be remembered that SPME is not an exhaustive extraction; therefore, the obtained proﬁle of volatile compounds will depend on the partition of compounds between the headspace phase and particular ﬁber; in the case of varied K values, the ‘proﬁles’ will differ substantially. This problem is illustrated in Figure 8, which shows the main groups of volatile compounds extracted from sweet, liquered Jutrzenka wine in the authors’ laboratory. Depending on the ﬁber used for extraction, mutual proportions of the extracted main groups of volatiles vary. Carboxene/PDMS (CAR/PDMS) ﬁber extracted alcohols as effectively as esters, whereas when polyacrylate ﬁber (PA) was used (polar ﬁber for polar compound applications), alcohols formed the dominant group. For the Carboxene/divinylbenzene/polydimethylsiloxane (CAR/DVB/PDMS) ﬁber, esters were the main group, followed by alcohols and carbonyl compounds, and for polydimethylsiloxane ﬁber (PDMS), similar to CAR/DVB/PDMS, esters dominated, but alcohols were few times less abundant than in this ﬁber and carbonyls formed a very small fraction. Therefore, the SPME ‘proﬁle’ reﬂected in peak areas may be misleading, as, for the real proﬁle of volatile compounds, exhaustive extraction should be used. To illustrate this issue in Figure 9, the main fractions of the same Jutrzenka for which various ﬁbers were tested are shown, when extracted with different techniques: HS-SPME, SPE, and SAFE (Solvent Assisted Flavor Evaporation – a high vacuum distillation which will be discussed later in this chapter). The pronounced differences between proﬁles of main ﬂavor groups are shown in the comparison between selective (SPME and SDE) and exhaustive extraction (SAFE). The inﬂuence of the extraction method on the proﬁle of isolated compounds can be observed in the majority of investigations where such comparison is made. As an example, the inﬂuence of various methods of extraction compared to SPME can be observed for garlic volatiles38 (Table 3). It illustrates again the problem of proper method selection for (often labile) ﬂavor compounds. Garlic volatiles were extracted in different conditions – in steam distillation (SD), the process lasted 5 h at 100 C, in the SDE procedure carried out in Likens–Nickerson apparatus it lasted for 8 (!) h and the volatiles extracted with dichloromethane. Solidphase trapping solvent extraction was performed at room temperature adsorbing volatiles for 2 h into Porapak-Q and compounds were eluted with dichloromethane. In HS-SPME, the sample was extracted for 60 min, at 20 C. All the injections were made at 250 C. This illustrates the complexity of food ﬂavor compound analysis. With compounds such as Allium (garlic, onion, shallots, leek, etc.) volatiles that are formed on the enzymatic pathway catalyzed by allinase and the intermediates (sulfenic acids) are extremely labile and decompose to more stable products (thiosulﬁnates decompose to disulﬁdes, polysulﬁdes or cepaenes, and zwiebellane depending on the plant). Therefore, care must be taken during the whole process of extraction and chromatographic analysis and often labile intermediates or ﬂavor compounds cannot be registered due to high temperatures in the injection port, in which they may undergo decomposition reactions. The other area in which SPME is used and in which it can achieve the most spectacular results is target analysis of food odorants and off-odorants. Due to its preconcentration capabilities, it offers high sensitivity and small volume of phase results in short extraction times, during which equilibrium can be achieved. It has found application both in determination of compounds contributing to the characteristic smell of a product, and, even more frequently – to the analysis of off-odors and taints. Several reviews summarize the analysis and extraction methods for off-ﬂavors; a review on application of SPME to the analysis of taints and off-ﬂavors has also been published.39

134

Extraction Techniques and Applications: Food and Beverage

8e+8

1.6e+8

Peak area [arb. units]

1.4e+8 Peak area [arb. units]

CAR/DVB/PDMS

CAR/PDMS

1.2e+8 1.0e+8 8.0e+7 6.0e+7 4.0e+7

6e+8

4e+8

2e+8

2.0e+7 0

0.0 A

E

C

M

A

T

E

C

M

T

Fiber coatings

8e+8

5e+7

PDMS

4e+7

Peak area [arb. units]

Peak area [arb. units]

PA

3e+7

2e+7

6e+8

4e+8

2e+8

1e+7 0

0 A

E

C

M

T

A

E

C

M

T

Fiber coatings

Figure 8 Proﬁles of volatile compounds obtained for Jutrzenka liquered wine using different SPME ﬁber coatings: (A) – alcohols; (E) – esters; (C) – carbonyl compounds; (M) – miscellaneous; and (T) – terpenes.

The examples of SPME applications to off-ﬂavors in literature refer to main groups of compounds that are usually analyzed in food: earthy–musty compounds: chloroanisoles, geosmin, and methylisoborneol, and also aldehydes, and volatile phenols, and more speciﬁc compounds. Chloroanisols are contaminants of vegetables, dried fruit, coffee, and, probably the most well-known example, wines, with its cork taint of earthy–musty–moldy notes. The main compound associated with these notes is 2,4,6trichloroanisole. Chloroanisoles are formed in a process of chlorophenol methylation by fungi. Musty–earthy ﬂavors are usually associated with the presence of geosmin and 2-methylisoborneol and are present in stored grain, catﬁsh, red beet, and vegetables coming into contact with soil, as the main source of geosmin is Actinomycetes inhabiting soil. Volatile phenols are mainly of microbial origin and are often formed in the bioconversion of hydroxycinnamic acids into corresponding phenols. Volatile lipid oxidation products are usually associated with rancid off-ﬂavors in products containing various amounts of fats and aldehydes for the biggest group of these compounds. As many aldehydes originating from the oxidation of (poly)unsaturated fatty acids are unsaturated, they can be a source of other volatile compounds. Moreover, the aldehydes formed are very labile. Even at room temperature, 2,4-decadienal oxidizes to a mixture of hexanal, 2-heptenal, 2-octenal, furan, pentane acrolein, glyoxal, benzaldehyde, hexanoic acid, and 2,4-decadienoic acid. Formed in the autooxidation process, vinylketones have a distinct off-odor of rancid or ﬁshy character. The main challenge in the analysis of off-ﬂavors is the low odor threshold for these compounds. The odor threshold of 2,4,6-trichloroanisol varies depending on the medium, from 0.001 mg m3 in air to 1.4–10 ng l1 (wine); geosmin and 2-methylisoborneol also have odor thresholds of 0.0038 mg l1 and 0.015 mg l1, respectively. The odor threshold usually changes with a change of matrix – for MIB in catﬁsh, the odor threshold is estimated at 0.7 mg l1. Vinylphenol and vinylguaiacol also have odor thresholds of 0.022 mg l1 and 0.012 mg l1, respectively. Lipid oxidation products have odor thresholds highly dependent on the class to which the compounds belong. The low odor thresholds make the analysis of these compounds especially challenging.39 Determination of target compounds – key odorants or off-odorants in low concentrations – requires careful optimization of the whole SPME method. Although solid-phase microextraction is robust, fast, and simple, method optimization is a multistep procedure and, for the analysis of volatile ﬂavor compounds present at low concentrations, all steps should be directed to enhance method sensitivity. Optimization of the SPME method involves several elements inﬂuencing analysis results: SPME ﬁber, matrix

Sample Preparation for Food Flavor Analysis (Flavors/Off-Flavors)

SAFE

SPME

135

SPE

ALCOHOLS CARBONYLS ESTERS MISCELLANEOU TERPENES ACIDS UNIDENTIFIED HYDROCARBON

Figure 9 Comparison of volatile compounds extracted from Jutrzenka liquered wine using different isolation methods: SAFE (300 m Torr, 30 min.), SPME (Car/DVB/PDMS, 30 min, 50 C), and SPE (C18, dichloromethane).

type and preparation, extraction parameters, and chromatographic parameters. In the following section, crucial steps for SPME method sensitivity for ﬂavor volatile compounds will be outlined: l l l l l l l

selection of ﬁber coating; selection of sampling mode; agitation method selection; optimization of matrix; analyte derivatization (if required); selection of the calibration method; and validation of the method.

Selection of ﬁber coating is the ﬁrst step in optimization of SPME parameters. The choice of ﬁber coating inﬂuences method performance, as the selectivity of the ﬁber for a given analyte can provide radical impact for the limits of detection. The suitability of a ﬁber for a particular analysis is determined by its polarity. Currently, two groups of ﬁbers are available: liquids, in which the mechanism is based on absorption, and solid coatings, where adsorption prevails. Two ﬁbers representing absorption type ﬁbers are polyacrylate (PA) and polydimethylsiloxane (PDMS). PDMS is a ﬁber providing a good starting point in many SPME experiments and is a ﬁber that provides good general sensitivity and linearity. Because of the absorption mechanism, there are no negative effects observed in polymer-based ﬁbers (compound displacement on the surface of the ﬁber). As with other extraction media, there is

Table 3

Flavor composition identiﬁed by GC-MS of garlic collected by different extraction methods, and peak area percentages38

No

Compound

SDE

SD

SPTE

HS-SPME

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Propylene sulﬁde Allyl methyl sulﬁde Dimethyl disulﬁde 4-heptenal Allyl sulﬁde 1-(1-propenylthio)propane 1,3-dithiane Methyl propyl disulﬁde 5-methyl 1,2,3-thiadiazole 2-vinyl-1,3-dithiine Diallyl disulﬁde 1,2-dithiolane Unknown Methyl 2-propynyl sulﬁde 1,3-dithiolane Propenyl 1-propynyl sulﬁde Thiirane 3-vinyl-1,2-dithiocyclohex-5-ene Diallyl trisulﬁde Diallyl tetrasulﬁde 2-methyl-3-pentanol

1.05 12.7 0.43 8.4 0.08 18.9 0.08 þ 16.9 23.59 2.1 0.26 10.7 1.28 7.8 0.01 13.8 0.02 14.2 0.07 6.9 57.88 0.6 0.68 10.2 1.07 3.3 0.98 4.3 0.02 6.5 0.09 9.3 0.06 8.9 0.36 5.7 11.40 0.4 0.54 7.8 0.05 8.2

– 0.04 20.5 0.06 7.6 0.06 18.5 2.43 12.3 – 0.65 14.8 0.04 25.4 – – 89.77 2.6 0.49 7.0 2.12 10.6 0.19 4.9 – – 0.03 17.2 0.07 13.5 3.89 3.6 0.17 18.8 –

– – – – 0.17 14.4 – 0.31 7.3 – 0.01 15.1 – 97.77 0.5 0.32 13.8 1.29 9.9 – – – – – 0.10 7.1 0.03 9.7 –

0.04 12.7 – – – 0.01 6.4 – 0.95 9.8 0.01 15.3 0.01 6.3 – 97.85 0.5 0.17 5.4 0.94 4.3 – – 0.01 27.5 – – 0.01 9.3 – –

SDE – simultaneous distillation/extraction; SD – steam distillation; SPTE – solid-phase trapping solvent extraction; HS-SPME – headspace – solid-phase microextraction. Peak area % is provided with RSD (%).

136

Extraction Techniques and Applications: Food and Beverage

a general indication that polar molecules should be extracted by polar ﬁbers and nonpolar compounds by nonpolar ones. As the analyte dissolves in the whole volume of the coating, in absorption ﬁbers, usually equilibrium is reached after a longer time. The time required for reaching equilibrium depends also on the coating thickness; therefore, for rapid analyses thinner coatings should be chosen. However, for the analysis of volatile ﬂavor compounds present in very low concentrations, thicker coatings provide better sensitivity (more analyte adsorbed). Fibers based on mixed phases (carboxene and divinylbenzene) usually provide adsorption in a shorter time than liquid coatings, which usually also provide better peak responses. The disadvantage of these types of ﬁbers is their worse linearity compared to absorption-type ﬁbers. Comparison of several ﬁber coatings for the analyte of interest should be the ﬁrst step. Figure 10 shows peak areas of the 2-methylisoborneol peak extracted from wheat grain spiked with a standard of this compound. Presented data indicate that the responses can be substantially different for various ﬁbers. When the literature is searched for the most commonly used ﬁbers in ﬂavor analysis, there are some indications regarding the choice of particular ﬁbers: when method sensitivity is an issue, usually adsorption-type ﬁbers are chosen – divinylbenzene/Carboxene/PDMS or Carboxene/ PDMS; PDMS is the next choice. Comparing applications in Table 2, out of 80 cited papers a Carboxene/DVB/PDMS ﬁber is used in 35, in 19 papers Carboxene/PDMS was the ﬁber of choice, followed by 16 papers in which PDMS was chosen for extraction. In the analysis of haloanisoles, PDMS and DVB/CAR/PDMS are used most frequently; however, CW/DVB ﬁbers are also chosen. In the analysis of geosmin and methylisoborneol, mixed ﬁbers are used, whereas for volatile phenols polar (PA) or mixed (CW/DVB) ﬁbers are employed.39 The sampling mode in the analysis of ﬂavor volatiles depends on the matrix. Generally, in food volatile analysis, direct extraction is rarely used (one in the 80 papers indicated in Table 2). The cause is the nature of liquid foods that generally contain substances interfering with the analyte usually in the desorption process. Sugars, which can be present in some liquid foods, will undergo a caramelization reaction. Other macromolecules (proteins, lipids, and polyphenols) will be detrimental for ﬁber coating when heated in the injection port. For these reasons, headspace SPME is the method usually used. For the improvement of mass transport between the sample and the ﬁber sample, agitation is recommended. Stirring shortens the time needed to reach equilibrium, diminishing the boundary layer. Agitation is usually performed in liquid samples by stirring, needle or vial vibration in autosamplers, or by sonication. As many food samples analyzed using SPME are solids, mixing is not an issue in such cases, when analysis is performed on solid matrix. Such matrices are often ground and analyzed as solutes or slurries and in that case stirring can be applied. The consequence for static (nonstirring conditions) is that the equilibrium will be reached in a longer time. Optimization of sample volume plays a role in SPME method development. Method sensitivity is proportional to the amount (number of moles) extracted from the analyzed sample at equilibrium. The amount of analyte extracted increases with the sample size to a certain point. Usually, the sample volume inﬂuence on the extraction rate is important for very small samples. For autosamplers, the amount of analyte extracted is inﬂuenced by the sample capacity. 500x103

40x103

400x103 30x103

300x103 20x103 200x103

10x103 100x103

0

20C 35C 50C 75C 100C

P

D/P

C/P

C/D/P

0

Figure 10 Inﬂuence of ﬁber choice and extraction temperature on the peak area of 2-methylisoborneol extracted from wheat grains; C/D/P – Carboxene/ divinylbenzene/PDMS; C/P – Carboxene/divinylbenzene; D/P – divinylbenzene/PDMS; P – PDMS. For the ﬁber choice experiment, extraction was performed at 50 C for 30 min. from wheat grains spiked with 6 mg kg1. For the temperature choice experiment sampling, was performed using Carboxene/divinylbenzene/PDMS ﬁber for 30 min.

Sample Preparation for Food Flavor Analysis (Flavors/Off-Flavors)

137

SPME is an equilibrium extraction technique (providing free concentration of analyte in the sample). It is a nonexhaustive extraction; however, it can be exhaustive for very small samples and low analyte concentrations. Sampling at equilibrium provides the highest sensitivity; however, sampling at equilibrium is often not chosen. As the choice of sampling time is usually a compromise, often pre-equilibrium sampling is undertaken. A factor that has to be taken into consideration is the sampling time vs. chromatographic run time, which also is a limiting step. Another factor that is considered is that, in the mixed type ﬁbers, equilibrium is achieved for different compounds at different times and, for many compounds, equilibrium is not reached in a reasonable time. Sampling can be done in a pre-equilibrium state; however, there is a higher probability for errors due to time measurement (time differences) – compounds are sampled on the slope of the extraction curve. When comparing methods used for ﬂavor volatile compounds listed in Table 2, usually extraction times not exceeding 45 min are used. Extraction of analytes from the matrix is time dependent and also relies on other factors inﬂuencing distribution constant such as temperature, pH, and ionic strength. Increasing temperature of extraction can signiﬁcantly reduce the equilibration time and accelerates the whole extraction process. However, similar to static headspace analysis, the extraction temperature has to be considered with matrix/analyte stability in mind. In Figure 10, the inﬂuence of temperature on the extraction of 2-methylsioborneol from grain is monitored. Raising the temperature of extraction to 75 C results in the increase of the 2-methylisoborneol peak area. Increasing temperature to 100 C (solid sample) resulted in decrease of the 2-methylisoborneol peak area. Increasing sample temperature is limited by several factors: (1) analyte instability – for labile compounds, decomposition of analyte can take place when extraction time is sufﬁcient for such changes; (2) matrix instability – the temperature of extraction for liquid samples is restricted by the boiling point of the solvent, and, in case of water, temperatures of extraction usually do not exceed 60 C; and (3) desorption of the analyte from the ﬁber – at elevated temperature, this phenomenon is important. Desorption from the ﬁber can be limited by cooling the ﬁber and, to respond to this phenomenon, internally cooled ﬁbers have been designed. The last phenomenon is responsible for the losses of analyte from the ﬁber, as in Figure 10. In applications related to ﬂavor volatile compounds, lipid oxidation is a main, most frequently occurring reaction that should be minimized. Due to prolonged heating, decomposition of secondary lipid oxidation products can result in formation of new compounds and decomposition of volatile lipid oxidation products – hydroperoxides – ends up in increased levels of aldehydes and other lipid oxidation secondary products. It can be eliminated by addition of antioxidants to samples prior to heating and extraction or sampling at ambient temperature. Extraction at elevated temperature can be performed on solid and liquid samples. The distribution constant can be inﬂuenced by other factors – salting out or manipulation of pH. However, these operations are restricted to liquid samples. Similar to headspace analysis discussed before, in headspace SPME undissociated molecules are extracted, so low pH will improve extraction of acidic compounds whereas high pH values will improve sensitivity for basic compounds. In solid samples, the only way to inﬂuence the ﬂavor volatile partition is temperature increase. Detectability of the compounds can be improved by means of their derivatization. The group of food aroma compounds that are derivatized the most often are aldehydes. They can impair the sensory properties of food products and not only those containing high levels of fat. Due to the dependence of the fatty acid oxidation rate on water activity, even dry products oxidize relatively easily; similarly, when the amount of fat is low, but simultaneously the surface contact with oxygen is large. Aldehydes also create off-odors in beverages and water. Short chain aldehydes give poor response on an FID detector; therefore, derivatization is a way to increase sensitivity of a method and it is performed in HPLC, GC, and SPME. Considering as an example (E)-2-nonenal, which contributes to cardboard off-ﬂavor in beer, on ﬁber derivatization of beer aldehydes with O-(2,3,4,5,6-pentaﬂuorobenzyl)hydroxylamine (PFBOA) is used with DVB/PDMS ﬁber and a mass spectrometer as a detector, allowing quantiﬁcation at 0.01 mg l1. Derivatization in SPME can be performed in a number of ways: a derivatization agent can be added to the sample before sample extraction, or derivatization can be performed at the moment of extraction or after extraction. In these approaches, the ﬁber is used for extraction of reaction products from the headspace, the ﬁber is exposed to the analyte and transferred to vial with derivatizing agent solution, or ﬁnally, the ﬁber is exposed ﬁrst to the derivatization agent and then to sample vapors. Quantitation of odorants is one of the key issues in analysis of aroma compounds because of the low concentrations, instability of matrix and compounds, and sometimes, a complex isolation procedure. Depending on the matrix, different approaches are used ranging from external standard calibration, internal standard and standard addition to analysis using the SIDA (stable isotope dilution analysis) approach. The limiting factor in these analyses is the lack of commercially available standards of many aroma compounds and their very high costs. Very often, standards for aroma quantitation using SIDA have to be synthesized. Due to the complexity of the matrix, the most recommended in the case of solid matrices is the standard addition method; in a liquid matrix the internal standard method can be used. For off-ﬂavor compounds, quantitation is usually performed in the ways described above: for TCA determination 2,4,6-tribromoanisole (TBA) can be used, while p-iodoanisole as an internal standard in MSD is used for detection of mixed chloro- and bromoanisoles using headspace SPME. Cis-decahydro-1-naphtol (DHN) at 1 ppb was used as the internal standard prior to extraction for geosmin in catﬁsh. Deuterated MIB (MIB-d3) and GEO (GEO-d3) as internal standards can also be used for evaluation of these compounds in ﬁsh. Hexanal is a compound causing off-odors that became a popular indicator of lipid oxidation in foods. It is a secondary oxidation product formed during oxidation of linoleic acid. The characteristic off-odor of hexanal is often described as grassy and its odor threshold is estimated in water at 4.5 mg kg1. Usually, for its analysis, solid matrix is mixed with water. There are various approaches to the quantiﬁcation of this compound in meat. To raw pork deionized water was added, samples heated for 30 min at 75 C, then removed and allowed to cool to room temperature prior to extraction of volatiles using CAR/PDMS ﬁber. Using external calibration, a standard curve from 0 to 2.442 ppm was linearly constructed for hexanal and a linearity of 0.999 was observed for a range of 0–0.814 mg l1. A different approach was used based on the

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Extraction Techniques and Applications: Food and Beverage

preparation of hexanal vapor standard from hexanal in squalene and allowed to evaporate in a ﬂask for the quantitation of hexanal in freeze-dried chicken myoﬁbrils. Determination of hexanal in potato chips was based on SPME-GC, where samples were ground and mixed with water, and sampled at 70 C for 20 min using a 50/30 mm DVD-CAR-PDMS ﬁber.

4.06.4

Methods for Isolation of Volatile Compounds for Gas Chromatography–Olfactometry

GC–O methods are generally based on snifﬁng efﬂuent from the chromatographic column. To judge the importance of particular odorants in the aroma of investigated food in the GC–O approach, methods based on the analysis of serial dilutions of extract are used the most frequently. Two methods utilizing this approach are AEDA (aroma extract dilution analysis) and Charm.1 For the extract dilution analysis methods, high vacuum distillation is favored as the technique that provides mild conditions for compound isolation. As high vacuum distillation often lacks robustness, developments in labware are important. Solvent-assisted ﬂavor evaporation (SAFE) is the development that enabled rapid, robust, and simple isolation of ﬂavor compounds from liquid and solid matrices providing no discrimination of extracted compounds (exhaustive extraction), mild conditions that do not alter the compounds or form artifacts and remove the nonvolatiles (Figure 11). The distillation using SAFE usually lasts 20–40 min, but depending on the method of extraction – if products containing much water are extracted, a frozen water concentrate is obtained which, after thawing, is extracted with diethyl ether, pentane or dichloromethane, dried over anhydrous Na2SO4, and then concentrated using a rotary evaporator and/or a Vigreaux column. When compared with ‘classical’ high vacuum transfer, SAFE provided higher yields of compounds from solvent or high fat matrices.40 When an extract of volatile compounds is obtained by SAFE or other high vacuum distillation methods, it is ﬁrst concentrated to a volume of a few hundreds ml, which is subsequently used for preparing serial dilutions. In case of extraction methods based on adsorption on polymers, such as purge trap or SPME sample dilution necessary for GC–O has to be resolved in a different way. It can be done either by dilution of the sample extracted, or by changing the split ratio, although the latter method can be limited by instrument settings in the case of very potent odorants with FD values of 2000–4000, which would require split ratios impossible to achieve. For the GC–O analysis, apart from SAFE (or other vacuum distillation method), static headspace is used, especially for the extraction of the most volatile compounds. Static headspace sampling provides compounds, which in normal conditions, reach our olfactory system, as consumers in fact sniff headspace, not the extract. Therefore, static headspace is often used as a supplemental method or used for the extraction as the only one. For the analysis of 2-methyl-3-furanthiol, methional, and b-damascenone for GC–O and subsequent identiﬁcation, SPME (snifﬁng) and thermal desorption in conjunction with the NIF SNIF detection of odorants was proposed.41 Tenax traps in the purge and trap were also used in the extraction of aroma compounds from blue cheese in the work where the authors provided comparison of AEDA with OSME – a technique where no serial dilution is required.42 The authors compared two methods of GC–O and noted a general agreement between AEDA and OSME values; however, AEDA provided a higher level of discrimination based on FD values. Polymer traps were also used by Campo for the extraction of Madeira ﬂavor

Figure 11 SAFE apparatus:40 1 – main part of safe apparatus, 2 – central head, 3a – head inlet, 3b – head outlet, 4 – dropping funnel, 5 – sample dropping Teﬂon valve, 6 – cooling trap for liquid nitrogen, 7 and 8 – glass joint, 9 – tube for sample inlet, 10 – tube for vacuum pump, 11 and 12 – ‘legs’, 13 – water inlet, 14 – water outlet, 15 – polyethylene tubes for water circulation, 16 – screw cup, 17 – ground joints, and 18 – diffusion pump outlet.

Sample Preparation for Food Flavor Analysis (Flavors/Off-Flavors)

139

compounds for subsequent GC–O snifﬁng.43 In this case, a cartridge of the LiChrolut EN resin (400 mg in a 3-ml, 0.8-cm-diameter cartridge) was used as a trap for volatiles purged from 80 ml of Madeira wine. Interestingly, the same material (800 mg) was used in a form of SPE cartridge to extract sotolone for quantitative purposes. Purge-and-trap was also used for the extraction of cheese volatiles by adsorbing them on a Tenax TA trap.44 The novel approach in achieving better sample transfer was to use three silica capillaries in parallel for cryofocusing, which increases the cryofocusing area by 1.6 compared to traditional solutions with single capillary. Very often, isolation of compounds for subsequent GC–O analysis requires more than one method and to enrich fractions for identiﬁcation is relatively complicated. Such a multistep procedure has been applied to the identiﬁcation of key odorants in peel oil extract from Pontianak oranges.45 The peels were frozen in liquid nitrogen, powdered in a blender, extracted with dichloromethane, concentrated on a Vigreaux column, and volatiles were isolated using SAFE distillation with subsequent concentration of distillate on the Vigreaux column. For the enrichment of volatiles, peels were extracted with diethyl ether, SAFE-distilled, concentrated, the solvent changed to pentane, fractionated on a glass column ﬁlled with silicagel using pentane/diethyl ether mixtures, and ﬁve fractions were subjected to GC–O analysis. To enrich the sample for (unknown at the moment of analysis) phenylethanethiol, simultaneous steam distillation–extraction (SDE) was performed with subsequent fractionation of compounds using column chromatography, followed by covalent chromatography on a cross-linked organomercurial agarose, followed by SAFE, Vigreaux concentration, and microdistillation. For the identiﬁcation of 1-phenylethanethiol, preparative HRGC followed by 1H-NMR was used, and for characterization of remaining key odorants chiral analysis, AEDA, static headspace aroma dilution analysis, and twodimensional chromatography–mass spectrometry has to be used.45 Isolation and identiﬁcation of 2-methoxy-3,5-dimethylpyrazine – a compound responsible for a fungal must taint in corks with an extremely low odor threshold in wine of 2.1 ng l1 – can serve as another example. To extract the compound responsible for this taint and identify it, 140 corks were placed in a ﬂask with water, steam distilled, distillate extracted with pentane, and concentrated on a Vigreaux column. Basic, neutral, and acid extracts were obtained, and microscale preparative GC used for preconcentration of 2-methoxy-3,5-dimethylpyrazine.46 To summarize, sample preparation for the determination of key odorants should provide exhaustive extraction of ﬂavor compounds and the extract should resemble the aroma of the analyzed product as already mentioned before. As one method of extraction is often insufﬁcient for the full characteristic of key odorants, usually several methods of extraction, including selective extraction and fractionation, are used.

4.06.5

Methods for Isolation of Volatile Compounds for Electronic Noses

Analysis of food ﬂavors and off-ﬂavors is performed routinely using gas chromatography as a separation tool. This approach enables identiﬁcation of particular compounds responsible for a characteristic aroma (key odorant), off-ﬂavor, or taint. The other approach in the analysis of food aroma is the utilization of electronic noses or quasi-electronic noses, which are able to differentiate and classify samples, without the identiﬁcation of particular compounds. The idea behind the invention of electronic devices was to mimic the human olfactory system, so mechanical olfaction is based on the response of a sensor array (usually multiple gas sensors that are nonselective or of varied selectivity toward speciﬁc classes of compounds) to volatiles from the headspace above the product. The signal is transformed using multivariate statistical methods or neural networks into results, which can be used for differentiation, classiﬁcation, and prediction of analyzed products based on their headspace proﬁle similarities. Using electronic noses, a ‘ﬁngerprint’ reﬂecting the volatiles of particular food product is generated and by data treatment using multivariate analysis, different samples can be classiﬁed. Electronic noses ﬁnd their use mainly in product classiﬁcation and quality control – mainly adulteration detection in olive oils, alcoholic beverages, and other products; traceability studies, and detection of ﬂavor defects, the result of undergoing microbial spoilage and chemical or enzymatic changes. There are also limited (due to the nature of the technique) applications describing analysis of particular compounds such as 2,4,6-trichloroanisole in wines.47 This approach is limited only to electronic noses based on mass spectrometers, where speciﬁc ions can be monitored for this purpose. Electronic noses can be divided into two groups: the group that uses chemical sensors based most frequently on metal oxide semiconductors (SnO2 doped with other metals (MOS and MOSFET sensors)), or conducting polymers (CP, usually aggregates of polypyrrole and polythiophene), or piezoelectric resonators and the other group where mass spectrometers serve as a ‘sensor array’ and an average spectrum is subjected to MVA analysis.48 Instruments of the second type can be standard GC/MS units where, after replacing the analytical column with a fused silica capillary (with no stationary phase), volatile compounds reach the mass spectrometer in a single peak. The response of the electronic nose and as a result its ability to differentiate samples is dependent on the amount of sample that reaches the sensor array. Therefore, extraction methods play an important role in food analysis by electronic noses. Usually, volatile compounds are introduced into an electronic nose by static headspace. The most popular electronic noses are equipped with a static headspace autosampler as a standard. It has been used in the analysis of Camembert cheeses, olive oils, wine, or hams using an MS-based electronic nose. Due to the nature of materials used as sensors for electronic noses, several precautions must be taken regarding the composition of the headspace entering the sensor chamber. Some volatile compounds can change the characteristics of the sensor; compounds present in high concentrations, such as ethanol in alcohols can inﬂuence the sensor responses. Therefore, samples are often diluted prior to analysis, which decreases the amount of particular analytes in the sensor chamber. The other option is to remove selectively compounds that may impair sensor performance. Such attempts have been made in the elimination of ethanol in the process of automated sample introduction into an electronic nose, achieved using for this purpose gas chromatography with a backﬂush technique, that changes the ﬂow in analytical column preventing the undesired compounds from reaching the detector.49 Volatiles can be introduced into electronic noses also by other sampling/preconcentration methods, such as

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purge-and-trap, although this technique is not used often, compared to static headspace. Solid-phase microextraction (SPME) is a very versatile extraction method, offering a high degree of preconcentration, and is an attractive alternative for sample introduction into electronic noses. It is especially popular in MS-based electronic noses, which are often transformed GC/MS systems with injection ports enabling thermal desorption of compounds adsorbed onto SPME ﬁber. The majority of electronic noses based on MOS or CP sensor arrays are not equipped with a hot injection port in a standard conﬁguration. SPME as an extraction technique for subsequent electronic nose analysis is also attractive regarding total analysis time. Sample analysis by electronic nose takes usually less than one to two minutes. It is the time required for the elution of a single peak of unresolved volatiles in MS-based electronic noses. For chemical sensor types of electronic noses, the intervals between injections can be longer due to the return of the sensor response to its initial state (background level). When SPME is considered as a sampling technique for electronic noses, there are several factors that have to be considered: the sample preparation procedure steps are similar to using SPME for extraction of volatile compounds prior to chromatographic analysis. In the process of sample optimization, usually ﬁber choice is performed, optimal extraction temperature and time, the inﬂuence of pH adjustment, salting out, etc. It has to be kept in mind that in SPME sampling, equilibrium due to the small volume of the ﬁber coating is reached in a relatively short time. Moreover, SPME can be used in a nonequilibrium state – before the equilibrium is attained and as result, sampling time can be comparable to the analysis time by MS. Often, multiple sampling can be performed using SPME-MS50

4.06.6

Methods for Isolation and Analysis of Bound Flavor Compounds

Compared to the analysis of aroma compounds present in plants, raw materials, and food in a free form, a relatively limited number of papers have been published on ﬂavor compounds in their bound forms. Aroma compounds bound as glycosides can be released from them as a result of hydrolysis during food preparation, processing, and storage. First reports on the analysis of precursors of aroma compounds were noted in the 1980s, although the bound form of geraniol (as b-D-glucoside) was discovered in 1913. Flavor precursors (nonodoriferous) appear usually as b-D-glycosides or O-diglycosides in a widespread manner in the plant kingdom. The few examples described are trisaccharide forms. Odoriferous aglycone is always bound to b-D-glucose, which can be bound with monosaccharides such as a-L-arabinofuranose, a-L-arabinopiranose (vicianosides), a-L-rhamnopyranose (rutinosides), b-D-glucopyranose (gentiobiosides), b-D-apiofuranose, and b-D-xylopyranose (primeverosides). Glycosides of aroma compounds have been identiﬁed in plants belonging to over 50 different families51 and there are reports of their presence in tropical fruits, like passiﬂora, lulo, papaya, lychee, granadilla, and mango and also plums, strawberries, and grapes. The majority of work done on bound ﬂavor compounds is related to grapes and wine. The methods of analysis of aroma compounds in their bound forms are based mainly on their extraction using selective adsorbents, such as C-18, XAD-2, or SDVB (styrenedivinylbenzene copolymer) from liquid matrices, as their polar character limits liquid/liquid extraction. Analysis of bound aroma compounds can be performed in two ways: (1) by analyzing whole glycosides or (2) by analyzing odoriferous aglycones after hydrolysis (Figure 12). Analysis of intact glycosides of aroma compounds performed by gas chromatography requires derivatization (due to their polarity and nonvolatility) and is performed rarely also because of lack of commercially available standards of glycosides and the need for their synthesis, usually using the methods of Koenig–Knorr and Schmidt.52 Compounds used most frequently for the derivatization are: N-methyl-bis-triﬂuoroacetamide; TMSi þ 1% TMCS; N,O-bis(trimethylsilyl)-triﬂuoroacetamide þ 1% TMCS; and 1-trimethylsilylimidazole. As an example, derivatization is performed on dry extract from 1 ml of cupacao juice with 20 ml N-methyl-bistriﬂuoroacetamide in pyridine for 20 min at 60 C.53 Table 4 shows approaches for the analysis of bound aroma compounds using different extraction procedures. Analysis of bound ﬂavor compounds after their hydrolysis is the more common approach. Usually, Wine, juice or plant extract

Adsorption on C-18, SDVB, XAD-2 Hydrolysis

Aglycone analysis

Glycosidic mixture Derivatized Preseparation

GC/MS, GC/FID, LC, HPLC, CCC, SEC GCxGC/ToF-MS

Derivatized GC/MS, GC/FID

Sub-Fractions

NMR, MS, FTIR, Chiroptical Methods,

Figure 12

Scheme of sample preparation and analysis of bound ﬂavor compounds.51

HPLC-MS

Table 4

Different approaches in sample preparation used for the analysis of bound aroma compounds Glycosides analysis

Adsorbent

Extraction procedure

Derivatization

Hydrolysis

Reference

SDVB

200 ml of wine or must extracted on a 1 g column. Free compounds eluted with 50 ml pentane: dichloromethane mixture (2:1), glycosides eluted with 50 ml of ethyl acetate. Ethyl acetate mixtures evaporated to dryness, dissolved in 1 ml of methanol, 500 ml of extract hydrolyzed enzymatically, the other 500 ml analyzed after derivatization.

500 ml evaporated under nitrogen. 20 ml of anhydrous pyridine and 20 ml MBTFA added. Internal standard: phenyl-b-Dglucopyranoside. Reaction time 30 min. at 60 C. Glycosides quantiﬁed: Glucosides: linalyl, geranyl, neryl, alphaterpinyl, linalyl oxides, diendiol, benzyl, 2phenylethyl, 3-OH-beta-damascenone, and C6 alcohols. Arabinoglicosides: geranyl, neryl, alphaterpinyl, linalyl oxide furan, diendiols, benzyl, 2-phenylethyl, and 1-hexanol Rutinosides : linalyl, geranyl, neryl, linalyl oxides furan, diendiols, benzyl, 2-phenylethyl Apiosides: geranyl, neryl, linalyl oxides, diendiols, 2-phenylethyl Methanolic extract after XAD-2 extraction dried under nitrogen at 60 C. Derivatized by addition of anhydrous pyridine (20 ml) and MBTFA (20 ml). Reaction time 20 min at 60 C.

500 ml evaporated to dryness under nitrogen. Dissolved in 100 ml of citric-phosphate buffer (pH ¼ 5). Enzymatic hydrolysis (18 h at 40 C).

Palomo, E.S i in. 2006

C-18

100 ml of clariﬁed cupacao juice extracted on a 9 1 cm. column ﬁlled with precleaned XAD-2 resin. Glycosides eluted with methanol.

15 ml of grape juice on a 300 mg/3 ml column. Free volatiles eluted with 25 ml dichloromethane, glycosides eluted using 10 ml methanolic solutions in water (20%, 30%, 40% and 100%). Methanol fractions evaporated to dryness and derivatized.

Glycosides quantiﬁed: Glycosides: 3-methyl-butyl, benzyl, linalyl oxides furan, octyl, R-linalyl, S-linalyl, 2phenylethyl, linalyl oxides pyran, geranyl. Rutinosides: 3-methyl-butyl, benzy, linalyl oxides furan, octyl, R-linalyl, S-linalyl, 2phenylethyl, a-terpinyl Vicianosides: 3-Methyl but-2-enyl Each fraction evaporated to dryness and derivatized by adding MTBSTFA with 1% of TMCS. Reaction performed for 45 min at 60 C. Glycosides quantiﬁed: Glycosides: linalyl, and geranyl Arabinoglycosides: linalyl and geranyl Rutinosides: linalyl and geranyl,

Extract obtained from 95 ml of juice evaporated to dryness under nitrogen, redissolved in 0.3 ml 0,2 M citric–phosphate buffer (pH ¼ 5). Sample washed the whole dried extract was washed 5 times with pentane/dichloromethane mixture (2:1 v/v). The residue was enzymatically hydrolyzed using hemicellulase REG 2 almond glycosidase REG 2 at pH 5 Volatile phenol compounds, vanillins, esters, monoterpenes, acids, and lactones

Boulanger R.; Crouzet J. (2000)

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Nasi et al. 2008

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Sample Preparation for Food Flavor Analysis (Flavors/Off-Flavors)

XAD-2

C-6 alcohols, monoterpenes, isoprenoids, acids, and aliphatic alcohols

Different approaches in sample preparation used for the analysis of bound aroma compoundsdcont'd

142

Table 4

Glycosides analysis Extraction procedure

Derivatization

Hydrolysis

Reference

XAD-2

25 g of grape leaves crushed in liquid nitrogen, mixed with methanol and left overnight, centrifuged, evaporated to dryness, redissolved in water, washed with pentane, and extracted on a 30 1 cm column. 100 ml of grape juice loaded directly on a column. After extract application column washed with water, then with 100 ml pentane: dichloromethane 2:1 v/v). Glycosidic fraction washed with 100 ml of 70% methanol. Extract evaporated to 4 ml. Extracts evaporated to dryness and dissolved in 100 ml 0,2 M citrate-phosphate buffer (pH 5). 20 g of freshly dried tea leaves ground, internal standard (b-D-glucopyranoside) added. Leaves extracted twice with boiling water, cooled, 10 g Polyclar AT added and mixed to remove ployphenols, ﬁltered and mixed with another portion of Polyclar AT. Filtrate concentrated in vacuo and centrifuged. Methanol added to separate proteins and ﬁltered. Fractions separated on a 28 4 cm column. After extraction column was washed (2 l water, then pentane : diethyl ether, glycosides were eluted with methanol.)

––––––

Extracted in buffer, washed with pentane/ dichloromethane (2:1) and enzyme solution, added 20 mg ml1 of pectolase 3PA (Grinsted) and 10 mg ml1 hemicellulose (Gist Brocades) in 0,2 M buffer (pH ¼ 5). Left to react for 16 h at 40 C. Liberated aglycones extracted 5 times with pentane dichloromethane (2:1). The organic layer was dried over anhydrous sodium sulfate, an internal standard added, and extracted on Vigreaus and Duftone columns. C6 alcohols, aliphatic alcohols, isoprenoids, monoterpenes, vanillines, volatile phenols, and benzene derivatives.

Wirth J. i in. 2001

The mixture of 16 synthesized glycosides was evaporated to dryness and derivatization was performed using 20 ml anhydrous glycerine (30 min. at 60 C)bezwodnej pirydyny i 20 ml MBTFA pod azotem. Czas reakcji 30 min w 60 C. Crude extract (8 mg) evaporated to dryness and 25 ml anhydrous pyridine and 30 ml of MBTFA was added . Reaction conditions: 50 min. 60 C. Czas reakcji 50 min w 60 C. Glycosides quantiﬁed: Glycosides: Z-3-hexanol, benzenol, 2-phenylethanol, linalol, geraniol, methyl salicylate, trans furanoid linalol oxide, cis furanoid linalol oxide, cis pyranoid linalol oxide, trans pyranoid linalol oxide. Primeverosides: Z-3-hexanol, 2-phenylethanol, linalol, trans furanoid linalol oxide, cis furanoid linalol oxide. Vicianosides: geraniol

–––––––

Wang et al. 2000

XAD-2

–––––––

Extraction Techniques and Applications: Food and Beverage

Adsorbent

Sample Preparation for Food Flavor Analysis (Flavors/Off-Flavors)

143

either enzymatic or acidic hydrolysis is performed. The latter one uses mainly buffers and elevated temperature.54 In acidic conditions and at high temperatures aglycones liberated, mainly terpenes are prone to rearrangements due to their unsaturated character, leading to artifacts. However, the character of changes caused by low pH is similar to that occurring in wine (pH around 3). Due to a complicated proﬁle of glycosides of aroma compounds present in wine, it is difﬁcult to ﬁnd one set of conditions optimal for all glycosides. Contrary to harsh conditions in acid hydrolysis, enzymatic hydrolysis occurs in a mild environment (40 C, 18 h) using speciﬁc enzymes such as b-D-glucosidase (bG), a-rhamnosidase (RHA), a-arabinosidase (ARAF), and a-apiosidase (API).55 One universal enzymatic preparation commercially available is AR-2000, having previously mentioned enzymes with activities of 5.6, 0.32, 9.2, and 1.08 nkat mg1, respectively. Both types of hydrolysis result in different proﬁles of obtained ﬂavor compounds.54 Separation of fractions containing free and bound ﬂavor compounds is usually performed using C18 and SDVB cartridges. The C18 cartridges are preconditioned with methanol and washed with deionized water, whereas SDVB is preconditioned with pentane: dichloromethane, followed by methanol and water. Weakly bound compounds are washed with water, then nonpolar free volatiles are eluted with pentane:dichloromethane followed by elution of the polar fraction (containing glycosidally bound aroma compounds) by methanol (C18) or ethyl acetate (SDVB). Effectiveness of compound adsorption varies depending on the columns/ cartridges used. Groups of compounds of different polarity can be fractionated, changing the polarity of the eluting solvent. In wine analysis, benzyl glycosides are followed by monohydroxylated alcohols, the last being geranic acid. Terpene diol, linalool oxide, and norisoprenoide precursors exhibit medium polarity. For the fractionation of bound ﬂavor compounds, Amberlite XAD-2 is frequently used. It is a polymeric adsorbent – cross-linked polystyrene copolymer resin – usually of 20–60 mesh size, with a broad pore size distribution, large surface area, and nonionic structure. Usually, it is cleaned by subsequent washing with methanol, diethyl ether, pentane/dichloromethane, and water. After loading the XAD-2 column with wine and elution with water, free compounds are washed out with pentane/dichloromethane (2/1), dried and concentrated using a Vigreaux column. Glycosides are washed out with ethyl acetate/methanol (9/1), which is dried, hydrolyzed enzymatically, and the released aromatic aglycones are washed with pentene/dichloromethane (2/1).56 Dziadas and Jele n57 proposed the use of SPME ﬁber for further preconcentration of volatile compounds released in acidic hydrolysis after SPE procedure of free and bound volatile compound fractionation. In this approach, bound aroma compounds hydrolyzed at 100 C were sampled using SPME directly after termination of this process. The vial was removed from the heating block and was left to cool down during sampling. Compared to the SPE approach (elution of released aroma compounds with dichloromethane, concentration and analysis by GC/MS), a remarkable increase in the number of detected compounds as well as in sensitivity of the method was achieved (Figure 13).

Figure 13 Chromatograms of volatile compounds released in acid hydrolysis of wine bound volatiles.57 (a) – isolation using SPE on a C18 cartridge; (b) – isolation using SPME extraction directly after hydrolysis termination.

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Extraction Techniques and Applications: Food and Beverage

4.06.7

Conclusions

Sample preparation plays a crucial role in the characterization of aroma of food products using speciﬁc gas chromatography– olfactometry techniques, as well as in target analysis of food taints and off-ﬂavors, and in the characterization of volatile compounds proﬁle. Due to the complexity of food ﬂavors, usually one method of extraction of aroma compounds is not sufﬁcient and several techniques have to be employed to characterize key odorants. Available extraction methods allow the isolation, identiﬁcation, and quantitation of aroma compounds present in trace concentrations in food.

See also: Headspace Sampling in Flavor and Fragrance Field; Sampling Techniques for the Determination of Volatile Components in Grape Juice, Wine and Alcoholic Beverages; Sampling and Sample Preparation Techniques for the Determination of the Volatile Components of Milk and Dairy Products; Sampling Techniques for the Determination of Volatile Components in Food of Animal Origin; Application of Stir Bar Sorptive Extraction in Food Analysis

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50.

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Relevant Websites Websites of some of ﬂavor research groups http://www.leb.chemie.tu-muenchen.de/tum/english/index.html http://www.molekulare-sensorik.de/index_e.html http://www.cals.cornell.edu/cals/grapesandwine/faculty-staff/index.cfm> http://ﬂavor.umn.edu/ http://fshn.illinois.edu/people/keith_cadwallader http://www.laae.es/ http://www.awri.com.au/research_and_development/grape_and_wine_composition/wine_aroma_and_ﬂavour/ http://www.reading.ac.uk/food/about/staff/d-s-mottram.aspx http://www.up.poznan.pl/zks Other interesting ﬂavor related sites http://www.ﬂavorscience.net/blog http://www.lefﬁngwell.com http://www.ﬂavorchemist.org