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Sampling Techniques for the Determination of the Volatile Fraction of Honey
4.05 Sampling Techniques for the Determination of the Volatile Fraction of Honey A Verzera and C Condurso, University of Messina, Messina, Italy Ó 2012 Elsevier Inc. All rights reserved.
4.05.1 4.05.2 4.05.2.1 4.05.2.2 4.05.2.3 4.05.2.3.1 4.05.2.3.2 4.05.2.4 4.05.2.5 4.05.3 4.05.4 References
4.05.1
87 87 87 88 92 97 107 107 111 114 114 116
Honey Volatiles Extraction Techniques Solvent Extraction and SDE Static and DHS Analysis SPME SPME for Floral and Geographic Determination SPME for Honey Authenticity USE USE Coupled with SPME E-nose Conclusion
Honey Volatiles
Honey is a popular natural product not only as a source of energy but also for its health-promoting properties provided by the prebiotic, antioxidant, antibacterial, and/or antimutagenic functionalities of certain constituents.1–4 European Union Legislation (2001/110/EC) defines honey as “the natural sweet substance produced by Apis mellifera bees from the nectar of plants or from secretions of living parts of plants or excretions of plant-sucking insects on the living parts of plants, which the bees collect, transform by combining with specific substances of their own, deposit, dehydrate, store and leave in honeycombs to ripen and mature.”5 Honey is composed mainly of monosaccharides (fructose and glucose), lesser amounts of water, and a great number of minor components such as organic acids, oligosaccharides, enzymes, vitamins, minerals, pigments, a wide range of aroma compounds, and solid particles derived from honey collection.6 At present, beekeepers throughout the world produce various types of honey, some expressing very distinct sensory properties that strongly influence consumer preferences and price of the honey; honey of unifloral origin usually commands higher prices than wildflower honey. The flavor/fragrance qualities of honey are greatly dependent on the volatile and semivolatile organic compounds present both in the sample matrix and headspace aroma. Several studies on the volatile compounds of honey have been carried out since 19627 and it is well known that these compounds may be derived from the plant or nectar, from the conversion of plant compounds or directly generated by the honeybees, from heating or handling during honey processing and storage, and from microbial or environmental contamination. Since most honey volatiles originate from the plants being visited by the bees, the honey sensory characteristics are closely related to its botanical origin and from this it follows that the volatile compounds have a potential role in distinguishing the floral origin. Honey is a very complex mixture with volatile components of different functionality and relatively low molecular weight, often present at very low concentrations; for their isolation, various methods, such as solvent extraction, simultaneous steam distillation extraction (SDE), static (SHS) and dynamic headspace (DHS) extraction, solid-phase microextraction (SPME), ultrasound-assisted extraction (USE), have been used. With regard to the determination of volatiles, gas chromatography (GC)–mass spectrometry (MS) is usually the technique of choice since it combines high separation efficiency and sensitivity and provides qualitative and quantitative data for these compounds. More recently, electronic noses (E-nose) based on mass spectrometry (MSE-nose), piezoelectric effects (zNose), and electrical resistance, have been tested with interesting results. The main analytical techniques used to extract and analyze the volatile fraction of honey are described. The advantages and drawbacks of each methodology, comparison of alternative reliable methods and results achieved in the field of honey quality determination are discussed. Special emphasis is given to the SPME technique, by far the most commonly used technique.
4.05.2
Extraction Techniques
4.05.2.1
Solvent Extraction and SDE
Solvent extraction has been widely used since the second half of the 1990s for honey volatiles.8–10 The success of this technique was due to its simplicity and because thermolabile compounds were retained; moreover, the commonly used solvents have low polarity so that the main compounds, water and sugars, are not extracted. Nevertheless this procedure also has some disadvantages, such as the long time involved, large volumes of solvents, environmental hazards, etc, so nowadays it is no longer used in favor of more suitable innovative techniques.
Comprehensive Sampling and Sample Preparation, Volume 4
doi:10.1016/B978-0-12-381373-2.10129-2
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Extraction Techniques and Applications: Food and Beverage
The simultaneous distillation–extraction (SDE) system developed by Likens and Nickerson11,12 and modified by Godefroot et al.13 is one of the currently used methods for isolation of honey volatile components; honeys of different floral origin were analyzed and a large number of volatiles were identified. This approach, unfortunately, determines the formation of artifacts, loss of analytes and provides results that are not easily compared; moreover, it needs a long sampling time prior to chromatographic separation and a large volume of solvent that influences trace analysis. However, interesting results were obtained on the floral origin of honey when the SDE method was carefully optimized. Following Bicchi et al.14 who first emphasized the importance of pre-extracting flavor compounds from sugars, in 1995 Bouseta and Collin15 proposed a two-step SDE protocol including preliminary solvent extraction under an inert atmosphere using dichloromethane as solvent. The method was applied to a commercial clover Canadian honey and the sensorial features of extracts closely matched those of the honey samples; the extracts were analyzed by GC-MS using an apolar capillary column. Unfortunately, using MS, authentic standard injection, and a mass spectra library, a limited number of volatile compounds, mainly furan and aromatic compounds, were identified (Table 1). In 1998, Guyot et al.16 applied the two-step SDE method15 to find reliable markers to ascertain the floral origin of chestnut and lime tree honeys. The unifloral honeys were selected from various countries and the screening for floral purity was based on pollen analyses, sensory tests, electrical conductivity, pH, titratable acidity, and sugar composition. The authors confirmed that about 400 aroma compounds were separated by GC although only 72 were identified (Table 1); chestnut and lime tree samples were authenticated on the basis of a few discriminate flavoring compounds, the content of which was significantly different from those of the other honeys; chestnut honeys were distinguishable by high concentrations of acetophenone, 1-phenylethanol (>88 ppb), and 2-aminoacetophenone (>154 ppb) and lime tree honeys by enhanced amounts of shikimate pathway derivatives, namely ethylmethylphenol isomer (>31 ppb), 4-tert-butylphenol, estragole (>51 ppb), and p-methylacetophenone, and by high concentrations of monoterpene-derived compounds such as menthol, thymol, 8-p-menthene-1,2-diol, and carvacrol. In the following years, the same authors17,18 applied their method to heather and lavender unifloral honeys from various countries (Calluna vulgaris from France, Belgium, United Kingdom, Norway, and Germany; Erica arborea from France, Greece, and Italy; Lavandula stoechas from Portugal, Lavandula angustifolia latifolia (lavandin), and Lavandula angustifolia (fine lavender) from the southwest of France). Forty-eight aroma compounds were identified by GC-MS in heather honey but less in lavender honeys (Table 1). The authors reported that phenylacetic acid, dehydrovomifoliol, and 4-(3-oxo-1-butynyl)-3,5,5-trimethylcyclohex-2-en1-one and high level of 3,5,5-trimethylcyclohexene derivatives were specific markers of Calluna vulgaris honeys. According to the authors, the presence of shikimate pathway derivatives such as 4-methoxybenzaldehyde, 4-methoxybenzoic acid, and methyl vanillate, in Erica arborea honey samples irrefutably proved their floral origin. The aromatic profiles of the L. angustifolia latifolia and L. angustifolia honeys were very similar and only phenylacetaldehyde and heptanoic acid were indicated as quantitative markers, respectively; French lavender honeys were easily authenticated from L. stoechas samples of various other origins, due to their high content of linear aldehydes, linear alcohols, and phenylacetaldehyde. Some recently published papers19,20 have made an important contribution to assessing the authenticity of honey combining the honey volatile data from SDE with those of descriptive sensory analysis. The difficulty in finding volatile compounds exclusively in honeys from a specific botanical origin justified the use of sensory analysis to make this differentiation possible. Commercial Spanish honey samples (citrus, rosemary, eucalyptus, lavender, thyme, and heather) were extracted using a microscale SDE apparatus using dichloromethane as solvent; all extracts were analyzed using GC-MS equipped with a polar capillary column. Peak identifications were based on spectral data; about 100 volatile compounds were identified in the analyzed honey samples (Table 1) and 12 different sensory descriptors were defined. The statistical elaboration of the chemical and sensorial data allowed the authors to distinguish the different honeys. Citrus honeys were characterized by higher amounts of linalool derivatives, limonyl alcohol, sinensal isomers, and a-4-dimethyl-3-cyclohexene-1-acetaldehyde, together with fresh fruit and citric aroma descriptors; eucalyptus honeys by hydroxyketones (acetoin, 5-hydroxy-2,7-dimethyl-4-octanone), p-cymene derivatives, 3-caren-2-ol, and spathulenol, cheese and hay aromas; lavender honeys by hexanal, nerolidol oxide, coumarin, important concentrations of hexanol and hotrienol and balsamic and aromatic herb aromas; heather honeys by high contents of benzene and phenolic compounds and ripe fruit and spicy aromas.
4.05.2.2
Static and DHS Analysis
Static headspace (SHS) analysis has not been widely applied to honey fractionation21 because of the low concentration of volatiles in honey and the low recovery obtained for semivolatile compounds. In contrast, applications of the dynamic headspace extraction technique (DHS) have been reported in the literature.22–27 This technique affords main advantages such as a high sensitivity for fractionation of high volatility compounds, the absence of extended heating times, and the reproducibility associated with a totally automated system; qualitative results on honey volatiles by different authors24–27 are reported in Table 2. One of the first applications was carried out by Bouseta et al.23 in 1992. By purging the volatile compounds from the honey matrix with a stream of nitrogen gas and concentrating them into a cooled trap, the authors extracted and analyzed unifloral honeys from different countries. Although most of the aldehydes and alcohols identified were related to microbiological activity, heat exposure, and honey aging, some linear aldehydes with defined characteristic compounds were associated with certain floral origins. In 2001, using a DHS-GC-MS analysis method, Radovick et al.24 studied the volatile profiles of authentic unifloral honey samples from different countries. In particular, acacia, chestnut, eucalyptus, heather, lavender, lime, rape, rosemary, and sunflower honeys were considered. A purified helium (60 ml min1) flow was used for the extraction; the entrained volatiles were adsorbed on
Sampling Techniques for the Determination of the Volatile Fraction of Honey
Table 1 Compounds
89
Volatile compounds in unifloral honeys (extraction technique: SDE) Clover
(Z)-3-Hexen-1-ol 1,3-Dimethylpyrazole 1,3-Diphenyl-2-propanone 1-Cyclopentyl-2-propanone 1-Heptanol 1-Hexanol 15 1-Hydroxy-2-butanone 1-Hydroxy-2-propanone 1-Methoxy-4-propylbenzene 1-Nonanol 1-Pentanol 1-Phenylethanol 1-Phenylethanol þ 1-phenyl1,2-propanedione 1-p-Menthen-9-ol 2(3H)-Furanone 2,3-Dimethyl-2-cyclopenten-1-one 2,3-Dimethyl-4-isopropenyl-1cyclopentanone 2,3-Dimethyl-4-isopropyl-1cyclopentanone 2,3-Dimethylphenol 2,5-Hexanediol 2,6,6-Trimethyl-2-vinyltetraidropyrane 2-Acetylfuran 2-Aminoacetophenone 2-Cyclopentene-1,4-diol 2-Furaldehyde 2-Furancarboxilic acid 2H-Pyran-2-one 2-Hydroxy-3,5,5-trimethyl-2-cyclohexen1,4-dione 2-Hydroxy-5-methyl-3-hexanone 2-Hydroxyacetophenone 2-Hydroxycineol (isomer I) 2-Hydroxycineol (isomer II) 2-Methoxy-6-methylpyrazine 2-Methyl propanoic acid 2-Methyl-1-butanol 2-Methyl-2-buten-1-ol 2-Methyl-3-(2H)-dihydrofuranone 2-Methylbutanoic acid 2-Phenylethanol 2-p-Methene-1,8-diol 3,4,5-Trimethyl phenol 3,5,5-Trimethylcyclohex-2-en-1-one (isophorone) 3,5-Dimethylphenol 3-caren-2-ol 3-Formyl-pyridine 3-Hexanol 3-Hydroxy-2-butanone 3-Hydroxy-2-pentanone 3-Hydroxy-5-methyl-2-hexanone 3-Methoxybenzene ethanol 3-Methyl thiopropanal 3-Methyl-1-butanol 3-Methyl-2-buten-1-ol 3-Methyl-3-(2H)-dihydrofuranone 3-Methyl-3-buten-1-ol
Chestnut
Lime
16 16
16
16
16
Heather
Erica
20 17
17
17 20 20
16
17
Citrus
Rosemary
Thyme
Eucalyptus
18 18,20 20 20
19,20 19,20
20 20 20
20 20 20
20 20 20
20
19,20
20
20
20
20
20
20
20
20
20
17 20
16 16
Lavender
16 16 20 19,20
16 16
16 16 16
16
16
16 16 16 16
16 16 16 16 16
16
16
17,20 20 20 17 20
17
20
17
18 20
19,20
19 20
20 20
20 20 20
20 16
16
16
16
20
16 16
16
16 16
17,20 20 17,20 17,20 20 20 17
17 17 17
20 20 20 18,20 20 20
19,20 20 19,20 19,20
20 20 20 20
19,20
20
20 20 20 20 20 20
20 20 20 20 20 20
17
16 20 20
16 16
16 16
16
16
16
16
16
16
17,20 20
17
20 20
19,20 20
20 20
20 20
20 20 20
17,20
17
20 18
19,20
20
20
20 20
17,20
17
20
19 19,20
20
20
20 (Continued)
90
Extraction Techniques and Applications: Food and Beverage
Table 1
Volatile compounds in unifloral honeys (extraction technique: SDE)dcont’d
Compounds 3-Methylbutanoic acid 3-Methylfuranoate 3-Phenyl-1-propanol 3-Phenyl-2-propen-1-ol 3-Pyridine carboxaldehyde 4-(3-Oxo-1-butynyl)-3,5,5trimethylcyclohex-2-en-1-one 4-(3-Oxobut-1-enylidene)-3,5,5trimethylcyclohex-2-en-1-one 4-Butyl-1,3-cyclopentanedione 4-Ethyl-3,4-dimethyl-2-cyclohexen-1-one 4-Hydroxy-3-methoxybenzoate methyl ester (methyl vanillate) 4-Hydroxy-4-(3-oxo-1-butenyl)-3,5,5trimethylcyclohex-2-en-1-one (dehydrovomifoliol) 4-Methoxybenzaldehyde (p-anisaldehyde) 4-Methoxybenzoic acid (p-anisic acid) 4-tert-Butylphenol 5-(Hydroxymethyl)furfural 5-Ethenyl-5-methyl-2(3H)-furanone 5-Methyl furfural 5-Methyl-2-(3H)furanone 5-Methylfuraldehyde 5-Methylfurfural 6-Methyl-3,5-heptadien-2-one 8-p-Menthene-1,2-diol Acetic acid Acetophenone Acetyl furan Benzaldehyde Benzoic acid Benzoic acid hydrazone Benzyl alcohol Butanoic acid Camphor Caproaldehyde Car-2-en-4-one Carvacrol Cinnamic acid Cinnamyl alcohol cis-Linalool oxide Coumarin Decanoic acid Dimethyl disulfide Dimethyl trisulfide Dodecanoic acid Epoxylinalool Epoxylinalool (isomer I) Epoxylinalool (isomer II) Eptadecane Estragole Ethylmethylphenol isomer Eugenol Furfural Furfuryl alcohol Guaiacol Heptadecane Heptanal Heptanoic acid Hexanal
Clover
Chestnut
Lime
16
16
16
16
16
Heather
Erica
17 17 17 20 17 17
17 17 17
17
17
Lavender
Citrus
Rosemary
20
Thyme
Eucalyptus
20
17
16 16 17 17
17 17 16 16 16
16
17 20 17,20 20
17
19,20
20 20 20
20 20 20
20 20 20
20
19,20 19,20
20
20
20
20
19,20
20
20
18,20
19,20
20
20
18 20
19 20
20
20
20
19,20 20
20 20
20 20
20
20 18,20 20 20 20 20
19,20
20
20
20
19,20 20 19,20 19,20 19
20 20 20 20
20 20 20 20
20 20 20 20
20 20
20 20
20 20
20 20
20 20
17
20 20 18,20
19,20 19,20
20 20
20 20
20 20
19,20
20
20
17
18,20 18,20 18,20
20 20 20
17
20 18,20 20
19,20
15
15 15
15
16
16
16
16
16
16 16
16 16
16 16
16
16
20 17
17
17,20 17 17 17 17,20
17 17 17 17 17
20
15 15
15
20 17 17 20
17 17
16 16
17,20 20 20 20
17
20 20 20 16 16 15 15
16 16
16
16 16 16 16 16
20 20 17,20 20
20 17
Sampling Techniques for the Determination of the Volatile Fraction of Honey
Table 1
91
Volatile compounds in unifloral honeys (extraction technique: SDE)dcont’d
Compounds Hexanoic acid Hotrienol Indole Isophorone Ketoisophorone Ketoisophorone (isomer I) Ketoisophorone (isomer II) Lilac alcohol (isomer I) Lilac alcohol (isomer II) Lilac alcohol (isomer III) Lilac alcohol (isomer IV) Lilac aldehyde (isomer I) Lilac aldehyde (isomer II) Lilac aldehyde (isomer III) Lilac aldehyde (isomer IV) Limonyl alcohol Linalool Menthol Methyl antranilate Methyl(1-methylethenyl)-benzene Methylfuran m-Xylene Nerolidol Nerolidol oxide Nonadecane Nonanal Nonane Nonanoic acid Octanal Octane Octanoic acid p-Anisaldehyde p-Cresol p-Cymen-8-ol (isomer I) p-Cymen-9-ol p-Cymene Pentadecane Pentanoic acid Phenethyl alcohol Phenol Phenylacetaldehyde Phenylacetic acid p-Mentha-(7),8-(10)-dien-9-ol p-Methylacetophenone Propylanisole Pyridine Sinensal (isomer I) Sinensal (isomer II) Spathulenol Terpineal Tetradecanoic acid Thymol Toluene trans-Caryophyllene trans-Linalool oxide Tetradecanoic acid Trimethoxybenzene (isomer) Vinylguaiacol a-4-Dimethyl-3-cyclohexen-1acetaldehyde
Clover
Chestnut
16
Lime
Heather
16
20 20 17 20
16
Erica
Lavender
Citrus
Rosemary
Thyme
Eucalyptus
18,20 20
19,20 19,20
20 20
20 20
20 20
20
19,20 19 20 20 19,20 19,20 19,20 19,20 19,20 19,20 19,20 19,20 19,20 19,20
20
20
20
20 20 20 20 20 20 20 20 20 20
20 20 20 20 20 20 20 20 20 20
20 20 20 20 20 20
20
20
20
20 19,20
20 20
20 20
20 20
19,20
20
20
20
19,20
20
20
20
20
20 19
20
20
20
20 20
20
20
20
20 20
18,20
19,20
20
20
20
19,20
20
20
20
17
20 20 20 20 20 20 20
20 20 20 20 20 20
20
20
16 19,20 16
16
15 15 19,20 20 20 16
15
16 16
16
15 15 15
16
16 16
16 16 16
16 16 16
16
16
16
16
17,20
17
17,20 20 20 20
17
20 20 17 17,20 17
17 17
20 20 18,20 18 20 18 18 20
20 18 19,20 19,20 20
16 16
16 16
20 20 17
20 20
19 19,20 19,20
20 20
20 20
20 20
20 20
20 20
17
15
16
19,20 19
20
19,20 20
20 20
16 20
20 20
(Continued)
92
Extraction Techniques and Applications: Food and Beverage
Table 1
Volatile compounds in unifloral honeys (extraction technique: SDE)dcont’d
Compounds a-Humulene a-Pinene a-Terpineol b-Damascenone b-Pinene g-Butyrolactone g-Terpinene g-Valerolactone
Clover
Chestnut
Lime
15
16 16
16 16
Heather
Erica
20 20 15
16
16
16 16
16 16
17,20
17
17,20
17
Lavender
Citrus
Rosemary
Thyme
Eucalyptus
20 20
19,20 19,20
20 20
20 20
20 20
20
19
20
20
20
19,20
20
15. Bouseta, A.; Collin, S. J. Agric. Food Chem. 1995, 43, 1890–1897; 16, Guyot, C.; Bouseta, A.; Scheirman V.; Collin, S. J. Agric. Food Chem. 1998, 46, 625–633; 17, Guyot, C.; Scheirmann, V.; Collin S. Food Chem. 1999, 64, 3–11; 18, Guyot-Declerck, C.; Renson, S.; Bouseta, A.; Collin, C. Food Chem. 2002, 79, 453–459; 19, Castro-Vázquez, L.; Díaz-Maroto, M.C.; Pérez-Coello, M.S. Food Chem. 2007, 103, 601–606; 20, Castro-Vázquez, L.; Díaz-Maroto, M.; González-Viñas, M.; Pérez-Coello, M. Food Chem. 2009, 112, 1022–1030.
a porous polymer resin, then thermally desorbed at 280 C under a helium flow, cryofocused in a glass lined tube at 120 C with liquid nitrogen, and finally injected into the GC capillary column. GC-MS analysis of the honey headspace was performed on a polar capillary column in a temperature gradient. A large number of volatiles were identified and the existence of certain marker compounds for the floral origins was assessed; e.g., the presence of either 2-methyldihydrofuranone or a-methylbenzyl alcohol or both 3-hexen-1-ol and dimethylstyrene were indicated for chestnut honeys (Table 2). Subsequently Bianchi et al.25 proposed a DHS extraction method coupled with GC-MS analysis as a valid alternative to pollen analysis for floral source detection, especially for products such as strawberry-tree honey characterized by low pollen content. Strawberry-tree (Arbutus unedo L.) honey samples with a certified floral origin, produced in Sardinia, were analyzed together with eucalyptus, heather, lavender, and thyme honey purchased from a local store. The method was similar to that reported above24 although a reduced amount of honey sample was used (1.5 g in a 50-ml round-bottomed flask at 40 C). A total of 28 aroma compounds were identified (Table 2), but only norisoprenoid compounds such as a-isophorone, b-isophorone, and 4-oxoisophorone, were recognized as markers of specific floral origin. The application of multivariate statistical analysis to the DHS-GC-MS data was first reported by Soria et al.26 in 2008; the authors demonstrated that the statistical elaboration of the volatile data was very promising to classify samples according to their botanical origin. Eucalyptus, thyme, citrus, rosemary, heather, lavender, and multiflower honeys were considered; the concentration of the honey solutions and temperature and time of purge were optimized to maximize the amount of volatiles. Volatiles swept by the helium flow were collected at ambient temperature on a porous polymer resin based on 2,6-diphenylene oxide, thermally desorbed at 220 C for 5 min and then concentrated at the end of a fused silica capillary transfer line, indirectly cooled with liquid nitrogen; the capillary transfer line and valves were heated to 200 C in order to avoid volatile compound condensation; chromatographic separations were carried out on a polar capillary column operating in a temperature gradient with helium as carrier gas. Qualitative analysis was based on spectra data and linear retention indices (LRI) while quantitative data were obtained using 5-nonanone as internal standard. One hundred volatile compounds (Table 2), including terpenes derived from the floral nectar, furan derivatives from honey processing and storage, and other compounds whose origin could be related to microbial or environmental contamination, were identified. Following discriminant analysis (DA), eucalyptus honeys were characterized by the amount of octane and diketones while lilac aldehydes were the most characteristic compounds of citrus honeys. An interesting application of DHS extraction was reported by Escriche et al.27 in 2009 with the aim of verifying if the honey volatile fraction was affected by the industrial thermal treatment processes. Four types of Spanish honey, i.e., citrus, rosemary, polyfloral, and honeydew, were studied. Each honey sample was analyzed untreated, liquefied (at 45 C for 48 h) and pasteurized (at 80 C for 4 min). Volatile compounds were extracted using purified helium as the stripping gas (30 g samples in a purging vessel flask at 45 C for 45 min) and volatile compounds were trapped on a porous polymer resin and subsequently thermally desorbed under a helium flow at 220 C for 16 min. The volatiles were then cryofocused in a cold trap at 30 C and transferred directly onto the head of the capillary column by heating the cold trap to 250 C. Volatile compounds were identified by mass spectra and LRI; quantitative results were obtained using camphor as internal standard. The volatile compounds identified (Table 2) allowed the authors to classify the honey by their botanical origin and to establish a clear differentiation between honeydew and nectar honey. Moreover, the authors demonstrated that honey type had a greater influence on the volatile fraction than heat treatment (liquefaction and pasteurization) under moderate industrial conditions.
4.05.2.3
SPME
SPME eliminates problems associated with DHS extraction while retaining the advantages: solvents are completely eliminated and extraction time can be reduced to a few minutes.28 In recent years, some efforts have been made to analyze honey volatiles using the SPME technique29 and different types of SPME fibers have been evaluated taking into consideration the polarity of fiber coatings and fiber coating film thickness. In addition, operating conditions that have a significant influence on the headspace equilibrium
Table 2
Volatile compounds in unifloral honeys (extraction technique: DHS)
Compounds
Rosemary
Strawberry
Citrus
Eucalyptus
Multiflower
Lavender
Heater
1-(2-Furanyl)-ethanone 1,3,8-Menthatriene 1-Butanol 1-Hexanol 1-Hydroxy-2-propanone 1-Methoxy-2-propyl acetate 1-Nonanol 1-Octen-3-ol 1-Octene 1-Pentanol 1-Penten-3-ol 2,2,6-Trimethylcyclohexanone 2,3,4-Trimethyl-2-cyclopenten-1-one 2,3-Butanedione 2,3-Dihydro-4-methylfuran 2,3-Heptanedione 2,3-Pentanedione 2,4,4-Trimethylcyclopentanone 2,5-Dimethylfuran 2,6,6-Trimethyl-2,4-cycloheptadien-1-one 2,6,6-Trimethylcyclohexan-1,4-dione 2,6-Dimethyl-4-heptanol 2- and 3-Methylbutanoic acid 2-Acetylfuran 2-Allyl-4-methyl phenol 2-Butanol 2-Butanone 2-Butenenitrile 2-Butoxyethanol 2-Cyclohexene-1-one 2-Decanone 2-Ethylfuran 2-Ethylhexanoic acid 2-Heptanone 2-Hexanol 2-Hydroxy-3-pentanone 2-Hydroxy-5-methyl-3-hexanone 2-Methyl- and 3-methyl-2-buten-1-ol 2-Methyl-1-butanol 2-Methyl-1-propanol 2-Methyl-2-butenal 2-Methyl-2-propanol
26
25
26,27
26
26
26
26
24,26
25
27 27 27
26
24
24 24 24
Rape
Sunflower
Acacia
Thyme 26
24,26 24,26
24,26 26
24
24
24
26
26 24 26 24
24,26 24 24,26
26
27
25 25
25 25 25
26
24 26
26
26
26
26
26,27 26
26 24,26 26 24,26
26,27 26
26 24,26
26 24,26
26 26
26
26
26
26
26
26
26 24
26
26 26
26 24
26
24 26
26
24 26,27 24,26
25
26 26 26
26 24,26
26 26 26
27
24
24 24
24 24
26 24,26
26 24,26
24
24 24 24
24 24
24
24
24
24
24
26 26
24 24 26 24
27 26
26
24,26 26 24 26
26 26,27 26 27
26 24,26 26
24
26 24,26 26,27 27
25
26
26
26
26
26 26,27 26,27 26,27 27
26 24,26 26
26 26 24,26 26
26 26 26 26
24
24
24
24
93
(Continued)
Sampling Techniques for the Determination of the Volatile Fraction of Honey
26 24
24,26 26
Lime
27
26
26,27 24,26 26
26
Chestnut
94
Table 2
Volatile compounds in unifloral honeys (extraction technique: DHS)dcont’d Rosemary
2-Methyl-3-buten-2-ol 2-Methylacetophenone 2-Methylbutanal 2-Methylbutanenitrile 2-Methyldihydrofuranone 2-Methylfuran 2-Methylhexanoic acid 2-Methyl-2-buten-1-ol 2-Methylpropanenitrile 2-Methylpropanoic acid 2-Pentanol 2-Pentanone 2-Propanol 3-(1-Methylethyl)-2-cyclopenten-1-one 3,5,5-Trimethylcyclohexan-1,4-dione 3-Aminoacetophenone 3-Butenenitrile 3-Cyclohexen-1-ol-5-methylene-6-isopropylene 3-Furancarboxaldehyde 3-Hexen-1-ol 3-Hexen-2-one 3-Hexenylformate 3-Hydroxy-2-butanone (acetoin) 3-Hydroxy-2-pentanone 3-Methyl-1-butanol 3-Methyl-1-undecene 3-Methyl-2-butanol 3-Methyl-2-butanone 3-Methyl-2-buten-1-ol 3-Methyl-2-butenal 3-Methyl-2-pentanone 3-Methyl-3-buten-1-ol 3-Methylbutanal 3-Methylbutanenitrile 3-Penten-2-one 4,5,7a-Hexahydrobenzofuran-3,6-dimethyl-2,3,3a 4-Acetyl-1-methylcyclohexene 4-Ethylphenylacetate 4-Methyl-1-pentanol 4-Methylacetophenone 4-Oxoisophorone (3,5,5-trimethylcyclohex-2-en-1,4-dione) 5-Methyl-1-hexanol Dihydro-5-methyl-2(3H)-furanone 5-Methylfurfural
26,27
Strawberry
Citrus
Eucalyptus
Multiflower
Lavender
Heater
26,27
26
26
26
26
24,26 26
26 26
24,26 26
26 26
24,26
24,26 26
26 27
26
26
26
26
26
26,27 26
26
26
26
Chestnut
Lime
24
24 24
Rape
Sunflower
Acacia
Thyme 26
24
24
24
26 26
24
26
24
26
24
26
24 26 24 24
26
26 24 24
26
24,26 24 24
24,26 24 24
26 24 24
24 24
24 24
24 24
24 24
25 25 26
26
24 26
24 26
26
26 24
25 24 26
24
27
26 24 24
24
24
24
26,27
24,26
24,26
24 24
24
26
26
26
26
24 24 24,26,27
27
24 24 24,26
25
26
26 24
24
24
26
24
24
24
26
24 24
26 26 26 26
24 24 26 27
27 26 24,26,27 24,26 26
26,27 26 26 26
27 24,26 24,26 26 26
26,27 26,27 26 26
24,26 24,26 26
24,26 24,26 26 26
24 24
24 24
24 24
24 24
24 24 24 24,26
26
25
26
24,26
26
24 24
24,26
26
26
24,26
24
24 24,26
24 24
24 24
24
24
Extraction Techniques and Applications: Food and Beverage
Compounds
24 26,27 24,26 26 24 26,27 27 24,27
25 25
25
26,27 26,27 26 26,27 27
26 24,26 26 26 24,26
27 26,27 26 26
24
26
26
24 26 24,26 26 24,26 24,26 24
24
24
24
24
24,26
24
24
24
24
24
24 26
24 24
24 24
24 24
24
24 24
24
24
24 24
24
24
24
26
26 26 26
26 24
26 24,26
26 26
24,26
26 26
26 26
26 26
27 26 26
24 26 26 26
26
26
26,27 27 27 24,26,27 26
24,27 26 26
25
26 26
24,26
24,26 24,26 24 24 26 26
24 24
26 26
24,26 26 24 24 26 26
24,26
26,27
26
24,26
24 24
26,27 27
26
26,27 27
26
26
26,27 26
24,26 26
26,27 26
24,26 24,26
26
26
26
24,26 24,26 24 26
26 26,27
26 24,26 24 26 24 26
26 26,27
26
24 24
24
24 24
24 24
24 24
26 26
24
24
24
24 26 26
25
25
26 26 24,26,27 26 24 26 27 26
25
25 26
26,27 26 27 26,27 26,27
24,26 24
26 24,26 24
26 24 26 24,26 24
24 24
24
26 26
24 24
24
24
24 24
24 24
26 26
24 24
24 24
24
24
24
24
24 24
24
26 26
26
26,27
26 26,27
24 26
26
24,26 24
24,26 24
24 26
24 24
24 24
24 24
24 24
24 24
26
26 26
95
(Continued)
Sampling Techniques for the Determination of the Volatile Fraction of Honey
6-Methyl-5-hepten-2-one Acetic acid Acetone Acetonitrile Acetophenone Benzaldehyde Benzene acetaldehyde Benzyl alcohol Bicyclo-3,2,1-octane-2,3-bis(methylene) Butanal Bicyclo 2,2,2-octan-1-ol-4-methyl Carbon tetrachloride Chloroform cis-Linalool oxide cis-Linalool oxide (furan ring) Cyclohexane Cyclopentenedione Decanal Dichloromethane Dihydro-2-methyl-3(2H)-furanone Dihydro-5-methyl-2(3H)-furanone Dihydroisophorone (3,3,5-trimethylcyclohexanone) Dimethyldisulfide Dimethylstyrene Dimethylsulfide D-Limonene Dodecane Ethanol Ethyl acetate Ethyl-2-hydroxypropanoate Ethylbenzene Ethylbenzoate Furan Furfural Furfuryl alcohol Geraniol Heptanal Heptane Hexanal Hexanoic acid Hotrienol (3,7-dimethyl-1,5,7-octatrien-3-ol) Hydroxyacetone (acetol) Isobutyl acetate Isopropyl myristate Lilac aldehyde (isomer I) Lilac aldehyde (isomer II)
96
Table 2
Volatile compounds in unifloral honeys (extraction technique: DHS)dcont’d
Compounds
Strawberry
Citrus
Eucalyptus
26,27 26,27
Multiflower
Heater
Chestnut
Lime
Rape
Sunflower
Acacia
26,27 26,27 24
27
Lavender
27 27
Thyme 26 26
24
24
24
24
27 24
27 26 24 24 26 26 26
25
24,27 24 26,27 26
25 25
26 26
26 27
26 26,27 26 26
24
26
24 24 26
24 24 26
24 24
24 24
24 24
24 24
24 24
26,27
26
26
26,27
26 26 24,26
26
26,27 26
26 26
26,27 26
24 24 26
26 27 26 26 26
24,26 24
26 27 26 26 26
24 24,26 24
24 24 26 26 24 24,26 24
26
26 26
26 26 26
26 26 26
26 26 26
26 26 26
26 26 26
24 24
24
24,26
24
24
24 24
26,27 26 26 26
25
24 24 26
26 26 26 26 26 26
26 26 24 24
24 24
24 24
24 24
24 24 26 26
24
24
24
24
24
24
24
24
24
24
26
24 24
24 24
24 24
24
24
24 24 24 27 25 27 24
24
24
24
24, Radovic, B.S.; Careri, M.; Mangia, A.; Musci, M.; Gerboles, M.; Anklam, E. Food Chem. 2001, 72, 511–520; 25, Bianchi, F.; Careri, M.; Musci, M. Food Chem. 2005, 89, 527–535; 26. Soria, A. C.; Martínez-Castro, I.; Sanz, J. Food Res. Int. 2008, 41, 838–848; 27. Escriche, I.; Visquert, M.; Juan-Borrás, M.; Fito, P. Food Chem. 2009, 112, 329–338.
Extraction Techniques and Applications: Food and Beverage
Lilac aldehyde (isomer III) Lilac aldehyde (isomer IV) Linalool (3,7-dimethyl-1,6-octadien-3-ol) Linalool oxide Methyl antranilate Methyl isopropyl benzene Methyl salicylate Methyl-1,3-pentadiene Methyl-2-butenale Methyl-2-butenol Methylcyclohexane Methylnaphthalene (isomer not identified) m-Xylene Nerol Nonanal Octanal Octane o-Xylene Pentanal Phenylacetaldehyde Phenylethyl alcohol p-Menth-1-en-9-al (isomers I and II) Propanenitrile p-Xylene Terpinen-4-ol Toluene trans-Linalool oxide (furan ring) Trichloroethylene a,a-Dimethylbenzyl alcohol a-4-Dimethyl-3-cyclohexe-1-acetaldehyde a-Cyclic ether a-Isophorone (3,5,5-trimethyl-2-cyclohexen-1-one) a-Methylbenzyl alcohol (sec-phenethyl alcohol) a-Methylbutenol a-Methylpropyl phenyl acetate a-Pinene a-Pinene oxide a-Terpinene b-Damascenone b-Isophorone (3,5,5-trimethyl-3-cyclohexen-1-one) b-Linalool g-Butyrolactone
Rosemary
Sampling Techniques for the Determination of the Volatile Fraction of Honey
97
and on fiber absorption capacity, such as vial size, salt addition, magnetic stirring, equilibrium, and extraction time and temperature, plus GC injector desorption time were taken into consideration. By using SPME-GC-MS methods, an important number of volatile compounds have been found as possible markers of different types of honeys since most of these are related to the botanical origin.30–39 Moreover, the application of SPME has allowed the authenticity of honey to be verified by the presence of extraneous substances.40–45 Recently, interesting perspectives for improving the study of honey volatiles have arisen from SPME sampling followed by comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC GC-TOF-MS) analysis; important information on honey traceability has been obtained.38–40
4.05.2.3.1
SPME for Floral and Geographic Determination
As previously mentioned, volatile compounds are related to the botanical and geographical origin of honey and many can be considered as reliable markers. In this context, by using SPME-GC-MS methods, an important number of organic compounds have been found as components of unifloral honeys coming from different countries; among these, the Spanish honeys are the most studied.30–32,36,37 The first applications of SPME for the extraction of honey volatile constituents were reported by Verzera et al.33 and Piasenzotto et al.31 who studied Italian unifloral honeys from different regions. These authors were able to optimize their SPME-GC-MS methods (Table 3) so that the identification of a large number of volatile constituents became possible (Table 4). The two methods differed mainly in the extraction temperature, which was lower in the method developed by Verzera et al.,33 and the selected fibers; Verzera et al.33 found better results with a polydimethylsiloxane/divinylbenzene (PDMS/DVB) fiber and Piasenzotto et al.31 used a polyacrylate (PA) fiber. In the unifloral Sicilian honeys,33 more than 100 compounds were identified which belonged to the following classes of substances: aliphatic and aromatic compounds, acyclic and monocyclic monoterpenes and their oxygenated derivates; furan derivates; sulfurated and nitrogen-containing compounds. From the data obtained, the authors33 identified borneol and dihydrocarveol as markers for eucalyptus honey along with a high amount of nonanol, nonanal, nonanoic acid, 5-hexen-2-ol, and 2,3-dimethyl-5-hexen-2-ol, whereas acetophenone, 2-aminoacetophenone and 1-phenylethanol were markers for chestnut honey. Sulla honey was characterized by high levels of hexanol, hexanoic acid linalol, nonanal, terpinen-4-ol, and a-terpineol. With regard to the unifloral Spanish honeys, one of the first SPME-GC-MS methods was developed by Perez et al.36 for the characterization of floral origin. The most significant aspects of the SPME technique, such as sampling, fiber, equilibration time, etc., were considered in order to optimize the method. Two SPME fibers were used and the best results were obtained with the carboxen/ polydimethylsiloxane (CAR/PDMS) fiber, using a homogenization time of 1 h at 70 C and a sampling period of 30 min (Table 3), The analyses were performed using GC-MS and volatiles were separated using an apolar capillary column with helium as carrier gas. A total of 35 compounds were detected, most of them identified by GC-MS (Table 4) and quantified using external standards. In 2003, Soria et al.32 considered some Spanish unifloral honeys when developing an SPME-GC-MS method for simple and rapid analysis of honey volatile compounds (Table 3). In this case, SPME fiber coatings of different polarity, the influence of sample temperature, equilibrium and exposure time, magnetic stirring, and salt addition on amount of volatiles were considered. GC-MS analyses were performed on a polar column operating in a temperature gradient, with helium as carrier gas. The authors proposed the use of a PA fiber (32 compounds identified) for distinguishing samples of different types and a CAR/PDMS fiber (24 compounds identified) for the characterization of honeys that were poor in volatile components (Table 4). Principal component analysis (PCA) using the amount of each volatile compound expressed as peak areas was considered essential for definition of the botanical origin. The same authors37 (Table 3) applied their method to a large number of honeys of different botanical origin and some honey blends. More than 100 volatile compounds were identified in all the samples analyzed and some of them were reported as possible markers. For example, high levels of lilac aldehydes, lilac alcohols, cis-linalool oxide, benzaldehyde, phenylacetaldehyde, p-menth1-en-9-al, p-menth-1-en-9-ol, hotrienol, and particularly p-menth-1(7),8(10)-dien-9-ol, were found for unifloral citrus honey; high contents of 2,6,6-trimethyl-2,4-cycloheptadien-1-one, 3,5,5-trimethylcyclohex-2-ene-1-one (isophorone), and 4-oxoisophorone were found for rosemary honey, whereas the high nitrile content (2-methylpropanenitrile, 2-methyl butanenitrile, 3-methyl butanenitrile, 2-butenenitrile (cis- or trans-isomers), 3-butenenitrile, 3-methylpentanenitrile, and benzeneacetonitrile) proved to be distinctive for dandelion honey. Following this research, de la Fuente et al.30 applied the method previously developed by Soria et al.32 (Table 3) to study the volatile composition of widely used Spanish honeys, such as eucalyptus, rosemary, heather, and citrus. GC-MS profiles of honey volatiles were complex and 83 volatile compounds were identified using MS and retention data (Table 4). Due to the lack of reference samples and the presence on the market of mixed honey types, a statistical elaboration processing the relative concentrations of volatiles was applied. PCA was used as a first step to indicate the existence of possible natural groups followed by stepwise discriminant (SDA) and regression analyses. By the statistical elaboration, the authors provided valid information for the characterization of eucalyptus and citrus samples. The SPME-GC-MS methods discussed above used conventional one-dimensional GC. which, even if typical analysis times of 30–90 min were required to achieve acceptable chromatographic resolution, can lead to coelution of volatile constituents. In this context, multidimensional gas chromatography (MDGC) represents a possible solution to obtain efficient separation of the entire sample. This analytical approach was first applied by Cajka et al.38 in 2007; then Cajka39 and Starimirova40 demonstrated the potential of this challenging technique for its application in various follow-up studies including traceability of honey origin and authentication. The authors developed an SPME-based procedure for the isolation of honey volatiles followed by their separation/ detection/identification by means of the GC-GC-TOF-MS technique. The authors analyzed more than 300 honey samples from
98
SPME methods: operation conditions for honey volatile extraction Equilibrium Extraction temperature Equilibrium temperature Extraction ( C) time (min) ( C) time (min)
Desorption temperature ( C)
Desorption time (min)
Splitless/split after 2 min
250
2
20
Splitless/split after 3 min
250
3
60
30
Splitless/split after 2 min
250
2
30
30
25
Splitless
220
3
40
60
40
30
Splitless
220
5
DVB/CAR/PDMS 50/30 mm
–
-
40
20
Splitless/split after 3 min
250
CAR/PDMS, PDMS/DVB
70
60
70
30
Splitless
Addition of 1 ml CAR/PDMS of water 75 mm; PA 85 mm
60
15
60
30
Splitless/split after 2 min
PDMS/DVB at 250, CAR/ PDMS at 270 250
Reference Floral source
Honey Sample provenance size (g)
Vial size (ml)
Sample treatment
Fiber type
30
Spain
1.5–2.0
5
Diluted with 1 ml water
CAR/PDMS 75 mm
60
15
60
30
Italy
3
10
Added to 0.5 g sodium sulfate
PDMS 100 mm, 70 PA 85 mm, CAR 75 mm
30
70
Spain
1.5–2
5
Addition of 1 ml CAR/PDMS 75 mm; 60 of water PA 85 mm
15
Italy
16
40
30
Poland
1–1.5
4
PDMS/DVB Diluted with 65 mm 7 ml of water, added to 2 g NaCl – PDMS/DVB 65 mm
Croatia
3.00
50
Spain
1
4
1.5–2.0
5
31
32
33
34
35
Eucalyptus, rosemary, heather, citrus Citrus, chestnut, eucalyptus, lime tree, thyme, dandelion Rosemary, heather, honeydew, orange blossom, lavender, multiflower Orange, sulla, chestnut, eucalyptus, wild flowers Multifloral, heather, buckwheat, limehoneydew Fir-honeydew, sage
36
Orange, eucalyptus, rosemary, lavender, thyme
37
Eucalyptus, citrus, Spain rosemary, thyme, lavender, chestnut, dandelion, rhododendron, Teide broom, acacia, heather, honeydew, multiflower
Added to 0.5 g sodium sulfate –
Injection mode
5
2
Extraction Techniques and Applications: Food and Beverage
Table 3
41 42
Chestnut Italy Nectar and honeydew Spain
20
43
Thyme
–
8
10
44
Dandelion
Italy
1.5–2
5
45
Chestnut, fir, acacia, Pyrenees, orange, lavender, eucalyptus, forest, oak
France, Italy,1 Hungary, Spain
10
50
Lavender
Croatia
-
15
51
Christ’s thorn
Croatia
-
15
52
Desert false indigo
Croatia
-
15
PDMS 100 mm Diluted with CAR/PDMS 1 ml of water 75 mm then sonicated for 5 min Diluted with PDMS 100 mm 3,5 ml of water, added to 1 g NaCl Addition of 1 CAR/PDMS ml of water 75 mm
– 60
– 15
45 60
20 30
Splitless Splitless
250 250
0.4 2
30
30
30
25
–
250
3
60
15
60
30
250
2
Diluted with PDMS 100 mm, 4 ml of water, PA 85 mm, CW/ added to 1 g DVB 70 mm, NaCl and CAR/PDMS 0.2 ml of 75 mm, PDMS/ acetic acid DVB 65 mm
–
–
–
30
Splitless/split valve opening at 2 min Splitless
2
5 ml of honey– DVB/CAR/PDMS NaCl sat. H2O solution 1:1 v/v 5 ml honey– CAR/DVB, DVB/ NaCl sat. H2O CAR/PDMS solution 1:1 v/v 5 ml honey– PDMS/DVB NaCl sat. H2O solution 1:1 v/v
60
15
60
40
Split
300 for PA and CAR/ PDMS; 250 for PDMS, CW/DVB, PDMS/ DVB 250
60
15
60
40
Split
250
6
60
15
60
40
Split
250
6
Abbreviations: CAR, carboxen; DVB, divinylbenzene; PA, polyacrylate; PDMS, polydimethylsiloxane.
6
Sampling Techniques for the Determination of the Volatile Fraction of Honey
1 1.5–2.0
99
Volatile compounds in unifloral honeys (extraction technique: SPME)
Compounds
Sage
Rosemary
Orange
Lime
Lavender
Heather
Eucalyptus
Buckwheat
Chestnut
Dandelion
Citrus
Sulla
Wild flower
33 33 33 33
33 33 33 33
33 33
33
33
33
33
33
33
33 33
33
33
33
33
33
a
32 33 33 33 35
33
35
34
34 33
35 30,32a,32b
32a,32b
35 35
32a,32b
30,32a,32b,34
30
34 34
30
35 33 33 33
36
30,32b 32a,36
33 33 32b 32b,36
32b,36 32b,36
30,32b 32b
32a
32a,34
33 30 36
33 30
35 35 31
32a
33 32a
33 31 33
34
33 31 33
30
30
30
30
32a 31 36
31,35 36 30 30 30
36
36
34 34 30 30 30
34
31,36 30 30 30 33 30
34 34
31
30
Extraction Techniques and Applications: Food and Beverage
(Dimethylphenyl)-ethanone (E)-2-Octenal (E)-2-Pentenol (E)-2-Undecenal (E)-3-Hexenol (E)-6,10-Dimethyl-5,3undecandien2-one (E)-Cinnamaldehyde (Z)-2-Pentenal (Z)-2-Pentenol (Z)-3-Hexenol 1-(2,4-Dimethylphenyl)ethanone 1-(2-Aminophenyl)-ethanone 1-(2-Furanyl)-ethanone 1-(2-Hydroxy-5-methylphenyl) ethanone 1-(4-Methylphenyl)ethanone 1,3,5,7-Cyclooctatetraene 1,3,8-p-Menthatriene 1,3-Butandiol 1,4-Cineole 1,8-Cineole 1-Hexanol 1-Hydroxy-2-propanone 1-Methyl-4-(1-methylethyl) benzene 1-Methyl-4-benzene 1-Methylethyl benzene 1-Nonanol 1-Octen-3-ol 1-Phenylethanol 1-Propyne 2- and 3-Methyl-2-buten-1-ol 2(H)-1-Benzopyran-2-one 2-(p-Methoxyphenyl)ethanol 2,2,4-Trimethyl-pentane 2,3-Butanediol 2,3-Butanediol (erythro) 2,3-Butanediol (threo) 2,3-Butanedione 2,3-Dimethyl-5-hexen-2-ol 2,3-Pentanedione 2,4,5-Trimethyl cumene
Thyme
100
Table 4
33 36
36
34 34
36
34 34
34 33 33
33
33
33
33
33
33
33
36 36 33 32a 30,36
36
32a
35
32a 36
32a 30,34
30
34
30
35 33
33
34 30
30
30
30
33 34
33 34
33 33 36 36 36
36
34
36
31,36
33
34
33
33
31
36 36
31,36
34 34
36 35 35 35 35
36
34 34
35 30,32a,32b,36
32a,32b,36
31 35
32a,32b,36
30,32a,32b,34
36
34 34
34 34 33 30,31,36
34
33 31
31
30,31
34 34
101
(Continued)
Sampling Techniques for the Determination of the Volatile Fraction of Honey
2,5-Dimethyl-3-hexanone 2,5-Furandicarboxaldehyde 2,6,6-Trimethyl-2-cyclohexene1, 4-dione 2,6-Dimethoxy-phenol (syringol) 2,6-Dimethyl-3,7-octadien-1,6diol 2,6-Dimethyl-3,7-octadien-2,6diol 2-Acetyl benzoic acid 2-Aminomethyl benzoate 2-Butanol 2-Decanol 2-Decanone 2-Ethyl hexanol 2-Furan methanol 2-Furanocarboxaldehyde 2-Heptanone 2-Hydroxy-3,5,5trimethylcyclohex-2-enone 2-Hydroxy-3,5,5trimethylcyclohexanone 2-Hydroxy-ethyl benzoate 2-Methyl butanoic acid 2-Methyl propanoic acid 2-Methyl,dihydro-2(3H)furanone 2-Methyl-1-butanol 2-Methyl-1-butanol 2-Methyl-1-propanol 2-Methyl-2-buten-1-ol 2-Methyl-2-butenal 2-Methyl-3-buten-2-ol 2-Methyl-3-phenyl-2-propenal 2-Methyl-6-(2-propenyl)phenol 2-Methylbenzene 2-Methyl butanal 2-Methyl propanoic acid 2-Nonanone 2-Phenylethanol 2-Xylylethanol 3,4,5-Trimethyl phenol 3,5,5-Trimethyl-2-cyclohexen-1one
102
Table 4
Volatile compounds in unifloral honeys (extraction technique: SPME)dcont’d
Compounds
Sage
Rosemary
Orange
Lime
Lavender
Heather
Eucalyptus
Buckwheat
Chestnut
Dandelion
Citrus
Sulla
Wild flower
34 36 31
31 33 35
36
30,32b,36
32b,36
32b,36
30,32b 30
30,31,36 30
36 36
30,32b,36
32b,33
32b
30,32b,34
30,33,36
32a,32b,36
36 32a,32b,36
31
34
33
30
30
33 33
32a,32b,36
36
30
32a,32b 34 30
33
31
33
31
33
34 30 33,36
30 33
33
33
33
33 33
34 35
35 35 35
35
31
31
31
31
36
35 35 35 31 35
31
35 33 35 35
34
34
Extraction Techniques and Applications: Food and Beverage
3,5-Dimethoxy benzaldehyde 3,7-Dimethyl-1,5,7-octatrien3-ol 3,9-Epoxy-p-menth-1-ene 3-Aminoacetophenone 3-Furanocarboxylicacid methyl ester 3-Hexanol 3-Hydroxy-2-butanone (acetoin) 3-Hydroxy-2-pentanone 3-Methyl butanal 3-Methyl-1-butanol 3-Methyl-1-hexanol 3-Methyl-2-buten-1-ol 3-Methyl-3-buten-1-ol 3-Methyl butanal 3-Methyl butanenitrile 3-Methyl butanoic acid 3-Phenyl-2-propen-1-ol 3-Pyridine carbonitrile 4-(1-Methylethyl)benzaldehyde 4-(1-Methylethyl) benzeneethanol 4-(3-Hydroxy-1-butenyl)-3,5,5trimethyl-2-cyclohexen-1-one 4-(3-Oxo-1-butenyl)-3,5,5trimethyl-2-cyclohexen-1-one 4,5,6,7-Tetrahydro-3,6dimethyl-benzofuran 4,7-Dimethyl-benzofuran 4-Hydroxy-4-methyl-2pentanone 4-Hydroxybenzenemethanol 4-Ketoisophorone 4-Methoxybenzaldehyde 4-Methyl,dihydro-2(3H)furanone 4-Methyl-1-(1-methylethyl)-3cyclohexen-1-ol 4-Methyl-1-(1-methylethyl)bicyclo[3.1.0]hexene-2-one 4-Methyl-1-pentanol 4-Methyl-3-pentenol 4-Methyl phenol
Thyme
30
33
35
30
30,33 33 31
33 33
30 31
35 33
33 31
33
33 31
31 35 36 36
33,36 36
36 36
34 34 32a 34
36 36
34
33
34
33 31
34 34
31,33
31
31,36 36
35 35 35
33
31
33
36 36
33
30,32a,32b,36 36
32a,32b,33,3631,35 36 35
32a 36
34 30,32a,32b,34 30,34 34
30,31,33,36 30,36
33
31
30 30
31
30
33
33
33
33
33 33
33 33
33
33
33 35
35 35
35 35
35 30,32a,32b
31
35 35
33 32a,32b,33
35 31
33
35 35
32a,32b
34 30,32a,32b,34 34
33 30,31,33 33 33
33
34 34 34 34
33 33
34
33
33
33 31 33
31,35
33 30,32a,32b
32a,32b,33 33 33
34 34
34 30,31,33 33
33 33 33
31 33 33
34 34
33 33
31 30
33
33
33 33
33 33 (Continued)
103
33 33 33
32a,32b
34 30,32a,32b
Sampling Techniques for the Determination of the Volatile Fraction of Honey
4-Oxoisophorone 5-Hexen-2-ol 5-Hydroxymethylfurfural 5-Methyl-2(5H)-furanone 5-Methyl-2furanocarboxaldehyde 5-Methyl-5-ethenyldihydro2(3H)-furanone 6-Methyl-5-hepten-2-one 8-p-Menthen-1,2-diol 9-Octadecanoic acid, methyl ester derivative Acetic acid Acetone Acetonitrile Acetophenone Benzenedicarboxylic acid, monobutyl ester Benzyl methyl ketone Benzaldehyde Benzeneacetaldehyde Benzeneacetic acid Benzeneacetonitrile Benzenedicarboxylic acid derivative Benzeneethanol Benzenemethanol Benzenepropanol Benzoic acid Benzyl alcohol Benzyl nitrile Borneol Butanoic acid Butanoic acid, 3-hexenyl ester Camphor Carvacrol Carvone cis-Carveol cis-Linalool oxide cis-Linalool oxide (furanoid) cis-Linalool oxide (pyranoid) cis-Rose oxide Damascenonne Decanal Decanoic acid Decanol
Volatile compounds in unifloral honeys (extraction technique: SPME)dcont’d
Compounds
Sage
Rosemary
Orange
Lime
Lavender
Heather
Eucalyptus
Buckwheat
Chestnut
Dandelion
Citrus
31
31
Sulla
Wild flower
33 33
33 33
33 33
33 33
33 33 33 33 33 33
33 33 33 33 33 33 33 33
35 31
31 34
31
31 34
36
31 34 34 34 34
31
34 33 33,36
33
31 31,35 36
36 31
36
36
36
33 33 36
36
34
36
34 33 33 33
36
34
32a,32b,36 32a,32b
30 30 34 32a,32b,34 32a,32b
36
34 31
30 32a,32b,36 32a,32b
36
32a,32b,33,36 32a,32b
30
30
31,36
34
33 33
34
31,33
31
31 33 35
33 33 33 33 33
35 30,32a
33 33 33
33 33 33 33 33 33 33
33 33
35
34 32a
30,32a
32b
34 32a,32b
33 33 33 33 33 30,33
34
31 32a,32b
32a,32b
33 33 33 31
33 33 33 30
34 33 34
31
33 33 33
31
Extraction Techniques and Applications: Food and Beverage
Dehydromethylene-2(3H)furanone Dehydrovomifolio Dibutyl phthalate Dihydro-2(3H)-furanone Dihydro-3-methyl-2(3H)furanone Dihydro-4-methyl-2(3H)furanone Dihydro-5-methyl-2(3H)furanone Dihydrocarveol Dimethyl disulfide Dimethyl styrene Dimethyl styrene isomer Dimethyl sulfide Dimethyltrisulfide Dodecanal Dodecanoic acid Ethanol Ethenyl phenylacetate Ethylbenzeneacetate Ethylbenzoate Formic acid Furfural Furfuryl alcohol Furfuryl n-butyrate Geranyl acetone Heptadecane Heptanal Heptanoic acid Heptanol Hexadecanoic acid Hexadecanol Hexanal Hexanedioic acid Hexanoic acid Hexanol Hotrienol Isoborneol Isobutyl phthalate Isophorone Isopropyl myristate Lilac alcohol
Thyme
104
Table 4
31
31
32b 32b 32b 32b
32a,32b 32b,32b 32a,32b 32a,32b
32a 32b 32b 32b
30,32a,32b 30,32a,32b 30,32a,32b 30,32a,32b
32a,32b,36 32a,32b,36 32a,32b,36 32a,32b 33
32b 32b 32b 32b
35
30,32a
32a,33
32b 32b 32b 32b 34 30,32b 30,32b 30,32b 30,32b
32a
30,32a
32b
34 32b
34 30 30 30 30 33 30,33
31 30 30 30 30 33
34
31 30
33
33
33
33
33
35 33 32a,32b
32b
33
32a,32b,33 33 33
33 33 32b
32b
34 32b 34
33
30,33
33
33 33 33
34
30,31
33 33 33
33 33
33 33
33 33
33 33 33
33
33
34
33 32a 32a 35
33
33 33
33
33 33
33
33
33 33
33 33 33 33
32a 33 31
31
33
31
34
33 33
35
34
35
34 34
33 35
33 33
31,33 33 31,33 33 33 33 33 33
34 34 34
34 34
33 33 31,33 33 33 33 33 33
31
33 33
33 33
105
(Continued)
Sampling Techniques for the Determination of the Volatile Fraction of Honey
Lilac alcohol Lilac alcohol (isomer I) Lilac alcohol (isomer II) Lilac alcohol (isomer III) Lilac alcohol (isomer VI) Lilac aldehyde Lilac aldehyde (isomer I) Lilac aldehyde (isomer II) Lilac aldehyde (isomer III) Lilac aldehyde (isomer VI) Limonene Limonene diol Linalool Maltol Menthofuran Methoxy-phenyl-oxime Methyl anthranilate Methyl decanoate Methyl decenoate Methyl heptanoate Methyl heptanoate Methyl octanoate Methyl salicylate Methyl-2-buten-1-ol Methyl-2-furoate Methylhexanoic acid Methylnaphthalene (isomer I) Methylnaphthalene (isomer II) Methylstyrene Myrcenol Myrtenol Naphthalene Neral Nerol Nerolidol Nonanal Nonane Nonanenitrile Nonanoic acid Nonanol Octadecanal Octanal Octane Octanoic acid Octanol
106
Table 4
Volatile compounds in unifloral honeys (extraction technique: SPME)dcont’d
Compounds
Sage
Rosemary
Orange
Lime
Lavender
Heather
31
Eucalyptus
Buckwheat
33
Chestnut
Dandelion
Citrus
31
Sulla
Wild flower
33
33
34 33 31
35
35 32a
32a,33 33
32a
34 32a
31 33 33
34
35 31 35 35 31 31 35
31
33 33
33 33
33 33
33
33
33
33
33
31
31
33 31
31 33 33
33 33
33
31 35 33
33
35 35 33
34
34
33
35 30,32a,32b 31 31
32a,32b,33
34 32a,32b
30,32a,32b
30
33
31
31 33
30
31
33 35 35 33
33
30
30
30 33
33 30 33
33
33
33 31,33 33
33 33 33
33
34 35 32a
33 33 33
31
32a
32a
33 33
30, de la Fuente, E.; Martõnez-Castro, I.; Sanz, J. J. Sep. Sci. 2005, 28, 1093–1100; 31, Piasenzotto, L.; Gracco, L.; Conte, L. J. Sci. Food Agric. 2003, 83, 1037–1044; 32, Soria, A. C.; Martõnez-Castro, I.; Sanz, J. J. Sep. Sci. 2003, 26, 793–801; 33, c, D.; Sabatini, A.G.; Conte, L.S. Food Verzera, A.; Campisi, S.; Zappala, M.; Bonaccorsi, I. Am. Lab. 2001, 33, 18–21; 34, Wolski, T.; Tambor, K.; Rybak-Chmielewska, H.; Kedzia, B. J. Apic. Sci. 2006, 50, 115–126; 35, Lusic, D.; Koprivnjak, O.; Curi Technol. Biotechnol. 2007, 45, 156–165; 36, Pérez, R.A.; Sánchez-Brunete, C.; Calvo, R.M.; Tadeo, J.L. J. Agric. Food Chem., 2002, 50, 2633–2637.
Extraction Techniques and Applications: Food and Beverage
p-Cymen-8-ol Pentanoic acid Pentanol Phenol Phenylacetaldehyde Phenylethyl alcohol p-Mentha-1,5-dien-8-ol p-Methylacetophenone Pulegone Rose oxide Rose oxide isomer Rose oxide isomer Teresantalol Terpinen-1-ol Terpinen-4-ol Terpinen-7-al Terpinene Tetradecanoic acid Tetrahydro-2,2,5,5tetramethylfuran Thymol Toluene trans-2-Caren-4-ol trans-Linalool oxide trans-linalool oxide (furanoid) trans-p-Mentha-2,8-dien-1-ol Trimethylphenol Undecane Verbenol a,a-4Trimethylbenzenemethanol a-4-Dimethyl-3-cyclohexen-1acetaldehyde a-Isophorone a-p-Dimethylstyrene a-Phenethyl alcohol a-Terpinen-7-al a-Terpineol b-Damascenone g-Nonalactone
Thyme
Sampling Techniques for the Determination of the Volatile Fraction of Honey
107
Corsica and different European countries with the emphasis on confirming the authenticity of the honeys labeled as Corsican. A DVB/CAR/PDMS 50/30 mm fiber provided the best absorption capacity and the broadest range of volatiles extracted from the headspace of a mixed honey sample. A combination of DB-5MS and SUPELCOWAX 10 columns gave the best resolution. This powerful analytical strategy led to the identification of 164 volatile compounds present in a honey mixture during a 19-min GC run. The absolute peak areas of some honey volatiles considered important markers were submitted to chemometric analysis. Interesting information was obtained and the authors were able to demonstrate that the GC GC-TOFMS combined with chemometric methods such as linear discriminant analysis (LDA), discriminant partial least squares (DPLS), and support vector machines (SVM) can be successfully applied to detect mislabeling of Corsican honeys.
4.05.2.3.2
SPME for Honey Authenticity
Interesting research regarding the determination of specific classes of substances that can be used for the authentication of honey are reported in the literature (Table 3). Two of these deal with the application of SPME-GC-MS methods developed to differentiate nectar and honeydew honeys.42,45 The interest of the authors arose from the consideration that this differentiation is very often difficult, not only because of the wide variability in composition and sensory properties among samples from the same source, but also because of the frequent existence of honeys resulting from a blend of nectar and honeydew. Soria et al.42 considered a set of Spanish nectar and honeydew honeys, whose floral source was confirmed by melissopalynology analysis. For SPME headspace sampling, a manual holder equipped with a CAR/PDMS fiber was used (Table 3) and chromatographic separation was carried out on a polar capillary column operating at programmed temperatures. Identifications were based on mass spectra, LRI, and standard injections. Stepwise regression (SRA) from volatiles data gave an estimation of honeydew and it was possible to determine which volatile compounds were related to the different honey sources. The floral origin of honey was related to the presence of several terpenoids such as borneol, cis- and trans-linalool oxide, lilac aldehydes, and lilac alcohols while acetic acid, erythro- and threo-2,3butanediol were indicative of honeydew honey. Later, Daher and Gulacar45 focused their attention on the presence of phenolic and other aromatic compounds to differentiate nectar and honeydew honeys. With regard to SPME, the different parameters affecting the efficiency of the extraction, such as the type of the fiber stationary phase, addition of NaCl and acetic acid, and extraction time, were optimized; the most appropriate fiber type appeared to be the polar PA fiber (Table 3). The extracts of honey and honeydew samples from different countries and floral origin were analyzed using an apolar column in a temperature gradient; a total of 31 compounds were detected, most of which were identified and quantified by GC-MS. Also in this case, PCA was applied to the data matrix which allowed for the differentiation between honeydew and nectar honeys on the basis of the salicylic acid concentration. Interesting results were obtained by Soria et al.44 in 2008 who reported that the nitrile compounds in Taraxacum Italian honeys were markers for the determination of floral origin. Fractionation of volatiles from honey headspace was carried out by using a CAR/PDS fiber as previously reported32 (Table 3) and the following nitriles were identified: 2-methylpropanenitrile, 2-methylbutanenitrile, 3-methylbutanenitrile, 2-butenenitrile, (E)-3-butenenitrile, 3-methylpentanenitrile, benzonitrile, and benzeneacetonitrile. Since nitrogen-containing compounds, such as nitriles, thiocyanates, and isothiocyanates, are hydrolysis products of glucosinolates present in Diplotaxis sp. and other Brassicaceae,46 the authors proposed the presence of nitriles in Taraxacum honey as the nectar contribution of species belonging to the Brassicaceae family. The application of SPME-GC-MS to verify the presence of environmental pollutants in honey was reported by Bentivenga et al.41 in 2004. They suggested that honey acts as a marker of environmental pollution of the area where the bees live. Italian honey samples (Basilicata, Southern Italy) were analyzed; the characterization of the pollens demonstrated that chestnut prevailed. A PDMS fiber (Table 3) was used and the volatiles were analyzed using GC-MS equipped with an apolar capillary column. From their mass spectra, some phenyl-substituted and hydrocarbons were identified and correlated to the presence of pollutants from anthropogenic activities in the honey production area. A SPME-GC-MS method for estimation of authenticity of thyme honey was developed by Mannas and Altu g in 2007.43 Thyme honey is considered to be one of the most delicious and high quality among the unifloral honeys; samples were analyzed following the SPME procedures previously developed by Verzera et al.33 and Piasenzotto et al.31 (Table 3). The results of GC-MS analysis showed that pure thyme honey involved volatile compounds that originate from the thyme plant, such as thymol and carvacrole, in low amounts; high amounts of these substances were considered as markers for detecting adulteration by thyme essential oil.
4.05.2.4
USE
USE is an extraction technique that does not use any heat. It is applied to the isolation of volatile compounds from natural products using organic solvents and a water bath with ultrasound assistance at room temperature. The first application of this technique for the study of honey volatile fraction goes back to 2003.47 The authors applied a USE methodology for Greek citrus honeys and flowers. The volatile fraction both of fresh flower and honey samples was extracted using an n-pentane/diethylether (1:2) mixture in an ultrasound water bath apparatus, maintained at 25 C for 10 min; the extracts were then analyzed directly by GC-FID and GC-MS using an apolar capillary column operating at programmed temperature with helium as carrier gas. The analysis of the flowers allowed the identification of 17 volatile compounds, mainly monoterpenes; among these, linalool was predominant in all citrus species, except for lemon, with eucalyptol the main compound. For the honey volatile extracts (Table 5), linalool derivatives accounted for more than 80% of the total volatile amount, in agreement with the flower extracts. Thus, through the quantification of linalool derivatives present in citrus honey volatiles, the authors expected to establish a threshold to distinguish citrus honeys from others of different floral origin.
108
Extraction Techniques and Applications: Food and Beverage
Table 5
Volatile and semivolatile honey compounds (extraction technique: USE)
Compounds
Citrus
(E)-2,6-Dimethyl-2,7-octadiene-1,6-diol (E)-2,6-Dimethyl-6-hydroxy-2,7-octadienal (Z)-2,6-Dimethyl-2,7-octadiene-1,6-diol (Z)-9-Tricosene (Z)-Octadec-9-en-1-ol 1-(4-Methoxyphenyl)propan-2-one 1,3-bis(1,1-Dimethyl)benzene 1,3-Di-tert-butylbenzene 1H-Pyrrole 1-Isocyanato-2-methylbenzene 1-Methoxy-4-propylbenzene 1-Methyl-2-pyrrolidinone 1-Octanol 1-Octanol 1-Phenyl-2,3-butanediol (isomer I) 1-Phenyl-2,3-butanediol (isomer II) 1-Phenyl-2,3-butanedione 2,2,6-Trimethylcyclohexane-1,4-dione 2,3,5-Trimethylphenol 2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one 2,3-Dihydro-benzofuran 2,4-Di-tert-butylphenol 2,5-Dimethyl-2,4-dihydroxy-3(2H)-furanone 2,6,6-Trimethyl-4-oxocyclohex-2-ene-1-carbaldehyde 2,6-Dimethoxyphenol 2,6-Dimethyl-1,7-octadien-3,6-diol 2,6-Dimethyl-3,7-octadiene-2,6-diol 2-Ethyl-3-hydroxyhexyl 2-methylpropanoate 2H-1-Benzopyran-2-one 2-Hydroxy-1-(4-methoxyphenyl)ethanone 2-Hydroxyacetophenone 2-Hydroxyphenylethanol 2-Methylbenzene-1,4-diol 2-Methylbutanoic acid 2-Methylpropanoic acid 2-Phenylacetaldehyde 2-Phenylacetic acid 2-Phenylethanol 3,4,5-Trimethoxybenzaic acid 3,4,5-Trimethoxybenzaldehyde 3,4,5-Trimethylphenol 3,5-Dihydroxy-2-methyl-4H-pyran-4one 3,7-Dimethyl-1,5-octadiene-3,7-diol 3,7-Dimethyl-1,6-octadiene-3,5-diol 3-Hydroxy-1-phenyl-2-butanone 3-Hydroxy-2-butanone 3-Hydroxy-2-methyl-4H-pyran-4-one 3-Hydroxy-4-phenyl-2-butanone 3-Hydroxy-4-phenyl-3-buten-2-one 3-Methyl-1-butanol 3-Oxo-a-ionone 4-(4-Hydroxy-2,2,6-trimethyl-7-oxabicyclo[4.1.0]hept-1-yl)-3-buten-2-one 4-Ethenyl-2-methoxyphenol 4-Hydroxy-3,5-dimethylbenzaldehyde 4-Hydroxyphenylacetonitrile 4-Hydroxyphenylethanol 4-Ketoisophorone 4-Methoxybenzaldehyde 4-Methoxybenzoic acid 4-Methoxyphenethyl alcohol 4-Methoxyphenylacetonitrile
47 47 47
Sage
Thyme
49 48 48 47 49 48 47 48 47 47 47 49 49 49 48 48 49 47 49 49 48 48 47 47 48 48 49 49 49 48 49 49
47
48 48 48 48 48
49 49 49
48 47 49 49 48 49 49 49 49 48 48 49 48 48 48
49
49 49
Sampling Techniques for the Determination of the Volatile Fraction of Honey
Table 5
109
Volatile and semivolatile honey compounds (extraction technique: USE)dcont’d
Compounds 5-(Hydroxymethyl)-furan-2-carbaldehyde Acetic acid Benzaldehyde Benzene-1,4-diol Benzeneacetaldehyde Benzeneacetic acid Benzoic acid Benzyl alcohol bis-(2-Ethylhexyl) adipate Butanoic acid Butyl acetate Caffeine Carvacrol cis-Linalool oxide (furanoid) cis-Linalool oxide (pyranoid) Coumaran Cyclohexanone Decanal Decane Decanoic acid Degraded carotenoid Dehydrovomifoliol Dibutylphthalate Dihydroanethole Diisobutylphthalate Docosane Dodecane Dodecanoic acid Eicosane Ethyl hexadecanoate Ethyl oleate Ethyl benzene Eugenol Furfural Guaiacol (2-methoxyphenol) Heneicosane Henicos-10-ene Heptadecane Heptane Hexadecane Hexadecanoic acid Hexanoic acid Hydroxymethylfurfural Hotrienol Indole Isophorone Isovaleric acid Lilac alcohol Lilac alcohol Lilac aldehyde Lilac aldehyde Lilac aldehyde Lilac aldehyde (isomer I) Lilac aldehyde (isomer II) Lilac aldehyde (isomer III) Limonene Linalool oxide B Linoleic acid m-(or p-)Xylene Methyl 4-methoxybenzoate
Citrus
47 47 47 47
Sage 48 48 48 48
Thyme
49
48 48
49 49
48
49 49
47
47 49 47 47 48 47 47 47 48
49 49 49
47 47
48 48
47
49 49 49 49 49 49
48 49 49 49 49 49 49 48 48 48 48 47 48 48 47 47
49 49 49 49 49 49 49 49 49 49
47 47 47 47 47 47 47 47 47
49 48 49
47 48 (Continued)
110
Extraction Techniques and Applications: Food and Beverage
Table 5
Volatile and semivolatile honey compounds (extraction technique: USE)dcont’d
Compounds
Citrus
Methyl anthranilate Methyl benzoate Methyl hexadecanoate Methyl syringate Methyl vanillate Methyl cyclohexane Nerolidol Nonadecane Nonanal Nonane Nonanoic acid Octadecane Octadecanoic acid Octanal Octane Octanoic acid Oleic acid o-Xylene p-Anisaldehyde p-Anisic acid Pentacosane Pentadecane Phenol Phenylacetaldehyde Phenylacetic acid Phenylacetonitrile Phenylethyl alcohol Propanoic acid p-Toluic acid p-Xylene Salicylic acid Syringaldehyde Tetracosane Tetradecane Tetradecanoic acid Toluene trans-Linalool oxide (furanoid) trans-Linalool oxide (pyranoid) Tricosane Tridecane Undecane Vanillic acid Vanillin Veratric acid a-4-Dimethyl-3-cyclohexene-1-acetaldehyde (isomer III) a-4-Dimethyl-3-cyclohexene-1-acetaldehyde (isomer VI) a-Isophorone
47
Sage
Thyme
48 49 49 49 47 47 48 47 48 48
47 48
49 49 49 49 49 49 49 49 49 49
47 49 49 48 48 48 47 47
48 47 47 47 47 47 47 47
48
49 49 49 49 49 49 49 49 49 49 49 49 49 49 49 49 49 49 49 49 49
47 47 48
47, Alissandrakis, E.; Daferera, D.; Tarantilis, P.A.; Polissiou, M.; Harizanis, P.C. Food Chem. 2003, 82, 575–582; 48, Jerkovic, I. Mastelic J., Marijanovic, Z. Chem Biodivers. 2006, 3, 1307–1316; 49, Alissandrakis, E.; Tarantilis, P.A.; Pappas, C.; Harizanis P.C.; Polissiou M. Eur. Food Res. Technol. 2009, 3, 365–373.
In 2006, the USE technique was used by Jerkovic et al.48 for the first characterization of the volatiles of sage (Salvia officinalis L.) honeys. Unifloral Salvia honey samples (ascertained by pollen analysis) were selected from various producers in South Croatia; 54 volatile compounds were identified (Table 5) by GC-FID and GC-MS in n-pentane/diethylether extracts after sonication at 25 C for 30 min and quantified using internal standards. Compounds such as benzoic acid, phenylacetic acid, p-anisaldehyde, a-isophorone, and 4-ketoisophorone, were proposed as markers for sage honey. A few years later, Alissandriks et al.49 presented their results on the composition of the volatile fraction of unifloral Greek thyme honey, the most marketed type in Greece. Using the experimental method previously developed,47 about 100 compounds were identified (Table 5) and quantified using b-ionone as internal standard. Phenolic compounds were the most abundant and were proposed as potent botanical markers for thyme honey.
Sampling Techniques for the Determination of the Volatile Fraction of Honey
4.05.2.5
111
USE Coupled with SPME
Recently examples of applications of USE and SPME, applied as complementary extraction techniques for the characterization of honey volatile fraction, have been reported in the literature.50–52 The use of both techniques allowed complete patterns of honey headspace to be obtained, since the HS-SPME method proved to be suitable for the isolation of low-molecular-weight aroma compounds, whereas the USE procedure enabled extraction of semivolatile compounds. The optimization and application of this comprehensive method was due exclusively to Jerkovic and co-workers who in 2009 reported the honey volatile composition of different Croatian unifloral honeys, namely Lavandula hybrida Reverchon II50 (Lavandula angustifolia Lavandula latifolia), Paliurus spina-christi,51 and Amorpha fruticosa52 honeys. For the HS-SPME analysis different fibers were considered (Table 3), whereas for the USE procedure three solvents, pentane, diethyl ether, and a mixture of pentane/ethyl ether (1:2 v/v) were used, under the same experimental conditions previously reported.48 The GC-MS analysis was performed on polar and apolar capillary columns at programmed temperatures using helium as carrier gas. With regard to Lavandula hybrida Reverchon II, HS-SPME made it possible to isolate short chain aliphatic compounds (Table 6), mainly hexan-1-ol, hexanal, acetic acid, hotrienol, and 2-phenylacetaldehyde, important for authentication of this honey, whereas the ultrasound pentane/ethyl ether extract contained the majority of the honey floral origin compounds and potential biomarkers (hexan-1-ol, acetic acid, butane-1,3diol, butane-2,3-diol, benzoic acid, coumarin, and 2-phenylacetic acid) (Table 6). In the Paliurus spina-christi headspace a total of 33 compounds were identified (Table 6); major constituents and possible markers were nonanal, four isomers of lilac aldehyde, decanal, methyl nonanoate, hexanoic, and 2-ethylhexanoic acids; in the USE extracts a total of 79 compounds were identified
Table 6
Volatile and semivolatile honey compounds (extraction techniques: USE and SPME) Lavender
Compounds USE (E)-b-Damascenone (Z)-Octadec-9-en-1-ol (Z)-Octadec-9-enoic acid (Z,Z)-9,12-Octadecadienal (Z,Z)-9,12-Octadecadienoic acid 1-(2-Furanyl)-2-hydroxyethanone 1-(2-Furanyl)-ethanone 1,3-Butanediol 1,3-Butanediol 1,3-Dimethylbenzene 1,4-Benzenediol 1,4-Dimethylbenzene 1,4-Di-tert-butylbenzene 1-Dodecanol 1-Hexadecanol 1-Hexanol 1H-Indole-3-acetic acid 1H-Pyrrole-2-carboxylic acid 1-Hydroxylinalool 1-Nonanol 1-Octadecanol 1-Phenylethan-1,3-diol 1-Tetradecanol 2-(4-Methoxyphenyl)ethanol 2-(p-Methoxyphenyl)-ethanol 2,3-Butanediol 2,3-Butanediol 2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one 2,3-Dihydro-3,5-dimethyl-4H-pyran-4-one 2,3-Dihydro-5-hydroxy-6-methyl-4H-pyran-4-one 2,3-Dihydrobenzofuran 2,4-Dimethyl-3,6-dihydro-2H-pyran 2,5-Dimethoxy-4-ethylbenzaldehyde 2-Ethylfuran 2-Ethylhexanoic acid 2-Ethylhexyl 2-ethylhexenoate 2-Furancarbaldehyde 2-Furancarboxylic acid
SPME
Christ’s thorn USE
SPME
Desert false indigo USE
SPME 52
50
51
52 52
51 51 52 52 51 50 52 52 52 52 52 52
51
51 50
50 51 51 52 52 52 52 52
50 51 51 50 50
50
52 51
50 50
51 52 52 52 51a,51b
50 50 50 (Continued)
112
Extraction Techniques and Applications: Food and Beverage
Table 6
Volatile and semivolatile honey compounds (extraction techniques: USE and SPME)dcont’d Lavender
Compounds USE 2-Furanmethanol 2-Hydroxy-3-methyl-4H-pyran-4-one (maltol) 2-Methoxy-6-methylpyrazine 2-Phenylacetaldehyde 2-Phenylacetamide 2-Phenylacetic acid 2-Phenylethanol 3-(4-Hydroxy-3-methoxyphenyl)-prop-2-enoic acid (ferulic acid) 3,5-Dihydroxy-2-methyl-4H-pyran-4-one 3,5-Dimethoxy-4-hydroxybenzoic acid (syrigic acid) 3,7-Dimethylocta-1,5-diene-3,7-diol 3-Hydroxy-4-methoxycinnamic acid (isoferulic acid) 3-Methyl-2-butanol 3-Methylpentanoic acid 3-Pyridinecarbonitrile 4,5-Dimethyl-2-formylfuran 4-Ethenyl-2-methoxyphenol 4-Ethylbenzaldehyde 4-Hydroxy-3,5,6-trimethyl-4-(3-oxo-1-butenyl)-cyclohex-2-en-1-one 4-Hydroxy-3,5-dimethoxybenzaldehyde (syringyl aldehyde) 4-Hydroxy-3,5-dimethoxybenzoic acid 4-Hydroxy-3,5-dimethylbenzaldehyde 4-Hydroxy-3-methoxybenzaldehyde (vanilline) 4-Hydroxy-3-methoxybenzoic acid (vanillic acid) 4-Hydroxybenzaldehyde 4-Hydroxybenzoic acid (p-salicylic acid) 4-Hydroxybenzyl alcohol 4-Hydroxycinnamic acid (3-(4-hydroxyphenyl)-prop-2-enoic acid) 4-Hydroxyphenylacetic acid 4-Ketoisophorone 4-Methoxybenzaldehyde 4-Methoxybenzoic acid 4-Methyl-2,6-bis (1,1-dimethylethyl)-phenol 4-Methylbenzyl alcohol 4-Methyloctane 4-Vinyl-2-methoxyphenol 4-Vinylphenol 5-(Hydroxymethyl)furan-2-carbaldehyde 5-Hydroxymethylfurfural 5-Methyl-1,4-benzenedione 5-Methylfurfural Acetic acid Benzaldehyde Benzoic acid Benzyl alcohol Butanoic acid Butoxyethoxyethyl acetate cis-Linalool oxide Coumarin Decanal Decanoic acid Diisobutyl phthalate Dimethyl disulfide Docosane Dodecane Ethylbenzene Formic acid Furfural
SPME
Christ’s thorn USE
SPME
Desert false indigo USE
SPME
51b 51 51 50 52 50 50
50
51
51a,51b
52 52
52
50 50
52 52 52
51 51 51 51
52 50 50 52 52 51 51 52 52 51a,51b 51
52 52 52 52 52 52
51 52 52 52 52 52
51
52
50 50
51
52 51a
50
50 50 50
50
51
51a,51b 51a,51b
51 51 51
50
51a
52 52 52 52 52
52 52
52
50 51 51
51a,51b 52 52
51 51
50
51a 52
50
50 50
51b
52
Sampling Techniques for the Determination of the Volatile Fraction of Honey
Table 6
113
Volatile and semivolatile honey compounds (extraction techniques: USE and SPME)dcont’d Lavender
Compounds USE Furfuryl alcohol Heneicosane Heptacosane Heptadecane Heptanal Heptanoic acid Hexacosane Hexadecan-1-ol Hexadecane Hexadecanoic acid Hexanal Hexanoic acid Hotrienol Isophorone Lilac aldehyde (isomer I) Lilac aldehyde (isomer II) Lilac aldehyde (isomer III) Lilac aldehyde (isomer not identified) Lilac aldehyde (isomer VI) Linalool Maltol Methoxybenzene Methyl 2-furancarboxilate Methyl 3,5-dimethoxy-4-hydroxybenzoate (methyl syringate) Methyl furan-2-carboxylate Methyl nonanoate Methyl octanoate Methyl sulfide Nananoic acid Nonacosane Nonadecane Nonanal Nonane Nonanoic acid Octacosane Octadecane Octadecanoic acid Octanal Octane Octanedioic acid Octanoic acid o-Ethylanisole Pantoic lactone Pentadecane Pentanoic acid Phenylacetaldehyde Phenylacetic acid Phenylacetonitrile Phytol Pinocarvone Propanoic acid Tetracosane Toluene trans-Linalool oxide Tetradecane Tricosane Tridecane Undecane
SPME
Christ’s thorn
Desert false indigo
USE
SPME
USE
51 51 51
51a
52
SPME
50 50
51a,51b
50 50 51 50 51 51
50 50 50
50 50 50
51
52 51a,51b
52 52 52
51a,51b 51a,51b 51a,51b 52 51a,51b 52 50
50 52 51 52
50
50 51a 51a 52 52 51 51 51
50
50
51 51 51 51 51
51a 51a,51b
52 52 52
51a,51b
51a 52
50
50
51 51
51a,51b 51a
50 51 51 51
51a
52
51a,51b
52 52
52 52
51 52 50
51 51
52 51b 52
52
51 51 51a 51a,51b
50, Jerkovic, I.; Marijanovic, Z. Chem Biodivers. 2009, 6(3), 421–430; 51, Jerkovic, I.; Tuberoso, C.I.G.; Marijanovic, Z.; Jelic, M.; Kasuma, A. Food Chem. 2009, 112(1), 239–245; 52, Jerkovic, I.; Marijanovic, Z.; Kezic, J.; Gugic, M. Molecules 2009, 14, 2717–2728.
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(Table 6) and benzene derivatives (4-hydroxy-3,5-dimethylbenzaldehyde, 4-hydroxybenzoic acid, and 4-methoxybenzoic acid) and aliphatic acids (butanoic, hexanoic, octanoic, and nonanoic) were considered interesting for floral determination. By HS-SPME in Amorpha fruticosa a total of 25 volatile constituents (Table 6) with 2-phenylethanol, cis- and trans-linalool oxides, benzaldehyde, and benzyl alcohol were identified as the main compounds, while by USE a total of 57 volatile and semivolatile compounds were identified, with 2-phenylethanol and methyl syringate the main compounds (Table 6). For each unifloral honey, the USE technique allowed the identification of a higher number of volatile constituents than SPME, as expected. Thus, using the two different extraction methods and combining the data obtained, interesting information can be obtained, especially when markers of honey floral origin include both volatile and semivolatile compounds.
4.05.3
E-nose
A new analytical tool for the identification of volatile compounds has been recently proposed to address the need for routine quality testing in the food industry; this is the so-called electronic nose (E-nose) consisting of an array of weakly specific or broad spectrum chemical sensors that intend to mimic the human olfactory system and convert sensor signals to data that can be analyzed with appropriate statistical software. The use of E-nose to characterize the origin of honey based on the volatile fraction introduced a different strategy that allows a profile or fingerprint to be obtained avoiding any separation into individual compounds. The first application of E-nose to honey volatiles was due to Benedetti et al.53 who applied an artificial neural network (ANN) to classify signals from an electronic nose smelling different types of honeys. Unifloral honey samples of specific botanical and geographical origins (Robinia pseudoacacia L. (Italy), Rhododendron spp. (Italy), Citrus spp (Italy), Robinia pseudoacacia L.(Hungary)) were analyzed; the honey gas headspace, sampled by an automatic syringe, was pumped for 30 s over the surface of 22 different gas sensors (10 metal oxide semiconductor field effect transistor sensors, 12 metal oxide semiconductor sensors). The data obtained from the sensor array were statistically analyzed by PCA and ANN and good results were obtained using a neural network model based on a multilayer perceptron (MLP) that learned using an algorithm called backpropagation. In the following years, this method53 was applied by Cacic et al.54 to geographic origin characterization of Robinia pseudoacacia L. and Castanea sativa Mill. honeys produced in Croatia. Volatile profile data obtained by E-nose were analyzed by PCA and it was found that honey samples from geographically close regions tended to be grouped together, while those from distant geographic regions showed differences although they were of the same botanical origin. In 2004, Ampuero et al.55 studied honey volatiles using a fast sensitive E-nose based on mass spectrometry (MS-nose). Three different sampling techniques, SHS, SPME, and inside needle dynamic extraction (INDEX) (also known as solid-phase dynamic extraction (SPDE)), were used for volatiles of Swiss honeys derived from dandelion, lime, acacia, chestnut, fir, and rape. For each sampling technique, the extracted volatiles were injected into the injector port of the E-nose. The responses of the detector (specific ionic masses), processed by PCA and discriminating factor, allowed the authors to classify the samples into different botanical groups in agreement with the results simultaneously obtained by a classic method, i.e., sensory, pollen, and physicochemical analysis. Among the different techniques, the SPME sampling method showed 98% correct classification of the model samples. Another fast, sensitive, and nondestructive electronic nose, the zNose, was tested by Lammertyn et al.56 on honeys (buckwheat, clover, orange blossom, black locust, mint, and carrot) from different geographic origins. The zNose is a fast GC technique which allows identification and fingerprinting of aroma as with regular GC but at the same time it operates at the speed of the E-nose using a surface acoustic wave sensor detector made of an uncoated piezoelectric quartz crystal. For the zNose measurements, a needle (provided by the zNose), inserted through the septa of the vials from an SHS system, was used for sampling headspace honey volatiles, which were thus released from the trap inside the system and carried over the column (DB-5) in a helium flow. Since the zNose is a combination sensor-based detector and regular GC analyzer, the data resulting from the zNose measurements were approached in two different ways: first, by comparing the different peaks and peak areas obtained considering only the positives values of the first derivative plot (chromatogram approach); and second, the positive and negative values of the first derivative profile were considered and treated as spectral data, analyzing the full frequency spectrum of each sample. The statistical treatment of the data by PCA and canonical discriminant analysis made it possible to discriminate among honeys of different floral origin and between honey and plain sugar.
4.05.4
Conclusion
In volatile fraction analysis, the choice of the extraction method depends on the type of food and the information needed, since there is a great variability in the aroma compounds obtained depending on the procedure used. In most cases, the extraction method selected should be able to provide an aromatic extract as representative as possible of the product. From this point of view, all the extraction methods that require heat are less suitable since the volatile profiles obtained contain heat-generated artifacts, whereas the most sensitive compounds could be missing, destroyed by the drastic conditions applied. The extraction techniques that make it possible to obtain a volatile profile comparable with the sensorial descriptors are those operating in the sample headspace, such as DHS and SPME, when carried out at low temperature. In the case of honey, the use of heat along with solvent, as required by the SDE technique, significantly modifies the volatile profile. For example, in an acid aqueous medium, terpenes easily lead to hydrated and oxidized compounds and substances from the Maillard reaction and/or sugar caramelization can arise
Sampling Techniques for the Determination of the Volatile Fraction of Honey
Table 7
115
Furan and pyran derivatives (mg kg1) in thyme honey volatiles sampled by different extraction techniques
Compounds 1-(2-Furanyl)-ethanone 2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one 2,3-Dihydro-4-methylfuran 2,5-Dimethyl-2,4-dihydroxy-3(2H)-furanone 2,5-Dimethylfurane 2-Acetylfuran 2-Furan methanol 2-Furancarboxilic acid 2-Methyl-3-(2H)-dihydrofuranone 2-Methylfuran 3,5-Dihydroxy-2-methyl-4H-pyran-4one 5-Ethenyl-5-methyl-2(3H)-furanone 5-Methyl furfural 5-Methyl-2-(3H)-furanone Furan derivative (95, 123, 124) Furfural Furfuryl alcohol Furfuryl n-butyrate Vinylguaiacol
SDE20
DHS26
SPME31,36
USE49
–a 33 327.0 86 13.4 95.8 6.0c 22.4 21.5 95 30.1 69.2 12.6 1558 19.8
495.4
35.6c
78 74
–b 19.5
a
Compounds identified but not quantified. Compounds identified by Piasenzotto et al.31 Compounds identified by Pe´rez et al.36
b c
Figure 1 HS-SPME-GC-MS (SIM) chromatogram of orange honey volatile fraction. Compounds: 1, limonene (LRI ¼ 1185); 2, a-pinene oxide (LRI ¼ 1198); 3, cis-limonene oxide (LRI ¼ 1230); 4, cis-geraniol (1241); 5, terpinolene (LRI ¼ 1256); 6, 6-methyl-5-epten-2-one (LRI ¼ 1324); 7, a-pdimethyl styrene (LRI ¼ 1417); 8, cis-linalool oxide (LRI ¼ 1420); 9, trans-linalool oxide (LRI ¼ 1450); 10, terpinene-4-acetate (LRI ¼ 1494); 11, lilac aldehyde isomer I (LRI ¼ 1501); 12, lilac aldehyde isomer II (LRI ¼ 1523); 13, lilac aldehyde isomer III (LRI ¼ 1533); 14, lilac aldehyde isomer IV (LRI ¼ 1555); 15, hotrienol (LRI ¼ 1581); 16, phenyl acetaldehyde (LRI ¼ 1618); 17, decatriene-2,2,5,8-tetramethyl (LRI ¼ 1631); 18, p-menthen-9-al (LRI ¼ 1644); 19, a-terpineol (LRI ¼ 1670); 20, b-damascenone (LRI ¼ 1788); 21, geranlyl acetone (LRI ¼ 1825); 22, cedrene (LRI ¼ 1831); 23, trans-nerolidol (LRI ¼ 2005); 24, limonene diepoxide (LRI ¼ 2189); 25, (E,E)-farnesol (LRI ¼ 2336).
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simultaneously, such as furan derivatives. Furan-derived compounds, such as furfural, furfuryl alcohol, and 5-methyl furfural, which are present in a low concentration in fresh honey, increase during storage and heating and the increase is greater the higher the temperature.57 Thus, they are considered as indicators of thermic processes and storage and are usually used in evaluating quality deterioration in food but they cannot be considered as appropriate floral markers. Table 7 reports the furan derivatives identified in thyme honey samples,20,26,31,36,49 extracted by different methods, along with their amount, where indicated. The SDE extracts were the richest in furan derivatives while the SPME extracts had less. Furfural, present in all the thyme honey samples analyzed, was the main compound together with phenylacetaldehyde (~1.5 mg kg1) in the SDE extracts. An high amount of furfural and 2,3dihydro-4-methylfuran was also present in thyme honeys extracted by DHS. The significant amount of these two substances could be associated with purge and trap fractionation that operated at a temperature of 80 C. In contrast, the amount of furan derivatives in the SPME and USE extracts was small even though a large number of volatile compounds were identified by both techniques. With regard to the possibility of verify the floral and geographic origin of honey, interesting information is obtained from primary aroma compounds, such as terpenes and their derivatives, that are associated with the floral nectar or honeydew gathered by honeybees. Figure 1 reports a SPME-GC-MS(SIM)58 of a citrus honey in which a large number of terpenes, sesquiterpenes, and their oxygenated compounds were identified. Among these, the markers for the floral origin can be defined, in agreement with other authors. From the above observations, SPME seems to be a very reliable technique since high reproducibility and sensibility have been achieved with respect to extraction and identification of honey volatile compounds without the complexity of traditional methods.
See also: Headspace Analysis; Solid-Phase Microextraction; Sample Preparation Automation for GC Injection; Headspace Sampling in Flavour and Fragrance Field; Theory of Extraction
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Mannas, D.; Altug, T. Int. J. Food Sci. Technol. 2007, 42, 133–138. Soria, A. C.; Martínez-Castro, I.; de Lorenzo, C.; Sanz, J. Food Chem. 2008, 107, 439–443. Daher, S.; Gulacar, F. O. J. Agric. Food Chem. 2008, 56, 5775–5780. Condurso, C.; Verzera, A.; Romeo, V.; Ziino, M.; Trozzi, A.; Ragusa, S. Planta Med. 2006, 72, 924–928. Alissandrakis, E.; Daferera, D.; Tarantilis, P. A.; Polissiou, M.; Harizanis, P. C. Food Chem. 2003, 82, 575–582. Jerkovic, I.; Mastelic, J.; Marijanovic, Z. Chem Biodivers. 2006, 3, 1307–1316. Alissandrakis, E.; Tarantilis, P. A.; Pappas, C.; Harizanis, P. C. Polissiou M. Eur. Food Res. Technol. 2009, 3, 365–373. Jerkovic, I.; Marijanovic, Z. Chem Biodivers. 2009, 6(3), 421–430. Jerkovic, I.; Tuberoso, C. I. G.; Marijanovic, Z.; Jelic, M.; Kasuma, A. Food Chem. 2009, 112(1), 239–245. Jerkovic, I.; Marijanovic, Z.; Kezic, J.; Gugic, M. Molecules 2009, 14, 2717–2728. Benedetti, S.; Mannino, S.; Sabatini, A. G.; Marcazzan, G. L. Apidologie 2004, 35, 397–402. Cacic, F.; Primorac, L.; Kenjeric, D.; Benedetti, S.; Mandic, M. J. Central Eur. Agric. 2009, 10(1), 19–26. Ampuero, S.; Bogdanov, S.; Bosset, J. O. Eur. Food Res. Technol. 2004, 218, 198–207. Lammertyn, J.; Veraverbeke, E. A.; Irudayaraj, J. Sensors Actuators B 2004, 98, 54–62. Castro-Vazquez, L.; Diaz Maroto, M. C.; Gonzalez Vinas, M. A.; de la Fuente, A.; Perez Coello, M. S. J. Agric. Food Chem. 2008, 56, 1999–2005. Verzera, A.; Condurso, C. Unpublished data.
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4.05.1 4.05.2 4.05.2.1 4.05.2.2 4.05.2.3 4.05.2.3.1 4.05.2.3.2 4.05.2.4 4.05.2.5 4.05.3 4.05.4 References
4.05.1
87 87 87 88 92 97 107 107 111 114 114 116
Honey Volatiles Extraction Techniques Solvent Extraction and SDE Static and DHS Analysis SPME SPME for Floral and Geographic Determination SPME for Honey Authenticity USE USE Coupled with SPME E-nose Conclusion
Honey Volatiles
Honey is a popular natural product not only as a source of energy but also for its health-promoting properties provided by the prebiotic, antioxidant, antibacterial, and/or antimutagenic functionalities of certain constituents.1–4 European Union Legislation (2001/110/EC) defines honey as “the natural sweet substance produced by Apis mellifera bees from the nectar of plants or from secretions of living parts of plants or excretions of plant-sucking insects on the living parts of plants, which the bees collect, transform by combining with specific substances of their own, deposit, dehydrate, store and leave in honeycombs to ripen and mature.”5 Honey is composed mainly of monosaccharides (fructose and glucose), lesser amounts of water, and a great number of minor components such as organic acids, oligosaccharides, enzymes, vitamins, minerals, pigments, a wide range of aroma compounds, and solid particles derived from honey collection.6 At present, beekeepers throughout the world produce various types of honey, some expressing very distinct sensory properties that strongly influence consumer preferences and price of the honey; honey of unifloral origin usually commands higher prices than wildflower honey. The flavor/fragrance qualities of honey are greatly dependent on the volatile and semivolatile organic compounds present both in the sample matrix and headspace aroma. Several studies on the volatile compounds of honey have been carried out since 19627 and it is well known that these compounds may be derived from the plant or nectar, from the conversion of plant compounds or directly generated by the honeybees, from heating or handling during honey processing and storage, and from microbial or environmental contamination. Since most honey volatiles originate from the plants being visited by the bees, the honey sensory characteristics are closely related to its botanical origin and from this it follows that the volatile compounds have a potential role in distinguishing the floral origin. Honey is a very complex mixture with volatile components of different functionality and relatively low molecular weight, often present at very low concentrations; for their isolation, various methods, such as solvent extraction, simultaneous steam distillation extraction (SDE), static (SHS) and dynamic headspace (DHS) extraction, solid-phase microextraction (SPME), ultrasound-assisted extraction (USE), have been used. With regard to the determination of volatiles, gas chromatography (GC)–mass spectrometry (MS) is usually the technique of choice since it combines high separation efficiency and sensitivity and provides qualitative and quantitative data for these compounds. More recently, electronic noses (E-nose) based on mass spectrometry (MSE-nose), piezoelectric effects (zNose), and electrical resistance, have been tested with interesting results. The main analytical techniques used to extract and analyze the volatile fraction of honey are described. The advantages and drawbacks of each methodology, comparison of alternative reliable methods and results achieved in the field of honey quality determination are discussed. Special emphasis is given to the SPME technique, by far the most commonly used technique.
4.05.2
Extraction Techniques
4.05.2.1
Solvent Extraction and SDE
Solvent extraction has been widely used since the second half of the 1990s for honey volatiles.8–10 The success of this technique was due to its simplicity and because thermolabile compounds were retained; moreover, the commonly used solvents have low polarity so that the main compounds, water and sugars, are not extracted. Nevertheless this procedure also has some disadvantages, such as the long time involved, large volumes of solvents, environmental hazards, etc, so nowadays it is no longer used in favor of more suitable innovative techniques.
Comprehensive Sampling and Sample Preparation, Volume 4
doi:10.1016/B978-0-12-381373-2.10129-2
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The simultaneous distillation–extraction (SDE) system developed by Likens and Nickerson11,12 and modified by Godefroot et al.13 is one of the currently used methods for isolation of honey volatile components; honeys of different floral origin were analyzed and a large number of volatiles were identified. This approach, unfortunately, determines the formation of artifacts, loss of analytes and provides results that are not easily compared; moreover, it needs a long sampling time prior to chromatographic separation and a large volume of solvent that influences trace analysis. However, interesting results were obtained on the floral origin of honey when the SDE method was carefully optimized. Following Bicchi et al.14 who first emphasized the importance of pre-extracting flavor compounds from sugars, in 1995 Bouseta and Collin15 proposed a two-step SDE protocol including preliminary solvent extraction under an inert atmosphere using dichloromethane as solvent. The method was applied to a commercial clover Canadian honey and the sensorial features of extracts closely matched those of the honey samples; the extracts were analyzed by GC-MS using an apolar capillary column. Unfortunately, using MS, authentic standard injection, and a mass spectra library, a limited number of volatile compounds, mainly furan and aromatic compounds, were identified (Table 1). In 1998, Guyot et al.16 applied the two-step SDE method15 to find reliable markers to ascertain the floral origin of chestnut and lime tree honeys. The unifloral honeys were selected from various countries and the screening for floral purity was based on pollen analyses, sensory tests, electrical conductivity, pH, titratable acidity, and sugar composition. The authors confirmed that about 400 aroma compounds were separated by GC although only 72 were identified (Table 1); chestnut and lime tree samples were authenticated on the basis of a few discriminate flavoring compounds, the content of which was significantly different from those of the other honeys; chestnut honeys were distinguishable by high concentrations of acetophenone, 1-phenylethanol (>88 ppb), and 2-aminoacetophenone (>154 ppb) and lime tree honeys by enhanced amounts of shikimate pathway derivatives, namely ethylmethylphenol isomer (>31 ppb), 4-tert-butylphenol, estragole (>51 ppb), and p-methylacetophenone, and by high concentrations of monoterpene-derived compounds such as menthol, thymol, 8-p-menthene-1,2-diol, and carvacrol. In the following years, the same authors17,18 applied their method to heather and lavender unifloral honeys from various countries (Calluna vulgaris from France, Belgium, United Kingdom, Norway, and Germany; Erica arborea from France, Greece, and Italy; Lavandula stoechas from Portugal, Lavandula angustifolia latifolia (lavandin), and Lavandula angustifolia (fine lavender) from the southwest of France). Forty-eight aroma compounds were identified by GC-MS in heather honey but less in lavender honeys (Table 1). The authors reported that phenylacetic acid, dehydrovomifoliol, and 4-(3-oxo-1-butynyl)-3,5,5-trimethylcyclohex-2-en1-one and high level of 3,5,5-trimethylcyclohexene derivatives were specific markers of Calluna vulgaris honeys. According to the authors, the presence of shikimate pathway derivatives such as 4-methoxybenzaldehyde, 4-methoxybenzoic acid, and methyl vanillate, in Erica arborea honey samples irrefutably proved their floral origin. The aromatic profiles of the L. angustifolia latifolia and L. angustifolia honeys were very similar and only phenylacetaldehyde and heptanoic acid were indicated as quantitative markers, respectively; French lavender honeys were easily authenticated from L. stoechas samples of various other origins, due to their high content of linear aldehydes, linear alcohols, and phenylacetaldehyde. Some recently published papers19,20 have made an important contribution to assessing the authenticity of honey combining the honey volatile data from SDE with those of descriptive sensory analysis. The difficulty in finding volatile compounds exclusively in honeys from a specific botanical origin justified the use of sensory analysis to make this differentiation possible. Commercial Spanish honey samples (citrus, rosemary, eucalyptus, lavender, thyme, and heather) were extracted using a microscale SDE apparatus using dichloromethane as solvent; all extracts were analyzed using GC-MS equipped with a polar capillary column. Peak identifications were based on spectral data; about 100 volatile compounds were identified in the analyzed honey samples (Table 1) and 12 different sensory descriptors were defined. The statistical elaboration of the chemical and sensorial data allowed the authors to distinguish the different honeys. Citrus honeys were characterized by higher amounts of linalool derivatives, limonyl alcohol, sinensal isomers, and a-4-dimethyl-3-cyclohexene-1-acetaldehyde, together with fresh fruit and citric aroma descriptors; eucalyptus honeys by hydroxyketones (acetoin, 5-hydroxy-2,7-dimethyl-4-octanone), p-cymene derivatives, 3-caren-2-ol, and spathulenol, cheese and hay aromas; lavender honeys by hexanal, nerolidol oxide, coumarin, important concentrations of hexanol and hotrienol and balsamic and aromatic herb aromas; heather honeys by high contents of benzene and phenolic compounds and ripe fruit and spicy aromas.
4.05.2.2
Static and DHS Analysis
Static headspace (SHS) analysis has not been widely applied to honey fractionation21 because of the low concentration of volatiles in honey and the low recovery obtained for semivolatile compounds. In contrast, applications of the dynamic headspace extraction technique (DHS) have been reported in the literature.22–27 This technique affords main advantages such as a high sensitivity for fractionation of high volatility compounds, the absence of extended heating times, and the reproducibility associated with a totally automated system; qualitative results on honey volatiles by different authors24–27 are reported in Table 2. One of the first applications was carried out by Bouseta et al.23 in 1992. By purging the volatile compounds from the honey matrix with a stream of nitrogen gas and concentrating them into a cooled trap, the authors extracted and analyzed unifloral honeys from different countries. Although most of the aldehydes and alcohols identified were related to microbiological activity, heat exposure, and honey aging, some linear aldehydes with defined characteristic compounds were associated with certain floral origins. In 2001, using a DHS-GC-MS analysis method, Radovick et al.24 studied the volatile profiles of authentic unifloral honey samples from different countries. In particular, acacia, chestnut, eucalyptus, heather, lavender, lime, rape, rosemary, and sunflower honeys were considered. A purified helium (60 ml min1) flow was used for the extraction; the entrained volatiles were adsorbed on
Sampling Techniques for the Determination of the Volatile Fraction of Honey
Table 1 Compounds
89
Volatile compounds in unifloral honeys (extraction technique: SDE) Clover
(Z)-3-Hexen-1-ol 1,3-Dimethylpyrazole 1,3-Diphenyl-2-propanone 1-Cyclopentyl-2-propanone 1-Heptanol 1-Hexanol 15 1-Hydroxy-2-butanone 1-Hydroxy-2-propanone 1-Methoxy-4-propylbenzene 1-Nonanol 1-Pentanol 1-Phenylethanol 1-Phenylethanol þ 1-phenyl1,2-propanedione 1-p-Menthen-9-ol 2(3H)-Furanone 2,3-Dimethyl-2-cyclopenten-1-one 2,3-Dimethyl-4-isopropenyl-1cyclopentanone 2,3-Dimethyl-4-isopropyl-1cyclopentanone 2,3-Dimethylphenol 2,5-Hexanediol 2,6,6-Trimethyl-2-vinyltetraidropyrane 2-Acetylfuran 2-Aminoacetophenone 2-Cyclopentene-1,4-diol 2-Furaldehyde 2-Furancarboxilic acid 2H-Pyran-2-one 2-Hydroxy-3,5,5-trimethyl-2-cyclohexen1,4-dione 2-Hydroxy-5-methyl-3-hexanone 2-Hydroxyacetophenone 2-Hydroxycineol (isomer I) 2-Hydroxycineol (isomer II) 2-Methoxy-6-methylpyrazine 2-Methyl propanoic acid 2-Methyl-1-butanol 2-Methyl-2-buten-1-ol 2-Methyl-3-(2H)-dihydrofuranone 2-Methylbutanoic acid 2-Phenylethanol 2-p-Methene-1,8-diol 3,4,5-Trimethyl phenol 3,5,5-Trimethylcyclohex-2-en-1-one (isophorone) 3,5-Dimethylphenol 3-caren-2-ol 3-Formyl-pyridine 3-Hexanol 3-Hydroxy-2-butanone 3-Hydroxy-2-pentanone 3-Hydroxy-5-methyl-2-hexanone 3-Methoxybenzene ethanol 3-Methyl thiopropanal 3-Methyl-1-butanol 3-Methyl-2-buten-1-ol 3-Methyl-3-(2H)-dihydrofuranone 3-Methyl-3-buten-1-ol
Chestnut
Lime
16 16
16
16
16
Heather
Erica
20 17
17
17 20 20
16
17
Citrus
Rosemary
Thyme
Eucalyptus
18 18,20 20 20
19,20 19,20
20 20 20
20 20 20
20 20 20
20
19,20
20
20
20
20
20
20
20
20
20
17 20
16 16
Lavender
16 16 20 19,20
16 16
16 16 16
16
16
16 16 16 16
16 16 16 16 16
16
16
17,20 20 20 17 20
17
20
17
18 20
19,20
19 20
20 20
20 20 20
20 16
16
16
16
20
16 16
16
16 16
17,20 20 17,20 17,20 20 20 17
17 17 17
20 20 20 18,20 20 20
19,20 20 19,20 19,20
20 20 20 20
19,20
20
20 20 20 20 20 20
20 20 20 20 20 20
17
16 20 20
16 16
16 16
16
16
16
16
16
16
17,20 20
17
20 20
19,20 20
20 20
20 20
20 20 20
17,20
17
20 18
19,20
20
20
20 20
17,20
17
20
19 19,20
20
20
20 (Continued)
90
Extraction Techniques and Applications: Food and Beverage
Table 1
Volatile compounds in unifloral honeys (extraction technique: SDE)dcont’d
Compounds 3-Methylbutanoic acid 3-Methylfuranoate 3-Phenyl-1-propanol 3-Phenyl-2-propen-1-ol 3-Pyridine carboxaldehyde 4-(3-Oxo-1-butynyl)-3,5,5trimethylcyclohex-2-en-1-one 4-(3-Oxobut-1-enylidene)-3,5,5trimethylcyclohex-2-en-1-one 4-Butyl-1,3-cyclopentanedione 4-Ethyl-3,4-dimethyl-2-cyclohexen-1-one 4-Hydroxy-3-methoxybenzoate methyl ester (methyl vanillate) 4-Hydroxy-4-(3-oxo-1-butenyl)-3,5,5trimethylcyclohex-2-en-1-one (dehydrovomifoliol) 4-Methoxybenzaldehyde (p-anisaldehyde) 4-Methoxybenzoic acid (p-anisic acid) 4-tert-Butylphenol 5-(Hydroxymethyl)furfural 5-Ethenyl-5-methyl-2(3H)-furanone 5-Methyl furfural 5-Methyl-2-(3H)furanone 5-Methylfuraldehyde 5-Methylfurfural 6-Methyl-3,5-heptadien-2-one 8-p-Menthene-1,2-diol Acetic acid Acetophenone Acetyl furan Benzaldehyde Benzoic acid Benzoic acid hydrazone Benzyl alcohol Butanoic acid Camphor Caproaldehyde Car-2-en-4-one Carvacrol Cinnamic acid Cinnamyl alcohol cis-Linalool oxide Coumarin Decanoic acid Dimethyl disulfide Dimethyl trisulfide Dodecanoic acid Epoxylinalool Epoxylinalool (isomer I) Epoxylinalool (isomer II) Eptadecane Estragole Ethylmethylphenol isomer Eugenol Furfural Furfuryl alcohol Guaiacol Heptadecane Heptanal Heptanoic acid Hexanal
Clover
Chestnut
Lime
16
16
16
16
16
Heather
Erica
17 17 17 20 17 17
17 17 17
17
17
Lavender
Citrus
Rosemary
20
Thyme
Eucalyptus
20
17
16 16 17 17
17 17 16 16 16
16
17 20 17,20 20
17
19,20
20 20 20
20 20 20
20 20 20
20
19,20 19,20
20
20
20
20
19,20
20
20
18,20
19,20
20
20
18 20
19 20
20
20
20
19,20 20
20 20
20 20
20
20 18,20 20 20 20 20
19,20
20
20
20
19,20 20 19,20 19,20 19
20 20 20 20
20 20 20 20
20 20 20 20
20 20
20 20
20 20
20 20
20 20
17
20 20 18,20
19,20 19,20
20 20
20 20
20 20
19,20
20
20
17
18,20 18,20 18,20
20 20 20
17
20 18,20 20
19,20
15
15 15
15
16
16
16
16
16
16 16
16 16
16 16
16
16
20 17
17
17,20 17 17 17 17,20
17 17 17 17 17
20
15 15
15
20 17 17 20
17 17
16 16
17,20 20 20 20
17
20 20 20 16 16 15 15
16 16
16
16 16 16 16 16
20 20 17,20 20
20 17
Sampling Techniques for the Determination of the Volatile Fraction of Honey
Table 1
91
Volatile compounds in unifloral honeys (extraction technique: SDE)dcont’d
Compounds Hexanoic acid Hotrienol Indole Isophorone Ketoisophorone Ketoisophorone (isomer I) Ketoisophorone (isomer II) Lilac alcohol (isomer I) Lilac alcohol (isomer II) Lilac alcohol (isomer III) Lilac alcohol (isomer IV) Lilac aldehyde (isomer I) Lilac aldehyde (isomer II) Lilac aldehyde (isomer III) Lilac aldehyde (isomer IV) Limonyl alcohol Linalool Menthol Methyl antranilate Methyl(1-methylethenyl)-benzene Methylfuran m-Xylene Nerolidol Nerolidol oxide Nonadecane Nonanal Nonane Nonanoic acid Octanal Octane Octanoic acid p-Anisaldehyde p-Cresol p-Cymen-8-ol (isomer I) p-Cymen-9-ol p-Cymene Pentadecane Pentanoic acid Phenethyl alcohol Phenol Phenylacetaldehyde Phenylacetic acid p-Mentha-(7),8-(10)-dien-9-ol p-Methylacetophenone Propylanisole Pyridine Sinensal (isomer I) Sinensal (isomer II) Spathulenol Terpineal Tetradecanoic acid Thymol Toluene trans-Caryophyllene trans-Linalool oxide Tetradecanoic acid Trimethoxybenzene (isomer) Vinylguaiacol a-4-Dimethyl-3-cyclohexen-1acetaldehyde
Clover
Chestnut
16
Lime
Heather
16
20 20 17 20
16
Erica
Lavender
Citrus
Rosemary
Thyme
Eucalyptus
18,20 20
19,20 19,20
20 20
20 20
20 20
20
19,20 19 20 20 19,20 19,20 19,20 19,20 19,20 19,20 19,20 19,20 19,20 19,20
20
20
20
20 20 20 20 20 20 20 20 20 20
20 20 20 20 20 20 20 20 20 20
20 20 20 20 20 20
20
20
20
20 19,20
20 20
20 20
20 20
19,20
20
20
20
19,20
20
20
20
20
20 19
20
20
20
20 20
20
20
20
20 20
18,20
19,20
20
20
20
19,20
20
20
20
17
20 20 20 20 20 20 20
20 20 20 20 20 20
20
20
16 19,20 16
16
15 15 19,20 20 20 16
15
16 16
16
15 15 15
16
16 16
16 16 16
16 16 16
16
16
16
16
17,20
17
17,20 20 20 20
17
20 20 17 17,20 17
17 17
20 20 18,20 18 20 18 18 20
20 18 19,20 19,20 20
16 16
16 16
20 20 17
20 20
19 19,20 19,20
20 20
20 20
20 20
20 20
20 20
17
15
16
19,20 19
20
19,20 20
20 20
16 20
20 20
(Continued)
92
Extraction Techniques and Applications: Food and Beverage
Table 1
Volatile compounds in unifloral honeys (extraction technique: SDE)dcont’d
Compounds a-Humulene a-Pinene a-Terpineol b-Damascenone b-Pinene g-Butyrolactone g-Terpinene g-Valerolactone
Clover
Chestnut
Lime
15
16 16
16 16
Heather
Erica
20 20 15
16
16
16 16
16 16
17,20
17
17,20
17
Lavender
Citrus
Rosemary
Thyme
Eucalyptus
20 20
19,20 19,20
20 20
20 20
20 20
20
19
20
20
20
19,20
20
15. Bouseta, A.; Collin, S. J. Agric. Food Chem. 1995, 43, 1890–1897; 16, Guyot, C.; Bouseta, A.; Scheirman V.; Collin, S. J. Agric. Food Chem. 1998, 46, 625–633; 17, Guyot, C.; Scheirmann, V.; Collin S. Food Chem. 1999, 64, 3–11; 18, Guyot-Declerck, C.; Renson, S.; Bouseta, A.; Collin, C. Food Chem. 2002, 79, 453–459; 19, Castro-Vázquez, L.; Díaz-Maroto, M.C.; Pérez-Coello, M.S. Food Chem. 2007, 103, 601–606; 20, Castro-Vázquez, L.; Díaz-Maroto, M.; González-Viñas, M.; Pérez-Coello, M. Food Chem. 2009, 112, 1022–1030.
a porous polymer resin, then thermally desorbed at 280 C under a helium flow, cryofocused in a glass lined tube at 120 C with liquid nitrogen, and finally injected into the GC capillary column. GC-MS analysis of the honey headspace was performed on a polar capillary column in a temperature gradient. A large number of volatiles were identified and the existence of certain marker compounds for the floral origins was assessed; e.g., the presence of either 2-methyldihydrofuranone or a-methylbenzyl alcohol or both 3-hexen-1-ol and dimethylstyrene were indicated for chestnut honeys (Table 2). Subsequently Bianchi et al.25 proposed a DHS extraction method coupled with GC-MS analysis as a valid alternative to pollen analysis for floral source detection, especially for products such as strawberry-tree honey characterized by low pollen content. Strawberry-tree (Arbutus unedo L.) honey samples with a certified floral origin, produced in Sardinia, were analyzed together with eucalyptus, heather, lavender, and thyme honey purchased from a local store. The method was similar to that reported above24 although a reduced amount of honey sample was used (1.5 g in a 50-ml round-bottomed flask at 40 C). A total of 28 aroma compounds were identified (Table 2), but only norisoprenoid compounds such as a-isophorone, b-isophorone, and 4-oxoisophorone, were recognized as markers of specific floral origin. The application of multivariate statistical analysis to the DHS-GC-MS data was first reported by Soria et al.26 in 2008; the authors demonstrated that the statistical elaboration of the volatile data was very promising to classify samples according to their botanical origin. Eucalyptus, thyme, citrus, rosemary, heather, lavender, and multiflower honeys were considered; the concentration of the honey solutions and temperature and time of purge were optimized to maximize the amount of volatiles. Volatiles swept by the helium flow were collected at ambient temperature on a porous polymer resin based on 2,6-diphenylene oxide, thermally desorbed at 220 C for 5 min and then concentrated at the end of a fused silica capillary transfer line, indirectly cooled with liquid nitrogen; the capillary transfer line and valves were heated to 200 C in order to avoid volatile compound condensation; chromatographic separations were carried out on a polar capillary column operating in a temperature gradient with helium as carrier gas. Qualitative analysis was based on spectra data and linear retention indices (LRI) while quantitative data were obtained using 5-nonanone as internal standard. One hundred volatile compounds (Table 2), including terpenes derived from the floral nectar, furan derivatives from honey processing and storage, and other compounds whose origin could be related to microbial or environmental contamination, were identified. Following discriminant analysis (DA), eucalyptus honeys were characterized by the amount of octane and diketones while lilac aldehydes were the most characteristic compounds of citrus honeys. An interesting application of DHS extraction was reported by Escriche et al.27 in 2009 with the aim of verifying if the honey volatile fraction was affected by the industrial thermal treatment processes. Four types of Spanish honey, i.e., citrus, rosemary, polyfloral, and honeydew, were studied. Each honey sample was analyzed untreated, liquefied (at 45 C for 48 h) and pasteurized (at 80 C for 4 min). Volatile compounds were extracted using purified helium as the stripping gas (30 g samples in a purging vessel flask at 45 C for 45 min) and volatile compounds were trapped on a porous polymer resin and subsequently thermally desorbed under a helium flow at 220 C for 16 min. The volatiles were then cryofocused in a cold trap at 30 C and transferred directly onto the head of the capillary column by heating the cold trap to 250 C. Volatile compounds were identified by mass spectra and LRI; quantitative results were obtained using camphor as internal standard. The volatile compounds identified (Table 2) allowed the authors to classify the honey by their botanical origin and to establish a clear differentiation between honeydew and nectar honey. Moreover, the authors demonstrated that honey type had a greater influence on the volatile fraction than heat treatment (liquefaction and pasteurization) under moderate industrial conditions.
4.05.2.3
SPME
SPME eliminates problems associated with DHS extraction while retaining the advantages: solvents are completely eliminated and extraction time can be reduced to a few minutes.28 In recent years, some efforts have been made to analyze honey volatiles using the SPME technique29 and different types of SPME fibers have been evaluated taking into consideration the polarity of fiber coatings and fiber coating film thickness. In addition, operating conditions that have a significant influence on the headspace equilibrium
Table 2
Volatile compounds in unifloral honeys (extraction technique: DHS)
Compounds
Rosemary
Strawberry
Citrus
Eucalyptus
Multiflower
Lavender
Heater
1-(2-Furanyl)-ethanone 1,3,8-Menthatriene 1-Butanol 1-Hexanol 1-Hydroxy-2-propanone 1-Methoxy-2-propyl acetate 1-Nonanol 1-Octen-3-ol 1-Octene 1-Pentanol 1-Penten-3-ol 2,2,6-Trimethylcyclohexanone 2,3,4-Trimethyl-2-cyclopenten-1-one 2,3-Butanedione 2,3-Dihydro-4-methylfuran 2,3-Heptanedione 2,3-Pentanedione 2,4,4-Trimethylcyclopentanone 2,5-Dimethylfuran 2,6,6-Trimethyl-2,4-cycloheptadien-1-one 2,6,6-Trimethylcyclohexan-1,4-dione 2,6-Dimethyl-4-heptanol 2- and 3-Methylbutanoic acid 2-Acetylfuran 2-Allyl-4-methyl phenol 2-Butanol 2-Butanone 2-Butenenitrile 2-Butoxyethanol 2-Cyclohexene-1-one 2-Decanone 2-Ethylfuran 2-Ethylhexanoic acid 2-Heptanone 2-Hexanol 2-Hydroxy-3-pentanone 2-Hydroxy-5-methyl-3-hexanone 2-Methyl- and 3-methyl-2-buten-1-ol 2-Methyl-1-butanol 2-Methyl-1-propanol 2-Methyl-2-butenal 2-Methyl-2-propanol
26
25
26,27
26
26
26
26
24,26
25
27 27 27
26
24
24 24 24
Rape
Sunflower
Acacia
Thyme 26
24,26 24,26
24,26 26
24
24
24
26
26 24 26 24
24,26 24 24,26
26
27
25 25
25 25 25
26
24 26
26
26
26
26
26,27 26
26 24,26 26 24,26
26,27 26
26 24,26
26 24,26
26 26
26
26
26
26
26
26
26 24
26
26 26
26 24
26
24 26
26
24 26,27 24,26
25
26 26 26
26 24,26
26 26 26
27
24
24 24
24 24
26 24,26
26 24,26
24
24 24 24
24 24
24
24
24
24
24
26 26
24 24 26 24
27 26
26
24,26 26 24 26
26 26,27 26 27
26 24,26 26
24
26 24,26 26,27 27
25
26
26
26
26
26 26,27 26,27 26,27 27
26 24,26 26
26 26 24,26 26
26 26 26 26
24
24
24
24
93
(Continued)
Sampling Techniques for the Determination of the Volatile Fraction of Honey
26 24
24,26 26
Lime
27
26
26,27 24,26 26
26
Chestnut
94
Table 2
Volatile compounds in unifloral honeys (extraction technique: DHS)dcont’d Rosemary
2-Methyl-3-buten-2-ol 2-Methylacetophenone 2-Methylbutanal 2-Methylbutanenitrile 2-Methyldihydrofuranone 2-Methylfuran 2-Methylhexanoic acid 2-Methyl-2-buten-1-ol 2-Methylpropanenitrile 2-Methylpropanoic acid 2-Pentanol 2-Pentanone 2-Propanol 3-(1-Methylethyl)-2-cyclopenten-1-one 3,5,5-Trimethylcyclohexan-1,4-dione 3-Aminoacetophenone 3-Butenenitrile 3-Cyclohexen-1-ol-5-methylene-6-isopropylene 3-Furancarboxaldehyde 3-Hexen-1-ol 3-Hexen-2-one 3-Hexenylformate 3-Hydroxy-2-butanone (acetoin) 3-Hydroxy-2-pentanone 3-Methyl-1-butanol 3-Methyl-1-undecene 3-Methyl-2-butanol 3-Methyl-2-butanone 3-Methyl-2-buten-1-ol 3-Methyl-2-butenal 3-Methyl-2-pentanone 3-Methyl-3-buten-1-ol 3-Methylbutanal 3-Methylbutanenitrile 3-Penten-2-one 4,5,7a-Hexahydrobenzofuran-3,6-dimethyl-2,3,3a 4-Acetyl-1-methylcyclohexene 4-Ethylphenylacetate 4-Methyl-1-pentanol 4-Methylacetophenone 4-Oxoisophorone (3,5,5-trimethylcyclohex-2-en-1,4-dione) 5-Methyl-1-hexanol Dihydro-5-methyl-2(3H)-furanone 5-Methylfurfural
26,27
Strawberry
Citrus
Eucalyptus
Multiflower
Lavender
Heater
26,27
26
26
26
26
24,26 26
26 26
24,26 26
26 26
24,26
24,26 26
26 27
26
26
26
26
26
26,27 26
26
26
26
Chestnut
Lime
24
24 24
Rape
Sunflower
Acacia
Thyme 26
24
24
24
26 26
24
26
24
26
24
26
24 26 24 24
26
26 24 24
26
24,26 24 24
24,26 24 24
26 24 24
24 24
24 24
24 24
24 24
25 25 26
26
24 26
24 26
26
26 24
25 24 26
24
27
26 24 24
24
24
24
26,27
24,26
24,26
24 24
24
26
26
26
26
24 24 24,26,27
27
24 24 24,26
25
26
26 24
24
24
26
24
24
24
26
24 24
26 26 26 26
24 24 26 27
27 26 24,26,27 24,26 26
26,27 26 26 26
27 24,26 24,26 26 26
26,27 26,27 26 26
24,26 24,26 26
24,26 24,26 26 26
24 24
24 24
24 24
24 24
24 24 24 24,26
26
25
26
24,26
26
24 24
24,26
26
26
24,26
24
24 24,26
24 24
24 24
24
24
Extraction Techniques and Applications: Food and Beverage
Compounds
24 26,27 24,26 26 24 26,27 27 24,27
25 25
25
26,27 26,27 26 26,27 27
26 24,26 26 26 24,26
27 26,27 26 26
24
26
26
24 26 24,26 26 24,26 24,26 24
24
24
24
24
24,26
24
24
24
24
24
24 26
24 24
24 24
24 24
24
24 24
24
24
24 24
24
24
24
26
26 26 26
26 24
26 24,26
26 26
24,26
26 26
26 26
26 26
27 26 26
24 26 26 26
26
26
26,27 27 27 24,26,27 26
24,27 26 26
25
26 26
24,26
24,26 24,26 24 24 26 26
24 24
26 26
24,26 26 24 24 26 26
24,26
26,27
26
24,26
24 24
26,27 27
26
26,27 27
26
26
26,27 26
24,26 26
26,27 26
24,26 24,26
26
26
26
24,26 24,26 24 26
26 26,27
26 24,26 24 26 24 26
26 26,27
26
24 24
24
24 24
24 24
24 24
26 26
24
24
24
24 26 26
25
25
26 26 24,26,27 26 24 26 27 26
25
25 26
26,27 26 27 26,27 26,27
24,26 24
26 24,26 24
26 24 26 24,26 24
24 24
24
26 26
24 24
24
24
24 24
24 24
26 26
24 24
24 24
24
24
24
24
24 24
24
26 26
26
26,27
26 26,27
24 26
26
24,26 24
24,26 24
24 26
24 24
24 24
24 24
24 24
24 24
26
26 26
95
(Continued)
Sampling Techniques for the Determination of the Volatile Fraction of Honey
6-Methyl-5-hepten-2-one Acetic acid Acetone Acetonitrile Acetophenone Benzaldehyde Benzene acetaldehyde Benzyl alcohol Bicyclo-3,2,1-octane-2,3-bis(methylene) Butanal Bicyclo 2,2,2-octan-1-ol-4-methyl Carbon tetrachloride Chloroform cis-Linalool oxide cis-Linalool oxide (furan ring) Cyclohexane Cyclopentenedione Decanal Dichloromethane Dihydro-2-methyl-3(2H)-furanone Dihydro-5-methyl-2(3H)-furanone Dihydroisophorone (3,3,5-trimethylcyclohexanone) Dimethyldisulfide Dimethylstyrene Dimethylsulfide D-Limonene Dodecane Ethanol Ethyl acetate Ethyl-2-hydroxypropanoate Ethylbenzene Ethylbenzoate Furan Furfural Furfuryl alcohol Geraniol Heptanal Heptane Hexanal Hexanoic acid Hotrienol (3,7-dimethyl-1,5,7-octatrien-3-ol) Hydroxyacetone (acetol) Isobutyl acetate Isopropyl myristate Lilac aldehyde (isomer I) Lilac aldehyde (isomer II)
96
Table 2
Volatile compounds in unifloral honeys (extraction technique: DHS)dcont’d
Compounds
Strawberry
Citrus
Eucalyptus
26,27 26,27
Multiflower
Heater
Chestnut
Lime
Rape
Sunflower
Acacia
26,27 26,27 24
27
Lavender
27 27
Thyme 26 26
24
24
24
24
27 24
27 26 24 24 26 26 26
25
24,27 24 26,27 26
25 25
26 26
26 27
26 26,27 26 26
24
26
24 24 26
24 24 26
24 24
24 24
24 24
24 24
24 24
26,27
26
26
26,27
26 26 24,26
26
26,27 26
26 26
26,27 26
24 24 26
26 27 26 26 26
24,26 24
26 27 26 26 26
24 24,26 24
24 24 26 26 24 24,26 24
26
26 26
26 26 26
26 26 26
26 26 26
26 26 26
26 26 26
24 24
24
24,26
24
24
24 24
26,27 26 26 26
25
24 24 26
26 26 26 26 26 26
26 26 24 24
24 24
24 24
24 24
24 24 26 26
24
24
24
24
24
24
24
24
24
24
26
24 24
24 24
24 24
24
24
24 24 24 27 25 27 24
24
24
24
24, Radovic, B.S.; Careri, M.; Mangia, A.; Musci, M.; Gerboles, M.; Anklam, E. Food Chem. 2001, 72, 511–520; 25, Bianchi, F.; Careri, M.; Musci, M. Food Chem. 2005, 89, 527–535; 26. Soria, A. C.; Martínez-Castro, I.; Sanz, J. Food Res. Int. 2008, 41, 838–848; 27. Escriche, I.; Visquert, M.; Juan-Borrás, M.; Fito, P. Food Chem. 2009, 112, 329–338.
Extraction Techniques and Applications: Food and Beverage
Lilac aldehyde (isomer III) Lilac aldehyde (isomer IV) Linalool (3,7-dimethyl-1,6-octadien-3-ol) Linalool oxide Methyl antranilate Methyl isopropyl benzene Methyl salicylate Methyl-1,3-pentadiene Methyl-2-butenale Methyl-2-butenol Methylcyclohexane Methylnaphthalene (isomer not identified) m-Xylene Nerol Nonanal Octanal Octane o-Xylene Pentanal Phenylacetaldehyde Phenylethyl alcohol p-Menth-1-en-9-al (isomers I and II) Propanenitrile p-Xylene Terpinen-4-ol Toluene trans-Linalool oxide (furan ring) Trichloroethylene a,a-Dimethylbenzyl alcohol a-4-Dimethyl-3-cyclohexe-1-acetaldehyde a-Cyclic ether a-Isophorone (3,5,5-trimethyl-2-cyclohexen-1-one) a-Methylbenzyl alcohol (sec-phenethyl alcohol) a-Methylbutenol a-Methylpropyl phenyl acetate a-Pinene a-Pinene oxide a-Terpinene b-Damascenone b-Isophorone (3,5,5-trimethyl-3-cyclohexen-1-one) b-Linalool g-Butyrolactone
Rosemary
Sampling Techniques for the Determination of the Volatile Fraction of Honey
97
and on fiber absorption capacity, such as vial size, salt addition, magnetic stirring, equilibrium, and extraction time and temperature, plus GC injector desorption time were taken into consideration. By using SPME-GC-MS methods, an important number of volatile compounds have been found as possible markers of different types of honeys since most of these are related to the botanical origin.30–39 Moreover, the application of SPME has allowed the authenticity of honey to be verified by the presence of extraneous substances.40–45 Recently, interesting perspectives for improving the study of honey volatiles have arisen from SPME sampling followed by comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC GC-TOF-MS) analysis; important information on honey traceability has been obtained.38–40
4.05.2.3.1
SPME for Floral and Geographic Determination
As previously mentioned, volatile compounds are related to the botanical and geographical origin of honey and many can be considered as reliable markers. In this context, by using SPME-GC-MS methods, an important number of organic compounds have been found as components of unifloral honeys coming from different countries; among these, the Spanish honeys are the most studied.30–32,36,37 The first applications of SPME for the extraction of honey volatile constituents were reported by Verzera et al.33 and Piasenzotto et al.31 who studied Italian unifloral honeys from different regions. These authors were able to optimize their SPME-GC-MS methods (Table 3) so that the identification of a large number of volatile constituents became possible (Table 4). The two methods differed mainly in the extraction temperature, which was lower in the method developed by Verzera et al.,33 and the selected fibers; Verzera et al.33 found better results with a polydimethylsiloxane/divinylbenzene (PDMS/DVB) fiber and Piasenzotto et al.31 used a polyacrylate (PA) fiber. In the unifloral Sicilian honeys,33 more than 100 compounds were identified which belonged to the following classes of substances: aliphatic and aromatic compounds, acyclic and monocyclic monoterpenes and their oxygenated derivates; furan derivates; sulfurated and nitrogen-containing compounds. From the data obtained, the authors33 identified borneol and dihydrocarveol as markers for eucalyptus honey along with a high amount of nonanol, nonanal, nonanoic acid, 5-hexen-2-ol, and 2,3-dimethyl-5-hexen-2-ol, whereas acetophenone, 2-aminoacetophenone and 1-phenylethanol were markers for chestnut honey. Sulla honey was characterized by high levels of hexanol, hexanoic acid linalol, nonanal, terpinen-4-ol, and a-terpineol. With regard to the unifloral Spanish honeys, one of the first SPME-GC-MS methods was developed by Perez et al.36 for the characterization of floral origin. The most significant aspects of the SPME technique, such as sampling, fiber, equilibration time, etc., were considered in order to optimize the method. Two SPME fibers were used and the best results were obtained with the carboxen/ polydimethylsiloxane (CAR/PDMS) fiber, using a homogenization time of 1 h at 70 C and a sampling period of 30 min (Table 3), The analyses were performed using GC-MS and volatiles were separated using an apolar capillary column with helium as carrier gas. A total of 35 compounds were detected, most of them identified by GC-MS (Table 4) and quantified using external standards. In 2003, Soria et al.32 considered some Spanish unifloral honeys when developing an SPME-GC-MS method for simple and rapid analysis of honey volatile compounds (Table 3). In this case, SPME fiber coatings of different polarity, the influence of sample temperature, equilibrium and exposure time, magnetic stirring, and salt addition on amount of volatiles were considered. GC-MS analyses were performed on a polar column operating in a temperature gradient, with helium as carrier gas. The authors proposed the use of a PA fiber (32 compounds identified) for distinguishing samples of different types and a CAR/PDMS fiber (24 compounds identified) for the characterization of honeys that were poor in volatile components (Table 4). Principal component analysis (PCA) using the amount of each volatile compound expressed as peak areas was considered essential for definition of the botanical origin. The same authors37 (Table 3) applied their method to a large number of honeys of different botanical origin and some honey blends. More than 100 volatile compounds were identified in all the samples analyzed and some of them were reported as possible markers. For example, high levels of lilac aldehydes, lilac alcohols, cis-linalool oxide, benzaldehyde, phenylacetaldehyde, p-menth1-en-9-al, p-menth-1-en-9-ol, hotrienol, and particularly p-menth-1(7),8(10)-dien-9-ol, were found for unifloral citrus honey; high contents of 2,6,6-trimethyl-2,4-cycloheptadien-1-one, 3,5,5-trimethylcyclohex-2-ene-1-one (isophorone), and 4-oxoisophorone were found for rosemary honey, whereas the high nitrile content (2-methylpropanenitrile, 2-methyl butanenitrile, 3-methyl butanenitrile, 2-butenenitrile (cis- or trans-isomers), 3-butenenitrile, 3-methylpentanenitrile, and benzeneacetonitrile) proved to be distinctive for dandelion honey. Following this research, de la Fuente et al.30 applied the method previously developed by Soria et al.32 (Table 3) to study the volatile composition of widely used Spanish honeys, such as eucalyptus, rosemary, heather, and citrus. GC-MS profiles of honey volatiles were complex and 83 volatile compounds were identified using MS and retention data (Table 4). Due to the lack of reference samples and the presence on the market of mixed honey types, a statistical elaboration processing the relative concentrations of volatiles was applied. PCA was used as a first step to indicate the existence of possible natural groups followed by stepwise discriminant (SDA) and regression analyses. By the statistical elaboration, the authors provided valid information for the characterization of eucalyptus and citrus samples. The SPME-GC-MS methods discussed above used conventional one-dimensional GC. which, even if typical analysis times of 30–90 min were required to achieve acceptable chromatographic resolution, can lead to coelution of volatile constituents. In this context, multidimensional gas chromatography (MDGC) represents a possible solution to obtain efficient separation of the entire sample. This analytical approach was first applied by Cajka et al.38 in 2007; then Cajka39 and Starimirova40 demonstrated the potential of this challenging technique for its application in various follow-up studies including traceability of honey origin and authentication. The authors developed an SPME-based procedure for the isolation of honey volatiles followed by their separation/ detection/identification by means of the GC-GC-TOF-MS technique. The authors analyzed more than 300 honey samples from
98
SPME methods: operation conditions for honey volatile extraction Equilibrium Extraction temperature Equilibrium temperature Extraction ( C) time (min) ( C) time (min)
Desorption temperature ( C)
Desorption time (min)
Splitless/split after 2 min
250
2
20
Splitless/split after 3 min
250
3
60
30
Splitless/split after 2 min
250
2
30
30
25
Splitless
220
3
40
60
40
30
Splitless
220
5
DVB/CAR/PDMS 50/30 mm
–
-
40
20
Splitless/split after 3 min
250
CAR/PDMS, PDMS/DVB
70
60
70
30
Splitless
Addition of 1 ml CAR/PDMS of water 75 mm; PA 85 mm
60
15
60
30
Splitless/split after 2 min
PDMS/DVB at 250, CAR/ PDMS at 270 250
Reference Floral source
Honey Sample provenance size (g)
Vial size (ml)
Sample treatment
Fiber type
30
Spain
1.5–2.0
5
Diluted with 1 ml water
CAR/PDMS 75 mm
60
15
60
30
Italy
3
10
Added to 0.5 g sodium sulfate
PDMS 100 mm, 70 PA 85 mm, CAR 75 mm
30
70
Spain
1.5–2
5
Addition of 1 ml CAR/PDMS 75 mm; 60 of water PA 85 mm
15
Italy
16
40
30
Poland
1–1.5
4
PDMS/DVB Diluted with 65 mm 7 ml of water, added to 2 g NaCl – PDMS/DVB 65 mm
Croatia
3.00
50
Spain
1
4
1.5–2.0
5
31
32
33
34
35
Eucalyptus, rosemary, heather, citrus Citrus, chestnut, eucalyptus, lime tree, thyme, dandelion Rosemary, heather, honeydew, orange blossom, lavender, multiflower Orange, sulla, chestnut, eucalyptus, wild flowers Multifloral, heather, buckwheat, limehoneydew Fir-honeydew, sage
36
Orange, eucalyptus, rosemary, lavender, thyme
37
Eucalyptus, citrus, Spain rosemary, thyme, lavender, chestnut, dandelion, rhododendron, Teide broom, acacia, heather, honeydew, multiflower
Added to 0.5 g sodium sulfate –
Injection mode
5
2
Extraction Techniques and Applications: Food and Beverage
Table 3
41 42
Chestnut Italy Nectar and honeydew Spain
20
43
Thyme
–
8
10
44
Dandelion
Italy
1.5–2
5
45
Chestnut, fir, acacia, Pyrenees, orange, lavender, eucalyptus, forest, oak
France, Italy,1 Hungary, Spain
10
50
Lavender
Croatia
-
15
51
Christ’s thorn
Croatia
-
15
52
Desert false indigo
Croatia
-
15
PDMS 100 mm Diluted with CAR/PDMS 1 ml of water 75 mm then sonicated for 5 min Diluted with PDMS 100 mm 3,5 ml of water, added to 1 g NaCl Addition of 1 CAR/PDMS ml of water 75 mm
– 60
– 15
45 60
20 30
Splitless Splitless
250 250
0.4 2
30
30
30
25
–
250
3
60
15
60
30
250
2
Diluted with PDMS 100 mm, 4 ml of water, PA 85 mm, CW/ added to 1 g DVB 70 mm, NaCl and CAR/PDMS 0.2 ml of 75 mm, PDMS/ acetic acid DVB 65 mm
–
–
–
30
Splitless/split valve opening at 2 min Splitless
2
5 ml of honey– DVB/CAR/PDMS NaCl sat. H2O solution 1:1 v/v 5 ml honey– CAR/DVB, DVB/ NaCl sat. H2O CAR/PDMS solution 1:1 v/v 5 ml honey– PDMS/DVB NaCl sat. H2O solution 1:1 v/v
60
15
60
40
Split
300 for PA and CAR/ PDMS; 250 for PDMS, CW/DVB, PDMS/ DVB 250
60
15
60
40
Split
250
6
60
15
60
40
Split
250
6
Abbreviations: CAR, carboxen; DVB, divinylbenzene; PA, polyacrylate; PDMS, polydimethylsiloxane.
6
Sampling Techniques for the Determination of the Volatile Fraction of Honey
1 1.5–2.0
99
Volatile compounds in unifloral honeys (extraction technique: SPME)
Compounds
Sage
Rosemary
Orange
Lime
Lavender
Heather
Eucalyptus
Buckwheat
Chestnut
Dandelion
Citrus
Sulla
Wild flower
33 33 33 33
33 33 33 33
33 33
33
33
33
33
33
33
33 33
33
33
33
33
33
a
32 33 33 33 35
33
35
34
34 33
35 30,32a,32b
32a,32b
35 35
32a,32b
30,32a,32b,34
30
34 34
30
35 33 33 33
36
30,32b 32a,36
33 33 32b 32b,36
32b,36 32b,36
30,32b 32b
32a
32a,34
33 30 36
33 30
35 35 31
32a
33 32a
33 31 33
34
33 31 33
30
30
30
30
32a 31 36
31,35 36 30 30 30
36
36
34 34 30 30 30
34
31,36 30 30 30 33 30
34 34
31
30
Extraction Techniques and Applications: Food and Beverage
(Dimethylphenyl)-ethanone (E)-2-Octenal (E)-2-Pentenol (E)-2-Undecenal (E)-3-Hexenol (E)-6,10-Dimethyl-5,3undecandien2-one (E)-Cinnamaldehyde (Z)-2-Pentenal (Z)-2-Pentenol (Z)-3-Hexenol 1-(2,4-Dimethylphenyl)ethanone 1-(2-Aminophenyl)-ethanone 1-(2-Furanyl)-ethanone 1-(2-Hydroxy-5-methylphenyl) ethanone 1-(4-Methylphenyl)ethanone 1,3,5,7-Cyclooctatetraene 1,3,8-p-Menthatriene 1,3-Butandiol 1,4-Cineole 1,8-Cineole 1-Hexanol 1-Hydroxy-2-propanone 1-Methyl-4-(1-methylethyl) benzene 1-Methyl-4-benzene 1-Methylethyl benzene 1-Nonanol 1-Octen-3-ol 1-Phenylethanol 1-Propyne 2- and 3-Methyl-2-buten-1-ol 2(H)-1-Benzopyran-2-one 2-(p-Methoxyphenyl)ethanol 2,2,4-Trimethyl-pentane 2,3-Butanediol 2,3-Butanediol (erythro) 2,3-Butanediol (threo) 2,3-Butanedione 2,3-Dimethyl-5-hexen-2-ol 2,3-Pentanedione 2,4,5-Trimethyl cumene
Thyme
100
Table 4
33 36
36
34 34
36
34 34
34 33 33
33
33
33
33
33
33
33
36 36 33 32a 30,36
36
32a
35
32a 36
32a 30,34
30
34
30
35 33
33
34 30
30
30
30
33 34
33 34
33 33 36 36 36
36
34
36
31,36
33
34
33
33
31
36 36
31,36
34 34
36 35 35 35 35
36
34 34
35 30,32a,32b,36
32a,32b,36
31 35
32a,32b,36
30,32a,32b,34
36
34 34
34 34 33 30,31,36
34
33 31
31
30,31
34 34
101
(Continued)
Sampling Techniques for the Determination of the Volatile Fraction of Honey
2,5-Dimethyl-3-hexanone 2,5-Furandicarboxaldehyde 2,6,6-Trimethyl-2-cyclohexene1, 4-dione 2,6-Dimethoxy-phenol (syringol) 2,6-Dimethyl-3,7-octadien-1,6diol 2,6-Dimethyl-3,7-octadien-2,6diol 2-Acetyl benzoic acid 2-Aminomethyl benzoate 2-Butanol 2-Decanol 2-Decanone 2-Ethyl hexanol 2-Furan methanol 2-Furanocarboxaldehyde 2-Heptanone 2-Hydroxy-3,5,5trimethylcyclohex-2-enone 2-Hydroxy-3,5,5trimethylcyclohexanone 2-Hydroxy-ethyl benzoate 2-Methyl butanoic acid 2-Methyl propanoic acid 2-Methyl,dihydro-2(3H)furanone 2-Methyl-1-butanol 2-Methyl-1-butanol 2-Methyl-1-propanol 2-Methyl-2-buten-1-ol 2-Methyl-2-butenal 2-Methyl-3-buten-2-ol 2-Methyl-3-phenyl-2-propenal 2-Methyl-6-(2-propenyl)phenol 2-Methylbenzene 2-Methyl butanal 2-Methyl propanoic acid 2-Nonanone 2-Phenylethanol 2-Xylylethanol 3,4,5-Trimethyl phenol 3,5,5-Trimethyl-2-cyclohexen-1one
102
Table 4
Volatile compounds in unifloral honeys (extraction technique: SPME)dcont’d
Compounds
Sage
Rosemary
Orange
Lime
Lavender
Heather
Eucalyptus
Buckwheat
Chestnut
Dandelion
Citrus
Sulla
Wild flower
34 36 31
31 33 35
36
30,32b,36
32b,36
32b,36
30,32b 30
30,31,36 30
36 36
30,32b,36
32b,33
32b
30,32b,34
30,33,36
32a,32b,36
36 32a,32b,36
31
34
33
30
30
33 33
32a,32b,36
36
30
32a,32b 34 30
33
31
33
31
33
34 30 33,36
30 33
33
33
33
33 33
34 35
35 35 35
35
31
31
31
31
36
35 35 35 31 35
31
35 33 35 35
34
34
Extraction Techniques and Applications: Food and Beverage
3,5-Dimethoxy benzaldehyde 3,7-Dimethyl-1,5,7-octatrien3-ol 3,9-Epoxy-p-menth-1-ene 3-Aminoacetophenone 3-Furanocarboxylicacid methyl ester 3-Hexanol 3-Hydroxy-2-butanone (acetoin) 3-Hydroxy-2-pentanone 3-Methyl butanal 3-Methyl-1-butanol 3-Methyl-1-hexanol 3-Methyl-2-buten-1-ol 3-Methyl-3-buten-1-ol 3-Methyl butanal 3-Methyl butanenitrile 3-Methyl butanoic acid 3-Phenyl-2-propen-1-ol 3-Pyridine carbonitrile 4-(1-Methylethyl)benzaldehyde 4-(1-Methylethyl) benzeneethanol 4-(3-Hydroxy-1-butenyl)-3,5,5trimethyl-2-cyclohexen-1-one 4-(3-Oxo-1-butenyl)-3,5,5trimethyl-2-cyclohexen-1-one 4,5,6,7-Tetrahydro-3,6dimethyl-benzofuran 4,7-Dimethyl-benzofuran 4-Hydroxy-4-methyl-2pentanone 4-Hydroxybenzenemethanol 4-Ketoisophorone 4-Methoxybenzaldehyde 4-Methyl,dihydro-2(3H)furanone 4-Methyl-1-(1-methylethyl)-3cyclohexen-1-ol 4-Methyl-1-(1-methylethyl)bicyclo[3.1.0]hexene-2-one 4-Methyl-1-pentanol 4-Methyl-3-pentenol 4-Methyl phenol
Thyme
30
33
35
30
30,33 33 31
33 33
30 31
35 33
33 31
33
33 31
31 35 36 36
33,36 36
36 36
34 34 32a 34
36 36
34
33
34
33 31
34 34
31,33
31
31,36 36
35 35 35
33
31
33
36 36
33
30,32a,32b,36 36
32a,32b,33,3631,35 36 35
32a 36
34 30,32a,32b,34 30,34 34
30,31,33,36 30,36
33
31
30 30
31
30
33
33
33
33
33 33
33 33
33
33
33 35
35 35
35 35
35 30,32a,32b
31
35 35
33 32a,32b,33
35 31
33
35 35
32a,32b
34 30,32a,32b,34 34
33 30,31,33 33 33
33
34 34 34 34
33 33
34
33
33
33 31 33
31,35
33 30,32a,32b
32a,32b,33 33 33
34 34
34 30,31,33 33
33 33 33
31 33 33
34 34
33 33
31 30
33
33
33 33
33 33 (Continued)
103
33 33 33
32a,32b
34 30,32a,32b
Sampling Techniques for the Determination of the Volatile Fraction of Honey
4-Oxoisophorone 5-Hexen-2-ol 5-Hydroxymethylfurfural 5-Methyl-2(5H)-furanone 5-Methyl-2furanocarboxaldehyde 5-Methyl-5-ethenyldihydro2(3H)-furanone 6-Methyl-5-hepten-2-one 8-p-Menthen-1,2-diol 9-Octadecanoic acid, methyl ester derivative Acetic acid Acetone Acetonitrile Acetophenone Benzenedicarboxylic acid, monobutyl ester Benzyl methyl ketone Benzaldehyde Benzeneacetaldehyde Benzeneacetic acid Benzeneacetonitrile Benzenedicarboxylic acid derivative Benzeneethanol Benzenemethanol Benzenepropanol Benzoic acid Benzyl alcohol Benzyl nitrile Borneol Butanoic acid Butanoic acid, 3-hexenyl ester Camphor Carvacrol Carvone cis-Carveol cis-Linalool oxide cis-Linalool oxide (furanoid) cis-Linalool oxide (pyranoid) cis-Rose oxide Damascenonne Decanal Decanoic acid Decanol
Volatile compounds in unifloral honeys (extraction technique: SPME)dcont’d
Compounds
Sage
Rosemary
Orange
Lime
Lavender
Heather
Eucalyptus
Buckwheat
Chestnut
Dandelion
Citrus
31
31
Sulla
Wild flower
33 33
33 33
33 33
33 33
33 33 33 33 33 33
33 33 33 33 33 33 33 33
35 31
31 34
31
31 34
36
31 34 34 34 34
31
34 33 33,36
33
31 31,35 36
36 31
36
36
36
33 33 36
36
34
36
34 33 33 33
36
34
32a,32b,36 32a,32b
30 30 34 32a,32b,34 32a,32b
36
34 31
30 32a,32b,36 32a,32b
36
32a,32b,33,36 32a,32b
30
30
31,36
34
33 33
34
31,33
31
31 33 35
33 33 33 33 33
35 30,32a
33 33 33
33 33 33 33 33 33 33
33 33
35
34 32a
30,32a
32b
34 32a,32b
33 33 33 33 33 30,33
34
31 32a,32b
32a,32b
33 33 33 31
33 33 33 30
34 33 34
31
33 33 33
31
Extraction Techniques and Applications: Food and Beverage
Dehydromethylene-2(3H)furanone Dehydrovomifolio Dibutyl phthalate Dihydro-2(3H)-furanone Dihydro-3-methyl-2(3H)furanone Dihydro-4-methyl-2(3H)furanone Dihydro-5-methyl-2(3H)furanone Dihydrocarveol Dimethyl disulfide Dimethyl styrene Dimethyl styrene isomer Dimethyl sulfide Dimethyltrisulfide Dodecanal Dodecanoic acid Ethanol Ethenyl phenylacetate Ethylbenzeneacetate Ethylbenzoate Formic acid Furfural Furfuryl alcohol Furfuryl n-butyrate Geranyl acetone Heptadecane Heptanal Heptanoic acid Heptanol Hexadecanoic acid Hexadecanol Hexanal Hexanedioic acid Hexanoic acid Hexanol Hotrienol Isoborneol Isobutyl phthalate Isophorone Isopropyl myristate Lilac alcohol
Thyme
104
Table 4
31
31
32b 32b 32b 32b
32a,32b 32b,32b 32a,32b 32a,32b
32a 32b 32b 32b
30,32a,32b 30,32a,32b 30,32a,32b 30,32a,32b
32a,32b,36 32a,32b,36 32a,32b,36 32a,32b 33
32b 32b 32b 32b
35
30,32a
32a,33
32b 32b 32b 32b 34 30,32b 30,32b 30,32b 30,32b
32a
30,32a
32b
34 32b
34 30 30 30 30 33 30,33
31 30 30 30 30 33
34
31 30
33
33
33
33
33
35 33 32a,32b
32b
33
32a,32b,33 33 33
33 33 32b
32b
34 32b 34
33
30,33
33
33 33 33
34
30,31
33 33 33
33 33
33 33
33 33
33 33 33
33
33
34
33 32a 32a 35
33
33 33
33
33 33
33
33
33 33
33 33 33 33
32a 33 31
31
33
31
34
33 33
35
34
35
34 34
33 35
33 33
31,33 33 31,33 33 33 33 33 33
34 34 34
34 34
33 33 31,33 33 33 33 33 33
31
33 33
33 33
105
(Continued)
Sampling Techniques for the Determination of the Volatile Fraction of Honey
Lilac alcohol Lilac alcohol (isomer I) Lilac alcohol (isomer II) Lilac alcohol (isomer III) Lilac alcohol (isomer VI) Lilac aldehyde Lilac aldehyde (isomer I) Lilac aldehyde (isomer II) Lilac aldehyde (isomer III) Lilac aldehyde (isomer VI) Limonene Limonene diol Linalool Maltol Menthofuran Methoxy-phenyl-oxime Methyl anthranilate Methyl decanoate Methyl decenoate Methyl heptanoate Methyl heptanoate Methyl octanoate Methyl salicylate Methyl-2-buten-1-ol Methyl-2-furoate Methylhexanoic acid Methylnaphthalene (isomer I) Methylnaphthalene (isomer II) Methylstyrene Myrcenol Myrtenol Naphthalene Neral Nerol Nerolidol Nonanal Nonane Nonanenitrile Nonanoic acid Nonanol Octadecanal Octanal Octane Octanoic acid Octanol
106
Table 4
Volatile compounds in unifloral honeys (extraction technique: SPME)dcont’d
Compounds
Sage
Rosemary
Orange
Lime
Lavender
Heather
31
Eucalyptus
Buckwheat
33
Chestnut
Dandelion
Citrus
31
Sulla
Wild flower
33
33
34 33 31
35
35 32a
32a,33 33
32a
34 32a
31 33 33
34
35 31 35 35 31 31 35
31
33 33
33 33
33 33
33
33
33
33
33
31
31
33 31
31 33 33
33 33
33
31 35 33
33
35 35 33
34
34
33
35 30,32a,32b 31 31
32a,32b,33
34 32a,32b
30,32a,32b
30
33
31
31 33
30
31
33 35 35 33
33
30
30
30 33
33 30 33
33
33
33 31,33 33
33 33 33
33
34 35 32a
33 33 33
31
32a
32a
33 33
30, de la Fuente, E.; Martõnez-Castro, I.; Sanz, J. J. Sep. Sci. 2005, 28, 1093–1100; 31, Piasenzotto, L.; Gracco, L.; Conte, L. J. Sci. Food Agric. 2003, 83, 1037–1044; 32, Soria, A. C.; Martõnez-Castro, I.; Sanz, J. J. Sep. Sci. 2003, 26, 793–801; 33, c, D.; Sabatini, A.G.; Conte, L.S. Food Verzera, A.; Campisi, S.; Zappala, M.; Bonaccorsi, I. Am. Lab. 2001, 33, 18–21; 34, Wolski, T.; Tambor, K.; Rybak-Chmielewska, H.; Kedzia, B. J. Apic. Sci. 2006, 50, 115–126; 35, Lusic, D.; Koprivnjak, O.; Curi Technol. Biotechnol. 2007, 45, 156–165; 36, Pérez, R.A.; Sánchez-Brunete, C.; Calvo, R.M.; Tadeo, J.L. J. Agric. Food Chem., 2002, 50, 2633–2637.
Extraction Techniques and Applications: Food and Beverage
p-Cymen-8-ol Pentanoic acid Pentanol Phenol Phenylacetaldehyde Phenylethyl alcohol p-Mentha-1,5-dien-8-ol p-Methylacetophenone Pulegone Rose oxide Rose oxide isomer Rose oxide isomer Teresantalol Terpinen-1-ol Terpinen-4-ol Terpinen-7-al Terpinene Tetradecanoic acid Tetrahydro-2,2,5,5tetramethylfuran Thymol Toluene trans-2-Caren-4-ol trans-Linalool oxide trans-linalool oxide (furanoid) trans-p-Mentha-2,8-dien-1-ol Trimethylphenol Undecane Verbenol a,a-4Trimethylbenzenemethanol a-4-Dimethyl-3-cyclohexen-1acetaldehyde a-Isophorone a-p-Dimethylstyrene a-Phenethyl alcohol a-Terpinen-7-al a-Terpineol b-Damascenone g-Nonalactone
Thyme
Sampling Techniques for the Determination of the Volatile Fraction of Honey
107
Corsica and different European countries with the emphasis on confirming the authenticity of the honeys labeled as Corsican. A DVB/CAR/PDMS 50/30 mm fiber provided the best absorption capacity and the broadest range of volatiles extracted from the headspace of a mixed honey sample. A combination of DB-5MS and SUPELCOWAX 10 columns gave the best resolution. This powerful analytical strategy led to the identification of 164 volatile compounds present in a honey mixture during a 19-min GC run. The absolute peak areas of some honey volatiles considered important markers were submitted to chemometric analysis. Interesting information was obtained and the authors were able to demonstrate that the GC GC-TOFMS combined with chemometric methods such as linear discriminant analysis (LDA), discriminant partial least squares (DPLS), and support vector machines (SVM) can be successfully applied to detect mislabeling of Corsican honeys.
4.05.2.3.2
SPME for Honey Authenticity
Interesting research regarding the determination of specific classes of substances that can be used for the authentication of honey are reported in the literature (Table 3). Two of these deal with the application of SPME-GC-MS methods developed to differentiate nectar and honeydew honeys.42,45 The interest of the authors arose from the consideration that this differentiation is very often difficult, not only because of the wide variability in composition and sensory properties among samples from the same source, but also because of the frequent existence of honeys resulting from a blend of nectar and honeydew. Soria et al.42 considered a set of Spanish nectar and honeydew honeys, whose floral source was confirmed by melissopalynology analysis. For SPME headspace sampling, a manual holder equipped with a CAR/PDMS fiber was used (Table 3) and chromatographic separation was carried out on a polar capillary column operating at programmed temperatures. Identifications were based on mass spectra, LRI, and standard injections. Stepwise regression (SRA) from volatiles data gave an estimation of honeydew and it was possible to determine which volatile compounds were related to the different honey sources. The floral origin of honey was related to the presence of several terpenoids such as borneol, cis- and trans-linalool oxide, lilac aldehydes, and lilac alcohols while acetic acid, erythro- and threo-2,3butanediol were indicative of honeydew honey. Later, Daher and Gulacar45 focused their attention on the presence of phenolic and other aromatic compounds to differentiate nectar and honeydew honeys. With regard to SPME, the different parameters affecting the efficiency of the extraction, such as the type of the fiber stationary phase, addition of NaCl and acetic acid, and extraction time, were optimized; the most appropriate fiber type appeared to be the polar PA fiber (Table 3). The extracts of honey and honeydew samples from different countries and floral origin were analyzed using an apolar column in a temperature gradient; a total of 31 compounds were detected, most of which were identified and quantified by GC-MS. Also in this case, PCA was applied to the data matrix which allowed for the differentiation between honeydew and nectar honeys on the basis of the salicylic acid concentration. Interesting results were obtained by Soria et al.44 in 2008 who reported that the nitrile compounds in Taraxacum Italian honeys were markers for the determination of floral origin. Fractionation of volatiles from honey headspace was carried out by using a CAR/PDS fiber as previously reported32 (Table 3) and the following nitriles were identified: 2-methylpropanenitrile, 2-methylbutanenitrile, 3-methylbutanenitrile, 2-butenenitrile, (E)-3-butenenitrile, 3-methylpentanenitrile, benzonitrile, and benzeneacetonitrile. Since nitrogen-containing compounds, such as nitriles, thiocyanates, and isothiocyanates, are hydrolysis products of glucosinolates present in Diplotaxis sp. and other Brassicaceae,46 the authors proposed the presence of nitriles in Taraxacum honey as the nectar contribution of species belonging to the Brassicaceae family. The application of SPME-GC-MS to verify the presence of environmental pollutants in honey was reported by Bentivenga et al.41 in 2004. They suggested that honey acts as a marker of environmental pollution of the area where the bees live. Italian honey samples (Basilicata, Southern Italy) were analyzed; the characterization of the pollens demonstrated that chestnut prevailed. A PDMS fiber (Table 3) was used and the volatiles were analyzed using GC-MS equipped with an apolar capillary column. From their mass spectra, some phenyl-substituted and hydrocarbons were identified and correlated to the presence of pollutants from anthropogenic activities in the honey production area. A SPME-GC-MS method for estimation of authenticity of thyme honey was developed by Mannas and Altu g in 2007.43 Thyme honey is considered to be one of the most delicious and high quality among the unifloral honeys; samples were analyzed following the SPME procedures previously developed by Verzera et al.33 and Piasenzotto et al.31 (Table 3). The results of GC-MS analysis showed that pure thyme honey involved volatile compounds that originate from the thyme plant, such as thymol and carvacrole, in low amounts; high amounts of these substances were considered as markers for detecting adulteration by thyme essential oil.
4.05.2.4
USE
USE is an extraction technique that does not use any heat. It is applied to the isolation of volatile compounds from natural products using organic solvents and a water bath with ultrasound assistance at room temperature. The first application of this technique for the study of honey volatile fraction goes back to 2003.47 The authors applied a USE methodology for Greek citrus honeys and flowers. The volatile fraction both of fresh flower and honey samples was extracted using an n-pentane/diethylether (1:2) mixture in an ultrasound water bath apparatus, maintained at 25 C for 10 min; the extracts were then analyzed directly by GC-FID and GC-MS using an apolar capillary column operating at programmed temperature with helium as carrier gas. The analysis of the flowers allowed the identification of 17 volatile compounds, mainly monoterpenes; among these, linalool was predominant in all citrus species, except for lemon, with eucalyptol the main compound. For the honey volatile extracts (Table 5), linalool derivatives accounted for more than 80% of the total volatile amount, in agreement with the flower extracts. Thus, through the quantification of linalool derivatives present in citrus honey volatiles, the authors expected to establish a threshold to distinguish citrus honeys from others of different floral origin.
108
Extraction Techniques and Applications: Food and Beverage
Table 5
Volatile and semivolatile honey compounds (extraction technique: USE)
Compounds
Citrus
(E)-2,6-Dimethyl-2,7-octadiene-1,6-diol (E)-2,6-Dimethyl-6-hydroxy-2,7-octadienal (Z)-2,6-Dimethyl-2,7-octadiene-1,6-diol (Z)-9-Tricosene (Z)-Octadec-9-en-1-ol 1-(4-Methoxyphenyl)propan-2-one 1,3-bis(1,1-Dimethyl)benzene 1,3-Di-tert-butylbenzene 1H-Pyrrole 1-Isocyanato-2-methylbenzene 1-Methoxy-4-propylbenzene 1-Methyl-2-pyrrolidinone 1-Octanol 1-Octanol 1-Phenyl-2,3-butanediol (isomer I) 1-Phenyl-2,3-butanediol (isomer II) 1-Phenyl-2,3-butanedione 2,2,6-Trimethylcyclohexane-1,4-dione 2,3,5-Trimethylphenol 2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one 2,3-Dihydro-benzofuran 2,4-Di-tert-butylphenol 2,5-Dimethyl-2,4-dihydroxy-3(2H)-furanone 2,6,6-Trimethyl-4-oxocyclohex-2-ene-1-carbaldehyde 2,6-Dimethoxyphenol 2,6-Dimethyl-1,7-octadien-3,6-diol 2,6-Dimethyl-3,7-octadiene-2,6-diol 2-Ethyl-3-hydroxyhexyl 2-methylpropanoate 2H-1-Benzopyran-2-one 2-Hydroxy-1-(4-methoxyphenyl)ethanone 2-Hydroxyacetophenone 2-Hydroxyphenylethanol 2-Methylbenzene-1,4-diol 2-Methylbutanoic acid 2-Methylpropanoic acid 2-Phenylacetaldehyde 2-Phenylacetic acid 2-Phenylethanol 3,4,5-Trimethoxybenzaic acid 3,4,5-Trimethoxybenzaldehyde 3,4,5-Trimethylphenol 3,5-Dihydroxy-2-methyl-4H-pyran-4one 3,7-Dimethyl-1,5-octadiene-3,7-diol 3,7-Dimethyl-1,6-octadiene-3,5-diol 3-Hydroxy-1-phenyl-2-butanone 3-Hydroxy-2-butanone 3-Hydroxy-2-methyl-4H-pyran-4-one 3-Hydroxy-4-phenyl-2-butanone 3-Hydroxy-4-phenyl-3-buten-2-one 3-Methyl-1-butanol 3-Oxo-a-ionone 4-(4-Hydroxy-2,2,6-trimethyl-7-oxabicyclo[4.1.0]hept-1-yl)-3-buten-2-one 4-Ethenyl-2-methoxyphenol 4-Hydroxy-3,5-dimethylbenzaldehyde 4-Hydroxyphenylacetonitrile 4-Hydroxyphenylethanol 4-Ketoisophorone 4-Methoxybenzaldehyde 4-Methoxybenzoic acid 4-Methoxyphenethyl alcohol 4-Methoxyphenylacetonitrile
47 47 47
Sage
Thyme
49 48 48 47 49 48 47 48 47 47 47 49 49 49 48 48 49 47 49 49 48 48 47 47 48 48 49 49 49 48 49 49
47
48 48 48 48 48
49 49 49
48 47 49 49 48 49 49 49 49 48 48 49 48 48 48
49
49 49
Sampling Techniques for the Determination of the Volatile Fraction of Honey
Table 5
109
Volatile and semivolatile honey compounds (extraction technique: USE)dcont’d
Compounds 5-(Hydroxymethyl)-furan-2-carbaldehyde Acetic acid Benzaldehyde Benzene-1,4-diol Benzeneacetaldehyde Benzeneacetic acid Benzoic acid Benzyl alcohol bis-(2-Ethylhexyl) adipate Butanoic acid Butyl acetate Caffeine Carvacrol cis-Linalool oxide (furanoid) cis-Linalool oxide (pyranoid) Coumaran Cyclohexanone Decanal Decane Decanoic acid Degraded carotenoid Dehydrovomifoliol Dibutylphthalate Dihydroanethole Diisobutylphthalate Docosane Dodecane Dodecanoic acid Eicosane Ethyl hexadecanoate Ethyl oleate Ethyl benzene Eugenol Furfural Guaiacol (2-methoxyphenol) Heneicosane Henicos-10-ene Heptadecane Heptane Hexadecane Hexadecanoic acid Hexanoic acid Hydroxymethylfurfural Hotrienol Indole Isophorone Isovaleric acid Lilac alcohol Lilac alcohol Lilac aldehyde Lilac aldehyde Lilac aldehyde Lilac aldehyde (isomer I) Lilac aldehyde (isomer II) Lilac aldehyde (isomer III) Limonene Linalool oxide B Linoleic acid m-(or p-)Xylene Methyl 4-methoxybenzoate
Citrus
47 47 47 47
Sage 48 48 48 48
Thyme
49
48 48
49 49
48
49 49
47
47 49 47 47 48 47 47 47 48
49 49 49
47 47
48 48
47
49 49 49 49 49 49
48 49 49 49 49 49 49 48 48 48 48 47 48 48 47 47
49 49 49 49 49 49 49 49 49 49
47 47 47 47 47 47 47 47 47
49 48 49
47 48 (Continued)
110
Extraction Techniques and Applications: Food and Beverage
Table 5
Volatile and semivolatile honey compounds (extraction technique: USE)dcont’d
Compounds
Citrus
Methyl anthranilate Methyl benzoate Methyl hexadecanoate Methyl syringate Methyl vanillate Methyl cyclohexane Nerolidol Nonadecane Nonanal Nonane Nonanoic acid Octadecane Octadecanoic acid Octanal Octane Octanoic acid Oleic acid o-Xylene p-Anisaldehyde p-Anisic acid Pentacosane Pentadecane Phenol Phenylacetaldehyde Phenylacetic acid Phenylacetonitrile Phenylethyl alcohol Propanoic acid p-Toluic acid p-Xylene Salicylic acid Syringaldehyde Tetracosane Tetradecane Tetradecanoic acid Toluene trans-Linalool oxide (furanoid) trans-Linalool oxide (pyranoid) Tricosane Tridecane Undecane Vanillic acid Vanillin Veratric acid a-4-Dimethyl-3-cyclohexene-1-acetaldehyde (isomer III) a-4-Dimethyl-3-cyclohexene-1-acetaldehyde (isomer VI) a-Isophorone
47
Sage
Thyme
48 49 49 49 47 47 48 47 48 48
47 48
49 49 49 49 49 49 49 49 49 49
47 49 49 48 48 48 47 47
48 47 47 47 47 47 47 47
48
49 49 49 49 49 49 49 49 49 49 49 49 49 49 49 49 49 49 49 49 49
47 47 48
47, Alissandrakis, E.; Daferera, D.; Tarantilis, P.A.; Polissiou, M.; Harizanis, P.C. Food Chem. 2003, 82, 575–582; 48, Jerkovic, I. Mastelic J., Marijanovic, Z. Chem Biodivers. 2006, 3, 1307–1316; 49, Alissandrakis, E.; Tarantilis, P.A.; Pappas, C.; Harizanis P.C.; Polissiou M. Eur. Food Res. Technol. 2009, 3, 365–373.
In 2006, the USE technique was used by Jerkovic et al.48 for the first characterization of the volatiles of sage (Salvia officinalis L.) honeys. Unifloral Salvia honey samples (ascertained by pollen analysis) were selected from various producers in South Croatia; 54 volatile compounds were identified (Table 5) by GC-FID and GC-MS in n-pentane/diethylether extracts after sonication at 25 C for 30 min and quantified using internal standards. Compounds such as benzoic acid, phenylacetic acid, p-anisaldehyde, a-isophorone, and 4-ketoisophorone, were proposed as markers for sage honey. A few years later, Alissandriks et al.49 presented their results on the composition of the volatile fraction of unifloral Greek thyme honey, the most marketed type in Greece. Using the experimental method previously developed,47 about 100 compounds were identified (Table 5) and quantified using b-ionone as internal standard. Phenolic compounds were the most abundant and were proposed as potent botanical markers for thyme honey.
Sampling Techniques for the Determination of the Volatile Fraction of Honey
4.05.2.5
111
USE Coupled with SPME
Recently examples of applications of USE and SPME, applied as complementary extraction techniques for the characterization of honey volatile fraction, have been reported in the literature.50–52 The use of both techniques allowed complete patterns of honey headspace to be obtained, since the HS-SPME method proved to be suitable for the isolation of low-molecular-weight aroma compounds, whereas the USE procedure enabled extraction of semivolatile compounds. The optimization and application of this comprehensive method was due exclusively to Jerkovic and co-workers who in 2009 reported the honey volatile composition of different Croatian unifloral honeys, namely Lavandula hybrida Reverchon II50 (Lavandula angustifolia Lavandula latifolia), Paliurus spina-christi,51 and Amorpha fruticosa52 honeys. For the HS-SPME analysis different fibers were considered (Table 3), whereas for the USE procedure three solvents, pentane, diethyl ether, and a mixture of pentane/ethyl ether (1:2 v/v) were used, under the same experimental conditions previously reported.48 The GC-MS analysis was performed on polar and apolar capillary columns at programmed temperatures using helium as carrier gas. With regard to Lavandula hybrida Reverchon II, HS-SPME made it possible to isolate short chain aliphatic compounds (Table 6), mainly hexan-1-ol, hexanal, acetic acid, hotrienol, and 2-phenylacetaldehyde, important for authentication of this honey, whereas the ultrasound pentane/ethyl ether extract contained the majority of the honey floral origin compounds and potential biomarkers (hexan-1-ol, acetic acid, butane-1,3diol, butane-2,3-diol, benzoic acid, coumarin, and 2-phenylacetic acid) (Table 6). In the Paliurus spina-christi headspace a total of 33 compounds were identified (Table 6); major constituents and possible markers were nonanal, four isomers of lilac aldehyde, decanal, methyl nonanoate, hexanoic, and 2-ethylhexanoic acids; in the USE extracts a total of 79 compounds were identified
Table 6
Volatile and semivolatile honey compounds (extraction techniques: USE and SPME) Lavender
Compounds USE (E)-b-Damascenone (Z)-Octadec-9-en-1-ol (Z)-Octadec-9-enoic acid (Z,Z)-9,12-Octadecadienal (Z,Z)-9,12-Octadecadienoic acid 1-(2-Furanyl)-2-hydroxyethanone 1-(2-Furanyl)-ethanone 1,3-Butanediol 1,3-Butanediol 1,3-Dimethylbenzene 1,4-Benzenediol 1,4-Dimethylbenzene 1,4-Di-tert-butylbenzene 1-Dodecanol 1-Hexadecanol 1-Hexanol 1H-Indole-3-acetic acid 1H-Pyrrole-2-carboxylic acid 1-Hydroxylinalool 1-Nonanol 1-Octadecanol 1-Phenylethan-1,3-diol 1-Tetradecanol 2-(4-Methoxyphenyl)ethanol 2-(p-Methoxyphenyl)-ethanol 2,3-Butanediol 2,3-Butanediol 2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one 2,3-Dihydro-3,5-dimethyl-4H-pyran-4-one 2,3-Dihydro-5-hydroxy-6-methyl-4H-pyran-4-one 2,3-Dihydrobenzofuran 2,4-Dimethyl-3,6-dihydro-2H-pyran 2,5-Dimethoxy-4-ethylbenzaldehyde 2-Ethylfuran 2-Ethylhexanoic acid 2-Ethylhexyl 2-ethylhexenoate 2-Furancarbaldehyde 2-Furancarboxylic acid
SPME
Christ’s thorn USE
SPME
Desert false indigo USE
SPME 52
50
51
52 52
51 51 52 52 51 50 52 52 52 52 52 52
51
51 50
50 51 51 52 52 52 52 52
50 51 51 50 50
50
52 51
50 50
51 52 52 52 51a,51b
50 50 50 (Continued)
112
Extraction Techniques and Applications: Food and Beverage
Table 6
Volatile and semivolatile honey compounds (extraction techniques: USE and SPME)dcont’d Lavender
Compounds USE 2-Furanmethanol 2-Hydroxy-3-methyl-4H-pyran-4-one (maltol) 2-Methoxy-6-methylpyrazine 2-Phenylacetaldehyde 2-Phenylacetamide 2-Phenylacetic acid 2-Phenylethanol 3-(4-Hydroxy-3-methoxyphenyl)-prop-2-enoic acid (ferulic acid) 3,5-Dihydroxy-2-methyl-4H-pyran-4-one 3,5-Dimethoxy-4-hydroxybenzoic acid (syrigic acid) 3,7-Dimethylocta-1,5-diene-3,7-diol 3-Hydroxy-4-methoxycinnamic acid (isoferulic acid) 3-Methyl-2-butanol 3-Methylpentanoic acid 3-Pyridinecarbonitrile 4,5-Dimethyl-2-formylfuran 4-Ethenyl-2-methoxyphenol 4-Ethylbenzaldehyde 4-Hydroxy-3,5,6-trimethyl-4-(3-oxo-1-butenyl)-cyclohex-2-en-1-one 4-Hydroxy-3,5-dimethoxybenzaldehyde (syringyl aldehyde) 4-Hydroxy-3,5-dimethoxybenzoic acid 4-Hydroxy-3,5-dimethylbenzaldehyde 4-Hydroxy-3-methoxybenzaldehyde (vanilline) 4-Hydroxy-3-methoxybenzoic acid (vanillic acid) 4-Hydroxybenzaldehyde 4-Hydroxybenzoic acid (p-salicylic acid) 4-Hydroxybenzyl alcohol 4-Hydroxycinnamic acid (3-(4-hydroxyphenyl)-prop-2-enoic acid) 4-Hydroxyphenylacetic acid 4-Ketoisophorone 4-Methoxybenzaldehyde 4-Methoxybenzoic acid 4-Methyl-2,6-bis (1,1-dimethylethyl)-phenol 4-Methylbenzyl alcohol 4-Methyloctane 4-Vinyl-2-methoxyphenol 4-Vinylphenol 5-(Hydroxymethyl)furan-2-carbaldehyde 5-Hydroxymethylfurfural 5-Methyl-1,4-benzenedione 5-Methylfurfural Acetic acid Benzaldehyde Benzoic acid Benzyl alcohol Butanoic acid Butoxyethoxyethyl acetate cis-Linalool oxide Coumarin Decanal Decanoic acid Diisobutyl phthalate Dimethyl disulfide Docosane Dodecane Ethylbenzene Formic acid Furfural
SPME
Christ’s thorn USE
SPME
Desert false indigo USE
SPME
51b 51 51 50 52 50 50
50
51
51a,51b
52 52
52
50 50
52 52 52
51 51 51 51
52 50 50 52 52 51 51 52 52 51a,51b 51
52 52 52 52 52 52
51 52 52 52 52 52
51
52
50 50
51
52 51a
50
50 50 50
50
51
51a,51b 51a,51b
51 51 51
50
51a
52 52 52 52 52
52 52
52
50 51 51
51a,51b 52 52
51 51
50
51a 52
50
50 50
51b
52
Sampling Techniques for the Determination of the Volatile Fraction of Honey
Table 6
113
Volatile and semivolatile honey compounds (extraction techniques: USE and SPME)dcont’d Lavender
Compounds USE Furfuryl alcohol Heneicosane Heptacosane Heptadecane Heptanal Heptanoic acid Hexacosane Hexadecan-1-ol Hexadecane Hexadecanoic acid Hexanal Hexanoic acid Hotrienol Isophorone Lilac aldehyde (isomer I) Lilac aldehyde (isomer II) Lilac aldehyde (isomer III) Lilac aldehyde (isomer not identified) Lilac aldehyde (isomer VI) Linalool Maltol Methoxybenzene Methyl 2-furancarboxilate Methyl 3,5-dimethoxy-4-hydroxybenzoate (methyl syringate) Methyl furan-2-carboxylate Methyl nonanoate Methyl octanoate Methyl sulfide Nananoic acid Nonacosane Nonadecane Nonanal Nonane Nonanoic acid Octacosane Octadecane Octadecanoic acid Octanal Octane Octanedioic acid Octanoic acid o-Ethylanisole Pantoic lactone Pentadecane Pentanoic acid Phenylacetaldehyde Phenylacetic acid Phenylacetonitrile Phytol Pinocarvone Propanoic acid Tetracosane Toluene trans-Linalool oxide Tetradecane Tricosane Tridecane Undecane
SPME
Christ’s thorn
Desert false indigo
USE
SPME
USE
51 51 51
51a
52
SPME
50 50
51a,51b
50 50 51 50 51 51
50 50 50
50 50 50
51
52 51a,51b
52 52 52
51a,51b 51a,51b 51a,51b 52 51a,51b 52 50
50 52 51 52
50
50 51a 51a 52 52 51 51 51
50
50
51 51 51 51 51
51a 51a,51b
52 52 52
51a,51b
51a 52
50
50
51 51
51a,51b 51a
50 51 51 51
51a
52
51a,51b
52 52
52 52
51 52 50
51 51
52 51b 52
52
51 51 51a 51a,51b
50, Jerkovic, I.; Marijanovic, Z. Chem Biodivers. 2009, 6(3), 421–430; 51, Jerkovic, I.; Tuberoso, C.I.G.; Marijanovic, Z.; Jelic, M.; Kasuma, A. Food Chem. 2009, 112(1), 239–245; 52, Jerkovic, I.; Marijanovic, Z.; Kezic, J.; Gugic, M. Molecules 2009, 14, 2717–2728.
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(Table 6) and benzene derivatives (4-hydroxy-3,5-dimethylbenzaldehyde, 4-hydroxybenzoic acid, and 4-methoxybenzoic acid) and aliphatic acids (butanoic, hexanoic, octanoic, and nonanoic) were considered interesting for floral determination. By HS-SPME in Amorpha fruticosa a total of 25 volatile constituents (Table 6) with 2-phenylethanol, cis- and trans-linalool oxides, benzaldehyde, and benzyl alcohol were identified as the main compounds, while by USE a total of 57 volatile and semivolatile compounds were identified, with 2-phenylethanol and methyl syringate the main compounds (Table 6). For each unifloral honey, the USE technique allowed the identification of a higher number of volatile constituents than SPME, as expected. Thus, using the two different extraction methods and combining the data obtained, interesting information can be obtained, especially when markers of honey floral origin include both volatile and semivolatile compounds.
4.05.3
E-nose
A new analytical tool for the identification of volatile compounds has been recently proposed to address the need for routine quality testing in the food industry; this is the so-called electronic nose (E-nose) consisting of an array of weakly specific or broad spectrum chemical sensors that intend to mimic the human olfactory system and convert sensor signals to data that can be analyzed with appropriate statistical software. The use of E-nose to characterize the origin of honey based on the volatile fraction introduced a different strategy that allows a profile or fingerprint to be obtained avoiding any separation into individual compounds. The first application of E-nose to honey volatiles was due to Benedetti et al.53 who applied an artificial neural network (ANN) to classify signals from an electronic nose smelling different types of honeys. Unifloral honey samples of specific botanical and geographical origins (Robinia pseudoacacia L. (Italy), Rhododendron spp. (Italy), Citrus spp (Italy), Robinia pseudoacacia L.(Hungary)) were analyzed; the honey gas headspace, sampled by an automatic syringe, was pumped for 30 s over the surface of 22 different gas sensors (10 metal oxide semiconductor field effect transistor sensors, 12 metal oxide semiconductor sensors). The data obtained from the sensor array were statistically analyzed by PCA and ANN and good results were obtained using a neural network model based on a multilayer perceptron (MLP) that learned using an algorithm called backpropagation. In the following years, this method53 was applied by Cacic et al.54 to geographic origin characterization of Robinia pseudoacacia L. and Castanea sativa Mill. honeys produced in Croatia. Volatile profile data obtained by E-nose were analyzed by PCA and it was found that honey samples from geographically close regions tended to be grouped together, while those from distant geographic regions showed differences although they were of the same botanical origin. In 2004, Ampuero et al.55 studied honey volatiles using a fast sensitive E-nose based on mass spectrometry (MS-nose). Three different sampling techniques, SHS, SPME, and inside needle dynamic extraction (INDEX) (also known as solid-phase dynamic extraction (SPDE)), were used for volatiles of Swiss honeys derived from dandelion, lime, acacia, chestnut, fir, and rape. For each sampling technique, the extracted volatiles were injected into the injector port of the E-nose. The responses of the detector (specific ionic masses), processed by PCA and discriminating factor, allowed the authors to classify the samples into different botanical groups in agreement with the results simultaneously obtained by a classic method, i.e., sensory, pollen, and physicochemical analysis. Among the different techniques, the SPME sampling method showed 98% correct classification of the model samples. Another fast, sensitive, and nondestructive electronic nose, the zNose, was tested by Lammertyn et al.56 on honeys (buckwheat, clover, orange blossom, black locust, mint, and carrot) from different geographic origins. The zNose is a fast GC technique which allows identification and fingerprinting of aroma as with regular GC but at the same time it operates at the speed of the E-nose using a surface acoustic wave sensor detector made of an uncoated piezoelectric quartz crystal. For the zNose measurements, a needle (provided by the zNose), inserted through the septa of the vials from an SHS system, was used for sampling headspace honey volatiles, which were thus released from the trap inside the system and carried over the column (DB-5) in a helium flow. Since the zNose is a combination sensor-based detector and regular GC analyzer, the data resulting from the zNose measurements were approached in two different ways: first, by comparing the different peaks and peak areas obtained considering only the positives values of the first derivative plot (chromatogram approach); and second, the positive and negative values of the first derivative profile were considered and treated as spectral data, analyzing the full frequency spectrum of each sample. The statistical treatment of the data by PCA and canonical discriminant analysis made it possible to discriminate among honeys of different floral origin and between honey and plain sugar.
4.05.4
Conclusion
In volatile fraction analysis, the choice of the extraction method depends on the type of food and the information needed, since there is a great variability in the aroma compounds obtained depending on the procedure used. In most cases, the extraction method selected should be able to provide an aromatic extract as representative as possible of the product. From this point of view, all the extraction methods that require heat are less suitable since the volatile profiles obtained contain heat-generated artifacts, whereas the most sensitive compounds could be missing, destroyed by the drastic conditions applied. The extraction techniques that make it possible to obtain a volatile profile comparable with the sensorial descriptors are those operating in the sample headspace, such as DHS and SPME, when carried out at low temperature. In the case of honey, the use of heat along with solvent, as required by the SDE technique, significantly modifies the volatile profile. For example, in an acid aqueous medium, terpenes easily lead to hydrated and oxidized compounds and substances from the Maillard reaction and/or sugar caramelization can arise
Sampling Techniques for the Determination of the Volatile Fraction of Honey
Table 7
115
Furan and pyran derivatives (mg kg1) in thyme honey volatiles sampled by different extraction techniques
Compounds 1-(2-Furanyl)-ethanone 2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one 2,3-Dihydro-4-methylfuran 2,5-Dimethyl-2,4-dihydroxy-3(2H)-furanone 2,5-Dimethylfurane 2-Acetylfuran 2-Furan methanol 2-Furancarboxilic acid 2-Methyl-3-(2H)-dihydrofuranone 2-Methylfuran 3,5-Dihydroxy-2-methyl-4H-pyran-4one 5-Ethenyl-5-methyl-2(3H)-furanone 5-Methyl furfural 5-Methyl-2-(3H)-furanone Furan derivative (95, 123, 124) Furfural Furfuryl alcohol Furfuryl n-butyrate Vinylguaiacol
SDE20
DHS26
SPME31,36
USE49
–a 33 327.0 86 13.4 95.8 6.0c 22.4 21.5 95 30.1 69.2 12.6 1558 19.8
495.4
35.6c
78 74
–b 19.5
a
Compounds identified but not quantified. Compounds identified by Piasenzotto et al.31 Compounds identified by Pe´rez et al.36
b c
Figure 1 HS-SPME-GC-MS (SIM) chromatogram of orange honey volatile fraction. Compounds: 1, limonene (LRI ¼ 1185); 2, a-pinene oxide (LRI ¼ 1198); 3, cis-limonene oxide (LRI ¼ 1230); 4, cis-geraniol (1241); 5, terpinolene (LRI ¼ 1256); 6, 6-methyl-5-epten-2-one (LRI ¼ 1324); 7, a-pdimethyl styrene (LRI ¼ 1417); 8, cis-linalool oxide (LRI ¼ 1420); 9, trans-linalool oxide (LRI ¼ 1450); 10, terpinene-4-acetate (LRI ¼ 1494); 11, lilac aldehyde isomer I (LRI ¼ 1501); 12, lilac aldehyde isomer II (LRI ¼ 1523); 13, lilac aldehyde isomer III (LRI ¼ 1533); 14, lilac aldehyde isomer IV (LRI ¼ 1555); 15, hotrienol (LRI ¼ 1581); 16, phenyl acetaldehyde (LRI ¼ 1618); 17, decatriene-2,2,5,8-tetramethyl (LRI ¼ 1631); 18, p-menthen-9-al (LRI ¼ 1644); 19, a-terpineol (LRI ¼ 1670); 20, b-damascenone (LRI ¼ 1788); 21, geranlyl acetone (LRI ¼ 1825); 22, cedrene (LRI ¼ 1831); 23, trans-nerolidol (LRI ¼ 2005); 24, limonene diepoxide (LRI ¼ 2189); 25, (E,E)-farnesol (LRI ¼ 2336).
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simultaneously, such as furan derivatives. Furan-derived compounds, such as furfural, furfuryl alcohol, and 5-methyl furfural, which are present in a low concentration in fresh honey, increase during storage and heating and the increase is greater the higher the temperature.57 Thus, they are considered as indicators of thermic processes and storage and are usually used in evaluating quality deterioration in food but they cannot be considered as appropriate floral markers. Table 7 reports the furan derivatives identified in thyme honey samples,20,26,31,36,49 extracted by different methods, along with their amount, where indicated. The SDE extracts were the richest in furan derivatives while the SPME extracts had less. Furfural, present in all the thyme honey samples analyzed, was the main compound together with phenylacetaldehyde (~1.5 mg kg1) in the SDE extracts. An high amount of furfural and 2,3dihydro-4-methylfuran was also present in thyme honeys extracted by DHS. The significant amount of these two substances could be associated with purge and trap fractionation that operated at a temperature of 80 C. In contrast, the amount of furan derivatives in the SPME and USE extracts was small even though a large number of volatile compounds were identified by both techniques. With regard to the possibility of verify the floral and geographic origin of honey, interesting information is obtained from primary aroma compounds, such as terpenes and their derivatives, that are associated with the floral nectar or honeydew gathered by honeybees. Figure 1 reports a SPME-GC-MS(SIM)58 of a citrus honey in which a large number of terpenes, sesquiterpenes, and their oxygenated compounds were identified. Among these, the markers for the floral origin can be defined, in agreement with other authors. From the above observations, SPME seems to be a very reliable technique since high reproducibility and sensibility have been achieved with respect to extraction and identification of honey volatile compounds without the complexity of traditional methods.
See also: Headspace Analysis; Solid-Phase Microextraction; Sample Preparation Automation for GC Injection; Headspace Sampling in Flavour and Fragrance Field; Theory of Extraction
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