4 05 Sampling Techniques for the Determination of the Volatile Fraction of Honey 2012 Reference Module in Chemistry Molecular Sciences and Chemical En

4.05 Sampling Techniques for the Determination of the Volatile Fractionof HoneyA Verzera and C Condurso, University of Messina, Messina, Italy

� 2012 Elsevier Inc. All rights reserved.

4.05.1 Honey Volatiles 874.05.2 Extraction Techniques 874.05.2.1 Solvent Extraction and SDE 874.05.2.2 Static and DHS Analysis 884.05.2.3 SPME 924.05.2.3.1 SPME for Floral and Geographic Determination 974.05.2.3.2 SPME for Honey Authenticity 1074.05.2.4 USE 1074.05.2.5 USE Coupled with SPME 1114.05.3 E-nose 1144.05.4 Conclusion 114References 116

4.05.1 Honey Volatiles

Honey is a popular natural product not only as a source of energy but also for its health-promoting properties provided by theprebiotic, 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 fromsecretions of living parts of plants or excretions of plant-sucking insects on the living parts of plants, which the bees collect, transformby 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 minorcomponents 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 ofunifloral origin usually commands higher prices than wildflower honey. The flavor/fragrance qualities of honey are greatlydependent on the volatile and semivolatile organic compounds present both in the sample matrix and headspace aroma. Severalstudies on the volatile compounds of honey have been carried out since 19627 and it is well known that these compounds may bederived from the plant or nectar, from the conversion of plant compounds or directly generated by the honeybees, from heating orhandling during honey processing and storage, and from microbial or environmental contamination. Since most honey volatilesoriginate from the plants being visited by the bees, the honey sensory characteristics are closely related to its botanical origin andfrom this it follows that the volatile compounds have a potential role in distinguishing the floral origin. Honey is a very complexmixture with volatile components of different functionality and relatively low molecular weight, often present at very lowconcentrations; 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 thetechnique of choice since it combines high separation efficiency and sensitivity and provides qualitative and quantitative data forthese 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 anddrawbacks of each methodology, comparison of alternative reliable methods and results achieved in the field of honey qualitydetermination 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 wasdue to its simplicity and because thermolabile compounds were retained; moreover, the commonly used solvents have low polarityso that the main compounds, water and sugars, are not extracted. Nevertheless this procedure also has some disadvantages, such asthe long time involved, large volumes of solvents, environmental hazards, etc, so nowadays it is no longer used in favor of moresuitable innovative techniques.

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88 Extraction Techniques and Applications: Food and Beverage

The simultaneous distillation–extraction (SDE) system developed by Likens and Nickerson11,12 and modified by Godefrootet al.13 is one of the currently used methods for isolation of honey volatile components; honeys of different floral origin wereanalyzed and a large number of volatiles were identified. This approach, unfortunately, determines the formation of artifacts, loss ofanalytes and provides results that are not easily compared; moreover, it needs a long sampling time prior to chromatographicseparation and a large volume of solvent that influences trace analysis. However, interesting results were obtained on the floralorigin of honey when the SDE method was carefully optimized. Following Bicchi et al.14 who first emphasized the importance ofpre-extracting flavor compounds from sugars, in 1995 Bouseta and Collin15 proposed a two-step SDE protocol includingpreliminary solvent extraction under an inert atmosphere using dichloromethane as solvent. The method was applied toa commercial clover Canadian honey and the sensorial features of extracts closely matched those of the honey samples; the extractswere analyzed by GC-MS using an apolar capillary column. Unfortunately, using MS, authentic standard injection, and a massspectra 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 chestnutand lime tree honeys. The unifloral honeys were selected from various countries and the screening for floral purity was based onpollen analyses, sensory tests, electrical conductivity, pH, titratable acidity, and sugar composition. The authors confirmed thatabout 400 aroma compounds were separated by GC although only 72 were identified (Table 1); chestnut and lime tree sampleswere authenticated on the basis of a few discriminate flavoring compounds, the content of which was significantly differentfrom 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 pathwayderivatives, 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, andcarvacrol.

In the following years, the same authors17,18 applied their method to heather and lavender unifloral honeys from variouscountries (Calluna vulgaris from France, Belgium, United Kingdom, Norway, and Germany; Erica arborea from France, Greece, andItaly; Lavandula stoechas from Portugal, Lavandula angustifolia � latifolia (lavandin), and Lavandula angustifolia (fine lavender) fromthe 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-en-1-one and high level of 3,5,5-trimethylcyclohexene derivatives were specific markers of Calluna vulgaris honeys. According to theauthors, the presence of shikimate pathway derivatives such as 4-methoxybenzaldehyde, 4-methoxybenzoic acid, and methylvanillate, in Erica arborea honey samples irrefutably proved their floral origin. The aromatic profiles of the L. angustifolia � latifoliaand 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 highcontent of linear aldehydes, linear alcohols, and phenylacetaldehyde.

Some recently published papers19,20 have made an important contribution to assessing the authenticity of honey combining thehoney volatile data from SDE with those of descriptive sensory analysis. The difficulty in finding volatile compounds exclusively inhoneys from a specific botanical origin justified the use of sensory analysis to make this differentiation possible. CommercialSpanish honey samples (citrus, rosemary, eucalyptus, lavender, thyme, and heather) were extracted using a microscale SDEapparatus using dichloromethane as solvent; all extracts were analyzed using GC-MS equipped with a polar capillary column. Peakidentifications 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 authorsto 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; eucalyptushoneys 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 hotrienoland balsamic and aromatic herb aromas; heather honeys by high contents of benzene and phenolic compounds and ripe fruit andspicy 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 volatilesin honey and the low recovery obtained for semivolatile compounds. In contrast, applications of the dynamic headspace extractiontechnique (DHS) have been reported in the literature.22–27 This technique affords main advantages such as a high sensitivity forfractionation of high volatility compounds, the absence of extended heating times, and the reproducibility associated with a totallyautomated 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 honeymatrix with a stream of nitrogen gas and concentrating them into a cooled trap, the authors extracted and analyzed unifloral honeysfrom different countries. Although most of the aldehydes and alcohols identified were related to microbiological activity, heatexposure, 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 honeysamples from different countries. In particular, acacia, chestnut, eucalyptus, heather, lavender, lime, rape, rosemary, and sunflowerhoneys were considered. A purified helium (60ml min�1) flowwas used for the extraction; the entrained volatiles were adsorbed on

Table 1 Volatile compounds in unifloral honeys (extraction technique: SDE)

Compounds Clover Chestnut Lime Heather Erica Lavender Citrus Rosemary Thyme Eucalyptus

(Z)-3-Hexen-1-ol 201,3-Dimethylpyrazole 17 171,3-Diphenyl-2-propanone 161-Cyclopentyl-2-propanone 16 161-Heptanol 181-Hexanol 15 16 16 17 17 18,20 20 20 201-Hydroxy-2-butanone 20 20 19,20 20 20 201-Hydroxy-2-propanone 20 20 19,20 20 20 201-Methoxy-4-propylbenzene 16 171-Nonanol 20 20 19,20 20 20 201-Pentanol 16 161-Phenylethanol 16 161-Phenylethanol þ 1-phenyl-

1,2-propanedione20

1-p-Menthen-9-ol 19,202(3H)-Furanone 16 162,3-Dimethyl-2-cyclopenten-1-one 16 162,3-Dimethyl-4-isopropenyl-1-

cyclopentanone16

2,3-Dimethyl-4-isopropyl-1-cyclopentanone

16 16

2,3-Dimethylphenol 16 162,5-Hexanediol 16 162,6,6-Trimethyl-2-vinyltetraidropyrane 162-Acetylfuran 16 16 17,20 17 20 19,20 20 20 202-Aminoacetophenone 16 16 202-Cyclopentene-1,4-diol 202-Furaldehyde 16 16 17 17 182-Furancarboxilic acid 20 20 20 20 202H-Pyran-2-one 192-Hydroxy-3,5,5-trimethyl-2-cyclohexen-

1,4-dione20 20

2-Hydroxy-5-methyl-3-hexanone 202-Hydroxyacetophenone 202-Hydroxycineol (isomer I) 202-Hydroxycineol (isomer II) 20 202-Methoxy-6-methylpyrazine 16 162-Methyl propanoic acid 202-Methyl-1-butanol 16 162-Methyl-2-buten-1-ol 17,20 17 20 19,20 20 20 202-Methyl-3-(2H)-dihydrofuranone 20 20 20 20 20 202-Methylbutanoic acid 16 16 17,20 17 20 19,20 20 20 202-Phenylethanol 16 16 17,20 17 18,20 19,20 20 20 202-p-Methene-1,8-diol 20 20 20 203,4,5-Trimethyl phenol 20 20 19,20 20 20 203,5,5-Trimethylcyclohex-2-en-1-one

(isophorone)17 17

3,5-Dimethylphenol 16 163-caren-2-ol 203-Formyl-pyridine 203-Hexanol 16 163-Hydroxy-2-butanone 16 16 17,20 17 20 19,20 20 20 203-Hydroxy-2-pentanone 20 20 20 20 20 203-Hydroxy-5-methyl-2-hexanone 203-Methoxybenzene ethanol 16 163-Methyl thiopropanal 203-Methyl-1-butanol 17,20 17 20 19,20 20 20 203-Methyl-2-buten-1-ol 16 16 183-Methyl-3-(2H)-dihydrofuranone 193-Methyl-3-buten-1-ol 16 16 17,20 17 20 19,20 20 20 20

(Continued)

Sampling Techniques for the Determination of the Volatile Fraction of Honey 89

Table 1 Volatile compounds in unifloral honeys (extraction technique: SDE)dcont’d


3-Methylbutanoic acid 17 173-Methylfuranoate 17 173-Phenyl-1-propanol 16 16 17 173-Phenyl-2-propen-1-ol 20 20 203-Pyridine carboxaldehyde 16 16 17 174-(3-Oxo-1-butynyl)-3,5,5-

trimethylcyclohex-2-en-1-one17

4-(3-Oxobut-1-enylidene)-3,5,5-trimethylcyclohex-2-en-1-one

17 17

4-Butyl-1,3-cyclopentanedione 16 164-Ethyl-3,4-dimethyl-2-cyclohexen-1-one 164-Hydroxy-3-methoxybenzoate methyl

ester (methyl vanillate)17

4-Hydroxy-4-(3-oxo-1-butenyl)-3,5,5-trimethylcyclohex-2-en-1-one(dehydrovomifoliol)

17

4-Methoxybenzaldehyde (p-anisaldehyde) 174-Methoxybenzoic acid (p-anisic acid) 174-tert-Butylphenol 165-(Hydroxymethyl)furfural 16 17 175-Ethenyl-5-methyl-2(3H)-furanone 20 20 19,20 20 20 205-Methyl furfural 16 16 17,20 17 18,20 20 20 205-Methyl-2-(3H)furanone 20 20 19,20 20 20 205-Methylfuraldehyde 155-Methylfurfural 19,206-Methyl-3,5-heptadien-2-one 20 19,20 20 20 208-p-Menthene-1,2-diol 16 16Acetic acid 20 20 19,20 20 20Acetophenone 16 16 17 17Acetyl furan 15Benzaldehyde 15 16 16 17,20 17 18,20 19,20 20 20 20Benzoic acid 16 17 17Benzoic acid hydrazone 17 17Benzyl alcohol 15 16 16 17 17 18 19Butanoic acid 16 16 17,20 17 20 20 20 20Camphor 15Caproaldehyde 15Car-2-en-4-one 19,20 20 20Carvacrol 16 16 20 20 20 20 20 20Cinnamic acid 17 17Cinnamyl alcohol 17 17cis-Linalool oxide 20 20 19,20 20 20 20Coumarin 15 16 18,20Decanoic acid 17,20 17 20 19,20 20 20 20Dimethyl disulfide 16 20 20 20 20 20 20Dimethyl trisulfide 20 20 19,20 20 20 20Dodecanoic acid 20 20 19,20 20 20 20Epoxylinalool 19Epoxylinalool (isomer I) 20Epoxylinalool (isomer II) 20 20 20 20 20 20Eptadecane 20 20 20 20 20 20Estragole 16Ethylmethylphenol isomer 16Eugenol 20 20Furfural 15 20 20 19,20 20 20 20Furfuryl alcohol 15 16 16 17,20 17 18,20 19,20 20 20 20Guaiacol 16 20Heptadecane 16 16Heptanal 16 18,20 20Heptanoic acid 20 18,20 19,20 20 20 20Hexanal 16 16 17 17 18,20 20




Hexanoic acid 16 20 18,20 19,20 20 20 20Hotrienol 20 20 19,20 20 20 20Indole 17 17Isophorone 16 16 20 20 19,20 20 20 20Ketoisophorone 19Ketoisophorone (isomer I) 20 20 20 20 20 20Ketoisophorone (isomer II) 20 20 20 20 20 20Lilac alcohol (isomer I) 20 20 19,20 20 20 20Lilac alcohol (isomer II) 20 20 19,20 20 20 20Lilac alcohol (isomer III) 20 20 19,20 20 20 20Lilac alcohol (isomer IV) 20 20 19,20 20 20 20Lilac aldehyde (isomer I) 20 19,20 20 20Lilac aldehyde (isomer II) 19,20 20 20Lilac aldehyde (isomer III) 19,20 20 20Lilac aldehyde (isomer IV) 19,20 20 20Limonyl alcohol 19,20Linalool 20 20 19,20 20 20 20Menthol 16Methyl antranilate 19,20Methyl(1-methylethenyl)-benzene 16 16Methylfuran 15m-Xylene 15Nerolidol 19,20Nerolidol oxide 20Nonadecane 20 20 20 20 20 20Nonanal 20 18,20 19,20 20 20 20Nonane 16 16 18Nonanoic acid 17,20 17 20 19,20 20 20 20Octanal 18Octane 15 16 18Octanoic acid 16 17,20 17 20 19,20 20 20 20p-Anisaldehyde 20p-Cresol 20p-Cymen-8-ol (isomer I) 20 20 20 20 20 20p-Cymen-9-ol 19p-Cymene 16 16 20Pentadecane 16 20 20 20 20 20 20Pentanoic acid 20 20Phenethyl alcohol 15Phenol 15 16 16 17 17Phenylacetaldehyde 15 16 16 17,20 17 18,20 19,20 20 20 20Phenylacetic acid 16 16 17p-Mentha-(7),8-(10)-dien-9-ol 19,20 20 20 20p-Methylacetophenone 16 16Propylanisole 20Pyridine 16 16 18Sinensal (isomer I) 19,20Sinensal (isomer II) 19,20Spathulenol 20Terpineal 19Tetradecanoic acid 20 20 19,20 20 20 20Thymol 16 16 20 20 19,20 20 20 20Toluene 16 16 17 17trans-Caryophyllene 15trans-Linalool oxide 19,20 20Tetradecanoic acid 19Trimethoxybenzene (isomer) 16 16Vinylguaiacol 20 20 19,20 20 20 20a-4-Dimethyl-3-cyclohexen-1-

acetaldehyde20 20 20 20 20

(Continued)




a-Humulene 16 16a-Pinene 15 16 16a-Terpineol 20 20 19,20 20 20 20b-Damascenone 20 20 19,20 20 20 20b-Pinene 15 16 16g-Butyrolactone 17,20 17 20 19 20 20 20g-Terpinene 16 16g-Valerolactone 16 16 17,20 17 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 withliquid nitrogen, and finally injected into the GC capillary column. GC-MS analysis of the honey headspace was performed ona polar capillary column in a temperature gradient. A large number of volatiles were identified and the existence of certain markercompounds for the floral origins was assessed; e.g., the presence of either 2-methyldihydrofuranone or a-methylbenzyl alcohol orboth 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 pollenanalysis 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 witheucalyptus, 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 aromacompounds were identified (Table 2), but only norisoprenoid compounds such as a-isophorone, b-isophorone, and 4-oxoiso-phorone, 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 authorsdemonstrated that the statistical elaboration of the volatile data was very promising to classify samples according to their botanicalorigin. Eucalyptus, thyme, citrus, rosemary, heather, lavender, and multiflower honeys were considered; the concentration of thehoney solutions and temperature and time of purge were optimized to maximize the amount of volatiles. Volatiles swept by thehelium flow were collected at ambient temperature on a porous polymer resin based on 2,6-diphenylene oxide, thermally desorbedat 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; chromatographicseparations were carried out on a polar capillary column operating in a temperature gradient with helium as carrier gas. Qualitativeanalysis was based on spectra data and linear retention indices (LRI) while quantitative data were obtained using 5-nonanone asinternal standard. One hundred volatile compounds (Table 2), including terpenes derived from the floral nectar, furan derivativesfrom honey processing and storage, and other compounds whose origin could be related to microbial or environmentalcontamination, were identified. Following discriminant analysis (DA), eucalyptus honeys were characterized by the amount ofoctane 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 honeyvolatile 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 vesselflask at 45 �C for 45 min) and volatile compounds were trapped on a porous polymer resin and subsequently thermally desorbedunder a helium flow at 220 �C for 16 min. The volatiles were then cryofocused in a cold trap at�30 �C and transferred directly ontothe 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 theauthors 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 (lique-faction 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 andextraction time can be reduced to a few minutes.28 In recent years, some efforts have been made to analyze honey volatiles using theSPME technique29 and different types of SPME fibers have been evaluated taking into consideration the polarity of fiber coatingsand 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 Chestnut Lime Rape Sunflower Acacia Thyme

1-(2-Furanyl)-ethanone 26 25 26,27 26 26 26 26 261,3,8-Menthatriene 241-Butanol 24,26 25 26 24,26 26 24,26 24,26 24 24 24 24 24 261-Hexanol 26 24,26 26 241-Hydroxy-2-propanone 27 271-Methoxy-2-propyl acetate 271-Nonanol 27 271-Octen-3-ol 241-Octene 26 26 26 26 24,26 261-Pentanol 24 241-Penten-3-ol 26 26 26 26 26 24,26 262,2,6-Trimethylcyclohexanone 242,3,4-Trimethyl-2-cyclopenten-1-one 252,3-Butanedione 26,27 25 26,27 26 26,27 26 26 262,3-Dihydro-4-methylfuran 24,26 26 24,26 26 24,26 24,26 262,3-Heptanedione 26 262,3-Pentanedione 25 26 24,26 26 26 26 262,4,4-Trimethylcyclopentanone 252,5-Dimethylfuran 26 25 26 26 26 26 26 262,6,6-Trimethyl-2,4-cycloheptadien-1-one 24 24 242,6,6-Trimethylcyclohexan-1,4-dione 242,6-Dimethyl-4-heptanol 26 262- and 3-Methylbutanoic acid 24 24 242-Acetylfuran 24 24 24 24 24 24 24 242-Allyl-4-methyl phenol 242-Butanol 26,27 26 26 26 26 26 262-Butanone 24,26 25 26 24,26 26 24,26 24,26 24 24 24 24 24 262-Butenenitrile 26 262-Butoxyethanol 27 242-Cyclohexene-1-one 242-Decanone 262-Ethylfuran 242-Ethylhexanoic acid 272-Heptanone 26 26 24,26 26 26 26 262-Hexanol 262-Hydroxy-3-pentanone 24 242-Hydroxy-5-methyl-3-hexanone 262-Methyl- and 3-methyl-2-buten-1-ol 26 26 262-Methyl-1-butanol 26 26 26 26,27 26 26 262-Methyl-1-propanol 24,26 25 26,27 24,26 26,27 24,26 24,26 24 24 24 24 262-Methyl-2-butenal 26,27 26 26 26,27 26 26 262-Methyl-2-propanol 27 27 27

(Continued)

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Table 2 Volatile compounds in unifloral honeys (extraction technique: DHS)dcont’d


2-Methyl-3-buten-2-ol 26,27 26,27 26 26 26 26 262-Methylacetophenone 242-Methylbutanal 24,26 26 24,26 26 24,26 24,26 24 24 24 24 24 262-Methylbutanenitrile 26 26 26 26 26 262-Methyldihydrofuranone 242-Methylfuran 26 26 26 26 26 26 24 262-Methylhexanoic acid 272-Methyl-2-buten-1-ol 26,272-Methylpropanenitrile 26 26 26 26 26 24 262-Methylpropanoic acid 242-Pentanol 26 26 26 26 24,26 24,26 262-Pentanone 24 24 24 24 24 24 24 24 242-Propanol 24 24 24 24 24 24 24 24 243-(1-Methylethyl)-2-cyclopenten-1-one 253,5,5-Trimethylcyclohexan-1,4-dione 253-Aminoacetophenone 24 243-Butenenitrile 26 26 26 26 26 263-Cyclohexen-1-ol-5-methylene-6-isopropylene 243-Furancarboxaldehyde 253-Hexen-1-ol 24 243-Hexen-2-one 26 26 26 26 26 26 263-Hexenylformate 243-Hydroxy-2-butanone (acetoin) 24 27 24 27 24 24 24 24 24 24 24 263-Hydroxy-2-pentanone 24 243-Methyl-1-butanol 24,26,27 25 26 24,26 26,27 24,26 24,26 24 24 24 24 263-Methyl-1-undecene 24 243-Methyl-2-butanol 243-Methyl-2-butanone 243-Methyl-2-buten-1-ol 263-Methyl-2-butenal 27 27 273-Methyl-2-pentanone 26 263-Methyl-3-buten-1-ol 24,26,27 26,27 24,26 26,27 24,26 24,26 24 24 24 24 24 263-Methylbutanal 24,26 26 24,26 26,27 24,26 24,26 24 24 24 24 24 263-Methylbutanenitrile 26 26 26 26 26 263-Penten-2-one 26 26 26 26 264,5,7a-Hexahydrobenzofuran-3,6-dimethyl-2,3,3a4-Acetyl-1-methylcyclohexene 244-Ethylphenylacetate 244-Methyl-1-pentanol 244-Methylacetophenone 244-Oxoisophorone (3,5,5-trimethylcyclohex-2-en-1,4-dione) 24,26 25 24,26 26 24 24,26 24 24 245-Methyl-1-hexanol 24Dihydro-5-methyl-2(3H)-furanone 24 245-Methylfurfural 26 26 24,26 26 26 24,26 24 24

94Extraction

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6-Methyl-5-hepten-2-one 24 25 24 24 24 24 24Acetic acid 26,27 26,27 26 27 26Acetone 24,26 25 26,27 24,26 26,27 24,26 24,26 24 24 24 24 24 26Acetonitrile 26 26 26 26 26 26Acetophenone 24 26 24,26 24 24 24 24 24Benzaldehyde 26,27 25 26,27 24,26 26 24,26 26 24 24 24 24 24 26Benzene acetaldehyde 27 27Benzyl alcohol 24,27 24 24 24 24 24 24 24 24Bicyclo-3,2,1-octane-2,3-bis(methylene) 24Butanal 26 26 26 26Bicyclo 2,2,2-octan-1-ol-4-methyl 24Carbon tetrachloride 26 26 26Chloroform 24,26 26 24,26 26 24,26 24,26 24 24 24 24 24 26cis-Linalool oxide 24 24 24 24cis-Linalool oxide (furan ring) 26 26 26 26 24,26 24,26 26Cyclohexane 26 26 26 26Cyclopentenedione 24 24 24Decanal 24,27 25 27 24 24 24 24 24 24 24 24Dichloromethane 26 26 26 26 26 26 26Dihydro-2-methyl-3(2H)-furanone 26 26 26 26 26 26 26Dihydro-5-methyl-2(3H)-furanone 26Dihydroisophorone (3,3,5-trimethylcyclohexanone) 25Dimethyldisulfide 26 26 24,26 26,27 26 24,26 24 24 24 26Dimethylstyrene 24 24Dimethylsulfide 26,27 26,27 26 26,27 26 26 26D-Limonene 27 27 27Dodecane 27Ethanol 24,26,27 25 26,27 24,26 26,27 24,26 24,26 24 24 24 24 24Ethyl acetate 26 26 26 26 24,26 24,26 24 24 24 26Ethyl-2-hydroxypropanoate 24Ethylbenzene 26 26 26 26 26 26 26Ethylbenzoate 24Furan 26 26 26 26 26 26Furfural 24,26,27 25 26,27 24,26 26,27 24,26 24,26 24 24 24 24 24 26Furfuryl alcohol 26 24 24 24 24 24 24Geraniol 26 26Heptanal 24 25 24 24 24 24 24 24Heptane 26 26 26 26 26 26 26HexanalHexanoic acid 27Hotrienol (3,7-dimethyl-1,5,7-octatrien-3-ol) 26 26,27 24,26 26,27 24,26 24,26 24 24 24 24 24 26Hydroxyacetone (acetol) 24 24 24 24 24 24 24 24Isobutyl acetate 26Isopropyl myristate 27Lilac aldehyde (isomer I) 26,27 26 26Lilac aldehyde (isomer II) 26,27 26,27 26

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Table 2 Volatile compounds in unifloral honeys (extraction technique: DHS)dcont’d


Lilac aldehyde (isomer III) 26,27 26,27 26Lilac aldehyde (isomer IV) 26,27 26,27 26Linalool (3,7-dimethyl-1,6-octadien-3-ol) 24 24 24 24 24Linalool oxide 27 27 27Methyl antranilate 27Methyl isopropyl benzene 24Methyl salicylate 27Methyl-1,3-pentadiene 26Methyl-2-butenale 24 25 24 24 24 24 24 24 24 24Methyl-2-butenol 24 24 24 24 24 24 24 24 24Methylcyclohexane 26 26 26 26 26 26Methylnaphthalene (isomer not identified) 26m-Xylene 26 26 26 26,27 26 26 26Nerol 26 26Nonanal 24,27 25 26,27 24,26 26 24 24 24 24 24 24 24Octanal 24 25 24 24 24 24 24 24 24Octane 26,27 26,27 26 26,27 26 26 26o-Xylene 26 26 26 26 26 26Pentanal 24 24 24 24 24 24 24Phenylacetaldehyde 26 26 24,26 26 24,26 24,26 26Phenylethyl alcohol 27 27 24 27 24 24 24 24 24 24 24p-Menth-1-en-9-al (isomers I and II) 26 26 26Propanenitrile 26 26 26 26p-Xylene 26 26 26 26 26 26 26Terpinen-4-ol 26Toluene 26,27 26,27 26 26 26 26 26trans-Linalool oxide (furan ring) 26 26 26 26 26 26 26Trichloroethylene 26 26 26 26 26 26 26a,a-Dimethylbenzyl alcohol 26 26a-4-Dimethyl-3-cyclohexe-1-acetaldehydea-Cyclic ether 24a-Isophorone (3,5,5-trimethyl-2-cyclohexen-1-one) 25 24 24 24,26a-Methylbenzyl alcohol (sec-phenethyl alcohol) 24a-Methylbutenol 24 24 24 24 24 24 24 24 24a-Methylpropyl phenyl acetate 24 24 24a-Pinene 24a-Pinene oxide 24a-Terpinene 24b-Damascenone 27b-Isophorone (3,5,5-trimethyl-3-cyclohexen-1-one) 25b-Linalool 27g-Butyrolactone 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.

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and on fiber absorption capacity, such as vial size, salt addition, magnetic stirring, equilibrium, and extraction time and temper-ature, plus GC injector desorption time were taken into consideration. By using SPME-GC-MS methods, an important number ofvolatile compounds have been found as possible markers of different types of honeys since most of these are related to the botanicalorigin.30–39 Moreover, the application of SPME has allowed the authenticity of honey to be verified by the presence of extraneoussubstances.40–45 Recently, interesting perspectives for improving the study of honey volatiles have arisen from SPME samplingfollowed 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 DeterminationAs previously mentioned, volatile compounds are related to the botanical and geographical origin of honey and many can beconsidered as reliable markers. In this context, by using SPME-GC-MS methods, an important number of organic compounds havebeen found as components of unifloral honeys coming from different countries; among these, the Spanish honeys are the moststudied.30–32,36,37

The first applications of SPME for the extraction of honey volatile constituents were reported by Verzera et al.33 and Piasenzottoet al.31 who studied Italian unifloral honeys from different regions. These authors were able to optimize their SPME-GC-MSmethods (Table 3) so that the identification of a large number of volatile constituents became possible (Table 4). The twomethods differed mainly in the extraction temperature, which was lower in the method developed by Verzera et al.,33 and theselected fibers; Verzera et al.33 found better results with a polydimethylsiloxane/divinylbenzene (PDMS/DVB) fiber and Pia-senzotto et al.31 used a polyacrylate (PA) fiber. In the unifloral Sicilian honeys,33 more than 100 compounds were identified whichbelonged to the following classes of substances: aliphatic and aromatic compounds, acyclic and monocyclic monoterpenes andtheir 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, nonanoicacid, 5-hexen-2-ol, and 2,3-dimethyl-5-hexen-2-ol, whereas acetophenone, 2-aminoacetophenone and 1-phenylethanol weremarkers 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 thecharacterization 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 andrapid analysis of honey volatile compounds (Table 3). In this case, SPME fiber coatings of different polarity, the influence of sampletemperature, equilibrium and exposure time, magnetic stirring, and salt addition on amount of volatiles were considered. GC-MSanalyses were performed on a polar column operating in a temperature gradient, with helium as carrier gas. The authors proposedthe use of a PA fiber (32 compounds identified) for distinguishing samples of different types and a CAR/PDMS fiber (24 compoundsidentified) 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 honeyblends. More than 100 volatile compounds were identified in all the samples analyzed and some of them were reported as possiblemarkers. For example, high levels of lilac aldehydes, lilac alcohols, cis-linalool oxide, benzaldehyde, phenylacetaldehyde, p-menth-1-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; highcontents of 2,6,6-trimethyl-2,4-cycloheptadien-1-one, 3,5,5-trimethylcyclohex-2-ene-1-one (isophorone), and 4-oxoisophoronewere found for rosemary honey, whereas the high nitrile content (2-methylpropanenitrile, 2-methyl butanenitrile, 3-methylbutanenitrile, 2-butenenitrile (cis- or trans-isomers), 3-butenenitrile, 3-methylpentanenitrile, and benzeneacetonitrile) proved to bedistinctive 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 thevolatile composition of widely used Spanish honeys, such as eucalyptus, rosemary, heather, and citrus. GC-MS profiles of honeyvolatiles were complex and 83 volatile compounds were identified using MS and retention data (Table 4). Due to the lack ofreference samples and the presence on the market of mixed honey types, a statistical elaboration processing the relative concen-trations of volatiles was applied. PCA was used as a first step to indicate the existence of possible natural groups followed bystepwise discriminant (SDA) and regression analyses. By the statistical elaboration, the authors provided valid information for thecharacterization of eucalyptus and citrus samples.

The SPME-GC-MS methods discussed above used conventional one-dimensional GC. which, even if typical analysis times of30–90 min were required to achieve acceptable chromatographic resolution, can lead to coelution of volatile constituents. In thiscontext, multidimensional gas chromatography (MDGC) represents a possible solution to obtain efficient separation of the entiresample. This analytical approach was first applied by Cajka et al.38 in 2007; then Cajka39 and Starimirova40 demonstrated thepotential of this challenging technique for its application in various follow-up studies including traceability of honey origin andauthentication. 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

Table 3 SPME methods: operation conditions for honey volatile extraction

Reference Floral source

Honey

provenance

Sample

size (g)

Vial

size (ml)

Sample

treatment Fiber type

Equilibrium

temperature

(�C)Equilibrium

time (min)

Extraction

temperature

(�C)Extraction

time (min) Injection mode

Desorption

temperature

(�C)Desorption

time (min)

30 Eucalyptus,rosemary, heather,citrus

Spain 1.5–2.0 5 Diluted with1 ml water

CAR/PDMS75 mm

60 15 60 30 Splitless/splitafter 2 min

250 2

31 Citrus, chestnut,eucalyptus, limetree, thyme,dandelion

Italy 3 10 Added to 0.5 gsodiumsulfate

PDMS 100 mm,PA 85 mm, CAR75 mm


250 3

32 Rosemary, heather,honeydew, orangeblossom, lavender,multiflower

Spain 1.5–2 5 Addition of 1 mlof water

CAR/PDMS 75 mm;PA 85 mm


250 2

33 Orange, sulla,chestnut,eucalyptus, wildflowers

Italy 16 40 Diluted with7 ml of water,added to 2 gNaCl

PDMS/DVB65 mm

30 30 30 25 Splitless 220 3

34 Multifloral, heather,buckwheat, lime-honeydew

Poland 1–1.5 4 – PDMS/DVB65 mm

40 60 40 30 Splitless 220 5

35 Fir-honeydew, sage Croatia 3.00 50 Added to 0.5 gsodiumsulfate

DVB/CAR/PDMS50/30 mm

– - 40 20 Splitless/splitafter 3 min

250

36 Orange, eucalyptus,rosemary,lavender, thyme

Spain 1 4 – CAR/PDMS,PDMS/DVB

70 60 70 30 Splitless PDMS/DVBat 250,CAR/PDMS at270

5

37 Eucalyptus, citrus,rosemary, thyme,lavender, chestnut,dandelion,rhododendron,Teide broom,acacia, heather,honeydew,multiflower

Spain 1.5–2.0 5 Addition of 1 mlof water

CAR/PDMS75 mm; PA85 mm


250 2

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41 Chestnut Italy 1 20 PDMS 100 mm – – 45 20 Splitless 250 0.442 Nectar and honeydew Spain 1.5–2.0 Diluted with

1 ml of waterthensonicated for5 min

CAR/PDMS75 mm

60 15 60 30 Splitless 250 2

43 Thyme – 8 10 Diluted with3,5 ml ofwater, addedto 1 g NaCl

PDMS 100 mm 30 30 30 25 – 250 3

44 Dandelion Italy 1.5–2 5 Addition of 1ml of water

CAR/PDMS75 mm

60 15 60 30 Splitless/splitvalveopening at2 min

250 2

45 Chestnut, fir, acacia,Pyrenees, orange,lavender,eucalyptus, forest,oak

France, Italy,Hungary,Spain

1 10 Diluted with4 ml of water,added to 1 gNaCl and0.2 ml ofacetic acid

PDMS 100 mm,PA 85 mm, CW/DVB 70 mm,CAR/PDMS75 mm, PDMS/DVB 65 mm

– – – 30 Splitless 300 for PAand CAR/PDMS;250 forPDMS,CW/DVB,PDMS/DVB

2

50 Lavender Croatia - 15 5 ml of honey–NaCl sat. H2Osolution 1:1v/v

DVB/CAR/PDMS 60 15 60 40 Split 250 6

51 Christ’s thorn Croatia - 15 5 ml honey–NaCl sat. H2Osolution 1:1v/v

CAR/DVB, DVB/CAR/PDMS

60 15 60 40 Split 250 6

52 Desert false indigo Croatia - 15 5 ml honey–NaCl sat. H2Osolution 1:1v/v

PDMS/DVB 60 15 60 40 Split 250 6

Abbreviations: CAR, carboxen; DVB, divinylbenzene; PA, polyacrylate; PDMS, polydimethylsiloxane.

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Table 4 Volatile compounds in unifloral honeys (extraction technique: SPME)

Compounds Thyme Sage Rosemary Orange Lime Lavender Heather Eucalyptus Buckwheat Chestnut Dandelion Citrus Sulla Wild flower

(Dimethylphenyl)-ethanone 32a

(E)-2-Octenal 33 33 33(E)-2-Pentenol 33 33 33 33 33(E)-2-Undecenal 33 33 33 33(E)-3-Hexenol 33 33(E)-6,10-Dimethyl-5,3-undecandien-2-one

35 35

(E)-Cinnamaldehyde 34 34(Z)-2-Pentenal 33 33 33(Z)-2-Pentenol 33(Z)-3-Hexenol 33 33 331-(2,4-Dimethylphenyl)ethanone 351-(2-Aminophenyl)-ethanone 341-(2-Furanyl)-ethanone 30,32a,32b 32a,32b 35 32a,32b 30,32a,32b,34 30 34 301-(2-Hydroxy-5-methylphenyl)ethanone

35

1-(4-Methylphenyl)ethanone 351,3,5,7-Cyclooctatetraene 331,3,8-p-Menthatriene 331,3-Butandiol 331,4-Cineole 33 33 331,8-Cineole 33 33 33 331-Hexanol 30,32b 32b 32b,36 30,32b 30 301-Hydroxy-2-propanone 36 32a,36 32b,36 32b,36 32b 361-Methyl-4-(1-methylethyl)benzene

35

1-Methyl-4-benzene 351-Methylethyl benzene 33 33 33 33 331-Nonanol 31 32a 32a 32a 32a,34 31 34 311-Octen-3-ol 33 33 331-Phenylethanol 331-Propyne2- and 3-Methyl-2-buten-1-ol 30 30 30 302(H)-1-Benzopyran-2-one 32a

2-(p-Methoxyphenyl)ethanol 31 31,352,2,4-Trimethyl-pentane 34 342,3-Butanediol 36 36 36 36 34 31,36 34 312,3-Butanediol (erythro) 30 30 302,3-Butanediol (threo) 30 30 302,3-Butanedione 30 30 30 302,3-Dimethyl-5-hexen-2-ol 332,3-Pentanedione 302,4,5-Trimethyl cumene 34

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2,5-Dimethyl-3-hexanone 332,5-Furandicarboxaldehyde 34 342,6,6-Trimethyl-2-cyclohexene-1,4-dione

36 36 34 36 34

2,6-Dimethoxy-phenol (syringol) 342,6-Dimethyl-3,7-octadien-1,6-diol

33

2,6-Dimethyl-3,7-octadien-2,6-diol

33 33 33

2-Acetyl benzoic acid 33 33 332-Aminomethyl benzoate 362-Butanol 362-Decanol 33 332-Decanone 332-Ethyl hexanol 32a 32a 32a 32a

2-Furan methanol 36 30,36 36 30,34 30 34 302-Furanocarboxaldehyde 35 352-Heptanone 33 332-Hydroxy-3,5,5-trimethylcyclohex-2-enone

34

2-Hydroxy-3,5,5-trimethylcyclohexanone

30 30 30 30

2-Hydroxy-ethyl benzoate 33 332-Methyl butanoic acid 34 342-Methyl propanoic acid 33 33 33 332-Methyl,dihydro-2(3H)-furanone

33

2-Methyl-1-butanol 36 36 34 31,36 34 312-Methyl-1-butanol 362-Methyl-1-propanol 36 36 362-Methyl-2-buten-1-ol 36 342-Methyl-2-butenal 342-Methyl-3-buten-2-ol 31,36 362-Methyl-3-phenyl-2-propenal 352-Methyl-6-(2-propenyl)phenol 352-Methylbenzene 352-Methyl butanal 34 342-Methyl propanoic acid 35 35 34 342-Nonanone 33 332-Phenylethanol 36 30,32a,32b,36 32a,32b,36 31 32a,32b,36 30,32a,32b,34 30,31,36 34 31 31 30,312-Xylylethanol 353,4,5-Trimethyl phenol 34 343,5,5-Trimethyl-2-cyclohexen-1-one

36 34 34

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Table 4 Volatile compounds in unifloral honeys (extraction technique: SPME)dcont’d


3,5-Dimethoxy benzaldehyde 343,7-Dimethyl-1,5,7-octatrien-3-ol

36

3,9-Epoxy-p-menth-1-ene 31 313-Aminoacetophenone 333-Furanocarboxylicacid methylester

35

3-Hexanol3-Hydroxy-2-butanone (acetoin) 36 30,32b,36 32b,36 32b,36 30,32b 30,31,36 31 303-Hydroxy-2-pentanone 30 303-Methyl butanal 363-Methyl-1-butanol 36 30,32b,36 32b,33 32b 30,32b,34 30,33,36 34 33 30 333-Methyl-1-hexanol 333-Methyl-2-buten-1-ol 363-Methyl-3-buten-1-ol 36 32a,32b,36 32a,32b,36 32a,32b,36 32a,32b 31 33 31 333-Methyl butanal 34 343-Methyl butanenitrile 30 30 30 303-Methyl butanoic acid 33 33,36 33 33 333-Phenyl-2-propen-1-ol 343-Pyridine carbonitrile 35 354-(1-Methylethyl)benzaldehyde 354-(1-Methylethyl)benzeneethanol

35

4-(3-Hydroxy-1-butenyl)-3,5,5-trimethyl-2-cyclohexen-1-one

31 31

4-(3-Oxo-1-butenyl)-3,5,5-trimethyl-2-cyclohexen-1-one

31 31

4,5,6,7-Tetrahydro-3,6-dimethyl-benzofuran

35 36

4,7-Dimethyl-benzofuran 354-Hydroxy-4-methyl-2-pentanone

35

4-Hydroxybenzenemethanol 354-Ketoisophorone 31 314-Methoxybenzaldehyde 35 354-Methyl,dihydro-2(3H)-furanone

33

4-Methyl-1-(1-methylethyl)-3-cyclohexen-1-ol

35

4-Methyl-1-(1-methylethyl)-bicyclo[3.1.0]hexene-2-one

35

4-Methyl-1-pentanol 33 334-Methyl-3-pentenol 334-Methyl phenol 34 34

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4-Oxoisophorone 30 33 30 30,33 33 30 33 335-Hexen-2-ol 33 335-Hydroxymethylfurfural 31 31 315-Methyl-2(5H)-furanone5-Methyl-2-

furanocarboxaldehyde35 35

5-Methyl-5-ethenyldihydro-2(3H)-furanone

33 33

6-Methyl-5-hepten-2-one 33 33 33 33 338-p-Menthen-1,2-diol 31 31 319-Octadecanoic acid, methyl

ester derivative35

Acetic acid 36 36 33,36 36 34 36 34 33Acetone 36 36 36 36 34 36Acetonitrile 32a

Acetophenone 34 34 33Benzenedicarboxylic acid,

monobutyl ester31 31

Benzyl methyl ketone 34Benzaldehyde 31,36 35 30,32a,32b,36 32a,32b,33,3631,35 32a 30,32a,32b,34 30,31,33,36 34 31,33 31 30 33 33Benzeneacetaldehyde 36 35 36 36 35 36 30,34 30,36 34 30Benzeneacetic acid 35 34Benzeneacetonitrile 33 33Benzenedicarboxylic acid

derivative35

Benzeneethanol 35 35Benzenemethanol 35 35Benzenepropanol 34Benzoic acid 35 33 35 34 33 34 33 33 33Benzyl alcohol 31 30,32a,32b 32a,32b,33 31 32a,32b 30,32a,32b,34 30,31,33 34 33 31 30 33 33Benzyl nitrile 34 34Borneol 35 33Butanoic acid 35 33 35 33 34 33Butanoic acid, 3-hexenyl ester 35Camphor 33 33 33 33 33Carvacrol 31,35 31Carvone 33cis-Carveol 33cis-Linalool oxide 34 34 31 31cis-Linalool oxide (furanoid) 30,32a,32b 32a,32b,33 32a,32b 30,32a,32b 30,31,33 33 30 33 33cis-Linalool oxide (pyranoid) 33 33 33cis-Rose oxide 33Damascenonne 34Decanal 33 34 33 34 33 33 33Decanoic acid 33 33 34 33 33 33Decanol 33 33

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Dehydromethylene-2(3H)-furanone

35

Dehydrovomifolio 31 31 31Dibutyl phthalate 34Dihydro-2(3H)-furanone 34 34Dihydro-3-methyl-2(3H)-

furanone34

Dihydro-4-methyl-2(3H)-furanone

31 31 34 31 31 31

Dihydro-5-methyl-2(3H)-furanone

34 34

Dihydrocarveol 33Dimethyl disulfide 36 33 33,36Dimethyl styrene 31Dimethyl styrene isomer 31,35Dimethyl sulfide 36 36 36 36 34 36 34Dimethyltrisulfide 33Dodecanal 33 33 33 33Dodecanoic acid 33 33 33 33Ethanol 36 36 36 36 34 36 34Ethenyl phenylacetate 31 31Ethylbenzeneacetate 30 30Ethylbenzoate 30 30 30Formic acid 34Furfural 36 32a,32b,36 32a,32b,33,36 32a,32b,36 32a,32b,34 31,36 34 31,33 33 33Furfuryl alcohol 32a,32b 32a,32b 32a,32b 32a,32b 33 33Furfuryl n-butyrate 31 31Geranyl acetone 33 34 33 33Heptadecane 33 33 33 33 33Heptanal 33 33 33Heptanoic acid 35 33 33 33 33 33Heptanol 33 33 33 33 33Hexadecanoic acid 33 33 33 33Hexadecanol 33 33 33 33Hexanal 33 33 33 33 33Hexanedioic acid 33Hexanoic acid 35 33 35 34 33 34 33 33 33Hexanol 33 33 33 33 33Hotrienol 30,32a 33 32a 30,32a 30,33 33 30 33Isoborneol 31 31Isobutyl phthalate 34 34Isophorone 32a,32b 32a,32b 32b 32a,32b 33Isopropyl myristate 34Lilac alcohol 31 31

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Lilac alcohol 31Lilac alcohol (isomer I) 32b 32a,32b 32a 32b

Lilac alcohol (isomer II) 32b 32b,32b 32b 32b

Lilac alcohol (isomer III) 32b 32a,32b 32b 32b

Lilac alcohol (isomer VI) 32b 32a,32b 32b 32b

Lilac aldehyde 31 35 34 34 31Lilac aldehyde (isomer I) 30,32a,32b 32a,32b,36 32b 30,32b 30 30Lilac aldehyde (isomer II) 30,32a,32b 32a,32b,36 32b 30,32b 30 30Lilac aldehyde (isomer III) 30,32a,32b 32a,32b,36 32b 30,32b 30 30Lilac aldehyde (isomer VI) 30,32a,32b 32a,32b 32b 30,32b 30 30Limonene 33 33 33 33 33Limonene diol 31Linalool 30,32a 32a,33 32a 30,32a 30,33 34 33 30 33 33Maltol 35Menthofuran 33 33 33Methoxy-phenyl-oxime 34Methyl anthranilate 32a,32b 32a,32b,33 32b 32b 30,33 33 30,31 33Methyl decanoate 33 33 33 33Methyl decenoate 33 33 33 33Methyl heptanoateMethyl heptanoate 33Methyl octanoate 33 33 33 33Methyl salicylate 33 34 33 34 33 33 33Methyl-2-buten-1-ol 32b 32b 32b 32b

Methyl-2-furoate 34 34Methylhexanoic acid 33 33 33Methylnaphthalene (isomer I) 32a

Methylnaphthalene (isomer II) 32a

Methylstyrene 35Myrcenol 33Myrtenol 33 33Naphthalene 32a

Neral 33 33Nerol 33 33Nerolidol 31Nonanal 33 31 34 31,33 34 33 31 33 33Nonane 34Nonanenitrile 33 33Nonanoic acid 31 33 35 34 31,33 34 31,33 33 33Nonanol 33 33 33 33 33Octadecanal 33 33 33Octanal 33 33 33 33 33Octane 34 34Octanoic acid 35 33 35 34 33 34 33 33 33Octanol 33 33 33 33

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p-Cymen-8-ol 31 33 31 33 33Pentanoic acid 34Pentanol 33Phenol 31 35 35 34 31 34 31Phenylacetaldehyde 32a 32a,33 32a 32a 33 33 31 33 33Phenylethyl alcohol 33 33 33 33 33p-Mentha-1,5-dien-8-ol 35p-Methylacetophenone 31 31Pulegone 35Rose oxide 35Rose oxide isomer 31 31Rose oxide isomer 31Teresantalol 35Terpinen-1-ol 33 33 33 33Terpinen-4-ol 33 33 33Terpinen-7-al 31 31Terpinene 35Tetradecanoic acid 33 33 33 33Tetrahydro-2,2,5,5-tetramethylfuran

35

Thymol 35Toluene 33 34 34 33trans-2-Caren-4-ol 35trans-Linalool oxide 34trans-linalool oxide (furanoid) 30,32a,32b 32a,32b,33 32a,32b 30,32a,32b 30 33 30 33 33trans-p-Mentha-2,8-dien-1-ol 31 31Trimethylphenol 31 31 31Undecane 33 33Verbenol 35a,a-4-Trimethylbenzenemethanol

35

a-4-Dimethyl-3-cyclohexen-1-acetaldehyde

33 33 33

a-Isophorone 30 30 30 30a-p-Dimethylstyrene 33 33 33 33a-Phenethyl alcohol 34a-Terpinen-7-al 35a-Terpineol 33 33 33 33 33b-Damascenone 32a 33 31 32a 32a 33 31,33 33g-Nonalactone 33 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,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, Lu�si�c, D.; Koprivnjak, O.; �Curi�c, D.; Sabatini, A.G.; Conte, L.S. FoodTechnol. 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.

106Extraction

Techniquesand

Applications:

Foodand

Beverage


Corsica and different European countries with the emphasis on confirming the authenticity of the honeys labeled as Corsican. ADVB/CAR/PDMS 50/30 mm fiber provided the best absorption capacity and the broadest range of volatiles extracted from theheadspace of a mixed honey sample. A combination of DB-5MS and SUPELCOWAX 10 columns gave the best resolution. Thispowerful 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. Interestinginformationwas obtained and the authors were able to demonstrate that theGC�GC-TOFMS combinedwith chemometricmethodssuch as linear discriminant analysis (LDA), discriminant partial least squares (DPLS), and support vector machines (SVM) canbe successfully applied to detect mislabeling of Corsican honeys.

4.05.2.3.2 SPME for Honey AuthenticityInteresting research regarding the determination of specific classes of substances that can be used for the authentication of honey arereported in the literature (Table 3). Two of these deal with the application of SPME-GC-MS methods developed to differentiatenectar and honeydew honeys.42,45 The interest of the authors arose from the consideration that this differentiation is very oftendifficult, not only because of the wide variability in composition and sensory properties among samples from the same source, butalso because of the frequent existence of honeys resulting from a blend of nectar and honeydew. Soria et al.42 considered a set ofSpanish nectar and honeydew honeys, whose floral source was confirmed by melissopalynology analysis. For SPME headspacesampling, a manual holder equipped with a CAR/PDMS fiber was used (Table 3) and chromatographic separation was carried outon a polar capillary column operating at programmed temperatures. Identifications were based on mass spectra, LRI, and standardinjections. Stepwise regression (SRA) from volatiles data gave an estimation of honeydew and it was possible to determine whichvolatile compounds were related to the different honey sources. The floral origin of honey was related to the presence of severalterpenoids such as borneol, cis- and trans-linalool oxide, lilac aldehydes, and lilac alcohols while acetic acid, erythro- and threo-2,3-butanediol were indicative of honeydew honey. Later, Daher and Gulacar45 focused their attention on the presence of phenolic andother aromatic compounds to differentiate nectar and honeydew honeys. With regard to SPME, the different parameters affectingthe 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 honeydewsamples from different countries and floral origin were analyzed using an apolar column in a temperature gradient; a total of 31compounds were detected, most of which were identified and quantified by GC-MS. Also in this case, PCA was applied to the datamatrix 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 honeyswere markers for the determination of floral origin. Fractionation of volatiles from honey headspace was carried out by using aCAR/PDS fiber as previously reported32 (Table 3) and the following nitriles were identified: 2-methylpropanenitrile, 2-methyl-butanenitrile, 3-methylbutanenitrile, 2-butenenitrile, (E)-3-butenenitrile, 3-methylpentanenitrile, benzonitrile, and benzeneace-tonitrile. Since nitrogen-containing compounds, such as nitriles, thiocyanates, and isothiocyanates, are hydrolysis products ofglucosinolates present inDiplotaxis sp. and other Brassicaceae,46 the authors proposed the presence of nitriles in Taraxacum honey asthe 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 honeysamples (Basilicata, Southern Italy) were analyzed; the characterization of the pollens demonstrated that chestnut prevailed. APDMS fiber (Table 3) was used and the volatiles were analyzed using GC-MS equipped with an apolar capillary column. From theirmass spectra, some phenyl-substituted and hydrocarbons were identified and correlated to the presence of pollutants fromanthropogenic 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 Thymehoney is considered to be one of the most delicious and high quality among the unifloral honeys; samples were analyzed followingthe SPME procedures previously developed by Verzera et al.33 and Piasenzotto et al.31 (Table 3). The results of GC-MS analysisshowed that pure thyme honey involved volatile compounds that originate from the thyme plant, such as thymol and carvacrole, inlow 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 productsusing organic solvents and a water bath with ultrasound assistance at room temperature. The first application of this technique forthe study of honey volatile fraction goes back to 2003.47 The authors applied a USE methodology for Greek citrus honeys andflowers. The volatile fraction both of fresh flower and honey samples was extracted using an n-pentane/diethylether (1:2) mixture inan ultrasound water bath apparatus, maintained at 25 �C for 10min; the extracts were then analyzed directly by GC-FID and GC-MSusing an apolar capillary column operating at programmed temperature with helium as carrier gas. The analysis of the flowersallowed the identification of 17 volatile compounds, mainly monoterpenes; among these, linalool was predominant in all citrusspecies, except for lemon, with eucalyptol the main compound. For the honey volatile extracts (Table 5), linalool derivativesaccounted for more than 80% of the total volatile amount, in agreement with the flower extracts. Thus, through the quantification oflinalool derivatives present in citrus honey volatiles, the authors expected to establish a threshold to distinguish citrus honeys fromothers of different floral origin.

Table 5 Volatile and semivolatile honey compounds (extraction technique: USE)

Compounds Citrus Sage Thyme

(E)-2,6-Dimethyl-2,7-octadiene-1,6-diol 47(E)-2,6-Dimethyl-6-hydroxy-2,7-octadienal 47(Z)-2,6-Dimethyl-2,7-octadiene-1,6-diol 47(Z)-9-Tricosene 49(Z)-Octadec-9-en-1-ol 481-(4-Methoxyphenyl)propan-2-one 481,3-bis(1,1-Dimethyl)benzene 471,3-Di-tert-butylbenzene 491H-Pyrrole 481-Isocyanato-2-methylbenzene 471-Methoxy-4-propylbenzene 481-Methyl-2-pyrrolidinone 471-Octanol 471-Octanol 471-Phenyl-2,3-butanediol (isomer I) 491-Phenyl-2,3-butanediol (isomer II) 491-Phenyl-2,3-butanedione 492,2,6-Trimethylcyclohexane-1,4-dione 482,3,5-Trimethylphenol 482,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one 492,3-Dihydro-benzofuran 472,4-Di-tert-butylphenol 492,5-Dimethyl-2,4-dihydroxy-3(2H)-furanone 492,6,6-Trimethyl-4-oxocyclohex-2-ene-1-carbaldehyde 482,6-Dimethoxyphenol 482,6-Dimethyl-1,7-octadien-3,6-diol 472,6-Dimethyl-3,7-octadiene-2,6-diol 472-Ethyl-3-hydroxyhexyl 2-methylpropanoate 482H-1-Benzopyran-2-one 482-Hydroxy-1-(4-methoxyphenyl)ethanone 492-Hydroxyacetophenone 492-Hydroxyphenylethanol 492-Methylbenzene-1,4-diol 482-Methylbutanoic acid 492-Methylpropanoic acid 492-Phenylacetaldehyde 482-Phenylacetic acid 482-Phenylethanol 47 483,4,5-Trimethoxybenzaic acid 493,4,5-Trimethoxybenzaldehyde 48 493,4,5-Trimethylphenol 483,5-Dihydroxy-2-methyl-4H-pyran-4one 493,7-Dimethyl-1,5-octadiene-3,7-diol 483,7-Dimethyl-1,6-octadiene-3,5-diol 473-Hydroxy-1-phenyl-2-butanone 493-Hydroxy-2-butanone 493-Hydroxy-2-methyl-4H-pyran-4-one 483-Hydroxy-4-phenyl-2-butanone 493-Hydroxy-4-phenyl-3-buten-2-one3-Methyl-1-butanol 493-Oxo-a-ionone 494-(4-Hydroxy-2,2,6-trimethyl-7-oxabicyclo[4.1.0]hept-1-yl)-3-buten-2-one 494-Ethenyl-2-methoxyphenol 484-Hydroxy-3,5-dimethylbenzaldehyde 484-Hydroxyphenylacetonitrile 494-Hydroxyphenylethanol4-Ketoisophorone 48 494-Methoxybenzaldehyde 484-Methoxybenzoic acid 484-Methoxyphenethyl alcohol 494-Methoxyphenylacetonitrile 49


Table 5 Volatile and semivolatile honey compounds (extraction technique: USE)dcont’d


5-(Hydroxymethyl)-furan-2-carbaldehyde 48Acetic acid 48Benzaldehyde 47 48 49Benzene-1,4-diol 48Benzeneacetaldehyde 47Benzeneacetic acid 47Benzoic acid 47 48 49Benzyl alcohol 48 49bis-(2-Ethylhexyl) adipate 47Butanoic acid 49Butyl acetate 48 49Caffeine 47Carvacrol 49cis-Linalool oxide (furanoid) 47cis-Linalool oxide (pyranoid) 47Coumaran 48Cyclohexanone 47Decanal 47 49Decane 47 49Decanoic acid 48 49Degraded carotenoid 47Dehydrovomifoliol 48 49Dibutylphthalate 47 48 49Dihydroanethole 49Diisobutylphthalate 49Docosane 49Dodecane 47 49Dodecanoic acid 48Eicosane 49Ethyl hexadecanoate 49Ethyl oleate 49Ethyl benzene 49Eugenol 49Furfural 49Guaiacol (2-methoxyphenol) 48Heneicosane 48 49Henicos-10-ene 48Heptadecane 48 49Heptane 47 49Hexadecane 49Hexadecanoic acid 48 49Hexanoic acid 48 49Hydroxymethylfurfural 49Hotrienol 47 49Indole 47Isophorone 49Isovaleric acid 49Lilac alcohol 47Lilac alcohol 47Lilac aldehyde 47Lilac aldehyde 47Lilac aldehyde 47Lilac aldehyde (isomer I) 47Lilac aldehyde (isomer II) 47Lilac aldehyde (isomer III) 47Limonene 47 49Linalool oxide B 48Linoleic acid 49m-(or p-)Xylene 47Methyl 4-methoxybenzoate 48

(Continued)


Table 5 Volatile and semivolatile honey compounds (extraction technique: USE)dcont’d


Methyl anthranilate 47Methyl benzoate 48Methyl hexadecanoate 49Methyl syringate 49Methyl vanillate 49Methyl cyclohexane 47Nerolidol 47Nonadecane 48 49Nonanal 49Nonane 47 49Nonanoic acid 48 49Octadecane 48 49Octadecanoic acid 49Octanal 49Octane 47 49Octanoic acid 48 49Oleic acid 49o-Xylene 47p-Anisaldehyde 49p-Anisic acid 49Pentacosane 48Pentadecane 48 49Phenol 48Phenylacetaldehyde 47 49Phenylacetic acid 47 49Phenylacetonitrile 49Phenylethyl alcohol 49Propanoic acid 49p-Toluic acid 49p-Xylene 49Salicylic acid 49Syringaldehyde 49Tetracosane 48 49Tetradecane 47 49Tetradecanoic acid 49Toluene 47 49trans-Linalool oxide (furanoid) 47 49trans-Linalool oxide (pyranoid) 47Tricosane 47 49Tridecane 47 48 49Undecane 47 49Vanillic acid 49Vanillin 49Veratric acid 49a-4-Dimethyl-3-cyclohexene-1-acetaldehyde (isomer III) 47a-4-Dimethyl-3-cyclohexene-1-acetaldehyde (isomer VI) 47a-Isophorone 48

47, Alissandrakis, E.; Daferera, D.; Tarantilis, P.A.; Polissiou, M.; Harizanis, P.C. Food Chem. 2003, 82, 575–582; 48, Jerkovi�c, I. Masteli�c J., Marijanovi�c, 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 Jerkovi�c 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; 54volatile compounds were identified (Table 5) by GC-FID and GC-MS in n-pentane/diethylether extracts after sonication at 25 �C for30min 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 thymehoney, the most marketed type in Greece. Using the experimental method previously developed,47 about 100 compounds wereidentified (Table 5) and quantified using b-ionone as internal standard. Phenolic compounds were the most abundant and wereproposed as potent botanical markers for thyme honey.


4.05.2.5 USE Coupled with SPME

Recently examples of applications of USE and SPME, applied as complementary extraction techniques for the characterization ofhoney volatile fraction, have been reported in the literature.50–52 The use of both techniques allowed complete patterns of honeyheadspace to be obtained, since the HS-SPME method proved to be suitable for the isolation of low-molecular-weight aromacompounds, whereas the USE procedure enabled extraction of semivolatile compounds.

The optimization and application of this comprehensive method was due exclusively to Jerkovi�c and co-workers who in 2009reported the honey volatile composition of different Croatian unifloral honeys, namely Lavandula hybrida Reverchon II50 (Lavandulaangustifolia � Lavandula latifolia), Paliurus spina-christi,51 and Amorpha fruticosa52 honeys. For the HS-SPME analysis different fiberswere considered (Table 3), whereas for the USE procedure three solvents, pentane, diethyl ether, and a mixture of pentane/ethylether (1:2 v/v) were used, under the same experimental conditions previously reported.48 The GC-MS analysis was performed onpolar and apolar capillary columns at programmed temperatures using helium as carrier gas. With regard to Lavandula hybridaReverchon II, HS-SPME made it possible to isolate short chain aliphatic compounds (Table 6), mainly hexan-1-ol, hexanal, aceticacid, hotrienol, and 2-phenylacetaldehyde, important for authentication of this honey, whereas the ultrasound pentane/ethyl etherextract contained the majority of the honey floral origin compounds and potential biomarkers (hexan-1-ol, acetic acid, butane-1,3-diol, butane-2,3-diol, benzoic acid, coumarin, and 2-phenylacetic acid) (Table 6). In the Paliurus spina-christi headspace a total of 33compounds 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)

CompoundsLavender Christ’s thorn Desert false indigo

USE SPME USE SPME USE SPME

(E)-b-Damascenone 52(Z)-Octadec-9-en-1-ol 50 51 52(Z)-Octadec-9-enoic acid 52(Z,Z)-9,12-Octadecadienal 51(Z,Z)-9,12-Octadecadienoic acid 511-(2-Furanyl)-2-hydroxyethanone 521-(2-Furanyl)-ethanone 521,3-Butanediol 511,3-Butanediol 501,3-Dimethylbenzene 521,4-Benzenediol 51 521,4-Dimethylbenzene 521,4-Di-tert-butylbenzene 521-Dodecanol 521-Hexadecanol 51 521-Hexanol 50 501H-Indole-3-acetic acid 511H-Pyrrole-2-carboxylic acid 511-Hydroxylinalool 521-Nonanol 521-Octadecanol 521-Phenylethan-1,3-diol 521-Tetradecanol 522-(4-Methoxyphenyl)ethanol 502-(p-Methoxyphenyl)-ethanol 512,3-Butanediol 512,3-Butanediol 502,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one 50 50 522,3-Dihydro-3,5-dimethyl-4H-pyran-4-one 512,3-Dihydro-5-hydroxy-6-methyl-4H-pyran-4-one 502,3-Dihydrobenzofuran 50 512,4-Dimethyl-3,6-dihydro-2H-pyran 522,5-Dimethoxy-4-ethylbenzaldehyde 522-Ethylfuran 522-Ethylhexanoic acid 51a,51b

2-Ethylhexyl 2-ethylhexenoate 502-Furancarbaldehyde 502-Furancarboxylic acid 50

(Continued)

Table 6 Volatile and semivolatile honey compounds (extraction techniques: USE and SPME)dcont’d



2-Furanmethanol 51b

2-Hydroxy-3-methyl-4H-pyran-4-one (maltol) 512-Methoxy-6-methylpyrazine 512-Phenylacetaldehyde 502-Phenylacetamide 522-Phenylacetic acid 502-Phenylethanol 50 50 51 51a,51b 52 523-(4-Hydroxy-3-methoxyphenyl)-prop-2-enoic acid (ferulic acid) 523,5-Dihydroxy-2-methyl-4H-pyran-4-one 503,5-Dimethoxy-4-hydroxybenzoic acid (syrigic acid) 523,7-Dimethylocta-1,5-diene-3,7-diol 50 51 523-Hydroxy-4-methoxycinnamic acid (isoferulic acid) 523-Methyl-2-butanol 513-Methylpentanoic acid 513-Pyridinecarbonitrile 514,5-Dimethyl-2-formylfuran 524-Ethenyl-2-methoxyphenol 504-Ethylbenzaldehyde 504-Hydroxy-3,5,6-trimethyl-4-(3-oxo-1-butenyl)-cyclohex-2-en-1-one 524-Hydroxy-3,5-dimethoxybenzaldehyde (syringyl aldehyde) 524-Hydroxy-3,5-dimethoxybenzoic acid 514-Hydroxy-3,5-dimethylbenzaldehyde 514-Hydroxy-3-methoxybenzaldehyde (vanilline) 524-Hydroxy-3-methoxybenzoic acid (vanillic acid) 524-Hydroxybenzaldehyde 51a,51b

4-Hydroxybenzoic acid (p-salicylic acid) 51 524-Hydroxybenzyl alcohol 524-Hydroxycinnamic acid (3-(4-hydroxyphenyl)-prop-2-enoic acid) 524-Hydroxyphenylacetic acid 524-Ketoisophorone 524-Methoxybenzaldehyde 524-Methoxybenzoic acid 514-Methyl-2,6-bis (1,1-dimethylethyl)-phenol 524-Methylbenzyl alcohol 524-Methyloctane 524-Vinyl-2-methoxyphenol 51 52 524-Vinylphenol 525-(Hydroxymethyl)furan-2-carbaldehyde 505-Hydroxymethylfurfural 50 51 525-Methyl-1,4-benzenedione 51a

5-Methylfurfural 50Acetic acid 50 50 51 51a,51b

Benzaldehyde 50 51a,51b 52 52Benzoic acid 50 51 52Benzyl alcohol 51 52 52Butanoic acid 50 51Butoxyethoxyethyl acetate 52cis-Linalool oxide 51a 52 52Coumarin 50Decanal 51 51a,51b

Decanoic acid 51Diisobutyl phthalate 52Dimethyl disulfide 52Docosane 51Dodecane 50 51 51a

Ethylbenzene 52Formic acid 50 50Furfural 50 51b 52


Table 6 Volatile and semivolatile honey compounds (extraction techniques: USE and SPME)dcont’d



Furfuryl alcohol 50Heneicosane 50 51 51a 52Heptacosane 51Heptadecane 51Heptanal 50 51a,51b

Heptanoic acid 50Hexacosane 51Hexadecan-1-ol 50Hexadecane 51Hexadecanoic acid 50 51 52Hexanal 50Hexanoic acid 50 50 51 51a,51b 52Hotrienol 50 50 52Isophorone 52Lilac aldehyde (isomer I) 51a,51b

Lilac aldehyde (isomer II) 51a,51b

Lilac aldehyde (isomer III) 51a,51b

Lilac aldehyde (isomer not identified) 52Lilac aldehyde (isomer VI) 51a,51b

Linalool 52Maltol 50 50Methoxybenzene 52Methyl 2-furancarboxilate 51Methyl 3,5-dimethoxy-4-hydroxybenzoate (methyl syringate) 52Methyl furan-2-carboxylate 50 50Methyl nonanoate 51a

Methyl octanoate 51a

Methyl sulfide 52Nananoic acid 52Nonacosane 51Nonadecane 51 51a

Nonanal 51 51a,51b 52Nonane 52Nonanoic acid 50 50 51 51a,51b 52Octacosane 51Octadecane 51Octadecanoic acid 51Octanal 51 51a

Octane 52Octanedioic acid 51Octanoic acid 50 50 51 51a,51b

o-Ethylanisole 51a

Pantoic lactone 50Pentadecane 51a 52Pentanoic acid 51Phenylacetaldehyde 51 51a,51b 52 52Phenylacetic acid 51 52Phenylacetonitrile 52Phytol 51Pinocarvone 52Propanoic acid 50 51Tetracosane 51 52Toluene 51b

trans-Linalool oxide 52 52Tetradecane 51Tricosane 51Tridecane 51a

Undecane 51a,51b

50, Jerkovi�c, I.; Marijanovi�c, Z. Chem Biodivers. 2009, 6(3), 421–430; 51, Jerkovi�c, I.; Tuberoso, C.I.G.; Marijanovi�c, Z.; Jeli�c, M.; Kasuma, A. Food Chem. 2009, 112(1), 239–245;52, Jerkovi�c, I.; Marijanovi�c, Z.; Kezi�c, J.; Gugi�c, M. Molecules 2009, 14, 2717–2728.



(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-SPMEin 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 wereidentified, 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, asexpected. Thus, using the two different extraction methods and combining the data obtained, interesting information can beobtained, 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 qualitytesting in the food industry; this is the so-called electronic nose (E-nose) consisting of an array of weakly specific or broad spectrumchemical sensors that intend to mimic the human olfactory system and convert sensor signals to data that can be analyzed withappropriate statistical software. The use of E-nose to characterize the origin of honey based on the volatile fraction introduceda different strategy that allows a profile or fingerprint to be obtained avoiding any separation into individual compounds. The firstapplication of E-nose to honey volatiles was due to Benedetti et al.53 who applied an artificial neural network (ANN) to classifysignals from an electronic nose smelling different types of honeys. Unifloral honey samples of specific botanical and geographicalorigins (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 (10metal oxide semiconductor field effect transistor sensors, 12metal oxide semiconductor sensors). The data obtained from the sensorarray were statistically analyzed by PCA and ANN and good results were obtained using a neural network model based ona 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 wasfound that honey samples from geographically close regions tended to be grouped together, while those from distant geographicregions 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). Threedifferent sampling techniques, SHS, SPME, and inside needle dynamic extraction (INDEX) (also known as solid-phase dynamicextraction (SPDE)), were used for volatiles of Swiss honeys derived from dandelion, lime, acacia, chestnut, fir, and rape. For eachsampling technique, the extracted volatiles were injected into the injector port of the E-nose. The responses of the detector (specificionic masses), processed by PCA and discriminating factor, allowed the authors to classify the samples into different botanicalgroups in agreement with the results simultaneously obtained by a classic method, i.e., sensory, pollen, and physicochemicalanalysis. 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 whichallows identification and fingerprinting of aroma as with regular GC but at the same time it operates at the speed of the E-nose usinga 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, wasused 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 fromthe zNose measurements were approached in two different ways: first, by comparing the different peaks and peak areas obtainedconsidering only the positives values of the first derivative plot (chromatogram approach); and second, the positive and negativevalues of the first derivative profile were considered and treated as spectral data, analyzing the full frequency spectrum of eachsample. The statistical treatment of the data by PCA and canonical discriminant analysis made it possible to discriminate amonghoneys 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, sincethere is a great variability in the aroma compounds obtained depending on the procedure used. In most cases, the extractionmethod 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 techniquesthat make it possible to obtain a volatile profile comparable with the sensorial descriptors are those operating in the sampleheadspace, such as DHS and SPME, when carried out at low temperature. In the case of honey, the use of heat along with solvent, asrequired by the SDE technique, significantly modifies the volatile profile. For example, in an acid aqueous medium, terpenes easilylead to hydrated and oxidized compounds and substances from the Maillard reaction and/or sugar caramelization can arise

Table 7 Furan and pyran derivatives (mg kg�1) in thyme honey volatiles sampled by different extraction techniques

Compounds SDE20 DHS26 SPME31,36 USE49

1-(2-Furanyl)-ethanone –a

2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one 332,3-Dihydro-4-methylfuran 327.02,5-Dimethyl-2,4-dihydroxy-3(2H)-furanone 862,5-Dimethylfurane 13.42-Acetylfuran 95.82-Furan methanol 6.0c

2-Furancarboxilic acid 22.42-Methyl-3-(2H)-dihydrofuranone 21.52-Methylfuran �3,5-Dihydroxy-2-methyl-4H-pyran-4one 955-Ethenyl-5-methyl-2(3H)-furanone 30.15-Methyl furfural 69.25-Methyl-2-(3H)-furanone 12.6Furan derivative (95, 123, 124) 78Furfural 1558 495.4 35.6c 74Furfuryl alcohol 19.8Furfuryl n-butyrate –b

Vinylguaiacol 19.5

aCompounds identified but not quantified.bCompounds identified by Piasenzotto et al.31cCompounds identified by Perez et al.36

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-p-dimethyl 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, lilacaldehyde 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).



simultaneously, such as furan derivatives. Furan-derived compounds, such as furfural, furfuryl alcohol, and 5-methyl furfural, whichare present in a low concentration in fresh honey, increase during storage and heating and the increase is greater the higher thetemperature.57 Thus, they are considered as indicators of thermic processes and storage and are usually used in evaluating qualitydeterioration in food but they cannot be considered as appropriate floral markers. Table 7 reports the furan derivatives identified inthyme honey samples,20,26,31,36,49 extracted by different methods, along with their amount, where indicated. The SDE extracts werethe richest in furan derivatives while the SPME extracts had less. Furfural, present in all the thyme honey samples analyzed, was themain compound together with phenylacetaldehyde (~1.5 mg kg�1) in the SDE extracts. An high amount of furfural and 2,3-dihydro-4-methylfuran was also present in thyme honeys extracted by DHS. The significant amount of these two substances couldbe associated with purge and trap fractionation that operated at a temperature of 80 �C. In contrast, the amount of furan derivativesin 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 primaryaroma compounds, such as terpenes and their derivatives, that are associated with the floral nectar or honeydew gathered byhoneybees. Figure 1 reports a SPME-GC-MS(SIM)58 of a citrus honey in which a large number of terpenes, sesquiterpenes, and theiroxygenated compounds were identified. Among these, the markers for the floral origin can be defined, in agreement with otherauthors. From the above observations, SPME seems to be a very reliable technique since high reproducibility and sensibility havebeen achieved with respect to extraction and identification of honey volatile compounds without the complexity of traditionalmethods.

See also: Headspace Analysis; Solid-Phase Microextraction; Sample Preparation Automation for GC Injection; Headspace Sampling inFlavour and Fragrance Field; Theory of Extraction

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4 05 Sampling Techniques for the Determination of the Volatile Fraction of Honey 2012 Reference Module in Chemistry Molecular Sciences and Chemical En