# Melissopalynological and Volatile Compounds Analysis of Buckwheat Honey from Different Geographical Origins and Their Role in Botanical Determination

Authors: Sara Panseri; Alessandra Manzo; Luca Maria Chiesa; Annamaria Giorgi
Journal: Journal of Chemistry
 (2013)

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## Abstract

Volatile organic compounds (VOCs) have been proposed as one of the main factors for differentiating honeys from different botanical/floral origins. In this work, we investigated the volatile profile of honeys, commercially labeled as buckwheat honeys, from the Alps and its relationship with melissopalynological investigation. The results showed that buckwheat honey samples that contained, to different extents, buckwheat pollen grains on melissopalynological analyses showed similar VOCs profiles, distinguishing them from the other honey floral types analyzed. Among VOCs identified, 3-methylbutanal, butanoic acid, pentanoic acid, and isovaleric acid were considerably greater in the buckwheat honey samples from the Alps. Other compounds were identified only in the honeys containing buckwheat pollen grains such as 3-methyl-2-buten-1-ol, 2-butanone, 2-hydroxy-3-pentanone, 4-methylpentanoic acid, 4-pentanoic acid, butanal, 2-methylbutanal, pentanal, dihydro-2-methyl-3(2H)-furanone, 5-methylfurfural, andcis-linalool oxide. These compounds give to buckwheat honey its characteristic aromatic and organoleptic properties and may be considered interesting as potential “variety markers” for botanical determination.


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## Body

## 1. Introduction

Honey is a highly energetic sweet food, produced by bees, used by human beings since ancient times, and is of significant economic value today.The organoleptic properties of honey, such as flavour, colour, aroma, and texture are essential factors in consumers’ estimation of honey quality. These factors are primarily determined by the type of plant species and flowers visited by bees in order to collect nectar or honeydew to produce honey. Climate conditions, bee physiology, honey harvesting and postcollection processing may also influence, to a lesser extent, honey quality. As a consequence, the botanical and, to some extent, also the geographical origins are important characteristics in the evaluation of honey quality [1].Melissopalynological analyses, consisting of the qualitative and quantitative microscopic examination of honey pollen grains, is, at present, the official test to determine the botanical and geographical origin of honey [2, 3]. Honey containing pollen mainly collected from a single species is classified as monofloral or unifloral, while multifloral honey contains pollen from lots of different species [4]. However, melissopalynology is time consuming, expensive, and highly influenced by the analyst’s subjective ability in interpreting data [1, 5]. Moreover, the quantity of pollen found in honey is not always directly correlated with the nectar contribution of a species, since, for example, when the honeys are derived from sterile plants, pollen analysis is of absolutely no use [6]. Considering all these limiting factors, efforts in the identification of alternative or complementary methods to replace or to integrate pollen analysis are particularly important in order to improve honey quality determination. Studies concerning quality characterization of honey on the basis of phytochemical content in relation with the physicochemical and pollen profile have been published [7–10].Among phytochemicals, volatile organic compounds (VOCs) have been proposed as one of the main factors for differentiating honeys from different botanical/floral origins [5]. In particular, some unifloral honeys, characterized by individual and specific sensory properties, have been proven to differ one from the other in, among other features, volatile organic composition [11]. Some VOCs are present in the nectar or honeydew collected by bees and could be related to plant characteristics, some others might be originated during honey processing and storage [12, 13].In this paper, the volatile profile of honeys commercially labeled as buckwheat honeys from the Italian Alps, Russia, Nepal, and Poland was analyzed by means of HS-SPME and GC/MS, a valuable method widely used for volatile extraction and analyses [1, 14, 15]. Buckwheat (Fagopyrum esculentum Moench) is an annual, dicotyledonous plant from the Polygonaceae family with a short growing season [16]. It is a multifood-use pseudocereal. Its inflorescence is formed by 7–9 white, pink, or red blossoms [17]. It is a hermaphroditic species which produces self-incompatible flowers pollinated by insects, including bees. The world’s largest producer of buckwheat nowadays is China, followed by Russia, Ukraine, and France. In Italy, buckwheat cultivation was introduced around the 16th century [18]. However, during the most recent decades cultivation of this crop has strongly declined and today it survives only in a few alpine valleys [19, 20]. Buckwheat honey, collected from the little pink flowers by honeybees during the summer, is characterized by a dark purple color, almost black [21]. Buckwheat monofloral honey is mainly produced in North America (Canada and California), China, and in some countries of Europe, such as Poland, Russia, Netherlands, and Germany. Because of the quite low cultivation of buckwheat plants, in Italy, the monofloral buckwheat honey is difficult to produce and it is usually found as a natural component of multifloral honeys [22]. Buckwheat honey is a high-quality product characterized by a sharp, sweet, and slightly biting taste, having beneficial effects on human health due to its antioxidant, bactericidal, and anti-inflammatory properties [23–25].The effectiveness of VOCs markers in the botanical determination of honeys labeled as buckwheat honeys and their relationship with melissopalynological investigation results are discussed.


## 2. Materials and Methods

### 2.1. Honey Samples

This study was carried out on eight types of honey samples (Table1). In 2012, 18 samples were bought from local small-scale beekeepers working in Valtellina, an alpine valley of the Lombardy region. In particular, samples B1–B6 (Table 1), labeled as buckwheat honeys, were produced in Teglio (851 m a.s.l.; min. 352–max. 2.911 m a.s.l.), a mountain village in Valtellina where buckwheat is still cultivated; M1–M6 were reported as multifloral, A1–A3 as acacia honey (produced in an altitudinal range included between 200 and 1000 m a.s.l.), and R1–R3 as rhododendron honey (produced over 1000 m a.s.l.). B7, B8, B9, and B10 honey samples, labeled as buckwheat honeys, from Russia, Poland, and Nepal, respectively, were bought from international traders (Gego Enterpriser Pvt. Ltd., Baneshwor, Kathmandu, Nepal; RATOS-NATURA S.C., Olszownica, 75, 27-552, Backowice, Swietokrzyskie, Poland; Dary Altaya, OOO, Moscow, Russia). The floral origins of buckwheat samples were verified by using melissopalynological analysis. All samples were stored in darkness at a temperature of 4–6°C prior to analysis.Table 1
List of samples. Codes, locations, and honey type reported on the commercial label.
Sample code
Origin
Site
Honey type on the label
B1–B6
Italy
TeglioValtellina-Lombardy region
Buckwheat
B7
Russia
—
Buckwheat
B8-B9
Poland
—
Buckwheat
B10
Nepal
—
Buckwheat
M1–M3
Italy
TeglioValtellina-Lombardy region
Multifloral
M4–M6
Italy
Valtellina-Lombardy region
Multifloral
A1–A3
Italy
Valtellina-Lombardy region
Acacia
R1–R3
Italy
Valtellina-Lombardy region
Rhododendron


### 2.2. Melissopalynological Analysis

Melissopalynological analysis was performed according to the techniques proposed by the International Commission for Bee Botany (ICBB) and published in 1978 [26]. In this study, all honey samples were analyzed to confirm their floral origin. The microscopic analysis of honey sediment composition provides the percentage of the specific pollen observed by microscopic comparison with known pollen grains (Table 1).It is necessary to count at least 300 pollen grains for an estimation of the relative frequencies of pollen types and 500 to 1000 pollen grains for the determination of relative frequencies [27]. The examination under the microscope was carried out at the magnification that was most suitable for identifying the various elements in the sediment (400 to 1000x). After a first general check to ascertain the main types and densities of pollen grains, the relative frequencies of each pollen type were determined. A count of abortive, irregular, or broken pollen grains, fungal spores, hyphae, and microscopic algae, if they could be identified, was performed.If the sediment contained a high percentage of over-represented pollen, a second count excluding the over-represented pollen was done in order to determine more precisely the relative abundance of the other pollen types. The pollen types present in the honey samples were identified, counted, and classified, according to their percentages, as dominant pollen (more than 45% of the total pollen grains counted), secondary pollen (from 16 to 45%), important minor pollen (from 3 to 15%), and minor pollen (less than 3%) [28].


### 2.3. HS-SPME Volatile Compounds Sampling from Honey Samples

All the samples were prepared by weighing exactly 5.00 g of honey in a 20 mL glass vial, fitted with cap and equipped with silicon/PTFE septa (Supelco, Bellefonte, PA, USA) and by adding 1 mL of the internal standard solution (IS) in water (1,4-cineol, 1μg/mL, CAS 470-67-7) to check the quality of the fibres. At the end of the sample equilibration period (1 h), a conditioned (1.5 h at 280°C) 50/30 μm Divinylbenzene/Carboxen/polydimethylsiloxane (CAR/PDMS/DVB) StableFlex fibre (Supelco, Bellefonte, PA) was exposed to the headspace of the sample for the extraction (180 min) by CombiPAL system injector autosampler (CTC analytics, Switzerland). The fibre and the time of extraction used in this study were selected after preliminary study, and the data were reported in Figure 1. The best adsorption of analyte was obtained using CAR/PDMS/DVB and 180 min as extraction time. The extraction temperature of 25°C was selected in order to prevent possible matrix alterations (oxidation of some compounds, particularly aldehydes and furans).Figure 1
Total absorption peak areas (arbitrary unit) for CAR/PDMS/DVB, CAR/PDMS, PDMS/DVB, PDMS, and PA fibres at different extraction time (15, 45, 60, 90, 120, 180, and 240 min).To keep a constant temperature during analysis, the vials were maintained on a heater plate (CTC Analytics, Zwingen, Switzerland). As demonstrated in other researches in which the VOCs profile of food is investigated, the use of high extraction temperature can lead toex novo formation of volatile compounds or to the production of artefacts [29, 30].


### 2.4. Gas Chromatography-Mass Spectrometry Analysis of VOCs

HS-SPME analysis was performed using a Trace GC Ultra (Thermo-Fisher Scientific, Waltham, MA, USA) Gas Chromatograph coupled to a quadrupole Mass Spectrometer Trace DSQ (Thermo-Fisher Scientific, Waltham, MA, USA) and equipped with an Rtx-Wax column (30 m; 0.25 mm i.d.; 0.25μm film thickness, Restek, USA). The oven temperature program was: from 35°C, hold 8 min, to 60°C at 4°C/min, then from 60°C to 160°C at 6°C/min, and finally from 160°C to 200°C at 20°C/min. Carryover and peaks originating from the fibre were regularly assessed by running blank samples. After each analysis, fibres were immediately thermally desorbed in the GC injector for 5 min at 250°C to prevent contamination. The injections were performed in splitless mode (5 min). The carrier gas was helium at a constant flow of 1 mL−1. The transfer line to the mass spectrometer was maintained at 230°C, and the ion source temperature was set at 250°C. The mass spectra were obtained by using a mass selective detector with the electronic impact at 70 eV, a multiplier voltage of 1456 V, and by collecting the data at rate of 1 scan s−1 over the m/z range of 30–350. Compounds were identified by comparing the retention times of the chromatographic peaks with those of authentic compounds analyzed under the same conditions when available. The identification of MS fragmentation patterns was performed either by comparison with those of pure compounds or using the National Institute of Standards and Technology (NIST) MS spectral database. Volatile compounds measurements from each headspace of honey extracts were carried out by peak area normalization (expressed in percentage). All analyses were done in duplicate.


## 2.1. Honey Samples

This study was carried out on eight types of honey samples (Table1). In 2012, 18 samples were bought from local small-scale beekeepers working in Valtellina, an alpine valley of the Lombardy region. In particular, samples B1–B6 (Table 1), labeled as buckwheat honeys, were produced in Teglio (851 m a.s.l.; min. 352–max. 2.911 m a.s.l.), a mountain village in Valtellina where buckwheat is still cultivated; M1–M6 were reported as multifloral, A1–A3 as acacia honey (produced in an altitudinal range included between 200 and 1000 m a.s.l.), and R1–R3 as rhododendron honey (produced over 1000 m a.s.l.). B7, B8, B9, and B10 honey samples, labeled as buckwheat honeys, from Russia, Poland, and Nepal, respectively, were bought from international traders (Gego Enterpriser Pvt. Ltd., Baneshwor, Kathmandu, Nepal; RATOS-NATURA S.C., Olszownica, 75, 27-552, Backowice, Swietokrzyskie, Poland; Dary Altaya, OOO, Moscow, Russia). The floral origins of buckwheat samples were verified by using melissopalynological analysis. All samples were stored in darkness at a temperature of 4–6°C prior to analysis.Table 1
List of samples. Codes, locations, and honey type reported on the commercial label.
Sample code
Origin
Site
Honey type on the label
B1–B6
Italy
TeglioValtellina-Lombardy region
Buckwheat
B7
Russia
—
Buckwheat
B8-B9
Poland
—
Buckwheat
B10
Nepal
—
Buckwheat
M1–M3
Italy
TeglioValtellina-Lombardy region
Multifloral
M4–M6
Italy
Valtellina-Lombardy region
Multifloral
A1–A3
Italy
Valtellina-Lombardy region
Acacia
R1–R3
Italy
Valtellina-Lombardy region
Rhododendron


## 2.2. Melissopalynological Analysis

Melissopalynological analysis was performed according to the techniques proposed by the International Commission for Bee Botany (ICBB) and published in 1978 [26]. In this study, all honey samples were analyzed to confirm their floral origin. The microscopic analysis of honey sediment composition provides the percentage of the specific pollen observed by microscopic comparison with known pollen grains (Table 1).It is necessary to count at least 300 pollen grains for an estimation of the relative frequencies of pollen types and 500 to 1000 pollen grains for the determination of relative frequencies [27]. The examination under the microscope was carried out at the magnification that was most suitable for identifying the various elements in the sediment (400 to 1000x). After a first general check to ascertain the main types and densities of pollen grains, the relative frequencies of each pollen type were determined. A count of abortive, irregular, or broken pollen grains, fungal spores, hyphae, and microscopic algae, if they could be identified, was performed.If the sediment contained a high percentage of over-represented pollen, a second count excluding the over-represented pollen was done in order to determine more precisely the relative abundance of the other pollen types. The pollen types present in the honey samples were identified, counted, and classified, according to their percentages, as dominant pollen (more than 45% of the total pollen grains counted), secondary pollen (from 16 to 45%), important minor pollen (from 3 to 15%), and minor pollen (less than 3%) [28].


## 2.3. HS-SPME Volatile Compounds Sampling from Honey Samples

All the samples were prepared by weighing exactly 5.00 g of honey in a 20 mL glass vial, fitted with cap and equipped with silicon/PTFE septa (Supelco, Bellefonte, PA, USA) and by adding 1 mL of the internal standard solution (IS) in water (1,4-cineol, 1μg/mL, CAS 470-67-7) to check the quality of the fibres. At the end of the sample equilibration period (1 h), a conditioned (1.5 h at 280°C) 50/30 μm Divinylbenzene/Carboxen/polydimethylsiloxane (CAR/PDMS/DVB) StableFlex fibre (Supelco, Bellefonte, PA) was exposed to the headspace of the sample for the extraction (180 min) by CombiPAL system injector autosampler (CTC analytics, Switzerland). The fibre and the time of extraction used in this study were selected after preliminary study, and the data were reported in Figure 1. The best adsorption of analyte was obtained using CAR/PDMS/DVB and 180 min as extraction time. The extraction temperature of 25°C was selected in order to prevent possible matrix alterations (oxidation of some compounds, particularly aldehydes and furans).Figure 1
Total absorption peak areas (arbitrary unit) for CAR/PDMS/DVB, CAR/PDMS, PDMS/DVB, PDMS, and PA fibres at different extraction time (15, 45, 60, 90, 120, 180, and 240 min).To keep a constant temperature during analysis, the vials were maintained on a heater plate (CTC Analytics, Zwingen, Switzerland). As demonstrated in other researches in which the VOCs profile of food is investigated, the use of high extraction temperature can lead toex novo formation of volatile compounds or to the production of artefacts [29, 30].


## 2.4. Gas Chromatography-Mass Spectrometry Analysis of VOCs

HS-SPME analysis was performed using a Trace GC Ultra (Thermo-Fisher Scientific, Waltham, MA, USA) Gas Chromatograph coupled to a quadrupole Mass Spectrometer Trace DSQ (Thermo-Fisher Scientific, Waltham, MA, USA) and equipped with an Rtx-Wax column (30 m; 0.25 mm i.d.; 0.25μm film thickness, Restek, USA). The oven temperature program was: from 35°C, hold 8 min, to 60°C at 4°C/min, then from 60°C to 160°C at 6°C/min, and finally from 160°C to 200°C at 20°C/min. Carryover and peaks originating from the fibre were regularly assessed by running blank samples. After each analysis, fibres were immediately thermally desorbed in the GC injector for 5 min at 250°C to prevent contamination. The injections were performed in splitless mode (5 min). The carrier gas was helium at a constant flow of 1 mL−1. The transfer line to the mass spectrometer was maintained at 230°C, and the ion source temperature was set at 250°C. The mass spectra were obtained by using a mass selective detector with the electronic impact at 70 eV, a multiplier voltage of 1456 V, and by collecting the data at rate of 1 scan s−1 over the m/z range of 30–350. Compounds were identified by comparing the retention times of the chromatographic peaks with those of authentic compounds analyzed under the same conditions when available. The identification of MS fragmentation patterns was performed either by comparison with those of pure compounds or using the National Institute of Standards and Technology (NIST) MS spectral database. Volatile compounds measurements from each headspace of honey extracts were carried out by peak area normalization (expressed in percentage). All analyses were done in duplicate.


## 3. Results and Discussion

### 3.1. Melissopalynological Analysis

The results of the melissopalynological analyses of the honeys labeled as buckwheat honeys are reported in Tables2 and 3. The results showed that the buckwheat honey samples B1, B2, and B3 could be classified as monofloral with 45.5%, 52%, and 46% of buckwheat pollen. In Valtellina, the maximum altitude at which buckwheat is cultivated is 1200 m a.s.l., and its flowering period is August, thus, giving an indication on the elevation and the period of honey samples B1, B2, and B3 production.Table 2
Relative frequencies of the main pollen types in honeys labeled as buckwheat honeys. B1, B2, and B3: buckwheat honey from Italy (Valtellina); B4, B5, and B6: buckwheat honey from Italy (Teglio).
Sample code
B1
B2
B3
B4
B5
B6
Dominant pollen(>45%)
F. esculentum 45.5%
F. esculentum 52%
F. esculentum 46%
—
Melilotus 79%
Secondary pollen(16–45%)
—
—
Robinia 16.5%
Rhododendron 36%Clematis 7.2%
Rhododendron 21%
Euphrasia officinalis 37%Compositae 18%Eryngium  alpinum 17.8%
Important minor pollen(3–15%)
Trifolium repens10.3%Robinia 8.5%Tilia 3%
Trifolium repens 8.7%Hedera 5.2%
Salix 8%Rubus 4.7%
Clematis 8.3%F. esculentum 5%Lotus alpinus 5.4%
F. esculentum 4.5%Rubus 4%Mentha 4%Eryngium alpinum 4%
F. esculentum 5.6%Mentha 8%Clematis 5%Rubus 4.3%
Minor pollen(<3%)
Sedum/Sempervivum 2%Prunus 2.8%Gleditsia 2.2%Pyrus/Malus 1.9%Acer 1.5%Salix 0.7% Clematis 0.4% Trifolium pratense <1%Umbelliferae <1%
Verbena 2%Castanea 1.8%Pyrus/Malus 1%Salix 0.4%Sedum/Sempervivum <1%Knautia/Scabiosa <1%
Tilia 2%Ericaceae 2%Compositae 1.8%Trifolium  repens 1.5%Umbelliferae <1%Achillea spp. <1%
Trifolium repens 3%Prunus 2.6%Ranunculaceae 1.7%Robinia 1%Campanulaceae 0.9%Trifolium pretense 0.7%Acer 0.7%Ranunculaceae 0.7%Umbelliferae 0.4%Salvia <1%Sedum/Sempervivum <1%
Compositae 2%Lotus alpinus 2%Prunus 1.8%Compositae 1%Trifolium repens <1%Salix <1%
Prunus 2%Umbelliferae 1.8%Hedera 0.9%Salvia 0.2%Table 3
Relative frequencies of the main pollen types in honeys labeled as buckwheat honeys. B7: buckwheat honey from Russia; B8 and B9: buckwheat honey from Poland; B10: buckwheat honey from Nepal.
Sample code
B7
B8
B9
B10
Dominant pollen(>45%)
—
—
—
Cruciferae 67%
Secondary pollen(16–45%)
Helianthus 30%Melilotus 24%
Fagopyrum esculentum 25%
Fagopyrum esculentum 30%Brassica napus 17.9%
Fagopyrum esculentum 16%
Important minor pollen(3–15%)
Cruciferae 9%Fagopyrum  esculentum 5%Trifolium repens 5%Echium 4%Verbascum 4%Robinia 3%
Brassica napus 14%Echium 8%Trifolium repens 4%Helianthus 3%Umbelliferae 3%
Echium 13%Gleditsia12.4%Helianthus 4.8%
Helianthus 4%Compositae 4%Eucalyptus 3%
Minor pollen(<3%)
Labiatae 2%Onobrychis 2%Cynoglossum 1%Prunus 1%Campanulaceae 1%Compositae 1%Lotus 1%Umbelliferae 1%
Lotus 2%Trifolium repens 1%Compositae <1%Sedum/Sempervivum <1%
Sedum/Sempervivum 3%Lamium 2.3%Trifolium  repens 1.9%Salix 1.5%Lotus  corniculatus 1.5%Umbelliferae 1.5%Salvia 1.5%Euphorbiaceae <1%
Rosaceae 2%Umbelliferae 2%Bombacaceae 1%Castanea 1%Labiatae <1%On the contrary, honey samples B4, B5, and B6, also labeled as buckwheat honeys, had to be classified as multifloral, with a very low (5%, 4.5%, and 5.6%, resp.) relative frequency of buckwheat pollen. The high presence of pollen grains of plants belonging to theRhododendron genus, growing at an elevation between 1600 and 2300 m a.s.l. and flowering from June to mid-July, suggested that honey samples B4 and B5 were produced in Valtellina at higher altitude and in a different season compared to honey samples B1, B2, and B3. The high presence of pollen grains of Eryngium alpinumL. and Euphrasia officinalis L. in honey sample B6 also suggested that it was produced at higher altitude compared to honey samples B1, B2, and B3.Finally, the presence of pollen grains of plants belonging to theClematis genus, flowering from May to July, and Lotus alpinus(DC.) Schleicher, growing from 1700 to 2700 m a.s.l. and flowering in July, seemed to confirm the different period and environment of production of honey samples B4, B5, and B6.The Polish (B8 and B9) and Nepali (B10) samples had to be classified as multifloral honeys with a prevalence of buckwheat pollen grain (25%, 30%, and 16%, resp.), while the Russian sample (B7) had to be classified as multifloral with 5% of buckwheat honey. The presence of pollen grains from plants of theBombacaceae genus in the Nepali honey sample confirmed its Asiatic origin, suggesting it was produced in a tropical or subtropical area of Nepal, where plants of this genus grow. Finally, as Poland is one of the major producers of canola in Europe, the presence of a relevant quantity of Brassica napusL. pollen grains in honey samples B9 and B10 was coherent with its declared origin.The results regarding the other honey samples (M1–M6, A1–A3, and R1–R3) confirmed the botanical classification reported on the commercial label (data not shown), no buckwheat pollen grain were recovered.


### 3.2. Analysis of VOCs in Honey Samples

Honey volatiles are a very complex mixture of substances frequently occurring at a very low concentration and with poor chemical stability. Thus, as reported by many authors [2, 31–33], the use of headspace solid-phase-microextraction (HS-SPME) and gas-chromatography/mass-spectrometry (GC/MS), a very sensible and solvent-free method for extraction and analyses of this chemical fraction, is particularly suitable. In our experimental conditions, 86 compounds have overall been identified in the honey samples analyzed, and they are summarized in Tables 4 and 5.Table 4
Volatile compounds identified in investigated honey samples expressed as percentages. B1–B6: buckwheat honey from Italy (Teglio-Valtellina); B7: buckwheat honey from Russia; B8 and B9: buckwheat honey from Poland; B10: Buckwheat honey from Russia.
Compounds
RTa
Identificationb
Sample code
B1c
B2c
B3c
B4c
B5c
B6c
B7d
B8d
B9d
B10d
Alcohols
Ethanol
3.97
MS, LRI
14.85
9.05
12.25
5.70
5.18
7.65
8.39
11.41
12.00
6.36
2,3-Butanedione
4.98
MS, LRI
0.07
0.09
0.06
0.07
0.07
0.09
0.42
0.13
0.11
2.78
2-Methyl-3-buten-2-ol
7.39
MS, LRI
0.03
0.04
0.02
0.35
0.42
0.48
0.27
nd
0.05
nd
1-Butanol
12.67
MS, LRI
0.36
0.49
0.30
0.09
0.11
0.13
0.08
0.17
0.19
0.06
2-Methylbutanol
14.46
MS, LRI
0.01
0.01
0.01
0.05
0.06
0.03
0.02
0.03
0.03
0.08
2-Methylpropanol
15.02
MS, LRI
0.01
0.02
0.01
nd
nd
0.01
0.02
nd
nd
nd
3-Methylbutanol
15.41
MS, LRI
0.87
1.17
0.72
0.95
1.12
1.29
1.03
1.00
0.92
0.90
2-Methyl-2-buten-1-ol
16.78
MS, LRI
0.07
0.09
0.05
0.23
0.28
0.32
0.42
nd
0.03
0.17
3-Methyl-2-buten-1-ol
16.94
MS, LRI
0.02
0.02
0.02
0.03
0.03
0.04
0.05
0.04
0.02
0.01
2-Methylbutenol
19.09
MS, LRI
0.01
0.01
0.01
0.03
0.03
0.03
nd
nd
0.03
nd
Total
16.30
11.00
13.45
7.50
7.29
10.07
10.68
12.95
13.38
10.36
Phenols
2-Caren-10-al
29.28
MS, LRI
0.01
0.01
0.01
0.03
0.06
0.07
0.05
nd
nd
0.14
p-Cymen-8-ol
30.46
MS, LRI
0.03
0.04
0.03
0.01
0.01
0.01
0.05
0.03
0.05
0.07
Phenol
32.53
MS, LRI
0.03
0.04
0.02
0.01
0.01
0.02
0.05
0.06
0.04
nd
p-Cresol
33.26
MS, LRI
0.19
0.26
0.16
0.01
0.02
0.03
0.20
0.01
0.09
nd
Thymol
34.34
MS, LRI
0.01
0.01
nd
nd
nd
nd
nd
nd
nd
nd
Total
0.27
0.36
0.21
0.06
0.11
0.12
0.35
0.11
0.18
0.21
Ketones
2-Butanone
6.61
MS, LRI
nd
nd
nd
0.33
0.78
0.70
0.35
nd
nd
0.08
3-Hydroxy-2-butanone
17.65
MS, LRI
1.06
1.44
0.88
0.44
0.52
0.60
1.09
0.80
1.23
1.26
Hydroxyacetone
18.08
MS, LRI
2.36
3.18
1.95
1.50
1.76
2.02
2.15
1.30
2.23
1.40
2-Hydroxy-3-pentanone
19.90
MS, LRI
0.02
0.03
0.02
0.03
0.04
0.04
0.04
0.04
0.02
0.03
1-Hydroxy-2-butanone
20.29
MS, LRI
0.09
0.12
0.07
0.32
0.38
0.44
0.24
0.02
0.15
0.02
2-Methyl-butyrolactone
25.20
MS, LRI
0.45
0.61
0.37
0.09
0.11
0.12
1.05
0.18
0.08
nd
4-Oxoisophorone
27.42
MS, LRI
0.06
0.04
0.05
0.05
0.06
0.07
0.28
0.13
0.08
0.03
Total
4.52
6.06
3.73
3.45
4.45
4.94
6.02
2.62
8.42
3.50
Free fatty acid
Acetic acid
22.14
MS, LRI
9.35
12.63
7.72
11.28
13.25
15.22
20.14
26.80
20.48
31.80
Formic acid
23.36
MS, LRI
6.65
8.98
5.49
nd
13.25
0.14
nd
6.29
5.90
4.41
Propanoic acid
24.28
MS, LRI
0.39
0.52
0.342
0.30
0.36
0.41
1.04
0.42
0.39
0.84
Isobutyric acid
24.95
MS, LRI
0.49
0.67
0.41
0.24
0.28
0.32
0.35
0.50
0.52
2.08
Butanoic acid
26.19
MS, LRI
2.70
3.64
2.22
0.63
0.74
0.85
1.99
1.01
3.01
1.55
2-Propanoic acid
26.28
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
Isovaleric acid
27.05
MS, LRI
10.49
14.17
8.66
3.02
3.55
4.08
3.35
7.00
11.26
3.96
Pentanoic acid
28.35
MS, LRI
1.46
1.98
1.21
0.15
0.18
0.20
1.51
0.18
1.62
0.66
2-Butenoic acid
28.95
MS, LRI
0.02
0.03
0.02
0.02
0.01
0.01
0.22
0.01
0.01
0.11
3-Methylpentanoic acid
29.40
MS, LRI
0.08
0.11
0.07
0.05
0.06
0.07
0.11
0.08
0.05
0.07
4-Methylpentanoic acid
29.58
MS, LRI
0.04
0.05
0.03
0.02
0.02
0.03
0.41
0.03
0.03
0.01
4-Pentanoic acid
30.84
MS, LRI
0.02
0.03
0.02
0.01
0.01
0.03
0.05
0.03
0.02
0.01
Benzyl nitrile
31.63
MS, LRI
0.01
0.01
0.01
0.01
0.01
0.01
0.01
nd
0.01
nd
Heptanoic acid
31.96
MS, LRI
0.01
0.02
0.01
0.03
0.03
0.04
0.02
0.07
0.02
0.12
Octanoic acid
33.05
MS, LRI
0.02
0.02
0.01
0.03
0.03
0.03
0.03
0.04
0.02
0.14
Nonanoic acid
34.12
MS, LRI
0.01
0.05
0.01
nd
nd
nd
nd
0.14
0.05
0.09
Benzoic acid
37.83
MS, LRI
0.02
0.02
0.01
0.03
0.03
0.03
0.01
0.09
0.04
0.08
Total
31.76
42.91
26.20
15.80
18.55
21.46
29.24
42.67
43.43
45.93
Esters
Ethyl acetate
3.04
MS, LRI
0.04
0.06
0.04
0.16
0.18
0.17
0.52
0.50
0.53
nd
Butanoic acid 3-methyl-ethyl ester
8.60
MS, LRI
nd
nd
0.01
nd
nd
nd
nd
nd
nd
nd
Ethyl lactate
19.65
MS, LRI
0.03
0.04
0.02
0.01
0.01
0.01
0.02
nd
0.03
nd
Vinyl 2,2-dimethyl pentanoate
21.70
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
0.13
Propylene carbonate
30.14
MS, LRI
0.01
nd
0.01
nd
nd
nd
nd
nd
nd
nd
Total
0.08
0.10
0.08
0.17
0.20
0.18
0.54
0.50
0.56
0.13
Aldehydes
Acetaldehyde
1.75
MS, LRI
0.10
0.13
0.08
0.71
0.84
0.96
0.09
0.39
0.17
0.54
Butanal
2.86
MS, LRI
0.75
1.01
0.62
0.02
0.05
0.08
0.61
0.20
0.53
0.02
2-Methylbutanal
3.37
MS, LRI
1.03
1.39
0.85
0.57
0.67
0.77
1.61
0.01
0.59
0.02
3-Methylbutanal
3.45
MS, LRI
6.70
9.05
5.53
3.29
3.86
4.44
6.76
4.42
2.46
nd
Pentanal
4.79
MS, LRI
0.16
0.22
0.13
0.05
0.05
0.09
0.20
0.70
0.84
0.1
Hexanal
9.01
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
0.03
2-Pentanal
11.54
MS, LRI
nd
nd
nd
0.08
0.09
0.08
nd
nd
nd
nd
Heptanal
14.09
MS, LRI
nd
nd
nd
nd
nd
nd
0.07
nd
nd
0.05
Nonanal
21.00
MS, LRI
0.01
0.01
nd
0.02
0.01
0.02
0.02
0.17
0.07
0.49
Benzaldehyde
23.89
MS, LRI
0.21
0.29
0.18
0.21
0.24
0.28
0.21
0.60
0.29
4.77
Lilac aldehyde A
24.88
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
Lilac aldehyde B
25.37
MS, LRI
nd
nd
nd
0.02
0.03
0.03
nd
0.04
0.05
nd
Benzeneacetaldehyde
26.42
MS, LRI
0.61
0.82
0.50
0.06
0.07
0.08
0.06
nd
0.08
0.09
Nicotinaldehyde
27.61
MS, LRI
nd
nd
nd
0.05
0.05
0.05
0.03
nd
nd
0.44
2-Methyl-2-octenedial
29.18
MS, LRI
nd
nd
nd
0.03
0.06
0.07
0.05
nd
nd
0.15
Phenylacetaldehyde
31.31
MS, LRI
nd
nd
nd
nd
nd
nd
0.05
nd
nd
0.05
2-Pyrrolecarboxaldehyde
32.69
MS, LRI
0.18
0.25
0.15
0.30
0.35
0.33
0.71
0.12
0.10
nd
Cinnamaldehyde
32.83
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
Total
9.75
13.16
8.04
5.41
6.37
7.30
10.48
6.65
5.18
6.63
Furans
Furan
2.19
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
0.68
Methylfuran
3.15
MS, LRI
nd
nd
nd
nd
nd
nd
0.28
nd
nd
1.19
Dihydro-2-methyl-3(2H)-furanone
17.04
MS, LRI
0.01
0.02
0.01
0.03
0.03
0.04
0.59
0.01
0.10
0.01
Furfural
22.61
MS, LRI
3.29
4.09
3.39
1.22
1.50
0.14
4.91
1.29
5.90
0.96
1-(2-Furanyl)-ethanone
23.54
MS, LRI
0.48
0.64
0.39
0.68
0.80
0.92
0.79
0.08
0.42
nd
5-Methylfurfural
25.03
MS, LRI
0.04
0.04
0.04
0.16
0.19
0.22
1.25
0.50
0.52
0.05
Dihydro-5-methyl-2(3H)-furanone
25.65
MS, LRI
nd
nd
nd
nd
nd
nd
0.03
0.07
4.21
nd
Dihydro-3-methyl-2(3H)-furanone
25.72
MS, LRI
nd
nd
nd
1.75
2.06
1.80
1.31
nd
nd
0.68
2(5H)-Furanone
28.49
MS, LRI
0.30
0.40
0.24
0.28
0.33
0.38
0.84
0.02
0.29
0.03
4,5-Dimethyl-2-furaldehyde
32.19
MS, LRI
0.05
0.06
0.04
0.09
0.11
0.13
0.15
0.04
0.03
nd
Total
3.69
5.25
3.72
2.31
4.23
2.70
9.37
1.93
11.05
3.60
Terpenes
Verbenene
10.74
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
0.03
α-Phellandrene
12.91
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
0.03
α-Terpinene
13.62
MS, LRI
nd
nd
nd
nd
nd
nd
nd
0.04
0.01
0.07
τ-Terpinene
16.45
MS, LRI
nd
nd
nd
0.02
0.02
0.01
0.03
nd
nd
0.07
Cymene
17.24
MS, LRI
nd
nd
nd
nd
nd
nd
0.65
nd
nd
0.08
cis-Linalool oxide
22.24
MS, LRI
0.67
0.90
0.55
4.4
5.18
5.95
3.31
6.80
11.50
2.15
trans-linalool oxide
22.87
MS, LRI
0.46
0.62
0.38
1.23
nd
1.66
3.23
0.28
0.25
nd
Menthofuran
23.06
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
0.25
Linalool
24.70
MS, LRI
0.13
0.17
0.11
0.11
0.13
0.15
0.13
0.20
0.29
0.35
Damascenone
29.96
MS, LRI
nd
nd
nd
0.01
0.28
0.02
0.09
nd
nd
nd
α-Terpinolene
31.77
MS, LRI
0.01
0.01
0.01
nd
nd
nd
nd
nd
nd
0.24
Carvacrol
34.66
MS, LRI
nd
0.06
nd
nd
nd
nd
nd
nd
nd
0.09
Total
1.26
1.77
1.04
5.77
5.61
7.78
7.43
7.32
12.05
3.36
Miscellaneous
Dimethyl sulfide
1.93
MS, LRI
1.94
2.61
1.60
0.76
0.89
0.83
0.90
0.62
1.16
0.92
Eugenol
34.45
MS, LRI
0.01
nd
nd
nd
nd
nd
nd
nd
nd
nd
Total
1.95
2.61
1.60
0.76
0.89
0.83
0.90
0.62
1.16
0.92
aRetention time; bMS: mass spectrum tentatively identified using NIST 05 and Wiley 275 libraries; LRI: linear retention index.
cNormalized amount of volatile compounds (percentage) (peak of volatile compound/total peak area of all volatile compounds) of buckwheat honeys from Italy (Valtellina).
dNormalized amount of volatile compounds (percentage) (peak of volatile compound/total peak area of all volatile compounds) of buckwheat honeys from Russia, Poland, and Nepal.
nd: not detected.Table 5
Volatile compounds identified in investigated honey samples expressed as percentages. M1–M3: multifloral honeys from Italy (Teglio-Valtellina); M4–M6: multifloral honeys from Italy (Valtellina); A1–A3: acacia honeys from Italy (Valtellina); R1–R3: rhododendron honeys from Italy (Valtellina).
Compounds
RTa
Identificationb
Sample code
M1c
M2c
M3c
M4c
M5c
M6c
A1c
A2c
A3c
R1c
R2c
R3c
Alcohols
Ethanol
3.97
MS, LRI
25.83
15.01
6.66
30.82
28.20
34.48
13.11
15.69
9.54
40.55
32.60
38.42
2,3-Butanedione
4.98
MS, LRI
2.97
nd
0.21
nd
0.78
0.39
0.48
0.27
0.60
0.25
0.30
0.22
2-Methyl-3-buten-2-ol
7.39
MS, LRI
0.03
0.05
nd
nd
0.10
0.05
0.04
nd
0.07
0.07
0.06
0.08
1-Butanol
12.67
MS, LRI
0.08
0.06
0.08
0.5
0.68
0.41
0.45
0.11
0.78
0.38
0.25
0.68
2-Methylbutanol
14.46
MS, LRI
0.05
0.02
0.04
0.01
0.03
0.02
0.04
0.02
0.06
0.03
0.02
0.05
2-Methylpropanol
15.02
MS, LRI
nd
nd
0.05
nd
nd
nd
0.02
0.02
0.01
nd
nd
nd
3-Methylbutanol
15.41
MS, LRI
0.58
0.47
0.56
0.39
0.51
0.55
0.54
0.25
0.92
0.51
0.35
0.60
2-Methyl-2-buten-1-ol
16.78
MS, LRI
0.14
0.06
0.10
0.01
0.10
0.09
0.10
0.07
0.14
0.12
0.08
0.10
3-Methyl-2-buten-1-ol
16.94
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
2-Methylbutenol
19.09
MS, LRI
0.02
0.01
0.04
0.01
0.02
nd
0.02
0.03
0.02
0.01
nd
0.01
Total
29.70
15.68
7.74
31.73
30.42
35.99
14.80
16.46
12.14
41.92
33.66
40.16
Phenols
2-Caren-10-al
29.28
MS, LRI
0.09
nd
0.19
0.10
nd
nd
nd
nd
nd
nd
nd
nd
p-Cymen-8-ol
30.46
MS, LRI
0.06
0.05
0.08
0.05
0.10
0.07
nd
nd
nd
0.03
0.03
0.02
Phenol
32.53
MS, LRI
0.04
0.03
0.04
0.01
0.02
0.01
0.01
nd
0.02
0.02
0.05
0.01
p-Cresol
33.26
MS, LRI
0.01
0.01
nd
nd
0.01
0.01
0.01
0.01
0.01
nd
nd
0.01
Thymol
34.34
MS, LRI
nd
0.01
nd
nd
0.02
0.01
0.01
0.01
0.01
0.02
0.03
nd
Total
0.20
0.10
0.31
0.16
0.15
0.10
0.03
0.02
0.043
0.07
0.11
0.04
Ketones
2-Butanone
6.61
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
3-Hydroxy-2-butanone
17.65
MS, LRI
1.04
1.47
0.62
1.44
0.44
0.84
1.10
1.72
1.50
0.47
0.49
0.44
Hydroxyacetone
18.08
MS, LRI
1.02
0.72
1.62
1.16
0.71
nd
2.92
1.27
2.57
1.65
2.55
1.56
2-Hydroxy-3-pentanone
19.90
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
1-Hydroxy-2-butanone
20.29
MS, LRI
0.04
0.07
nd
0.01
0.10
0.05
0.05
0.02
0.10
0.11
0.08
0.10
2-Methyl-butyrolactone
25.20
MS, LRI
nd
0.05
nd
0.15
0.27
0.21
0.16
0.14
0.13
0.06
0.05
0.04
4-Oxoisophorone
27.42
MS, LRI
0.16
0.07
0.06
0.04
0.01
nd
0.02
0.01
0.01
0.06
0.01
nd
Total
2.36
2.49
2.35
2.87
1.68
1.21
4.40
3.26
4.51
3.29
3.55
2.48
Free fatty acid
Acetic acid
22.14
MS, LRI
27.4
31.32
30.48
18.50
40.64
32.56
48.12
41.40
44.83
25.63
21.53
29.74
Formic acid
23.36
MS, LRI
2.58
3.13
3.03
12.08
5.49
6.29
8.29
5.56
5.03
7.53
12.25
6.82
Propanoic acid
24.28
MS, LRI
0.72
0.75
0.79
0.16
0.41
0.78
0.71
0.43
0.99
0.40
0.41
0.40
Isobutyric acid
24.95
MS, LRI
1.00
1.20
1.80
1.22
2.30
1.26
1.30
0.49
1.11
0.49
0.32
0.35
Butanoic acid
26.19
MS, LRI
1.30
1.26
1.34
0.43
0.46
0.94
1.22
1.30
1.20
1.20
1.50
1.90
2-Propanoic acid
26.28
MS, LRI
0.05
0.02
0.08
nd
0.01
nd
0.05
0.02
0.08
0.01
0.01
nd
Isovaleric acid
27.05
MS, LRI
2.73
1.70
3.74
1.41
1.47
2.94
3.50
3.17
3.82
1.71
1.43
1.99
Pentanoic acid
28.35
MS, LRI
0.43
0.03
0.23
0.07
0.18
0.18
0.24
0.25
0.28
nd
0.02
nd
2-Butenoic acid
28.95
MS, LRI
0.02
0.03
0.02
nd
0.01
nd
nd
nd
nd
0.02
0.01
0.03
3-Methylpentanoic acid
29.40
MS, LRI
0.04
0.02
0.04
0.01
0.03
0.07
0.10
0.08
0.10
0.11
0.02
0.10
4-Methylpentanoic acid
29.58
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
4-Pentanoic acid
30.84
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
Benzyl nitrile
31.63
MS, LRI
nd
0.02
nd
nd
nd
0.01
nd
nd
nd
0.02
0.03
nd
Heptanoic acid
31.96
MS, LRI
0.10
0.08
0.10
0.02
0.05
0.04
0.10
0.11
0.18
0.12
0.11
0.14
Octanoic acid
33.05
MS, LRI
0.07
0.03
0.03
nd
0.01
0.01
0.03
0.04
0.04
0.01
0.02
nd
Nonanoic acid
34.12
MS, LRI
0.03
0.02
0.03
nd
0.02
nd
0.02
nd
0.02
0.01
0.01
nd
Benzoic acid
37.83
MS, LRI
0.07
0.08
0.07
0.03
0.05
0.04
0.07
0.07
0.07
0.08
0.08
0.08
Total
36.54
39.69
41.78
33.93
51.13
45.12
63.75
53.37
57.76
37.34
37.75
41.55
Esters
Ethyl acetate
3.04
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
0.04
0.08
0.05
Butanoic acid 3-methyl-ethyl ester
8.60
MS, LRI
nd
0.02
0.01
0.05
nd
0.03
nd
nd
0.03
nd
nd
nd
Ethyl lactate
19.65
MS, LRI
0.03
0.04
nd
0.01
nd
0.01
nd
0.02
nd
0.02
nd
0.04
Vinyl 2,2-dimethyl pentanoate
21.70
MS, LRI
0.13
0.10
0.25
nd
0.1
nd
nd
nd
nd
nd
nd
nd
Propylene carbonate
30.14
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
0.01
0.02
0.01
Total
0.16
0.16
0.26
0.06
0.10
0.04
nd
0.02
0.03
0.07
0.10
0.10
Aldehydes
Acetaldehyde
1.75
MS, LRI
0.46
0.52
0.40
0.91
0.41
0.45
nd
nd
nd
0.37
0.20
0.46
Butanal
2.86
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
2-Methylbutanal
3.37
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
3-Methylbutanal
3.45
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
0.05
0.10
0.05
Pentanal
4.79
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
Hexanal
9.01
MS, LRI
nd
nd
nd
nd
nd
nd
0.04
nd
0.07
nd
nd
0.01
2-Pentanal
11.54
MS, LRI
nd
nd
nd
nd
nd
nd
0.01
0.01
nd
nd
nd
nd
Heptanal
14.09
MS, LRI
0.03
0.01
0.02
nd
0.03
0.02
nd
nd
nd
nd
nd
nd
Nonanal
21.00
MS, LRI
0.06
0.02
0.05
0.05
nd
nd
0.08
0.15
nd
nd
nd
nd
Benzaldehyde
23.89
MS, LRI
1.19
1.06
0.70
0.40
0.96
0.53
0.86
0.60
1.11
1.70
1.10
1.30
Lilac aldehyde A
24.88
MS, LRI
1.00
1.20
0.80
0.22
1.30
1.26
0.02
0.02
0.01
nd
nd
nd
Lilac aldehyde B
25.37
MS, LRI
0.10
0.08
nd
0.02
0.06
0.04
0.07
0.09
0.07
0.16
0.03
0.10
Benzeneacetaldehyde
26.42
MS, LRI
nd
0.01
nd
0.01
0.02
0.04
0.05
0.04
0.07
0.06
0.03
0.04
Nicotinaldehyde
27.61
MS, LRI
0.50
0.34
0.60
nd
0.04
nd
nd
nd
nd
0.22
0.10
0.14
2-Methyl-2-octenedial
29.18
MS, LRI
0.10
0.03
0.10
0.01
0.10
0.11
nd
nd
nd
0.02
0.03
0.01
Phenylacetaldehyde
31.31
MS, LRI
0.03
nd
0.07
nd
nd
nd
nd
0.02
nd
nd
nd
nd
2-Pyrrolecarbaldehyde
32.69
MS, LRI
nd
0.03
nd
0.01
nd
0.01
0.02
0.02
0.01
0.01
0.02
nd
Cinnamaldehyde
32.83
MS, LRI
0.03
nd
0.02
0.03
0.05
0.01
0.01
nd
0.02
nd
0.02
nd
Total
3.50
3.30
2.76
1.65
2.97
2.47
1.16
0.96
1.37
2.59
1.63
2.11
Furans
Furan
2.19
MS, LRI
0.18
nd
0.03
nd
0.02
nd
nd
nd
nd
nd
nd
nd
Methylfuran
3.15
MS, LRI
0.81
0.27
0.30
0.25
0.15
nd
nd
nd
nd
0.13
0.10
0.25
Dihydro-2-methyl-3(2H)-furanone
17.04
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
Furfural
22.61
MS, LRI
1.42
1.71
1.13
1.07
1.30
2.19
3.41
2.06
4.77
2.56
2.53
2.50
1-(2-Furanyl)-ethanone
23.54
MS, LRI
0.05
0.10
nd
0.05
0.10
0.08
0.10
0.05
0.15
0.5
0.08
0.01
5-Methylfurfural
25.03
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
Dihydro-5-methyl-2(3H)-furanone
25.65
MS, LRI
0.75
nd
0.09
0.01
0.05
0.03
0.05
0.05
0.05
nd
nd
0.01
Dihydro-3-methyl-2(3H)-furanone
25.72
MS, LRI
0.40
0.38
0.70
0.04
0.09
0.07
nd
nd
nd
0.44
0.87
0.50
2(5H)-Furanone
28.49
MS, LRI
nd
0.03
nd
nd
0.04
0.02
nd
nd
0.02
0.01
0.02
nd
4,5-Dimethyl-2-furaldehyde
32.19
MS, LRI
nd
0.02
nd
0.02
0.03
0.02
0.04
0.02
0.05
0.07
0.08
0.05
Total
3.56
2.41
2.25
1.39
1.68
2.33
3.50
2.13
4.89
3.25
3.60
3.31
Terpenes
Verbenene
10.74
MS, LRI
nd
nd
nd
nd
0.03
0.01
nd
nd
nd
nd
nd
nd
α-Phellandrene
12.91
MS, LRI
0.03
nd
0.05
nd
0.03
nd
nd
nd
nd
nd
nd
nd
α-Terpinene
13.62
MS, LRI
0.06
0.01
0.03
0.04
0.02
nd
nd
0.05
0.07
nd
nd
nd
τ-Terpinene
16.45
MS, LRI
nd
nd
nd
nd
nd
nd
nd
0.01
nd
nd
nd
nd
Cymene
17.24
MS, LRI
0.05
0.01
0.05
nd
0.01
nd
nd
nd
nd
nd
nd
nd
cis-Linalool oxide
22.24
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
trans-Linalool oxide
22.87
MS, LRI
0.19
0.10
0.25
0.03
nd
0.01
0.11
0.08
0.04
0.14
0.06
0.10
Menthofuran
23.06
MS, LRI
0.26
nd
0.50
nd
0.10
nd
nd
nd
nd
nd
nd
nd
Linalool
24.70
MS, LRI
0.75
0.20
0.23
0.12
0.18
nd
0.47
0.65
0.20
0.91
0.12
0.67
Damascenone
29.96
MS, LRI
0.02
0.02
nd
0.01
nd
0.01
nd
0.02
nd
0.13
0.10
0.12
α-Terpinolene
31.77
MS, LRI
0.14
nd
0.25
nd
0.11
0.05
nd
nd
0.03
0.01
0.02
nd
Carvacrol
34.66
MS, LRI
nd
nd
0.01
nd
0.07
0.03
nd
nd
nd
nd
nd
nd
Total
1.5
0.34
1.37
0.20
0.55
0.11
0.58
0.00
0.34
1.19
0.30
0.89
Miscellanous
Dimethyl sulfide
1.93
MS, LRI
1.07
1.14
0.70
nd
1.00
nd
nd
nd
nd
0.72
0.15
1.20
Eugenol
34.45
MS, LRI
nd
0.01
nd
nd
0.01
0.01
0.01
0.02
0.01
nd
nd
nd
Total
1.07
1.15
0.70
nd
1.01
0.01
0.01
0.02
0.01
0.72
0.15
1.20
aRetention time; bMS: mass spectrum tentatively identified using NIST 05 and Wiley 275 libraries; LRI: linear retention index.
cNormalized amount of volatile compounds (percentage) (peak of volatile compound/total peak area of all volatile compounds) of multifloral, acacia, and rhododendron honey from Italy (Valtellina).
nd: not detected.These compounds belonged to different major chemical classes as follows: alcohols, phenols, ketones, free fatty acid, esters, aldehydes, furans, and terpenes.This paper is the first investigation on the VOCs profile of a buckwheat monofloral honey from Valtellina (North of Italy). Remarkable differences in VOCs profiles were observed when comparing honey samples of different floral origins. Consistent with other authors [34–36], most of the compounds were identified in all of the analyzed honeys, but the proportion in which they occurred appeared very different taking into account the different floral/botanical origin. Similarly, in each of the analyzed samples there were compounds which were not present in other types of honeys to be evaluated as potential “floral markers”.The VOCs profile of honeys labeled as buckwheat honeys (B1–B10) was similar, particularly for samples containing a relevant quantity of buckwheat pollen grains despite the different geographical origin. In addition, comparing it with the VOCs profile of the other honey samples, not containing buckwheat pollen grains (M1–M6, A1–A3, and R1–R3), some differences were identified, particularly regarding the composition and the concentration of some chemical classes such as alcohols, aldehydes, free fatty acids, furans, and terpenes as shown in Tables4 and 5.Buckwheat honey is characterized by a sharp, sweet, and slightly biting taste, and its organoleptic characteristics have been proven to be reflected in its composition and concentrations of volatile compounds [33]. Moreover, among the aldehydes, methylbutanals have been reported to be responsible for the characteristic pungent, sweetish, and malty flavour of buckwheat honey [22, 37].In our experimental condition, 3-methylbutanal was found in highest concentration in buckwheat honey, and 2-methylbutanal was found to be present only in the honeys containing buckwheat pollen grains. These compounds are commonly found in barley malt [38]. They are known to be Strecker aldehydes, and their presence in honeys is usually associated with the Maillard browning reactions.The extremely high amounts of methylbutanals in buckwheat honeys compared with some other honeys suggested that this type of honey contains a higher abundance of Strecker degradation precursors, such as amino acids, which would result in a honey with an aroma resembling that which develops upon heat-promoted chemical reactions that occur during the malting of barley [22]. The presence of other Maillard reaction products such as phenylacetaldehyde and dimethyl sulfide supports this hypothesis.As reported in the literature [33], besides aldehydes, also the concentrations of free fatty acids, like butanoic acid and pentanoic acid, were considerably greater in the honey samples B1, B2, B3, and B9, those containing the higher quantity of buckwheat pollen grains. Butanoic acid gives buckwheat honey its typical pungent smell and pentanoic acid has a rancid smell and an acid taste [37]. Such chemical compounds, characterized by high concentrations in a honey type and specific sensory properties, are to be considered interesting as potential “variety markers” [33].Wolski et al. [32] reported butanal, phenol, trans-linalool oxide, 3,4,5-trimethylphenol as buckwheat honey marker compounds because they were not found in other honey types. In this study, only butanal was confirmed to be such a marker, being present only in the two honey samples containing a relevant quantity of buckwheat pollen grains, corresponding to the Italian monofloral buckwheat honey (samples B1, B2, and B3) and the Polish honeys (samples B8 and B9).In the same honey samples, we also found significantly great quantities of isovaleric acid, never reported before in buckwheat honey. Isovaleric acid is a potent, odorant, volatile compound, exhibiting an unpleasant odor associated with the rank smell of perspiring feet and has been considered to be an important off-flavor compound in honeys [39]. Isovaleric acid, has been found to be an important odorant for Anarcardium occidentaleL. and Croton sp. honeys from Brazil [40].Finally, we have identified additional characteristic volatile compounds of buckwheat honey such as 3-methyl-2-buten-1-ol, 2-butanone, 2-hydroxy-3-pentanone, 4-methylpentanoic acid, 4-pentanoic acid, butanal, 2-methylbutanal, pentanal, dihydro-2-methyl-3(2H)-furanone, 5-methylfurfural, andcis-linalool oxide. Pasini et al. [21] reported 5-methylfurfural and other furans as important buckwheat honey marker compounds. In the literature, furanic compounds were reported to derive from sugar degradation and considered to be indicators of thermal processes and storage [41].


## 3.1. Melissopalynological Analysis

The results of the melissopalynological analyses of the honeys labeled as buckwheat honeys are reported in Tables2 and 3. The results showed that the buckwheat honey samples B1, B2, and B3 could be classified as monofloral with 45.5%, 52%, and 46% of buckwheat pollen. In Valtellina, the maximum altitude at which buckwheat is cultivated is 1200 m a.s.l., and its flowering period is August, thus, giving an indication on the elevation and the period of honey samples B1, B2, and B3 production.Table 2
Relative frequencies of the main pollen types in honeys labeled as buckwheat honeys. B1, B2, and B3: buckwheat honey from Italy (Valtellina); B4, B5, and B6: buckwheat honey from Italy (Teglio).
Sample code
B1
B2
B3
B4
B5
B6
Dominant pollen(>45%)
F. esculentum 45.5%
F. esculentum 52%
F. esculentum 46%
—
Melilotus 79%
Secondary pollen(16–45%)
—
—
Robinia 16.5%
Rhododendron 36%Clematis 7.2%
Rhododendron 21%
Euphrasia officinalis 37%Compositae 18%Eryngium  alpinum 17.8%
Important minor pollen(3–15%)
Trifolium repens10.3%Robinia 8.5%Tilia 3%
Trifolium repens 8.7%Hedera 5.2%
Salix 8%Rubus 4.7%
Clematis 8.3%F. esculentum 5%Lotus alpinus 5.4%
F. esculentum 4.5%Rubus 4%Mentha 4%Eryngium alpinum 4%
F. esculentum 5.6%Mentha 8%Clematis 5%Rubus 4.3%
Minor pollen(<3%)
Sedum/Sempervivum 2%Prunus 2.8%Gleditsia 2.2%Pyrus/Malus 1.9%Acer 1.5%Salix 0.7% Clematis 0.4% Trifolium pratense <1%Umbelliferae <1%
Verbena 2%Castanea 1.8%Pyrus/Malus 1%Salix 0.4%Sedum/Sempervivum <1%Knautia/Scabiosa <1%
Tilia 2%Ericaceae 2%Compositae 1.8%Trifolium  repens 1.5%Umbelliferae <1%Achillea spp. <1%
Trifolium repens 3%Prunus 2.6%Ranunculaceae 1.7%Robinia 1%Campanulaceae 0.9%Trifolium pretense 0.7%Acer 0.7%Ranunculaceae 0.7%Umbelliferae 0.4%Salvia <1%Sedum/Sempervivum <1%
Compositae 2%Lotus alpinus 2%Prunus 1.8%Compositae 1%Trifolium repens <1%Salix <1%
Prunus 2%Umbelliferae 1.8%Hedera 0.9%Salvia 0.2%Table 3
Relative frequencies of the main pollen types in honeys labeled as buckwheat honeys. B7: buckwheat honey from Russia; B8 and B9: buckwheat honey from Poland; B10: buckwheat honey from Nepal.
Sample code
B7
B8
B9
B10
Dominant pollen(>45%)
—
—
—
Cruciferae 67%
Secondary pollen(16–45%)
Helianthus 30%Melilotus 24%
Fagopyrum esculentum 25%
Fagopyrum esculentum 30%Brassica napus 17.9%
Fagopyrum esculentum 16%
Important minor pollen(3–15%)
Cruciferae 9%Fagopyrum  esculentum 5%Trifolium repens 5%Echium 4%Verbascum 4%Robinia 3%
Brassica napus 14%Echium 8%Trifolium repens 4%Helianthus 3%Umbelliferae 3%
Echium 13%Gleditsia12.4%Helianthus 4.8%
Helianthus 4%Compositae 4%Eucalyptus 3%
Minor pollen(<3%)
Labiatae 2%Onobrychis 2%Cynoglossum 1%Prunus 1%Campanulaceae 1%Compositae 1%Lotus 1%Umbelliferae 1%
Lotus 2%Trifolium repens 1%Compositae <1%Sedum/Sempervivum <1%
Sedum/Sempervivum 3%Lamium 2.3%Trifolium  repens 1.9%Salix 1.5%Lotus  corniculatus 1.5%Umbelliferae 1.5%Salvia 1.5%Euphorbiaceae <1%
Rosaceae 2%Umbelliferae 2%Bombacaceae 1%Castanea 1%Labiatae <1%On the contrary, honey samples B4, B5, and B6, also labeled as buckwheat honeys, had to be classified as multifloral, with a very low (5%, 4.5%, and 5.6%, resp.) relative frequency of buckwheat pollen. The high presence of pollen grains of plants belonging to theRhododendron genus, growing at an elevation between 1600 and 2300 m a.s.l. and flowering from June to mid-July, suggested that honey samples B4 and B5 were produced in Valtellina at higher altitude and in a different season compared to honey samples B1, B2, and B3. The high presence of pollen grains of Eryngium alpinumL. and Euphrasia officinalis L. in honey sample B6 also suggested that it was produced at higher altitude compared to honey samples B1, B2, and B3.Finally, the presence of pollen grains of plants belonging to theClematis genus, flowering from May to July, and Lotus alpinus(DC.) Schleicher, growing from 1700 to 2700 m a.s.l. and flowering in July, seemed to confirm the different period and environment of production of honey samples B4, B5, and B6.The Polish (B8 and B9) and Nepali (B10) samples had to be classified as multifloral honeys with a prevalence of buckwheat pollen grain (25%, 30%, and 16%, resp.), while the Russian sample (B7) had to be classified as multifloral with 5% of buckwheat honey. The presence of pollen grains from plants of theBombacaceae genus in the Nepali honey sample confirmed its Asiatic origin, suggesting it was produced in a tropical or subtropical area of Nepal, where plants of this genus grow. Finally, as Poland is one of the major producers of canola in Europe, the presence of a relevant quantity of Brassica napusL. pollen grains in honey samples B9 and B10 was coherent with its declared origin.The results regarding the other honey samples (M1–M6, A1–A3, and R1–R3) confirmed the botanical classification reported on the commercial label (data not shown), no buckwheat pollen grain were recovered.


## 3.2. Analysis of VOCs in Honey Samples

Honey volatiles are a very complex mixture of substances frequently occurring at a very low concentration and with poor chemical stability. Thus, as reported by many authors [2, 31–33], the use of headspace solid-phase-microextraction (HS-SPME) and gas-chromatography/mass-spectrometry (GC/MS), a very sensible and solvent-free method for extraction and analyses of this chemical fraction, is particularly suitable. In our experimental conditions, 86 compounds have overall been identified in the honey samples analyzed, and they are summarized in Tables 4 and 5.Table 4
Volatile compounds identified in investigated honey samples expressed as percentages. B1–B6: buckwheat honey from Italy (Teglio-Valtellina); B7: buckwheat honey from Russia; B8 and B9: buckwheat honey from Poland; B10: Buckwheat honey from Russia.
Compounds
RTa
Identificationb
Sample code
B1c
B2c
B3c
B4c
B5c
B6c
B7d
B8d
B9d
B10d
Alcohols
Ethanol
3.97
MS, LRI
14.85
9.05
12.25
5.70
5.18
7.65
8.39
11.41
12.00
6.36
2,3-Butanedione
4.98
MS, LRI
0.07
0.09
0.06
0.07
0.07
0.09
0.42
0.13
0.11
2.78
2-Methyl-3-buten-2-ol
7.39
MS, LRI
0.03
0.04
0.02
0.35
0.42
0.48
0.27
nd
0.05
nd
1-Butanol
12.67
MS, LRI
0.36
0.49
0.30
0.09
0.11
0.13
0.08
0.17
0.19
0.06
2-Methylbutanol
14.46
MS, LRI
0.01
0.01
0.01
0.05
0.06
0.03
0.02
0.03
0.03
0.08
2-Methylpropanol
15.02
MS, LRI
0.01
0.02
0.01
nd
nd
0.01
0.02
nd
nd
nd
3-Methylbutanol
15.41
MS, LRI
0.87
1.17
0.72
0.95
1.12
1.29
1.03
1.00
0.92
0.90
2-Methyl-2-buten-1-ol
16.78
MS, LRI
0.07
0.09
0.05
0.23
0.28
0.32
0.42
nd
0.03
0.17
3-Methyl-2-buten-1-ol
16.94
MS, LRI
0.02
0.02
0.02
0.03
0.03
0.04
0.05
0.04
0.02
0.01
2-Methylbutenol
19.09
MS, LRI
0.01
0.01
0.01
0.03
0.03
0.03
nd
nd
0.03
nd
Total
16.30
11.00
13.45
7.50
7.29
10.07
10.68
12.95
13.38
10.36
Phenols
2-Caren-10-al
29.28
MS, LRI
0.01
0.01
0.01
0.03
0.06
0.07
0.05
nd
nd
0.14
p-Cymen-8-ol
30.46
MS, LRI
0.03
0.04
0.03
0.01
0.01
0.01
0.05
0.03
0.05
0.07
Phenol
32.53
MS, LRI
0.03
0.04
0.02
0.01
0.01
0.02
0.05
0.06
0.04
nd
p-Cresol
33.26
MS, LRI
0.19
0.26
0.16
0.01
0.02
0.03
0.20
0.01
0.09
nd
Thymol
34.34
MS, LRI
0.01
0.01
nd
nd
nd
nd
nd
nd
nd
nd
Total
0.27
0.36
0.21
0.06
0.11
0.12
0.35
0.11
0.18
0.21
Ketones
2-Butanone
6.61
MS, LRI
nd
nd
nd
0.33
0.78
0.70
0.35
nd
nd
0.08
3-Hydroxy-2-butanone
17.65
MS, LRI
1.06
1.44
0.88
0.44
0.52
0.60
1.09
0.80
1.23
1.26
Hydroxyacetone
18.08
MS, LRI
2.36
3.18
1.95
1.50
1.76
2.02
2.15
1.30
2.23
1.40
2-Hydroxy-3-pentanone
19.90
MS, LRI
0.02
0.03
0.02
0.03
0.04
0.04
0.04
0.04
0.02
0.03
1-Hydroxy-2-butanone
20.29
MS, LRI
0.09
0.12
0.07
0.32
0.38
0.44
0.24
0.02
0.15
0.02
2-Methyl-butyrolactone
25.20
MS, LRI
0.45
0.61
0.37
0.09
0.11
0.12
1.05
0.18
0.08
nd
4-Oxoisophorone
27.42
MS, LRI
0.06
0.04
0.05
0.05
0.06
0.07
0.28
0.13
0.08
0.03
Total
4.52
6.06
3.73
3.45
4.45
4.94
6.02
2.62
8.42
3.50
Free fatty acid
Acetic acid
22.14
MS, LRI
9.35
12.63
7.72
11.28
13.25
15.22
20.14
26.80
20.48
31.80
Formic acid
23.36
MS, LRI
6.65
8.98
5.49
nd
13.25
0.14
nd
6.29
5.90
4.41
Propanoic acid
24.28
MS, LRI
0.39
0.52
0.342
0.30
0.36
0.41
1.04
0.42
0.39
0.84
Isobutyric acid
24.95
MS, LRI
0.49
0.67
0.41
0.24
0.28
0.32
0.35
0.50
0.52
2.08
Butanoic acid
26.19
MS, LRI
2.70
3.64
2.22
0.63
0.74
0.85
1.99
1.01
3.01
1.55
2-Propanoic acid
26.28
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
Isovaleric acid
27.05
MS, LRI
10.49
14.17
8.66
3.02
3.55
4.08
3.35
7.00
11.26
3.96
Pentanoic acid
28.35
MS, LRI
1.46
1.98
1.21
0.15
0.18
0.20
1.51
0.18
1.62
0.66
2-Butenoic acid
28.95
MS, LRI
0.02
0.03
0.02
0.02
0.01
0.01
0.22
0.01
0.01
0.11
3-Methylpentanoic acid
29.40
MS, LRI
0.08
0.11
0.07
0.05
0.06
0.07
0.11
0.08
0.05
0.07
4-Methylpentanoic acid
29.58
MS, LRI
0.04
0.05
0.03
0.02
0.02
0.03
0.41
0.03
0.03
0.01
4-Pentanoic acid
30.84
MS, LRI
0.02
0.03
0.02
0.01
0.01
0.03
0.05
0.03
0.02
0.01
Benzyl nitrile
31.63
MS, LRI
0.01
0.01
0.01
0.01
0.01
0.01
0.01
nd
0.01
nd
Heptanoic acid
31.96
MS, LRI
0.01
0.02
0.01
0.03
0.03
0.04
0.02
0.07
0.02
0.12
Octanoic acid
33.05
MS, LRI
0.02
0.02
0.01
0.03
0.03
0.03
0.03
0.04
0.02
0.14
Nonanoic acid
34.12
MS, LRI
0.01
0.05
0.01
nd
nd
nd
nd
0.14
0.05
0.09
Benzoic acid
37.83
MS, LRI
0.02
0.02
0.01
0.03
0.03
0.03
0.01
0.09
0.04
0.08
Total
31.76
42.91
26.20
15.80
18.55
21.46
29.24
42.67
43.43
45.93
Esters
Ethyl acetate
3.04
MS, LRI
0.04
0.06
0.04
0.16
0.18
0.17
0.52
0.50
0.53
nd
Butanoic acid 3-methyl-ethyl ester
8.60
MS, LRI
nd
nd
0.01
nd
nd
nd
nd
nd
nd
nd
Ethyl lactate
19.65
MS, LRI
0.03
0.04
0.02
0.01
0.01
0.01
0.02
nd
0.03
nd
Vinyl 2,2-dimethyl pentanoate
21.70
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
0.13
Propylene carbonate
30.14
MS, LRI
0.01
nd
0.01
nd
nd
nd
nd
nd
nd
nd
Total
0.08
0.10
0.08
0.17
0.20
0.18
0.54
0.50
0.56
0.13
Aldehydes
Acetaldehyde
1.75
MS, LRI
0.10
0.13
0.08
0.71
0.84
0.96
0.09
0.39
0.17
0.54
Butanal
2.86
MS, LRI
0.75
1.01
0.62
0.02
0.05
0.08
0.61
0.20
0.53
0.02
2-Methylbutanal
3.37
MS, LRI
1.03
1.39
0.85
0.57
0.67
0.77
1.61
0.01
0.59
0.02
3-Methylbutanal
3.45
MS, LRI
6.70
9.05
5.53
3.29
3.86
4.44
6.76
4.42
2.46
nd
Pentanal
4.79
MS, LRI
0.16
0.22
0.13
0.05
0.05
0.09
0.20
0.70
0.84
0.1
Hexanal
9.01
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
0.03
2-Pentanal
11.54
MS, LRI
nd
nd
nd
0.08
0.09
0.08
nd
nd
nd
nd
Heptanal
14.09
MS, LRI
nd
nd
nd
nd
nd
nd
0.07
nd
nd
0.05
Nonanal
21.00
MS, LRI
0.01
0.01
nd
0.02
0.01
0.02
0.02
0.17
0.07
0.49
Benzaldehyde
23.89
MS, LRI
0.21
0.29
0.18
0.21
0.24
0.28
0.21
0.60
0.29
4.77
Lilac aldehyde A
24.88
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
Lilac aldehyde B
25.37
MS, LRI
nd
nd
nd
0.02
0.03
0.03
nd
0.04
0.05
nd
Benzeneacetaldehyde
26.42
MS, LRI
0.61
0.82
0.50
0.06
0.07
0.08
0.06
nd
0.08
0.09
Nicotinaldehyde
27.61
MS, LRI
nd
nd
nd
0.05
0.05
0.05
0.03
nd
nd
0.44
2-Methyl-2-octenedial
29.18
MS, LRI
nd
nd
nd
0.03
0.06
0.07
0.05
nd
nd
0.15
Phenylacetaldehyde
31.31
MS, LRI
nd
nd
nd
nd
nd
nd
0.05
nd
nd
0.05
2-Pyrrolecarboxaldehyde
32.69
MS, LRI
0.18
0.25
0.15
0.30
0.35
0.33
0.71
0.12
0.10
nd
Cinnamaldehyde
32.83
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
Total
9.75
13.16
8.04
5.41
6.37
7.30
10.48
6.65
5.18
6.63
Furans
Furan
2.19
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
0.68
Methylfuran
3.15
MS, LRI
nd
nd
nd
nd
nd
nd
0.28
nd
nd
1.19
Dihydro-2-methyl-3(2H)-furanone
17.04
MS, LRI
0.01
0.02
0.01
0.03
0.03
0.04
0.59
0.01
0.10
0.01
Furfural
22.61
MS, LRI
3.29
4.09
3.39
1.22
1.50
0.14
4.91
1.29
5.90
0.96
1-(2-Furanyl)-ethanone
23.54
MS, LRI
0.48
0.64
0.39
0.68
0.80
0.92
0.79
0.08
0.42
nd
5-Methylfurfural
25.03
MS, LRI
0.04
0.04
0.04
0.16
0.19
0.22
1.25
0.50
0.52
0.05
Dihydro-5-methyl-2(3H)-furanone
25.65
MS, LRI
nd
nd
nd
nd
nd
nd
0.03
0.07
4.21
nd
Dihydro-3-methyl-2(3H)-furanone
25.72
MS, LRI
nd
nd
nd
1.75
2.06
1.80
1.31
nd
nd
0.68
2(5H)-Furanone
28.49
MS, LRI
0.30
0.40
0.24
0.28
0.33
0.38
0.84
0.02
0.29
0.03
4,5-Dimethyl-2-furaldehyde
32.19
MS, LRI
0.05
0.06
0.04
0.09
0.11
0.13
0.15
0.04
0.03
nd
Total
3.69
5.25
3.72
2.31
4.23
2.70
9.37
1.93
11.05
3.60
Terpenes
Verbenene
10.74
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
0.03
α-Phellandrene
12.91
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
0.03
α-Terpinene
13.62
MS, LRI
nd
nd
nd
nd
nd
nd
nd
0.04
0.01
0.07
τ-Terpinene
16.45
MS, LRI
nd
nd
nd
0.02
0.02
0.01
0.03
nd
nd
0.07
Cymene
17.24
MS, LRI
nd
nd
nd
nd
nd
nd
0.65
nd
nd
0.08
cis-Linalool oxide
22.24
MS, LRI
0.67
0.90
0.55
4.4
5.18
5.95
3.31
6.80
11.50
2.15
trans-linalool oxide
22.87
MS, LRI
0.46
0.62
0.38
1.23
nd
1.66
3.23
0.28
0.25
nd
Menthofuran
23.06
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
0.25
Linalool
24.70
MS, LRI
0.13
0.17
0.11
0.11
0.13
0.15
0.13
0.20
0.29
0.35
Damascenone
29.96
MS, LRI
nd
nd
nd
0.01
0.28
0.02
0.09
nd
nd
nd
α-Terpinolene
31.77
MS, LRI
0.01
0.01
0.01
nd
nd
nd
nd
nd
nd
0.24
Carvacrol
34.66
MS, LRI
nd
0.06
nd
nd
nd
nd
nd
nd
nd
0.09
Total
1.26
1.77
1.04
5.77
5.61
7.78
7.43
7.32
12.05
3.36
Miscellaneous
Dimethyl sulfide
1.93
MS, LRI
1.94
2.61
1.60
0.76
0.89
0.83
0.90
0.62
1.16
0.92
Eugenol
34.45
MS, LRI
0.01
nd
nd
nd
nd
nd
nd
nd
nd
nd
Total
1.95
2.61
1.60
0.76
0.89
0.83
0.90
0.62
1.16
0.92
aRetention time; bMS: mass spectrum tentatively identified using NIST 05 and Wiley 275 libraries; LRI: linear retention index.
cNormalized amount of volatile compounds (percentage) (peak of volatile compound/total peak area of all volatile compounds) of buckwheat honeys from Italy (Valtellina).
dNormalized amount of volatile compounds (percentage) (peak of volatile compound/total peak area of all volatile compounds) of buckwheat honeys from Russia, Poland, and Nepal.
nd: not detected.Table 5
Volatile compounds identified in investigated honey samples expressed as percentages. M1–M3: multifloral honeys from Italy (Teglio-Valtellina); M4–M6: multifloral honeys from Italy (Valtellina); A1–A3: acacia honeys from Italy (Valtellina); R1–R3: rhododendron honeys from Italy (Valtellina).
Compounds
RTa
Identificationb
Sample code
M1c
M2c
M3c
M4c
M5c
M6c
A1c
A2c
A3c
R1c
R2c
R3c
Alcohols
Ethanol
3.97
MS, LRI
25.83
15.01
6.66
30.82
28.20
34.48
13.11
15.69
9.54
40.55
32.60
38.42
2,3-Butanedione
4.98
MS, LRI
2.97
nd
0.21
nd
0.78
0.39
0.48
0.27
0.60
0.25
0.30
0.22
2-Methyl-3-buten-2-ol
7.39
MS, LRI
0.03
0.05
nd
nd
0.10
0.05
0.04
nd
0.07
0.07
0.06
0.08
1-Butanol
12.67
MS, LRI
0.08
0.06
0.08
0.5
0.68
0.41
0.45
0.11
0.78
0.38
0.25
0.68
2-Methylbutanol
14.46
MS, LRI
0.05
0.02
0.04
0.01
0.03
0.02
0.04
0.02
0.06
0.03
0.02
0.05
2-Methylpropanol
15.02
MS, LRI
nd
nd
0.05
nd
nd
nd
0.02
0.02
0.01
nd
nd
nd
3-Methylbutanol
15.41
MS, LRI
0.58
0.47
0.56
0.39
0.51
0.55
0.54
0.25
0.92
0.51
0.35
0.60
2-Methyl-2-buten-1-ol
16.78
MS, LRI
0.14
0.06
0.10
0.01
0.10
0.09
0.10
0.07
0.14
0.12
0.08
0.10
3-Methyl-2-buten-1-ol
16.94
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
2-Methylbutenol
19.09
MS, LRI
0.02
0.01
0.04
0.01
0.02
nd
0.02
0.03
0.02
0.01
nd
0.01
Total
29.70
15.68
7.74
31.73
30.42
35.99
14.80
16.46
12.14
41.92
33.66
40.16
Phenols
2-Caren-10-al
29.28
MS, LRI
0.09
nd
0.19
0.10
nd
nd
nd
nd
nd
nd
nd
nd
p-Cymen-8-ol
30.46
MS, LRI
0.06
0.05
0.08
0.05
0.10
0.07
nd
nd
nd
0.03
0.03
0.02
Phenol
32.53
MS, LRI
0.04
0.03
0.04
0.01
0.02
0.01
0.01
nd
0.02
0.02
0.05
0.01
p-Cresol
33.26
MS, LRI
0.01
0.01
nd
nd
0.01
0.01
0.01
0.01
0.01
nd
nd
0.01
Thymol
34.34
MS, LRI
nd
0.01
nd
nd
0.02
0.01
0.01
0.01
0.01
0.02
0.03
nd
Total
0.20
0.10
0.31
0.16
0.15
0.10
0.03
0.02
0.043
0.07
0.11
0.04
Ketones
2-Butanone
6.61
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
3-Hydroxy-2-butanone
17.65
MS, LRI
1.04
1.47
0.62
1.44
0.44
0.84
1.10
1.72
1.50
0.47
0.49
0.44
Hydroxyacetone
18.08
MS, LRI
1.02
0.72
1.62
1.16
0.71
nd
2.92
1.27
2.57
1.65
2.55
1.56
2-Hydroxy-3-pentanone
19.90
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
1-Hydroxy-2-butanone
20.29
MS, LRI
0.04
0.07
nd
0.01
0.10
0.05
0.05
0.02
0.10
0.11
0.08
0.10
2-Methyl-butyrolactone
25.20
MS, LRI
nd
0.05
nd
0.15
0.27
0.21
0.16
0.14
0.13
0.06
0.05
0.04
4-Oxoisophorone
27.42
MS, LRI
0.16
0.07
0.06
0.04
0.01
nd
0.02
0.01
0.01
0.06
0.01
nd
Total
2.36
2.49
2.35
2.87
1.68
1.21
4.40
3.26
4.51
3.29
3.55
2.48
Free fatty acid
Acetic acid
22.14
MS, LRI
27.4
31.32
30.48
18.50
40.64
32.56
48.12
41.40
44.83
25.63
21.53
29.74
Formic acid
23.36
MS, LRI
2.58
3.13
3.03
12.08
5.49
6.29
8.29
5.56
5.03
7.53
12.25
6.82
Propanoic acid
24.28
MS, LRI
0.72
0.75
0.79
0.16
0.41
0.78
0.71
0.43
0.99
0.40
0.41
0.40
Isobutyric acid
24.95
MS, LRI
1.00
1.20
1.80
1.22
2.30
1.26
1.30
0.49
1.11
0.49
0.32
0.35
Butanoic acid
26.19
MS, LRI
1.30
1.26
1.34
0.43
0.46
0.94
1.22
1.30
1.20
1.20
1.50
1.90
2-Propanoic acid
26.28
MS, LRI
0.05
0.02
0.08
nd
0.01
nd
0.05
0.02
0.08
0.01
0.01
nd
Isovaleric acid
27.05
MS, LRI
2.73
1.70
3.74
1.41
1.47
2.94
3.50
3.17
3.82
1.71
1.43
1.99
Pentanoic acid
28.35
MS, LRI
0.43
0.03
0.23
0.07
0.18
0.18
0.24
0.25
0.28
nd
0.02
nd
2-Butenoic acid
28.95
MS, LRI
0.02
0.03
0.02
nd
0.01
nd
nd
nd
nd
0.02
0.01
0.03
3-Methylpentanoic acid
29.40
MS, LRI
0.04
0.02
0.04
0.01
0.03
0.07
0.10
0.08
0.10
0.11
0.02
0.10
4-Methylpentanoic acid
29.58
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
4-Pentanoic acid
30.84
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
Benzyl nitrile
31.63
MS, LRI
nd
0.02
nd
nd
nd
0.01
nd
nd
nd
0.02
0.03
nd
Heptanoic acid
31.96
MS, LRI
0.10
0.08
0.10
0.02
0.05
0.04
0.10
0.11
0.18
0.12
0.11
0.14
Octanoic acid
33.05
MS, LRI
0.07
0.03
0.03
nd
0.01
0.01
0.03
0.04
0.04
0.01
0.02
nd
Nonanoic acid
34.12
MS, LRI
0.03
0.02
0.03
nd
0.02
nd
0.02
nd
0.02
0.01
0.01
nd
Benzoic acid
37.83
MS, LRI
0.07
0.08
0.07
0.03
0.05
0.04
0.07
0.07
0.07
0.08
0.08
0.08
Total
36.54
39.69
41.78
33.93
51.13
45.12
63.75
53.37
57.76
37.34
37.75
41.55
Esters
Ethyl acetate
3.04
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
0.04
0.08
0.05
Butanoic acid 3-methyl-ethyl ester
8.60
MS, LRI
nd
0.02
0.01
0.05
nd
0.03
nd
nd
0.03
nd
nd
nd
Ethyl lactate
19.65
MS, LRI
0.03
0.04
nd
0.01
nd
0.01
nd
0.02
nd
0.02
nd
0.04
Vinyl 2,2-dimethyl pentanoate
21.70
MS, LRI
0.13
0.10
0.25
nd
0.1
nd
nd
nd
nd
nd
nd
nd
Propylene carbonate
30.14
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
0.01
0.02
0.01
Total
0.16
0.16
0.26
0.06
0.10
0.04
nd
0.02
0.03
0.07
0.10
0.10
Aldehydes
Acetaldehyde
1.75
MS, LRI
0.46
0.52
0.40
0.91
0.41
0.45
nd
nd
nd
0.37
0.20
0.46
Butanal
2.86
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
2-Methylbutanal
3.37
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
3-Methylbutanal
3.45
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
0.05
0.10
0.05
Pentanal
4.79
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
Hexanal
9.01
MS, LRI
nd
nd
nd
nd
nd
nd
0.04
nd
0.07
nd
nd
0.01
2-Pentanal
11.54
MS, LRI
nd
nd
nd
nd
nd
nd
0.01
0.01
nd
nd
nd
nd
Heptanal
14.09
MS, LRI
0.03
0.01
0.02
nd
0.03
0.02
nd
nd
nd
nd
nd
nd
Nonanal
21.00
MS, LRI
0.06
0.02
0.05
0.05
nd
nd
0.08
0.15
nd
nd
nd
nd
Benzaldehyde
23.89
MS, LRI
1.19
1.06
0.70
0.40
0.96
0.53
0.86
0.60
1.11
1.70
1.10
1.30
Lilac aldehyde A
24.88
MS, LRI
1.00
1.20
0.80
0.22
1.30
1.26
0.02
0.02
0.01
nd
nd
nd
Lilac aldehyde B
25.37
MS, LRI
0.10
0.08
nd
0.02
0.06
0.04
0.07
0.09
0.07
0.16
0.03
0.10
Benzeneacetaldehyde
26.42
MS, LRI
nd
0.01
nd
0.01
0.02
0.04
0.05
0.04
0.07
0.06
0.03
0.04
Nicotinaldehyde
27.61
MS, LRI
0.50
0.34
0.60
nd
0.04
nd
nd
nd
nd
0.22
0.10
0.14
2-Methyl-2-octenedial
29.18
MS, LRI
0.10
0.03
0.10
0.01
0.10
0.11
nd
nd
nd
0.02
0.03
0.01
Phenylacetaldehyde
31.31
MS, LRI
0.03
nd
0.07
nd
nd
nd
nd
0.02
nd
nd
nd
nd
2-Pyrrolecarbaldehyde
32.69
MS, LRI
nd
0.03
nd
0.01
nd
0.01
0.02
0.02
0.01
0.01
0.02
nd
Cinnamaldehyde
32.83
MS, LRI
0.03
nd
0.02
0.03
0.05
0.01
0.01
nd
0.02
nd
0.02
nd
Total
3.50
3.30
2.76
1.65
2.97
2.47
1.16
0.96
1.37
2.59
1.63
2.11
Furans
Furan
2.19
MS, LRI
0.18
nd
0.03
nd
0.02
nd
nd
nd
nd
nd
nd
nd
Methylfuran
3.15
MS, LRI
0.81
0.27
0.30
0.25
0.15
nd
nd
nd
nd
0.13
0.10
0.25
Dihydro-2-methyl-3(2H)-furanone
17.04
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
Furfural
22.61
MS, LRI
1.42
1.71
1.13
1.07
1.30
2.19
3.41
2.06
4.77
2.56
2.53
2.50
1-(2-Furanyl)-ethanone
23.54
MS, LRI
0.05
0.10
nd
0.05
0.10
0.08
0.10
0.05
0.15
0.5
0.08
0.01
5-Methylfurfural
25.03
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
Dihydro-5-methyl-2(3H)-furanone
25.65
MS, LRI
0.75
nd
0.09
0.01
0.05
0.03
0.05
0.05
0.05
nd
nd
0.01
Dihydro-3-methyl-2(3H)-furanone
25.72
MS, LRI
0.40
0.38
0.70
0.04
0.09
0.07
nd
nd
nd
0.44
0.87
0.50
2(5H)-Furanone
28.49
MS, LRI
nd
0.03
nd
nd
0.04
0.02
nd
nd
0.02
0.01
0.02
nd
4,5-Dimethyl-2-furaldehyde
32.19
MS, LRI
nd
0.02
nd
0.02
0.03
0.02
0.04
0.02
0.05
0.07
0.08
0.05
Total
3.56
2.41
2.25
1.39
1.68
2.33
3.50
2.13
4.89
3.25
3.60
3.31
Terpenes
Verbenene
10.74
MS, LRI
nd
nd
nd
nd
0.03
0.01
nd
nd
nd
nd
nd
nd
α-Phellandrene
12.91
MS, LRI
0.03
nd
0.05
nd
0.03
nd
nd
nd
nd
nd
nd
nd
α-Terpinene
13.62
MS, LRI
0.06
0.01
0.03
0.04
0.02
nd
nd
0.05
0.07
nd
nd
nd
τ-Terpinene
16.45
MS, LRI
nd
nd
nd
nd
nd
nd
nd
0.01
nd
nd
nd
nd
Cymene
17.24
MS, LRI
0.05
0.01
0.05
nd
0.01
nd
nd
nd
nd
nd
nd
nd
cis-Linalool oxide
22.24
MS, LRI
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
trans-Linalool oxide
22.87
MS, LRI
0.19
0.10
0.25
0.03
nd
0.01
0.11
0.08
0.04
0.14
0.06
0.10
Menthofuran
23.06
MS, LRI
0.26
nd
0.50
nd
0.10
nd
nd
nd
nd
nd
nd
nd
Linalool
24.70
MS, LRI
0.75
0.20
0.23
0.12
0.18
nd
0.47
0.65
0.20
0.91
0.12
0.67
Damascenone
29.96
MS, LRI
0.02
0.02
nd
0.01
nd
0.01
nd
0.02
nd
0.13
0.10
0.12
α-Terpinolene
31.77
MS, LRI
0.14
nd
0.25
nd
0.11
0.05
nd
nd
0.03
0.01
0.02
nd
Carvacrol
34.66
MS, LRI
nd
nd
0.01
nd
0.07
0.03
nd
nd
nd
nd
nd
nd
Total
1.5
0.34
1.37
0.20
0.55
0.11
0.58
0.00
0.34
1.19
0.30
0.89
Miscellanous
Dimethyl sulfide
1.93
MS, LRI
1.07
1.14
0.70
nd
1.00
nd
nd
nd
nd
0.72
0.15
1.20
Eugenol
34.45
MS, LRI
nd
0.01
nd
nd
0.01
0.01
0.01
0.02
0.01
nd
nd
nd
Total
1.07
1.15
0.70
nd
1.01
0.01
0.01
0.02
0.01
0.72
0.15
1.20
aRetention time; bMS: mass spectrum tentatively identified using NIST 05 and Wiley 275 libraries; LRI: linear retention index.
cNormalized amount of volatile compounds (percentage) (peak of volatile compound/total peak area of all volatile compounds) of multifloral, acacia, and rhododendron honey from Italy (Valtellina).
nd: not detected.These compounds belonged to different major chemical classes as follows: alcohols, phenols, ketones, free fatty acid, esters, aldehydes, furans, and terpenes.This paper is the first investigation on the VOCs profile of a buckwheat monofloral honey from Valtellina (North of Italy). Remarkable differences in VOCs profiles were observed when comparing honey samples of different floral origins. Consistent with other authors [34–36], most of the compounds were identified in all of the analyzed honeys, but the proportion in which they occurred appeared very different taking into account the different floral/botanical origin. Similarly, in each of the analyzed samples there were compounds which were not present in other types of honeys to be evaluated as potential “floral markers”.The VOCs profile of honeys labeled as buckwheat honeys (B1–B10) was similar, particularly for samples containing a relevant quantity of buckwheat pollen grains despite the different geographical origin. In addition, comparing it with the VOCs profile of the other honey samples, not containing buckwheat pollen grains (M1–M6, A1–A3, and R1–R3), some differences were identified, particularly regarding the composition and the concentration of some chemical classes such as alcohols, aldehydes, free fatty acids, furans, and terpenes as shown in Tables4 and 5.Buckwheat honey is characterized by a sharp, sweet, and slightly biting taste, and its organoleptic characteristics have been proven to be reflected in its composition and concentrations of volatile compounds [33]. Moreover, among the aldehydes, methylbutanals have been reported to be responsible for the characteristic pungent, sweetish, and malty flavour of buckwheat honey [22, 37].In our experimental condition, 3-methylbutanal was found in highest concentration in buckwheat honey, and 2-methylbutanal was found to be present only in the honeys containing buckwheat pollen grains. These compounds are commonly found in barley malt [38]. They are known to be Strecker aldehydes, and their presence in honeys is usually associated with the Maillard browning reactions.The extremely high amounts of methylbutanals in buckwheat honeys compared with some other honeys suggested that this type of honey contains a higher abundance of Strecker degradation precursors, such as amino acids, which would result in a honey with an aroma resembling that which develops upon heat-promoted chemical reactions that occur during the malting of barley [22]. The presence of other Maillard reaction products such as phenylacetaldehyde and dimethyl sulfide supports this hypothesis.As reported in the literature [33], besides aldehydes, also the concentrations of free fatty acids, like butanoic acid and pentanoic acid, were considerably greater in the honey samples B1, B2, B3, and B9, those containing the higher quantity of buckwheat pollen grains. Butanoic acid gives buckwheat honey its typical pungent smell and pentanoic acid has a rancid smell and an acid taste [37]. Such chemical compounds, characterized by high concentrations in a honey type and specific sensory properties, are to be considered interesting as potential “variety markers” [33].Wolski et al. [32] reported butanal, phenol, trans-linalool oxide, 3,4,5-trimethylphenol as buckwheat honey marker compounds because they were not found in other honey types. In this study, only butanal was confirmed to be such a marker, being present only in the two honey samples containing a relevant quantity of buckwheat pollen grains, corresponding to the Italian monofloral buckwheat honey (samples B1, B2, and B3) and the Polish honeys (samples B8 and B9).In the same honey samples, we also found significantly great quantities of isovaleric acid, never reported before in buckwheat honey. Isovaleric acid is a potent, odorant, volatile compound, exhibiting an unpleasant odor associated with the rank smell of perspiring feet and has been considered to be an important off-flavor compound in honeys [39]. Isovaleric acid, has been found to be an important odorant for Anarcardium occidentaleL. and Croton sp. honeys from Brazil [40].Finally, we have identified additional characteristic volatile compounds of buckwheat honey such as 3-methyl-2-buten-1-ol, 2-butanone, 2-hydroxy-3-pentanone, 4-methylpentanoic acid, 4-pentanoic acid, butanal, 2-methylbutanal, pentanal, dihydro-2-methyl-3(2H)-furanone, 5-methylfurfural, andcis-linalool oxide. Pasini et al. [21] reported 5-methylfurfural and other furans as important buckwheat honey marker compounds. In the literature, furanic compounds were reported to derive from sugar degradation and considered to be indicators of thermal processes and storage [41].


## 4. Conclusion

Honey samples labeled as buckwheat honey, found to contain, to different extents, buckwheat pollen grains on melissopalynological analyses, show similar VOCs profiles, distinguishing them from the other honey floral types analyzed. In particular, the honey samples from the Italian Alps, classified as monofloral buckwheat honey, and the two samples from Poland, classified as multifloral honey but containing a significant level of buckwheat pollen grains, were found to have a very similar volatile organic compounds profile, despite the different geographical origin. Thus, the VOCs profile analyses seemed to be useful in distinguishing honeys containing buckwheat pollen grains from those of different botanical origin.Many volatile compounds were identified in all honey types, but in the honeys containing buckwheat pollen grains there were components that were not present in other honey types such as 3-methyl-2-buten-1-ol, 2-butanone, 2-hydroxy-3-pentanone, 4-methylpentanoic acid, 4-pentanoic acid, butanal, 2-methylbutanal, pentanal, dihydro-2-methyl-3(2H)-furanone, 5-methylfurfural, andcis-linalool oxide. Among them, butanal and 2-methylbutanal have been proposed as buckwheat honey markers also by other authors [22, 32, 33].According to the literature, butanoic acid and pentanoic acid were considerably greater in the buckwheat honey samples particularly in those from Italy and Poland containing the higher level of buckwheat pollen grains. These compounds have been reported to give buckwheat honey its characteristic aromatic and organoleptic properties and are to be considered interesting as potential “variety markers”. Finally, isovaleric acid, whose presence is reported to have a negative sensorial impact, was for the first time detected in buckwheat honey, particularly in the Italian monofloral buckwheat honeys and in the Polish samples.



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*Source: 904202-2013-09-26.xml*