Characterization of Aroma-Active Compounds in Italian Tomatoes with Emphasis on New Odorants

Article abstract of DOI:10.1021/acs.jafc.7b01108

An aroma distillate was prepared by solvent extraction and subsequent SAFE distillation from Italian vine-ripe tomatoes eliciting an intense overall aroma. Application of gc/olfactometry and the aroma extract dilution analysis revealed 44 odor-active compounds, 42 of which could be identified. The highest odor activity value of 2048 was established for the green, grassy (Z)-3-hexenal, the metallic smelling trans-4,5-epoxy-(E)-2-decenal, the potato-like 3-(methylthio)propanal, and the caramel-like 4-hydroxy-2,5-dimethyl-3(2H)-furanone. Of the further odorants, 13 compounds have previously not been reported as tomato odorants. Although most of these showed lower FD-factors, in particular, the coconut/dill-like smelling wine lactone ((3S,3aS,7aR)-3a,4,5,7a-tetrahydro-3,6-dimethylbenzofuran-2(3H)-one) appeared with a quite high FD factor. In addition, a fruity, almond-like odorant (6) with an FD factor of 1024 was detected. By application of high resolution mass spectrometry and polarity considerations, the structure of a methyl-2-ethoxytetrahydropyran isomer was suggested for 6. Four of the five possible isomers, the 3-methyl-, 4-methyl-, 5-methyl-, and 6-methyl-2-ethoxytetrahydropyran were synthesized and showed similar mass spectrometric patterns. However, these were excluded by their different retention indices. Although the synthesis of the remaining 2-methyl-2-ethoxytetrahydropyran resulted in only small yields, which were not sufficient for NMR measurements, this structure is very likely for 6. This compound was never reported as a food constituent before. Finally, quantitation of 23 odorants by stable isotope dilution assays allowed for the preparation of an aroma recombinate resembling the overall aroma of the tomatoes.

Full text of DOI:10.1021/acs.jafc.7b01108

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Article
Characterization of Aroma-Active Compounds in
Italian Tomatoes with Emphasis on New Odorants
Johanna Kreissl, and Peter Schieberle
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Page 1 of 39
Journal of Agricultural and Food Chemistry
Characterization of Aroma-Active Compounds in
Italian Tomatoes with Emphasis on New Odorants
Johanna Kreissl and Peter Schieberle*
Deutsche Forschungsanstalt für Lebensmittelchemie,
LiseꢀMeitnerꢀStr. 34, Dꢀ85354 Freising, Germany
*Corresponding author:
Phone
Fax
+49 8161 71 2994
+49 8161 71 2970
Eꢀmail
Peter.Schieberle@ch.tum.de
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ABSTRACT
2
3
An aroma distillate was prepared by solvent extraction and subsequent SAFE
distillation from Italian vineꢀripe tomatoes eliciting an intense overall aroma.
Application of gc/olfactometry and the aroma extract dilution analysis revealed 44
odorꢀactive compounds, 42 of which could be identified. The highest odor activity
value of 2048 was established for the green, grassy (Z)ꢀ3ꢀhexenal, the metallic
4
5
6
7
smelling
transꢀ4,5ꢀepoxyꢀ(E)ꢀ2ꢀdecenal,
the
potatoꢀlike
smelling
3ꢀ
8
(methylthio)propanal and the caramelꢀlike smelling 4ꢀhydroxyꢀ2,5ꢀdimethylꢀ3(2H)ꢀ
furanone. Of the further odorants, thirteen compounds have previously not been
reported as tomato odorants. Although most of these showed lower FDꢀfactors, in
particular, the coconut/dillꢀlike smelling wine lactone ((3S,3aS,7aR)ꢀ3a,4,5,7aꢀ
tetrahydroꢀ3,6ꢀdimethylꢀbenzofuranꢀ2(3H)ꢀone) appeared with a quite high FD factor.
In addition, a fruity, almondꢀlike odorant with an FD factor of 1024 (6) was detected.
By application of high resolution mass spectrometry and polarity considerations the
structure of a methylꢀ2ꢀethoxytetrahydropyran isomer was suggested for 6. Four of
the five possible isomers, the 3ꢀmethylꢀ, 4ꢀmethylꢀ, 5ꢀmethylꢀ, and 6ꢀmethylꢀ2ꢀ
ethoxytetrahydropyran were synthesized and showed similar mass spectrometric
patterns. However, these were excluded by their different retention indices. Although
the synthesis of the remaining 2ꢀmethylꢀ2ꢀethoxytetrahydropyran resulted in only
small yields, which were not sufficient for NMR measurements, this structure is very
likely for 6. This compound was never reported as a food constituent before. Finally,
quantitation of twentyꢀthree odorants by stable isotope dilution assays allowed the
preparation of an aroma recombinate resembling the overall aroma of the tomatoes.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
Key words: stable isotope dilution assay; 2ꢀmethylꢀ2ꢀethoxytetrahydropyran, 3ꢀ
methylꢀ2ꢀethoxytetrahydropyran,
5ꢀmethylꢀ2ꢀethoxytetrahydropyran,
6ꢀmethylꢀ2ꢀ
ethoxytetrahydropyran, wine lactone
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28 Introduction
29
With a total consumption of 21 kg per person/per year1 tomatoes or tomato
products are among the most appreciated vegetable based foods in Germany. While
about one third of the tomatoes is consumed fresh, two thirds are used commercially
in the manufacturing of either tomato juices, sauces or purées. In 2014 the world
production of tomatoes was about 171 million tons,2 with one tenth produced in the
EU.
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31
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33
34
35
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40
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42
43
44
45
46
47
48
49
50
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52
53
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55
56
57
58
59
Tomatoes are much appreciated by consumers due to their characteristic aroma
and, therefore, several studies were already undertaken to identify the tomato
volatiles and also to address changes in volatile composition during ripening.3ꢀ7 Thus,
today, more than 400 tomato volatiles are known.
Buttery et al.8 were the first to undertake experiments identifying those aroma
compounds responsible for the aroma of unprocessed tomatoes. The authors
determined the concentrations of selected volatiles and calculated their odor units
(ratio of concentration to odor threshold). Sixteen compounds, among them (Z)ꢀ3ꢀ
hexenal, βꢀionone, hexanal, βꢀdamascenone and 1ꢀpentenꢀ3ꢀone, were found to
either reach or exceed their odor threshold.8 However, because no gas
chromatography/olfactometry was used in this study, potent odorants occurring in
trace amounts might have been overlooked at that time.
By applying the molecular sensory science concept (Sensomics), the complex
odor image of a food can be elucidated. 9 This bioactivityꢀguided approach allows one
to identify all odorants interacting with the odorant receptors in the human nose.
Following this approach, Mayer et al.10 previously reported the highest odor activity
values for (Z)ꢀ3ꢀhexenal, transꢀ4,5ꢀepoxyꢀ(E)ꢀ2ꢀdecenal, βꢀdamascenone, βꢀionone
and 1ꢀoctenꢀ3ꢀone in five different unprocessed tomato varieties. Further attempts to
characterize odorꢀactive compounds in unprocessed tomatoes with similar results
were also reported by other groups.10ꢀ14
Compared to investigations on raw, unprocessed tomatoes, studies on the aroma
compounds of thermally processed tomato products are rather scarce. Because our
group is currently studying chemical changes in key aroma compounds on the way
from raw tomatoes to processed tomato products, the need appeared to verify
whether the complete set of odorꢀactive compounds of tomatoes is already available
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64
in the current literature. Thus, using a Italian vineꢀripe tomato from Sicily eliciting a
very intense aroma, the aim of this study was to characterize the aroma active
compounds by means of the Sensomics concept9 in order to provide a basis for
further investigations on chemical changes in tomato odorants during a largeꢀscale
commercial process for tomato juice, sauce and paste.
65 MATERIALS AND METHODS
66
67
68
69
70
Materials. Italian vineꢀripe tomatoes (Solanum lycopersicum L.) from Sicily were
purchased at a local store. Based on their intense red color and intense aroma,
tomatoes were considered as full ripe. Sensory evaluations of six tomato samples
from different origins by a sensory panel pointed to the Sicilian tomato as the one
showing the most intense overall aroma.
71
72
73
74
75
76
77
78
79
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83
84
85
86
Chemicals. The following compounds were obtained from the sources given in
parentheses: 4ꢀallylꢀ2ꢀmethoxyphenol (eugenol) and acetic acid (Merck, Darmstadt,
Germany). 2,3ꢀButanedione (diacetyl), butanoic acid, (E,E)ꢀ2,4ꢀdecadienal, decanoic
acid, ethyl butanoate, 4ꢀethyloctanoic acid, (E,E)ꢀ2,4ꢀheptadienal, heptanoic acid,
hexanal, hexanol, hexanoic acid, (E)ꢀ2ꢀhexenal, 3ꢀhydroxyꢀ4,5ꢀdimethylꢀ2(5H)ꢀ
furanone (3ꢀHDF; sotolon), 4ꢀhydroxyꢀ2,5ꢀdimethylꢀ3(2H)ꢀfuranone (4ꢀHDF), 4ꢀ
hydroxyꢀ3ꢀmethoxybenzaldehyde (vanillin), 2ꢀisobutylthiazole, linalool (3,7ꢀ
dimethyloctaꢀ1,6ꢀdienꢀ3ꢀol), methyl salicylate, 3ꢀ(methylthio)propanal (methional), 3ꢀ
(methylthio)propanol (methionol), 3ꢀmethylbutanal, 3ꢀmethylbutanoic acid, 3ꢀ
methylindole (skatole), 4ꢀmethylphenol (pꢀcresol), (E,E)ꢀ2,4ꢀnonadienal, (E,Z)ꢀ2,6ꢀ
nonadienal, nonanal, (E)ꢀ2ꢀnonenal, octanal, (E)ꢀ2ꢀoctenal, 1ꢀpentenꢀ3ꢀone,
phenylacetaldehyde and phenylacetic acid (SigmaꢀAldrich Chemie, Taufkirchen,
Germany). (E)ꢀβꢀDamascenone, cisꢀ4,5ꢀepoxyꢀ(E)ꢀ2ꢀdecenal and (Z)ꢀ4ꢀheptenal
were gifts from Symrise (Holzminden, Germany). 2ꢀMethoxyphenol (guaiacol)
(Serva, Heidelberg, Germany), 1ꢀoctenꢀ3ꢀone (Lancaster, Mühlheim/Main, Germany)
and 3ꢀmethylꢀ2,4ꢀnonanedione (Chemos GmbH, Ratisbon, Germany).
87
88
89
The following odorants were synthesized as previously reported: transꢀ4,5ꢀepoxyꢀ(E)ꢀ
2ꢀdecenal,15 (Z)ꢀ1,5ꢀoctadienꢀ3ꢀone,16 and wine lactone ((3S,3aS,7aR)ꢀ3a,4,5,7aꢀ
tetrahydroꢀ3,6ꢀdimethylꢀbenzofuranꢀ2(3H)ꢀone).17
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(Z)ꢀ3ꢀHexenal. The compound was synthesized following closely an approach
previously reported.18 DessꢀMartin periodinane (520 mg) (SigmaꢀAldrich Chemie)
was added to dichloromethane (15 mL, distilled, Merck), flushed with argon, and
sealed with a septum. (Z)ꢀ3ꢀHexenol (100 mg, 1 mmol) (SigmaꢀAldrich Chemie) was
dissolved in dichloromethane (5 mL), then added to the suspension, and stirred for
20 h at room temperature. The organic phase was subsequently washed with an
aqueous sodium thiosulfateꢀsolution (1 M, 50 mL) saturated with sodium hydrogen
carbonate, followed by an aqueous sodium hydrogen carbonate solution (0.5 M,
50 mL), and finally with distilled water (50 mL). The organic phase was dried over
sodium sulfate and contained 30% of the target compound.
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93
94
95
96
97
98
99
100
101
MS (EI): m/z (%) 41 (100), 39 (58), 42 (49), 55 (46), 69 (42), 83 (36), 57 (32), 98
(M+, 10).
102
Syntheses of methyl-2-ethoxytetrahydropyran isomers.
103
104
105
106
107
108
3ꢀMethylꢀ2ꢀethoxytetrahydropyran (3ꢀmethylꢀisomer). Synthesis was performed as
reported previously19 with some modifications. 2ꢀPropenal (acrolein, 0.56 g, 10 mmol)
was added to ethyl propenyl ether (1.08 g, 12.5 mmol). After addition of
hydroquinone (11.0 mg, 0.1 mmol) and heating for 2 h at 185 °C in a sealed glass
vessel, platinum (IV) oxide was added and hydrogen was introduced. The mixture
was stirred for 4 days under normal pressure until the reduction was complete.
109
110
EIꢀMS: m/z (%): 75 (100), 42 (68), 47 (53), 55 (50), 99 (44), 70 (40), 144 (M+, 6),
143 (M+ꢀ1, 4).
111
112
113
4ꢀMethylꢀ2ꢀethoxytetrahydropyran (4ꢀmethylꢀisomer). 2ꢀButenal (0.7 g, 10 mmol)
was added to ethyl vinyl ether (0.9 g, 12.5 mmol). The further procedure was
performed as described above for the 3ꢀmethylꢀisomer.
114
115
EIꢀMS: m/z (%): 99 (100), 55 (59), 75 (49), 42 (46), 47 (34), 43 (31), 70 (26), 143
(M+ꢀ1, 13), 144 (M+, 2).
116
117
118
5ꢀMethylꢀ2ꢀethoxytetrahydropyran (5ꢀmethylꢀisomer). Synthesis followed the
procedure for the 4ꢀmethyl isomer, but replacing 2ꢀbutenal by 2ꢀmethylꢀ2ꢀpropenal
(0.7 g, 10 mmol).
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MSꢀEI; m/z (%): 99 (100), 55 (90), 75 (85), 47 (63), 42 (56), 72 (55), 81 (52), 70
(48), 143 (M+ꢀ1, 15), 144 (M+, 5).
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122
123
124
1H NMR (COSY, 500 MHz; chloroformꢀd, 300 K): δ (ppm) 0.82 (d, J = 6.8 Hz, 3H,
HꢀC8), 1.22 (t, 7.2 Hz, 3H, HꢀC1), 1.41ꢀ1.59 (m, 2H, HꢀC5), 1.66ꢀ1.73 (m, 2H, HꢀC4),
1.70ꢀ1.77 (m, 1H, HꢀC6), 3.34ꢀ3.40 (m, 1H, HαꢀC7), 3.41ꢀ3.46 (m, 1H, HαꢀC2), 3.41ꢀ
3.48 (m, 1H, HβꢀC7), 3.69ꢀ3.76 (m, 1H, HβꢀC2), 4.74 (t, J = 2.87 Hz, 1H, HꢀC3).
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126
127
13C NMR (HSQC, HMBC, 125 MHz; chloroformꢀd, 300 K): δ (ppm) 15.1 (CH3, C1),
17.4 (CH3, C8), 26.7 (CH2, C5), 30.0 (CH2, C4), 30.4 (CH, C6), 62.3 (CH2, C2), 66.2
(CH2, C7), 96.2(CH, C3).
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129
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6ꢀMethylꢀ2ꢀethoxytetrahydropyran (6ꢀmethylꢀisomer). Starting from butenone
(0.7 g, 10 mmol), the procedure described above for the 3ꢀmethylꢀisomer was
followed.
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132
MSꢀEI; m/z (%): 75 (100), 47 (63), 72 (53), 42 (51), 55 (42), 70 (36), 99 (20), 143
(M+ꢀ1, 9), 144 (M+,5).
133
Isotopically Labeled Internal Standards.
134
135
[2H6ꢀ7]ꢀ4ꢀMethylphenol and [13C2]ꢀphenylacetic acid were supplied by Sigmaꢀ
Aldrich Chemie.
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143
144
145
The following isotopically labeled internal standards were synthesized as
previously reported: [13C4]ꢀ2,3ꢀButanedione,20 [2H2]ꢀbutanoic acid,21 [2H2ꢀ6]ꢀ(E)ꢀβꢀ
damascenone,22 [2H3ꢀ6]ꢀ(E,E)ꢀ2,4ꢀdecadienal,23 [2H3ꢀ4]ꢀhexanal and [2H2]ꢀhexanoic
acid,24 [2H2]ꢀ(E)ꢀ2ꢀhexenal,25 [2H2]ꢀ(Z)ꢀ3ꢀhexenal,23 [13C2]ꢀ3ꢀhydroxyꢀ4,5ꢀdimethylꢀ
2(5H)ꢀfuranone,26 [13C2]ꢀ4ꢀhydroxyꢀ2,5ꢀdimethylꢀ3(2H)ꢀfuranone,27 [2H3]ꢀ4ꢀhydroxyꢀ3ꢀ
methoxybenzaldehyde,28
methylbutanoic
acid,31
[2H2]ꢀlinalool,29
[2H3]ꢀ2ꢀmethoxyphenol,30
[2H2]ꢀ3ꢀ
[2H3]ꢀ3ꢀ
[2H3]ꢀ3ꢀ(methylthio)propanal
and
(methylthio)propanol,32 [2H2]ꢀ(E,Z)ꢀ2,6ꢀnonadienal, [2H2]ꢀ(E)ꢀ2ꢀnonenal and [2H2ꢀ4]ꢀ1ꢀ
octenꢀ3ꢀone,23 [2H2]ꢀpentanoic acid,24 [2H2ꢀ3]ꢀ1ꢀpentenꢀ3ꢀone,33 [13C2]ꢀ
phenylacetaldehyde,34 [2H2ꢀ4]ꢀ4ꢀpropylꢀ2ꢀmethoxyphenol35 and [2H3]ꢀwine lactone.17
146
147
[2H4]ꢀtransꢀ4,5ꢀEpoxyꢀ(E)ꢀ2ꢀdecenal. The epoxide was prepared in a fourꢀstep
synthesis as follows:
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(a). [2H4]ꢀHexanol. Wilkinson’s catalyst (tris(triphenylphosphine)ꢀrhodium(I)
chloride, 0.5 g, 0.5 mmol) was suspended in toluene under a nitrogen atmosphere.
After replacing nitrogen by deuterium, the suspension was stirred for 30 min under a
slightly increased pressure until the color changed from dark red to orange. Then,
hexynꢀ1ꢀol (2.5 g, 25 mmol) dissolved in toluene (15 mL) was added to the activated
catalyst. The mixture was flushed with deuterium and stirred for 18 hours. To purify
the product, pentane (50 mL) was added, and toluene was removed by flushing the
reaction mixture over silica gel (G60, 100 g, waterꢀcooled column, diameter 3.5 cm).
The [2H4]ꢀhexanol formed was purified by highꢀvacuum distillation (10 mPa, 45 °C).
149
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(b). [2H4]ꢀHexanal. The oxidation of [2H4]ꢀhexanol to [2H4]ꢀhexanal was performed
36
as described in
suspension of
with some modifications. Under an argon atmosphere a
DessꢀMartin periodinane (1,1,1ꢀtriacetoxyꢀ1,1ꢀdihydroꢀ1,2ꢀ
benziodoxolꢀ3(1H)ꢀone, 3.5 g, 8.2 mmol) in dichloromethane was added dropwise to
[2H4]ꢀhexanol (0.8 g, 8 mmol) dissolved in dichloromethane (30 mL). After 1 h the
organic layer was washed with a sodium thiosulfate solution saturated with sodium
hydrogen carbonateꢀ (1 mol/L, 2 × 75 mL), followed by an aqueous sodium hydrogen
carbonate solution (100 mL) and finally with tap water (100 mL). The organic layer
was dried over anhydrous sodium sulfate to yield [2H4]ꢀhexanal.
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174
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(c). [2H4]ꢀ(E,E)ꢀ2,4ꢀDecadienal. [2H4]ꢀ(E,E)ꢀ2,4ꢀDecadienal was synthesized as
23
previously reported
with some variations. 1ꢀMethoxyꢀ1ꢀbutenꢀ3ꢀin (0.84 g,
10 mmol) dissolved in anhydrous tetrahydrofuran (THF, 5 mL) was added dropwise
to ethyl magnesium bromide (3 mL, 3.2 M) in anhydrous THF, and the mixture was
kept at 40 °C. After stirring for 1 hour at room temperature and cooling, [2H4]ꢀhexanal
(0.38 g, 3.65 mmol) dissolved in THF (5 mL) was added within 20 min. After 2 hours
at room temperature the reaction was cooled, treated with dry ethanol (0.2 mL), and
then lithium aluminum hydride (5.3 mL, 2.0 M) was added in small portions. The
mixture was stirred for another 2 hours and then washed with sulfuric acid (25 mL,
2 mol/L), followed by water (10 mL), sodium carbonate solution (10 mL, 10%) and
finally water (2 × 10 mL). The organic layer was dried over anhydrous sodium sulfate
and concentrated by means of a Vigreux column. The target compound was purified
by chromatography over silica gel (G60, 30 g, column diameter 2 cm).
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(d). [2H4]ꢀtransꢀ4,5ꢀEpoxyꢀ(E)ꢀ2ꢀdecadienal. The final product was obtained by
oxidation of [2H4]ꢀ(E,E)ꢀ2,4ꢀdecadienal with 3ꢀchloroperoxybenzoic acid.23 To a
solution
of
[2H4]ꢀ(E,E)ꢀ2,4ꢀdecadienal
(0.36 g,
0.8 mmol)
dissolved
in
dichloromethane (5 mL), small portions of 3ꢀchloroperoxybenzoic acid (0.16 mg,
1 mmol) in dichloromethane (5 mL) were added within 30 min. The reaction was
stirred for 12 hours at room temperature and then washed with an aqueous sodium
hydrogen carbonate solution (0.5 mol/L, 2 × 20 mL). The organic layer was dried over
anhydrous sodium sulfate and concentrated by means of a Vigreux column.
Purification of the compound was achieved by chromatography over silica gel (G60,
30 g, diameter 2 cm).
189
190
EIꢀMS: m/z (%): 68 (100), 81 (32), 40 (24), 39 (21), 44 (18), 57 (14), 41 (13), 55
(13), 43 (12), 45 (12), 69 (11), 46 (10), 107 (9), 143 (6), 156 (3).
191
192
MSꢀCI (methanol); m/z (%): 68 (100), 143 (15), 103 (8), 83 (7), 110 (6), 155 (3),
174 (3).
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200
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202
Isolation of Volatiles. Tomatoes (100 g) were puréed using a Multiquick 5 stick
blender (Braun, Kronberg, Germany). After 5 min, enzymes were inhibited by adding
saturated aqueous calcium chloride solution (100 mL) as suggested by Buttery et al.3
The tomato/CaCl2 mixture was extracted with dichloromethane (100 mL) by stirring
for 15 min at room temperature. To break the emulsion, the mixture was centrifuged
for 10 min at 4500 rpm. The organic layer was separated, the aqueous phase was
extracted twice with dichloromethane (2 × 50 mL) and the combined organic layers
were dried over anhydrous sodium sulfate. Volatiles were separated from the nonꢀ
volatile material by SAFE distillation,37 and the resulting distillate was concentrated to
200 ꢁL at 45 °C using a micro distillation apparatus.
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208
209
Gas Chromatography/Olfactometry (GC/O). GC/O was performed by means of
a GC 8000 gas chromatograph (Carlo Erba Instruments, Hofheim, Germany), using
the following fused silica capillary columns and temperature programs. DBꢀFFAP: 30
m × 0.32 mm i.D., 0.25 ꢂm (J&W Scientific, Folsom, USA), the initial temperature of
40 °C was held for 2 min, then raised at 6 °C/min to 230 °C and held for 5 min. CP Sil
8 CB: 30 m × 0.32 mm i.D., 0.25 ꢂm (Varian, Darmstadt, Germany), the initial
temperature of 40 °C was held for 2 min, then raised at 6 °C/min to 250 °C and held
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for 5 min. Samples were injected at 40 °C by means of the cold on column technique.
For GC/O, the effluent was evenly split at the end of the column between a flame
ionization detector (FID, 250 °C) and a heated sniffing port (200 °C) using a Yꢀ
shaped glass splitter and two deactivated fused silica capillaries (500 mm × 0.2 mm
i.D.). Helium adjusted to a flow rate of 1.5 mL/min served as the carrier gas.
Retention indices (RI) of the odorants were calculated from the retention times of nꢀ
alkanes by linear interpolation.
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Aroma Extract Dilution Analysis (AEDA). The undiluted distillate (100 ꢁL) was
subjected to GC/O to detect odorꢀactive regions. This was done by three trained
panelists. Following, the flavor dilution factors (FD factors) of the odorꢀactive
compounds were assigned by stepwise dilution of the distillate 1:1 (v+v) using
dichloromethane as solvent to obtain dilutions of 1:1, 1:2, 1:4, 1:8, 1:16, and up to
1:2048 of the original extract. Sniffing of the dilutions was continued until no odorant
could be detected. Each odorant was thus assigned an FD factor representing the
last dilution in which the odorant was still detectable. The results of the three
panelists, which differed to not more than plus/minus one dilution step, were
averaged.
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Static Headspace-Gaschromatography-Olfactometry/Mass Spectrometry
(SH-GC-O/MS). A 1:1 mixture of tomatoes (30 g) and a saturated CaCl2ꢀsolution (30
mL) was placed in a 120 mL flask, sealed and equilibrated at room temperature for
30 min. 10 mL of the headspace were withdrawn by means of a gastight syringe and
injected via an onꢀcolumn injector onto a deactivated capillary (250 mm × 0.53 mm
i.d.) placed in a cold trap 915 (Thermo Fisher Scientific, Dreieich, Germany). During
the injection, the volatiles were cryofocused at ꢀ150 °C, and the air was purged
through a flow controlled (20 mL/min) valve. After the injection, the valve was closed
and the trap was immediately heated to 250 °C to transfer the compounds onto a
fused silica capillary (30 m × 0.25 mm i.d., 1 ꢂm, DBꢀ5) (J&W Scientific). An initial
temperature of 0 °C was held for 2 min, then the temperature was raised at 6 °C/min
to 230 °C and held for 5 min. Helium was used as the carrier gas. At the end of the
column the effluent was transferred to an FID, a sniffingꢀport, and a Saturn 2100 T
ion trap mass spectrometer (Varian, Darmstadt, Germany). Spectra were generated
in the chemical ionization mode (CIꢀMS) with methanol as the reagent gas.
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Gas Chromatography/Mass Spectrometry (GC/MS). For the identification and
high resolution (HRGCꢀMS) experiments, mass spectra were recorded using a 5890
series II gas chromatograph (HewlettꢀPackard, Waldbronn, Germany) connected to
an MAT 95 S sector field mass spectrometer (Finnigan, Bremen, Germany) at 70 eV
in the electron impact mode (EIꢀMS) and at 115 eV in the chemical ionization mode
(CIꢀMS; reagent gas: isobutane) using the capillaries and the oven programs
described above.
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Two Dimensional (Heart Cut) Gas Chromatography-Olfactometry/Mass
Spectrometry (GC-GC/O/MS). To obtain unequivocal mass spectra, e.g., in case of
the trace wine lactone, GCꢀGC/O/MS was performed. An FFAP column (30 m × 0.32
mm i.d., 0.25 ꢂm (J&W Scientific), installed in the first Mega 2 Series GC (Fisons
Instruments, Mainz, Germany), was connected to the Moving Column Stream
Switching (MCSS) system (Fisons Instruments, Mainz, Germany). This was
connected to an FID and the first sniffingꢀport by a Yꢀtype splitter, and to a transfer
line via a cold trap with the column in the second Mega 2 Series GC (Carlo Erba,
Hofheim, Germany). Volatiles were cryofocused in the trap with liquid nitrogen at ꢀ80
°C. The end of the second capillary was coupled via a Yꢀtype splitter to an ITD 800
ion trap mass spectrometer (Finnigan, Bremen, Germany) and a sniffingꢀport. The
trap was quickly heated to 250 °C, and the volatiles were transferred to the second
GC equipped with a DBꢀ5 capillary (30 m × 0.25 mm i.d., 0.25 ꢂm) (J&W Scientific).
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Quantitation by Two Dimensional (Heart Cut) Gas Chromatography/Mass
Spectrometry (GC-GC/MS). On a Trace Ultra gas chromatograph (Thermo Fisher
Scientific) a Combi PAL autosampler (CTC Analytics) was mounted. The GC was
equipped with a cold onꢀcolumn injector and an FFAP capillary (30 m × 0.32 mm i.D.,
0.25 ꢂm) (J&W Scientific). The end of the capillary was connected via the MCSS by a
Yꢀtype splitter either to an FID, a sniffingꢀport or a heated transfer line (250 °C)
leading to a cold trap cooled with liquid nitrogen located in the CPꢀ3800 second GC
(Varian, Darmstadt, Germany). The transfer line led into a Saturn 2200 ion trap mass
spectrometer (Varian) operated in the CIꢀMS mode with methanol as the reactant
gas.
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Quantitation by Stable Isotope Dilution Assays (SIDA).
Volatile Isolation.
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Several tomatoes were puréed, weighed and enzymes were inhibited after 5 min
by using the same volume of an aqueous saturated calcium chloride solution.
Depending on the concentrations of the aroma compounds determined in preliminary
experiments, different amounts of the sample (from 2 to 100 g) were extracted to
obtain concentrations between 1 and 5 ꢂg/mL of the respective analyte. The internal
standards dissolved in dichloromethane were added and the mixture was equilibrated
for 30 min. The isolation followed the procedure described above for GC/O analysis,
and the extracts obtained were analyzed by means of GCꢀGC/MS. Concentrations
were calculated from the area counts of selected MS fragments for the analyte and
the internal standard (Table 1) as recently reported for linalool.29
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Because of the fast degradation of (Z)ꢀ3ꢀhexenal during freezing/thawing, this
compound was always analyzed in the tomatoes before freezing the fruits. For all
other odorants, frozen fruits were crushed and after 5 min the saturated CaCl2
solution was added. The further procedure was performed as described above.
289
Quantitation by Mass Spectrometry.
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A response factor (Table 1) was determined for each compound by analyzing
mixtures containing defined amounts of the unlabeled odorants and the respective
labeled standard in five different ratios (5:1, 3:1, 1:1, 1:3, and 1:5).
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Time Course of the Formation of Hexanal and (Z)ꢀ3ꢀHexenal during Storage of
Tomato Purée.
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After crushing the tomatoes, the purée was stored for 5, 10, 60, 120, 180 or
300 min, respectively, and 10 mL of a saturated calcium chloride solution was added
to 10 mg purée after the storage period. Volatiles were then extracted by adding
20 mL dichloromethane containing [2H3ꢀ4]ꢀhexanal and [2H2]ꢀ(Z)ꢀ3ꢀhexenal and
isolated as described above. One intact tomato was immediately puréed with an
equal volume of saturated calcium chloride to obtain a zero value.
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For freezing experiments, several tomatoes were stored at ꢀ25 °C for 24 hours.
The frozen tomatoes were puréed for 5 min and worked up as described above.
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Odor Thresholds. For the calculation of odor activity values odor thresholds in
water were either determined by means of the triangle test in increasing
concentrations, or were used from the literature.38
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Aroma Recombinate. All odorants showing an OAV ≥ 1 in tomatoes were mixed
in an aqueous buffer (5 mmol citric acid, pH 4.4) in the concentrations measured in
the tomato. A sensory panel of 20 participants in the age of 22ꢀ45 rated the
intensities of selected aroma attributes on a sevenꢀpoint scale from 0 (not
perceivable) to 3 (strongly perceivable). Samples were presented in covered Teflon
vessels (15 g each), and solutions of single aroma compounds reflecting each aroma
attribute were presented in concentrations about 100ꢀfold above their odor threshold.
The following odorants were chosen: (Z)ꢀ3ꢀhexenal (grassy and green), 1ꢀpentenꢀ3ꢀ
one (pungent), (E,E)ꢀ2,4ꢀdecadienal (fatty), 4ꢀhydroxyꢀ2,5ꢀdimethylꢀ3(2H)ꢀfuranone
(caramelꢀlike), transꢀ4,5ꢀepoxyꢀ(E)ꢀ2ꢀdecenal (metallic) and 2,3ꢀbutanedione (butterꢀ
like). The panel was trained weekly to perform a comparative aroma profile analysis,
which showed a quite good similarity in the overall rating of the descriptors during
training with model mixtures. The evaluation of the descriptors showed a good day to
day variation among the panel members of about 20%.
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Nuclear Magnetic Resonance Experiments. 1D and 2DꢀNMR experiments (1H,
1
13C, Hꢀ1H COSY, HSQC, and HMBC) were performed on an Avance III 500 MHz
spectrometer with a cryoꢀTCI probe (Bruker, Rheinstetten, Germany) at 300 K.
Benzeneꢀd6, chloroformꢀd, and methanolꢀd4 were obtained from EuroisoꢀTop
(Saarbrücken, Germany).
325 RESULTS AND DISCUSSION
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In a preliminary experiment, the overall aroma of seven tomatoes from the trade
and from different origins was evaluated, and the panel was asked to rank the
samples according to the intensity of the aroma. An Italian wineꢀripe tomato was
rated to elicit the most intense aroma and was therefore selected for the analyses.
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Identification of Odor-Active Compounds. A fresh tomato purée was treated
with saturated CaCl2 solution,39 extracted with dichloromethane and the nonꢀvolatile
components were removed by SAFE distillation.37 By adding a drop of the extract on
a strip of filter paper and comparing the smell to the original sample it could be
verified that the total set of aroma compounds was extracted. Application of GC/O on
the aroma extract and subsequent application of the AEDA revealed 44 aromaꢀactive
regions in the flavor dilution (FD) factor range between 4 and 2048 (Figure 1). Among
them, three odorants 7, 17, and 35 with a grassy/green, a cooked potatoꢀlike, and a
metallic note, respectively, showed the highest FD factors.
To identify the compounds responsible for the odorꢀactive regions, first the
retention indices (RI), and odor qualities were compared to an inꢀhouse database
containing data of about 1000 food odorants and their specific characteristics. In
addition, mass spectra were recorded. Finally, the structure of an odorant was
confirmed by comparing the analytical and sensory characteristics with that of the
respective reference compound. Thus, the three compounds with the highest FD
factors were identified as (Z)ꢀ3ꢀhexenal, 7, 3ꢀ(methylthio)propanal, 17, and transꢀ4,5ꢀ
epoxyꢀ(E)ꢀ2ꢀdecenal, 35, followed by 4ꢀhydroxyꢀ2,5ꢀdimethylꢀ3(2H)ꢀfuranone, 36, 1ꢀ
pentenꢀ3ꢀone, 3, (E)ꢀβꢀdamascenone, 32, and 4ꢀhydroxyꢀ3ꢀmethoxybenzaldehyde,
44. Additionally, the cooked potatoꢀlike 3ꢀ(methylthio)propanol, 30, and the geraniumꢀ
like (Z)ꢀ1,5ꢀoctadienꢀ3ꢀone, 14, contributed with a quite high FD factor to the overall
aroma (Table 2). These findings confirmed results of previous studies done on the
aroma compounds of tomatoes of other origins.5,8ꢀ11
However, as indicated in Table 2, thirteen compounds were previously not
reported as tomato aroma compounds. In particular 6 eliciting a fruity, almondꢀlike
odor, was of interest, because it appeared with a high FD factor of 1024 which was
close to the FD factors of the wellꢀknown odorants, such as (Z)ꢀ3ꢀhexenal or 3ꢀ
(methylthio)propanal. As a first hint on its structure, it was observed that during
chromatography of the tomato distillate on silica gel in an nꢀpentane/diethyl ether
gradient, 6 was eluted with a very low diethyl ether concentration indicating that the
compound is quite unpolar. Its mass spectrum (Figure 2) showed a molecular mass
of m/z 144, which was confirmed by CIꢀMS (data not shown). To gain a better insight
its structure, the molecular mass was determined by high resolution mass
spectrometry and was calculated as C8H16O2. This molecular formula indicated one
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double bond equivalent or a ring system, respectively. Due to its low polarity, and
assuming that one oxygen is part of a ring system this consideration allows only an
ether bond for the second oxygen. A comparison of the mass spectrum (Figure 2)
with data in the NIST database proposed 4ꢀmethylꢀ2ꢀethoxytetrahydropyran as a
possible structure. This structure would be in agreement with the fragments m/z 100
and m/z 99, respectively, in its mass spectrum indicating a loss of the ethoxy group
from the molecular ion (Figure 2). Therefore, 4ꢀmethylꢀ2ꢀethoxytetrahydrofuran was
synthesized. Its mass spectrum (Figure 3 B) contained a very similar set of fragments
as compared to 6, however with different intensities. Furthermore, although the odor
quality during GC/O was in agreement, the retention index (Table 3) was clearly
different from that of 6. Therefore, it was assumed that 6 is a positional isomer of 4ꢀ
methylꢀ2ꢀethoxytetrahydropyran, and thus three further compounds, i.e., the 3ꢀ
methylꢀ, 5ꢀmethylꢀ, and 6ꢀmethylꢀ2ꢀethoxytetrahydropyran were synthesized.
Because the syntheses followed the same principle, only the NMR data were
recorded for the 5ꢀmethylꢀisomer. The NMR clearly confirmed the success of the
synthesis (see experimental part). The mass spectra of the three isomers are shown
in Figure 3 A, C and D. All isomers showed similar fragments in their mass spectrum
compared to compound 6, but from all isomers the intensities of the fragments were
different (Figure 3). A spectrum closest to that of 6 was shown by the 6ꢀmethylꢀ
isomer (Figure 3 D). However, also for these three isomers, the retention indices on
two stationary GC phases were different, and in particular the 6ꢀmethyl isomer
showed the largest difference (Table 3). It should be mentioned that with the
exception of the 4ꢀmethylꢀisomer, the other three isomers were characterized for the
first time in literature.
The last possible isomer would be the 2ꢀmethylꢀ2ꢀethoxytetrahydropyran (Figure
4). However, this compound was scarcely mentioned in the literature.40,41
Unfortunately, the synthesis route could not follow an easy 1,4ꢀcycloaddition (Dielsꢀ
Alder reaction) as used in the synthesis of the other isomers. Thus, alternative routes
were thought of and a possible synthesis is shown in Figure 5. While a nucleophilic
addition via Grignard reaction from δꢀvalerolactone and methylmagnesium bromide
did not result in the desired intermediate 2ꢀhydroxyꢀ2ꢀmethyltetrahydropyran, finally
by adding methyllithium to δꢀvalerolactone ꢀ78 °C, a mixture of 2ꢀhydroxyꢀ2ꢀ
methyltetrahydropyran and 6ꢀhydroxyꢀ2ꢀhexanone was obtained.42 However, the
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chemical equilibrium of the tautomers always shifted towards the openꢀchain keto
form, in particular under acidic conditions for the substitution of the hydroxylꢀ by the
ethoxyꢀgroup. In the NMR spectrum the equilibrium of both tautomers is illustrated. In
benzeneꢀd6 (unipolar solvent) the openꢀchain tautomer already existed in a ratio of
4:1, but in methanolꢀd4 this was even fortified towards an excess of 20:1. Therefore,
the last step (Figure 5) did not yield the desired 2ꢀmethylꢀ2ꢀethoxytetrahydropyran in
great quantities. However, a small peak showing the same mass spectrum, retention
indices and aroma qualities as 6 was obtained. But, the amounts were not sufficient
for NMR experiments. Due to the very low concentrations in the tomato distillate and
close eluting peaks such as (Z)ꢀ3ꢀhexenal it was also not possible to purify the
compound from the tomato distillate for NMR analysis. However, taking all data into
account, it seems very likely that compound 6 is 2ꢀmethylꢀ2ꢀethoxytetrahydropyran.
Because the amounts were estimated to be about 20 ꢁg per kg, it can be assumed
from the high FD factor (Table 2) that the odorant has a very low odor threshold. The
compound has never been reported before in tomatoes or any food, respectively.
There are also no hints on its formation pathway by a known biochemical reaction
mechanism.
42 reaching an FD factor of 128 (Table 2) with a coconut/dillꢀlike scent is another
compound previously not reported in tomato. To obtain a clear mass spectrum, a
heartꢀcut GC/GC procedure was applied. During the first injection, the coconutꢀ, dillꢀ
like odor was located by GC/O at 26.8 min. In a second GC run the effluent between
26.2 to 27.2 min was transferred to the second GC and again analyzed by GC/O and
mass spectrometry to obtain the mass spectrum shown in Figure 6. A comparison
with the literature suggested wine lactone earlier identified in white wine.17 With the
aid of the reference compound, 42 was identified as one of the eight possible
isomers of wine lactone as (3S,3aS,7aR)ꢀ3a,4,5,7aꢀtetrahydroꢀ3,6ꢀdimethylꢀ
benzofuranꢀ2(3H)ꢀone.
The further identification experiments revealed 1ꢀoctenꢀ3ꢀone (11; mushroomꢀlike),
2ꢀ and 3ꢀmethylbutanoic acid (28; sweaty), hexanal (4; grassy, green), (E,E)ꢀ2,4ꢀ
decadienal (31; fatty), 2ꢀmethoxyphenol (33; smoky), 3ꢀhydroxyꢀ4,5ꢀdimethylꢀ2(5H)ꢀ
furanone (41; seasoning), and phenylacetic acid (43; honeyꢀlike) with somewhat
lower FD factors (Table 2). The application of a GC/Oꢀheadspace revealed no further
odorants (data not shown).
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As mentioned in the introduction, only one previous study also applied the
molecular sensory science concept on fresh tomatoes before. While 16 odorants
identified in our study were also found by Mayer et al.,10 thirteen odorants, such as
2,3ꢀbutanedione, linalool, (E)ꢀ2ꢀnonenal, 3ꢀhydroxyꢀ4,5ꢀdimethylꢀ2(5H)ꢀfuranone,
wine lactone or 2ꢀphenylacetic acid, were not reported in the tomato varieties
investigated by this group. This indicates that aroma differences in tomato varieties
cannot only be explained by odorants with high odor activity, but may also by caused
by differences in the set of odorants with lower FD factors.
Quantitation of aroma compounds.
Freezing Experiments.
Since a batch of the same tomatoes had to be stored at ꢀ25 °C prior to analysis,
stability experiments on (Z)ꢀ3ꢀhexenal ꢀ25 °C were performed, due to the known
instability of this compound, i.e., during freezing.43ꢀ45 (E)ꢀ2ꢀHexenal was also
determined, because it is a known isomerization product of (Z)ꢀ3ꢀhexenal.46 The
concentration of (Z)ꢀ3ꢀhexenal was 2219 ꢁg/kg before and 152 ꢁg/kg after the
freezing/thawing process. Hence, only 7% of the original amounts were left. A
different behavior was found for (E)ꢀ2ꢀhexenal, which was not affected by
freezing/thawing. However, recently it was reported that even storage temperatures
of 4 °C for 30 days led to a clear decrease in (Z)ꢀ3ꢀhexenal, hexanal and (E)ꢀ2ꢀ
hexenal.43
Time course of odorant formation.
(Z)ꢀ3ꢀhexenal was earlier shown to be released from its precursor linolenic acid via
a hydroperoxide already during grinding of tomatoes when the cell tissue is
destroyed.46
To illustrate the degree of (Z)ꢀ3ꢀhexenal generation in the Italian tomatoes, the
concentration of both aldehydes was determined over a period of 5 hours (Figure 7).
For both aldehydes an immediate formation was observed. (Z)ꢀ3ꢀhexenal increased
to more than 1400 ꢁg/kg during the first 5 min reaching its maximum (1510 ꢁg/kg)
after 10 min and finally decreased to its initial value after 3 hours. The amount of (E)ꢀ
2ꢀhexenal peaked after 5 min up to 106 ꢁg/kg and decreased over the next 2 hours.
Thus, the endogenous enzymes were always inhibited5,8 by CaCl2 after 5 min before
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the following workups. This procedure compromised the time needed for enzymatic
aroma generation, but also saved time for the throughput of a larger number of
samples.
Quantitation of aroma compounds. Twentyꢀfour aroma compounds were
quantitated by means of stable isotope dilution assays (SIDA).
The highest concentrations were determined for hexanal (1,510 ꢁg/kg) and (Z)ꢀ3ꢀ
hexenal (943 ꢁg/kg) (Table 4), followed by 4ꢀhydroxyꢀ2,5ꢀdimethylꢀ3(2H)ꢀfuranone
(142 ꢁg/kg),
1ꢀpentenꢀ3ꢀone
(92.6 ꢁg/kg),
transꢀ4,5ꢀepoxyꢀ(E)ꢀ2ꢀdecenal
(61.5 ꢁg/kg), 3ꢀmethylbutanoic acid (40.1 ꢁg/kg), 3ꢀ(methylthio)propanol (29.3 ꢁg/kg),
butanoic acid (17.1 ꢁg/kg), 4ꢀhydroxyꢀ3ꢀmethoxybenzaldehyde (12.1 ꢁg/kg), and 3ꢀ
(methylthio)propanal (11.7 ꢁg/kg). Many compounds, however, were only present in
trace amounts, for example the wine lactone (5.4 ꢁg/kg), (E)ꢀβꢀdamascenone
(3.1 ꢁg/kg), 1ꢀoctenꢀ3ꢀone (1.1 ꢁg/kg) or 4ꢀallylꢀ2ꢀmethoxyphenol (0.5 ꢁg/kg). The
amount of the newly identified 2ꢀmethylꢀ2ꢀethoxytetrahydropyran was estimated to be
below 20 ꢁg/kg.
Calculation of OAVs. To get further insight into the contribution of the odorants to
the overall aroma, OAVs (ratio of odor threshold to concentration) were calculated.
Because tomatoes contain 94% water, this was chosen as an adequate matrix for
threshold determinations. Most of the thresholds used, have previously been
published by our group.38
(Z)ꢀ3ꢀHexenal showed by far the highest OAV of 7,900, followed by transꢀ4,5ꢀ
epoxyꢀ(E)ꢀ2ꢀdecenal (1,600), hexanal (630), wine lactone (290), (E)ꢀβꢀdamascenone
(240), 1ꢀpentenꢀ3ꢀone (99), 1ꢀoctenꢀ3ꢀone (66), (E,E)ꢀ2,4ꢀdecadienal (54), 3ꢀ
(methylthio)propanal (27), and (E)ꢀ2ꢀnonenal (12), suggesting a major impact of
these compounds to the overall aroma of fresh tomatoes (Table 5).
Recombinate of Sicilian Tomatoes. To verify the quantitative data, a
recombinate was prepared in aqueous buffer including all 15 odorants with an OAV
≥ 1 in their natural concentrations (Table 4). The pH was adjusted to 4.4 as
measured in the fresh tomatoes. The sensory evaluation showed the same aroma
qualities in the recombinate and the tomato purée, but with slight differences in the
intensities. Overall the similarity was rated with 2.4 on a scale from 0 to 3. This is
probably due to the fact that 2ꢀmethylꢀ2ꢀethoxytetrahydropyran could not be used in
the recombinate. But also other odorants may have driven the overall similarity closer
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to that of the tomatoes. However, for the work on processed tomato products, the
results are very helpful to clarify, which compounds are degraded or newly formed
during processing. Further studies on this issue will be published soon.
498 ASSOCIATED CONTENT
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Supporting Information. The Supporting Information is available free of charge
on the ACS Publications website.
NMR spectra of the tautomers 6ꢀhydroxyꢀ2ꢀhexanone and 2ꢀhydroxyꢀ2ꢀ
methyltetrahydropyran in benzeneꢀd6 (Figures S1 and S2).
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1
Bayerische Landesanstalt für Landwirtschaft (LfL) Institut für Ernährungswissenschaft
und Markt; http://www.lfl.bayern.de/mam/cms07/iem/dateien/06_gem%C3%BCse_by
_.pdf (Accessed: 23. January 2017).
2
3
4
FAOSTAT. Food and Agriculture Organization of the United Nations. Production, Crops;
http://www.fao.org/faostat/en/#data/QC (Accessed: 23. January 2017).
Buttery, R. G.; Teranishi, R.; Ling, L. C.; Flath, R. A.; Stern, D. J. Quantitative studies on
origins of fresh tomato aroma volatiles, J. Agric. Food Chem., 1988, 36, 1247ꢀ1250.
Baldwin, E. A.; NisperosꢀCarriedo, M. O.; Moshonas, M. G. Quantiative analysis of flavor
and other volatiles for certain constituents of two tomato cultivars during ripening, J.
Amer. Soc. Hort. Sci., 1991, 116, 265ꢀ269.
5
6
Buttery, R. G.; Ling, L. Enzymic production of volatiles in tomatoes. In Prog. Flavour
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635
636
637
638
639
Figure Captions.
Figure 1. Flavor dilution (FD) chromatogram of the volatile fraction isolated from the
Italian tomatoes.
Figure 2. Mass spectrum (EIꢀMS) of compound 6.
Figure 3. Mass spectra of 3ꢀmethylꢀ (A), 4ꢀmethylꢀ (B), 5ꢀmethylꢀ (C), and 6ꢀmethylꢀ
2ꢀethoxytetrahydropyran (D).
640
641
Figure 4. Structure of 2ꢀmethylꢀ2ꢀethoxytetrahydropyran.
Figure 5. Synthetic route proposed for the synthesis of 2ꢀmethylꢀ2ꢀ
642
643
644
645
646
647
648
ethoxytetrahydropyran from δꢀvalerolactone via 2ꢀhydroxyꢀ2ꢀmethyltetrahydropyran.
Figure 6. (A): Chemical structure of wine lactone and detailed chomatogram (EIꢀMS)
of the second dimension. (B): EIꢀMS of the coconut/dillꢀlike smelling region.
Figure 7. Changes in the concentrations of (Z)ꢀ3ꢀhexenal and (E)ꢀ2ꢀhexenal in a
fresh tomato purée (Sicilian tomatoes) stored for 5 h.
Figure 8. Comparative aroma profile analyses of the tomato puree (black) and the
aroma recombinate (gray) consisting of 15 odorants.
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Table 1. Isotopically Labeled Internal Standards, Selected Ions, and Response Factors Used in the Stable Isotope Dilution Assays
odorant
ion (m/z)a
165
87
labeled standard
ion (m/z)a
169ꢀ171c
91
Rfb
4ꢀallylꢀ2ꢀmethoxyphenol
2,3ꢀbutanedione
[2H2ꢀ4]ꢀpropylꢀ2ꢀmethoxyphenol
[13C4]ꢀ2,3ꢀbutanedione
[2H2]ꢀbutanoic acid
1.09
0.97
0.99
0.96
0.76
0.88
0.53
0.80
1.03
0.78
0.57
1.12
0.95
0.88
0.80
0.99
0.81
0.99
1.14
0.86
0.71
0.91
butanoic acid
103
109
191
153
139
83
105
4ꢀmethylphenol
[2H6ꢀ7]ꢀ4ꢀmethylphenol
115/116
195ꢀ197c
156ꢀ159c
143
(E)ꢀβꢀdamascenone
(E,E)ꢀ2,4ꢀdecadienal
transꢀ4,5ꢀepoxyꢀ(E)2ꢀdecenal
hexanal
[2H6]ꢀ(E)ꢀβꢀdamascenone
[2H4]ꢀ(E,E)ꢀ2,4ꢀdecadienal
[2H4]ꢀtransꢀ4,5ꢀepoxyꢀ(E)2ꢀdecenal
[2H4]ꢀhexanal
[2H3]ꢀhexanoic acid
[2H2]ꢀ(E)ꢀ2ꢀhexenal
86/87c
hexanoic acid
131
99
134
(E)ꢀ2ꢀhexenal
101
(Z)ꢀ3ꢀhexenal
81
[2H2]ꢀ(Z)ꢀ3ꢀhexenal
83
3ꢀhydroxyꢀ4,5ꢀdimethylꢀ2(5H)ꢀfuranone
4ꢀhydroxyꢀ2,5ꢀdimethylꢀ3(2H)ꢀfuranone
4ꢀhydroxyꢀ3ꢀmethoxybenzaldehyde
linalool
129
129
153
137
125
117
105
89
[13C2]ꢀ3ꢀhydroxyꢀ4,5ꢀdimethylꢀ2(5H)ꢀfuranone
[13C2]ꢀ4ꢀhydroxyꢀ2,5ꢀdimethylꢀ3(2H)ꢀfuranone
[2H3]ꢀ4ꢀhydroxyꢀ3ꢀmethoxybenzaldehyde
[2H2]ꢀlinalool
131
131
156
139
2ꢀmethoxyphenol
[2H3]ꢀ2ꢀmethoxyphenol
128
3ꢀmethylbutanoic acid
3ꢀ(methylthio)propanal
3ꢀ(methylthio)propanol
(E,Z)ꢀ2,6ꢀnonadienal
(E)ꢀ2ꢀnonenal
[2H2]ꢀ3ꢀmethylbutanoic acid
[2H3]ꢀ3ꢀ(methylthio)propanal
[2H3]ꢀ3ꢀ(methylthio)propanol
[2H2]ꢀ(E,Z)ꢀ2,6ꢀnonadienal
[2H2]ꢀ(E)ꢀ2ꢀnonenal
119
108
92
139
123
127
141
125
129ꢀ131c
1ꢀoctenꢀ3ꢀone
[2H2]ꢀ1ꢀoctenꢀ3ꢀone
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Table 1 (continued). Isotopically Labeled Internal Standards, Selected Ions, and Response Factors Used in the Stable Isotope Dilution Assays
odorant
ion (m/z)a
labeled standard
ion (m/z)a
Rfb
pentanoic acid
1ꢀpentenꢀ3ꢀone
phenylacetaldehyde
phenylacetic acid
wine lactone
117
[2H3]ꢀpentanoic acid
[2H2ꢀ2]ꢀ1ꢀpentenꢀ3ꢀone
[13C2]ꢀphenylacetaldehyde
[13C2]ꢀphenylacetic acid
[2H3]ꢀwine lactone
120
0.97
0.99
0.95
0.90
1.0d
85
87/88c
123
121
137
139
167
170
aIons used to monitor the analyte or the internal standard, respectively (MSꢀCI). bMS response factor. cInternal standard was used as a mixture of isotopologs. dA response factor of
1.0 was assumed.
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Table 2. Compounds (FD ≥ 4) Identified in a Distillate Prepared from Italian Tomatoes
RIc on
CP Sil C8 CB
no. aroma compounda
1 3ꢀmethylbutanal
2 2,3ꢀbutanedione
3 1ꢀpentenꢀ3ꢀone
4 hexanal
aroma qualityb
malty
FFAP
976
FD factord
lite
39
n.d.
625
8
4
buttery
993
ꢀ
39
pungent
1024
1086
1094
1100
1124
1207
1242
1284
1287
1328
1367
1373
1400
1423
1459
1483
1509
1517
1543
1545
1591
1605
1624
684
512
32
32
1024
2048
32
16
8
39
grasy, green
fruity
800
5 unknown
n.d.
929
ꢀ
6 2ꢀmethylꢀ2ꢀethoxytetrahydropyran
7 (Z)ꢀ3ꢀhexenal
8 (E)ꢀ2ꢀhexenal
9 (Z)ꢀ4ꢀheptenalf
10 octanal
fruity, almondꢀlike
grassy, green
grassy, appleꢀlike
fishy
ꢀ
39
800
39
n.d.
900
ꢀ
47
citrusꢀlike
n.d.
978
10
11 1ꢀoctenꢀ3ꢀone
12 hexanol
mushroomꢀlike
grassy, fruity
fatty
64
4
869
ꢀ
10
13 nonanal
n.d.
979
4
14 (Z)ꢀ1,5ꢀoctadienꢀ3ꢀonef
15 2ꢀisobutylthiazol
16 (E)ꢀ2ꢀoctenal
17 3ꢀ(methylthio)propanal
18 unknown
geraniumꢀlike
pungent
128
4
13
39
14
48
1033
1058
905
citrusꢀlike, fatty
cooked potatoꢀlike
cheeseꢀlike, fruity
fatty
64
2048
4
n.d.
1147
n.d.
1160
1106
1155
1243
806
ꢀ
ꢀ
ꢀ
19 (Z)ꢀ2ꢀnonenalf
20 unknown
4
fatty, moldy, pungent
fatty, moldy
flowery
4
21 (E)ꢀ2ꢀnonenal
22 linalool
23 (E,Z)ꢀ2,6ꢀnonadienalf
24 (Z)ꢀ2ꢀdecenal
25 butanoic acid
32
16
4
ꢀ
10
10
fatty, cucumberꢀlike
fatty
4
ꢀ
47
sweaty
4
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Table 2 (continued). Most Odorꢀactive Compounds (FD ≥ 4) Identified in a Distillate Prepared from Italian Tomatoes
RIc on
no. aroma compounda
aroma qualityb
honeyꢀlike, beeswaxꢀlike
fatty
FFAP
1649
1668
1671
1710
1725
1816
1826
1868
1992
2025
2038
2071
2076
2179
2182
2206
2255
2541
2581
CP Sil C8 CB
n.d.
FD factord
lite
8
26 phenylacetaldehyde
8
4
27 (E,Z)ꢀ2,4ꢀnonadienalf
28 2ꢀ and 3ꢀmethylbutanoic acidg
29 (E,E)ꢀ2,4ꢀnonadienalf
30 3ꢀ(methylthio)propanol
31 (E,E)ꢀ2,4ꢀdecadienal
32 (E)ꢀβꢀdamascenone
1191
876
ꢀ
12
sweaty
64
fatty
1210
980
4
ꢀ
48
cooked potatoꢀlike, tallowy
fatty
256
32
10
8
1313
1385
1086
1350
1379
1067
n.d.
grape juiceꢀlike
smoky
512
32
48
49
12
49
33 2ꢀmethoxyphenol
34 cisꢀ4,5ꢀepoxyꢀ(E)ꢀ2ꢀdecenal
35 transꢀ4,5ꢀepoxyꢀ(E)ꢀ2ꢀdecenal
36 4ꢀhydroxyꢀ2,5ꢀdimethylꢀ3(2H)ꢀfuranone
37 unknown
metallic
4
metallic
2048
1024
32
caramelꢀlike
metallic
ꢀ
38 4ꢀmethylphenol
horse stableꢀlike, phenolic
cloveꢀlike
n.d.
4
ꢀ
12
39 4ꢀallylꢀ2ꢀmethoxyphenol
40 4ꢀethyloctanoic acid
n.d.
8
fecal, moldy
seasoning
1345
1108
1459
n.d.
16
ꢀ
12
41 3ꢀhydroxyꢀ4,5ꢀdimethylꢀ2(5H)ꢀfuranone
42 wine lactoneg
32
coconutꢀlike, dillꢀlike
honeyꢀlike
128
32
ꢀ
43 2ꢀphenylacetic acid
ꢀ
6
44 4ꢀhydroxyꢀ3ꢀmethoxybenzaldehyd
vanillaꢀlike
1407
512
aIdentification of the compounds was performed by comparing their mass spectra (MSꢀEI, MSꢀCI), retention indices on capillaries FFAP and CP
Sil 8 CB as well as the odor quality and the odor intensity perceived during sniffing with data of reference compounds. bOdorꢀquality perceived at
the sniffingꢀport. cRetention index. Flavor dilution factor, n.d. not determined (odorꢀquality of the compound was not detected during HRGCꢀO
d
on this capillary column). eReference reporting the compound for the first time as a fresh tomato odorant. fNo unequivocal mass spectrum was
obtained. Tentative identification is based on the remaining criteria given in footnote a. gStereochemistry was not determined. Odor qualities are
given for the racemate.
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Table 3. Odor quality and Retention Indices of Compound 6 and Four Methylꢀ2ꢀ
ethoxytetrahydropyran Isomers
RIb on
compound
aroma qualitya
fruity, almondꢀlike
fruity, almondꢀlike
fruity, almondꢀlike
fruity, almondꢀlike
fruity, almondꢀlike
FFAP
1100
1116
1130
1151
1198
CP Sil C8 CB
6
929
926
958
956
967
3ꢀmethylꢀ2ꢀethoxyꢀisomer
4ꢀmethylꢀ2ꢀethoxyꢀisomer
5ꢀmethylꢀ2ꢀethoxyꢀisomer
6ꢀmethylꢀ2ꢀethoxyꢀisomer
aOdorꢀquality perceived at the sniffingꢀport. bRetention index.
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Table 4. Concentrations of 24 Important Aroma Compounds in Italian Tomatoes
concna (
ꢀg/kg)
aroma compound
(fresh weight)
1514
943
range (ꢀg/kg)
hexanal
1414 – 1614
501 – 1554
138 – 149
88.3 – 96.0
56.0 – 69.3
37.8 – 42.4
20.7 – 37.8
15.6 – 18.6
11.2 – 13.0
11.4 – 11.8
5.3 – 5.5
(Z)ꢀ3ꢀhexenal
4ꢀhydroxyꢀ2,5ꢀdimethylꢀ3(2H)ꢀfuranone c
1ꢀpentenꢀ3ꢀone
142
92.6
61.5
40.1
29.3
17.1
12.1
11.7
5.4
transꢀ4,5ꢀepoxyꢀ(E)ꢀ2ꢀdecenal
3ꢀmethylbutanoic acid
3ꢀ(methylthio)propanol
butanoic acid
4ꢀhydroxyꢀ3ꢀmethoxybenzaldehyde
3ꢀ(methylthio)propanal c
wine lactone
(E)ꢀβꢀdamascenone
2,3ꢀbutanedione
3.1
2.6 – 3.6
2.9
1.8 – 3.9
(E)ꢀ2ꢀnonenal
2.3
2.1 – 2.5
linalool
1.7
1.6 – 1.8
(E,E)ꢀ2,4ꢀdecadienal
1ꢀoctenꢀ3ꢀone c
1.5
1.3 – 1.6
1.1
0.9 – 1.2
2ꢀmethoxyphenol c
phenylacetaldehyde
2ꢀphenylacetic acid
4ꢀallylꢀ2ꢀmethoxyphenol c
pꢀcresol
0.9
0.7 – 1.0
0.8
0.7 – 1.0
0.6
0.54 – 0.66
0.4 – 0.5
0.5
0.3
0.31 – 0.34
0.08 – 0.09
ꢀ
3ꢀhydroxyꢀ4,5ꢀdimethylꢀ2(5H)ꢀfuranone
(E,Z)ꢀ2,6ꢀnonadienal
aCalculated as mean value of three different workups. LoD: limit of detection.
0.1
< LoD
29
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