Identification, Synthesis, and Characterization of Novel Sulfur

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Identification, Synthesis, and Characterization of Novel SulfurContaining Volatile Compounds from the In-depth Analysis of Lisbon Lemon Peels (Citrus limon L. Burm. f., cv. Lisbon) Robert Cannon, Arkadiusz Kazimierski, Nicole L Curto, Jing Li, Laurence Trinnaman, Adam J Janczuk, David Agyemang, Neil C. Da Costa, and Michael Z. Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf505177r • Publication Date (Web): 31 Jan 2015 Downloaded from http://pubs.acs.org on February 7, 2015

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Journal of Agricultural and Food Chemistry

Identification, Synthesis, and Characterization of Novel Sulfur-Containing Volatile Compounds from the In-depth Analysis of Lisbon Lemon Peels (Citrus limon L. Burm. f., cv. Lisbon) Robert J. Cannon,* Arkadiusz Kazimierski, Nicole L. Curto, Jing Li, Laurence Trinnaman, Adam J. Jańczuk, David Agyemang, Neil C. Da Costa, Michael Z. Chen

International Flavors & Fragrances Inc., Research & Development 1515 State Highway 36, Union Beach, NJ 07735, USA

*Contact information for corresponding author (Phone) 732-335-2668 (Fax) 732-335-2591 [email protected]

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ABSTRACT

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Lemons (Citrus limon) are a desirable citrus fruit grown and used globally in a wide range of

3

applications. The main constituents of this sour tasting fruit have been well quantitated and

4

characterized. However, additional research is still necessary to better understand the trace, volatile

5

compounds that may contribute to the overall aroma of the fruit. In this study, Lisbon lemons (Citrus

6

limon L. Burm. f., cv. Lisbon) were purchased from a grove in California, USA and extracted by liquid-

7

liquid extraction. Fractionation and multidimensional gas-chromatography mass spectrometry were

8

utilized to separate, focus, and enhance unidentified compounds. In addition, these methods were

9

employed to more accurately assign flavor dilution factors by aroma extract dilution analysis. Numerous

10

compounds were identified for the first time in lemons, including a series of branched aliphatic

11

aldehydes and several novel sulfur-containing structures. Rarely reported in citrus peels, sulfur

12

compounds are known to contribute significantly to the aroma profile of the fruit and were found to be

13

aroma active in this particular study on lemons. This paper discusses the identification, synthesis, and

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organoleptic properties of these novel volatile sulfur compounds.

15 16 17

KEYWORDS: lemons, GC-O, volatile sulfur compounds, GC-MS

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INTRODUCTION

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Native to Asia, lemons (Citrus limon) have been used for centuries in a variety of applications including

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medicine, perfumery, and the food industry. The juice of lemons is quite sour in taste, mainly due to the

29

higher levels of citric acid in comparison to other citrus fruits. However, the peel or zest of a lemon,

30

used in beverage and cooking applications, can provide a bright, citrusy flavor without the sourness.

31

Lemon oil can be industrially obtained from the peel by two main processes: cold-pressing or

32

distillation. These processes yield approximately 1.5-3% non-volatile constituents, which consist mainly

33

of antioxidants such as tocopherols and furanocoumarins that help to stabilize the oil. Therefore, most of

34

the scientific research on lemons has focused on the volatile compounds. Early efforts were mainly

35

dedicated to analyzing and categorizing cold-pressed California lemon oils,1,2 as well as isolating and

36

evaluating well-known contributors to the aroma of lemon oil, including citronellal, neral, geranial, and

37

linear aliphatic aldehydes.3 Different varieties of lemon oils were also studied, including Meyer lemon

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(Citrus meyeri), where separation by column chromatography helped to identify thymol, a key odorant

39

and marker chemical for this particular lemon type.4 As modern technology and instrumentation

40

developed at the end of the 20th century and into the beginning of the 21st century, extensive analyses

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were conducted on many different lemon peel oils.5-9 The fractionation process of citrus peel oils has

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become an indispensable technique for the separation and enrichment of chemical classes of

43

compounds.10-12 This technique also aids in providing an accurate link to the aroma active constituents

44

of the peel oil.

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Natural products contain volatile sulfur compounds (VSCs) often at trace concentrations, many

46

of which contribute to the characteristic aroma of that natural product. The concentration of VSCs in

47

citrus fruits is significantly lower than most other natural products, which has made them a major

48

challenge for researchers to identify. Also, the VSCs from the juice and peel oil from one specific fruit

49

can differ. For example, 1-p-menthene-8-thiol and 4-mercapto-4-methyl-2-pentanone are odor active

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compounds found in grapefruit juice,13,14 but were not identified in the fruit’s peel oil.15 Over the years, 3 ACS Paragon Plus Environment


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VSCs from a variety of oranges and grapefruits have led to a better understanding of the chemistry and

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flavor impact these compounds have on their respective fruit type.11,16-22 However, for lemons, there is

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considerably less data on VSCs. Hydrogen sulfide has been quantitated in the headspace of Eureka

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lemon juice,23 and dimethyl sulfide has been quantitated in four Italian lemon juices.24

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In this work, Lisbon lemon peels (Citrus limon L. Burm. f., cv. Lisbon), a variety known for its

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high juice and acid concentrations compared to other lemon varieties, were extracted and further

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fractionated to separate and enhance trace VSCs. Incorporating gas chromatography-mass spectrometry

58

(GC-MS) and multidimensional gas chromatography-mass spectrometry (MDGC-MS) techniques, novel

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VSCs were synthesized and characterized. In addition, aroma extract dilution analysis (AEDA) by gas

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chromatography-olfactometry (GC-O) was applied to assign flavor dilution (FD) factors to the odor

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active compounds in the lemon extract.

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MATERIALS AND METHODS

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Materials. Conventional Lisbon lemons (SK Choice Grade – large size) were ordered from Saticoy

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Lemon Associates and Sunkist Growers in Oxnard, California. Upon arrival, the lemons were worked up

65

immediately. A microplane zester (OXO, Chambersburg, PA) was used to zest the peel.

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Chemicals. The following reagents were purchased from Sigma-Aldrich (St. Louis, MO):

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dichloromethane (DCM), methanol (MeOH), hexane, anhydrous magnesium sulfate (MgSO4), 1,5-

68

dibromopentane, 1,3-dibromopropane, Li2CuCl4, 2-pentylmagnesium bromide-diethyl ether solution,

69

and 2-propionylthiophene. The following compounds were purchased from the FCH Group (Ukraine):

70

3-methylundecanal and 3-methyldodecanal. Finally, the following Grignard reagents were purchased

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from Novel Chemical Solutions (Crete, NE): 2-hexylmagnesium bromide-THF solution, 2-

72

heptylmagnesium bromide-THF solution, 2-octylmagnesium bromide-THF solution, 2-nonylmagnesium

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bromide-THF solution, 2-decylmagnesium bromide-THF solution, and 2-undecylmagnesium bromide-

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THF solution.

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Synthesis of 6-methylnonanal 136. The synthesis of 136 involved a four-step pathway (Figure 1) and is 4 ACS Paragon Plus Environment

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described in detail as an example of the 6-methyl and 4-methylalkyl aldehydes synthesized in this

77

report:

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1-bromo-6-methylnonane (3a). Under N2, a 1 L, four-necked flask, equipped with a mechanical stirrer,

79

was charged with 1,5-dibromopentane (1a; 5.1 g, 25 mmol), THF (50 mL), and Li2CuCl4 (2.5 mL, 250

80

mmol) in 0.1 M THF and cooled to 0 ºC. A 2-pentylmagnesium bromide-diethyl ether solution (2a; 100

81

mL, 25 mmol) was added to the reaction and stirred for 45 min at 0 ºC under argon. The mixture was

82

allowed to reach ambient temperature and quenched with water (200 mL) and 1 M HCl (25 mmol). The

83

aqueous layer was extracted with hexane (3 x 100 mL), and the combined organic phases were washed

84

with a 5% solution of NaHCO3 (100 mL) and brine (100 mL). The organic phase was dried over MgSO4

85

and 3a was concentrated under partial vacuum to remove the solvent and used as is in the next step.

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6-methylnonyl acetate (4a). A 250 mL, four-necked flask was charged with the crude mixture of 3a

87

(16.0 g, 61 mmol) and DMF (50 mL). To this mixture, sodium acetate (9.9 g, 120 mmol) was added in

88

excess and the reaction was run for 10 h at 100 ºC. The reaction mixture was allowed to reach ambient

89

temperature, followed by the addition of water (300 mL). The mixture was then extracted with hexane (3

90

x 100 mL), and the combined organic fractions were washed with water (2 x 100 mL), brine (100 mL),

91

and dried over MgSO4. The solvent was evaporated under reduced pressure to produce 4a and used as is

92

in the next step.

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6-methylnonanol (5a). A 1 L, four-necked flask was charged with the crude mixture of 4a (10.0 g, 41

94

mmol), along with MeOH (200 mL) and a catalytic amount of K2CO3 (1.0 g, 7 mmol), and run for 5 h at

95

ambient temperature. The MeOH was evaporated off and hexane (100 mL) and water (100 mL) were

96

added to the residue. The phases were allowed to separate, and the organic phase was washed with water

97

(50 mL) and brine (50 mL), dried over MgSO4, and evaporated. The raw product mixture was purified

98

by column chromatography (silica; hexane/ethyl acetate) giving 5a (2.0 g) as a clear oil.

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6-methylnonanal 136. A 1 L, four-necked flask was charged with 5a (2.0 g, 10 mmol), KBr (0.1 g, 1

100

mmol), NaHCO3 (1.7 g, 20 mmol), and DCM (150 mL). 2,2,6,6-Tetramethyl-1-oxylpiperidine 5 ACS Paragon Plus Environment


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(TEMPO; 16 mg, 100 mmol) was added to the flask, followed by water (5 mL) and allowed to cool to 0

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°C. A 15 % solution of NaClO (8.9 g, 13 mmol, d = 1.2) was added dropwise at 5 °C and stirred for 1 h

103

at 10 °C. A 5 g solution of L-ascorbic acid (30% in water) was added and heated to reflux for 2 h. After

104

cooling, the phases were allowed to separate and the organic phase was washed with water (100 mL)

105

and brine (100 mL) and dried over MgSO4. After filtration and concentration, 136 (0.4 g, 20%) was

106

obtained by column chromatography (silica; ethyl acetate/hexanes). The total yield based on the four-

107

step synthesis beginning with 1a was 10%. 1H NMR (CDCl3, 500 MHz): 9.76 (t, J=1.9 Hz, 1H), 2.42

108

(td, J=7.4 Hz, 1.9 Hz, 2H), 1.56-1.67 (m, 2H), 1.21-1.42 (m, 7H), 1.04-1.17 (m, 2H), 0.88 (t, J=7.3 Hz,

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3H), 0.84 (d, J=6.6 Hz, 3H). EI-MS: 156 (0, M+), 95 (100), 43 (78), 71 (63), 70 (60), 69 (60), 41 (54), 57

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(49), 55 (47), 96 (42), 84 (38).

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The synthesis of 6-methyldecanal 215 followed a similar procedure but instead used a 2-

112

hexylmagnesium bromide-THF solution as the Grignard reagent. The 4-methylalkyl aldehydes were

113

prepared in a similar fashion except using 1,3-dibromopropane instead of 1,5-dibromopentane, along

114

with the associated Grignard reagent.

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6-methyldecanal 215. Yield (14%) 1H NMR (CDCl3, 400 MHz): 9.77 (t, J=1.9 Hz, 1H), 2.39-2.48 (m,

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2H), 1.53-1.74 (m, 2H), 1.19-1.40 (m, 9H), 1.06-1.16 (m, 2H), 0.88 (t, J=6.8 Hz, 3H), 0.84 (d, J=6.5 Hz,

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3H). EI-MS: 170 (0, M+), 43 (100), 95 (84), 69 (67), 41 (67), 57 (66), 85 (61), 84 (59), 55 (46), 96 (44),

118

29 (39).

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4-methyloctanal 86. Yield (25%) 1H NMR (CDCl3, 400 MHz): 9.77 (t, J=1.9 Hz, 1H), 2.35-2.49 (m,

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2H), 1.55-1.72 (m, 1H), 1.36-1.49 (m, 2H), 1.19-1.34 (m, 5H), 1.04-1.19 (m, 1H), 0.89 (t, J=6.6 Hz,

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3H), 0.88 (d, J=6.4 Hz, 3H). EI-MS: 142 (0, M+), 56 (100), 57 (56), 43 (49), 70 (44), 41 (43), 29 (36), 85

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(33), 55 (27), 27 (22).

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4-methylnonanal 140. Yield (20%) 1H NMR (CDCl3, 500 MHz): 9.77 (t, J=1.9 Hz, 1H), 2.30-2.48 (m,

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2H), 1.57-1.73 (m, 1H), 1.35-1.50 (m, 2H), 1.21-1.34 (m, 7H), 1.04-1.20 (m, 1H), 0.89 (t, J=6.6 Hz,

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3H), 0.88 (d, J=6.4 Hz, 3H). EI-MS: 156 (0, M+), 57 (100), 56 (83), 41 (47), 55 (33), 43 (31), 29 (23), 85 6 ACS Paragon Plus Environment

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(20), 69 (19), 84 (17) 112 (16).

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4-methyldecanal 217. Yield (20%) 1H NMR (CDCl3, 400 MHz): 9.77 (t, J=1.9 Hz, 1H), 2.35-2.48 (m,

128

2H), 1.55-1.71 (m, 1H), 1.35-1.50 (m, 2H), 1.22-1.34 (m, 9H), 1.04-1.20 (m, 1H), 0.89 (t, J=6.6 Hz,

129

3H), 0.88 (d, J=6.4 Hz, 3H). EI-MS: 170 (0, M+), 56 (100), 57 (97), 41 (53), 43 (52), 69 (43), 29 (43), 55

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(43), 71 (43), 85 (34), 82 (28).

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4-methylundecanal 274. Yield (18%) 1H NMR (CDCl3, 500 MHz): 9.77 (t, J=1.9 Hz, 1H), 2.32-2.50 (m,

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2H), 1.56-1.76 (m, 1H), 1.38-1.56 (m, 2H), 1.22-1.33 (m, 11H), 1.03-1.20 (m, 1H), 0.89 (t, J=6.6 Hz,

133

3H), 0.88 (d, J=6.4 Hz, 3H). EI-MS: 184 (0, M+), 56 (100), 57 (87), 85 (56), 43 (55), 41 (53), 69 (49), 29

134

(46), 55 (45), 71 (39), 82 (39).

135

4-methyldodecanal 321. Yield (20%) 1H NMR (CDCl3, 500 MHz): 9.77 (t, J=1.9 Hz, 1H), 2.32-2.50 (m,

136

2H), 1.56-1.76 (m, 1H), 1.38-1.56 (m, 2H), 1.22-1.33 (m, 13H), 1.03-1.20 (m, 1H), 0.89 (t, J=6.6 Hz,

137

3H), 0.88 (d, J=6.4 Hz, 3H). EI-MS: 198 (0, M+), 56 (100), 57 (98), 41 (57), 43 (56), 85 (50), 55 (49), 69

138

(44), 82 (37), 29 (31), 81 (28).

139

4-Methyltridecanal 370. Yield (15%) 1H NMR (CDCl3, 400 MHz): 9.77 (t, J=1.9 Hz, 1H), 2.35-2.48 (m,

140

2H), 1.56-1.76 (m, 1H), 1.38-1.48 (m, 2H), 1.21-1.33 (m, 15H), 1.04-1.20 (m, 1H), 0.89 (t, J=6.6 Hz,

141

3H), 0.88 (d, J=6.4 Hz, 3H). EI-MS: 212 (0, M+), 57 (100), 56 (87), 85 (58), 43 (47), 55 (47), 41 (45), 82

142

(44), 69 (43), 95 (31), 81 (31).

143

Synthesis of 3-mercapto-3,7-dimethyl-6-octenyl acetate 378. The multi-step synthesis of 378 is

144

shown in Figure 4:

145

S-(3,7-dimethyl-1-oxooct-6-en-3-yl)-ethanethioate (2b). Under N2, a 1 L, four-necked flask was charged

146

with citral (1b; 50.0 g, 330 mmol) and piperidine (2.8 g, 30 mmol). Thioacetic acid (37.5 g, 490 mmol)

147

was added dropwise to the mixture and heated to 60 °C for 3 h. The mixture was then cooled to RT and

148

methyl tert-butyl ether (MTBE; 200 mL) was added followed by a wash with brine (100 mL), a 5%

149

solution of NaHCO3 (100 mL) and brine (100 mL) successively. The solvent was removed under

150

vacuum and the crude of 2b (10.0 g) was used as is for the next step. 7 ACS Paragon Plus Environment


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3-mercapto-3,7-dimethyl-6-octenyl acetate 378. Under N2, a 1 L, four-necked flask was charged with

152

NaBH4 (2.5 g, 66 mmol) and dissolved in ethanol (40 mL) and water (10 mL). The temperature of the

153

reaction was lowered to 0 °C and 2b (10.0 g, 44 mmol) was added dropwise to the reaction flask,

154

keeping the temperature below 10 °C. After addition, the reaction mixture was stirred at RT for 30 min.

155

The mixture was then poured into a 10% solution of NH4Cl (100 mL), transferred to a separatory funnel,

156

and extracted with MTBE (200 mL). The organic layer was washed with brine (2 x 100 mL). The

157

solvent was removed under vacuum, and 378 (8.2 g, 81%) was obtained by column chromatography

158

(silica; ethyl acetate/hexanes). 1H NMR (CDCl3, 400 MHz): 5.00-5.14 (m, 1H), 4.21-4.30 (m, 2H), 2.07-

159

2.15 (m, 2H), 2.04 (s, 3H), 1.92-1.96 (m, 2H), 1.68 (s, 3H), 1.62 (s, 3H) ,1.56-1.70 (m, 3H), 1.38 (s,

160

3H). EI-MS: 230 (3, M+), 121 (100), 69 (31), 93 (20), 109 (15), 136 (13), 81 (13), 101 (13), 122 (12), 67

161

(10), 43 (9).

162

Synthesis of 2-(5-isopropyl-2-methyltetrahydrothiophen-2-yl)-ethanol 304 and 307. The multi-step

163

synthesis of 304 and 307 is shown in Figure 4, where intermediate 2b was synthesized in a similar

164

fashion as above and was used as is for the next step:

165

3-Mercapto-3,7-dimethyl-6-octenol (3b). Under N2, a 1 L, four-necked flask was charged with LiAlH4

166

(3.3 g, 88 mmol) and dissolved in THF (200 mL) at RT. The temperature of the reaction mixture was

167

then lowered to 0 °C and 2b (20.0 g, 88 mmol) was added dropwise to the reaction flask, maintaining

168

the temperature below 10 °C. After filtration and concentration, the crude of 3b (15.0 g) was used as is

169

for the next step.

170

2-(5-Isopropyl-2-methyltetrahydrothiophen-2-yl)-ethanol 304 and 307. Crude 3b (15.0 g, 80 mmol) was

171

dissolved in THF (200 mL), refluxed for 1 h, and poured into a 10% solution of NH4Cl (200 mL). The

172

mixture was then transferred to a separatory funnel and extracted with MTBE (400 mL). The organic

173

layer was washed with brine (2 x 100 mL), and the solvent was removed under vacuum. 304 and 307

174

(8.3 g, 50%) were obtained by vacuum distillation (bp 130 °C / 1.0 mmHg). 1H NMR (CDCl3, 500

175

MHz): 3.72-3.86 (m, 2H), 3.24-3.32 (m, 1H), 2.59 (br, 1H), 1.56-2.18 (m, 7H), 1.41 (s, ~ 50% of 3H), 8 ACS Paragon Plus Environment

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1.38 (s, ~ 50% of 3H), 0.92-0.98 (m, 6H). EI-MS: 188 (20, M+), 101 (100), 69 (42), 93 (41), 41 (27), 81

177

(21), 143 (21), 55 (20), 99 (16), 145 (15), 67 (13).

178

Synthesis

179

Compounds 304 and 307 (0.6 g, 3 mmol) were added to DCM (5 mL) at RT. The reaction mixture was

180

cooled to 0 °C and triethylamine (0.5 g, 5 mmol) and acetyl chloride (0.3 g, 4 mmol) were added as

181

shown in Figure 4. After 1 h, 1 M HCl (5 mL) was added and the reaction was extracted with DCM (2 x

182

5 mL), washed with water (10 mL) and brine (10 mL) and dried over MgSO4. The solvent was removed

183

under vacuum, and 373 and 375 (0.5 g, 90%) were obtained by column chromatography (silica;

184

hexanes/ethyl acetate). 1H NMR (CDCl3, 500 MHz): 4.12-4.33 (m, 2H), 3.17-3.36 (m, 1H), 2.07-2.20

185

(m, 1H), 2.04 (s, 3H), 1.64-2.03 (m, 6H), 1.42 (s, ~ 50% of 3H), 1.40 (s, ~ 50% of 3H), 0.91-0.98 (m,

186

6H). EI-MS: 230 (22, M+), 93 (100), 43 (84), 127 (83), 69 (66), 41 (39), 99 (36), 81 (34), 143 (30), 55

187

(26), 67 (23).

188

Synthesis of 2-(2-methyltetrahydrothiophen-2-yl)-ethyl acetate 281. The multi-step synthesis of 281

189

is shown in Figure 5:

190

Methyl 2-(2-methyltetrahydrothiophen-2-yl)-acetate (2c). 2-(2-Methyltetrahydrothiophen-2-yl)-acetic

191

acid (1c; 5.1 g, 32 mmol), synthesized according to the procedure described by Bunce et. al.,25 was

192

dissolved in MeOH (100 mL). p-Toluenesulfonic acid monohydrate (0.6 g, 3 mmol) was added and the

193

mixture was refluxed for 3 h. The solvent was removed under vacuum and ethyl acetate (100 mL) was

194

added to the residue. The organic layer was washed with a 5% solution of NaHCO3 (100 mL), brine

195

(100 mL), and dried over MgSO4. After filtration and concentration, the crude of 2c (5.0 g) was obtained

196

and used as is in the next step.

197

2-(2-Methyltetrahydrothiophen-2-yl)-ethanol (3c). LiAlH4 (1.1 g, 29 mmol) was dissolved in THF (100

198

mL). Under N2, a solution of 2c (5.0 g, 29 mmol) in THF (40 mL) was added dropwise to the reaction

199

flask. After addition, the mixture was allowed to stir for an additional 2 h at RT. The mixture was then

200

added to a 10% solution of NH4Cl (200 mL) and extracted with ether (200 mL). The organic layer was

of

2-(5-isopropyl-2-methyltetrahydrothiophen-2-yl)-ethyl


acetate

373

and

375.


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201

washed with brine (100 mL) and dried over MgSO4. After filtration and concentration, 3c (3.8 g, 81%)

202

was obtained by column chromatography (silica; ethyl acetate/hexanes).

203

2-(2-methyltetrahydrothiophen-2-yl)-ethyl acetate 281. 3c (3.8 g, 26 mmol) and triethylamine (3.2 g, 32

204

mmol) were dissolved in DCM (100 mL) and cooled to 0 °C. With stirring, a solution of acetyl chloride

205

(2.5 g, 32 mmol) in DCM (20 mL) was added dropwise to the reaction flask. After addition, the reaction

206

was stirred for an additional 2 h at RT. The mixture was transferred to a separatory funnel and washed

207

with a 10% solution of NH4Cl (100 mL), a 5% solution of NaHCO3 (100 mL), brine (100 mL), and dried

208

over MgSO4. After filtration and concentration, 281 (3.4 g, 70%) was obtained by column

209

chromatography (silica; ethyl acetate/hexanes). 1H NMR (CDCl3, 400 MHz): 4.17-4.29 (m, 2H), 2.88-

210

3.01 (m, 2H), 1.98-2.12 (m, 4H), 2.05 (s, 3H), 1.73-1.89 (m, 2H), 1.42 (s, 3H). EI-MS: 188 (17, M+),

211

101 (100), 43 (27), 113 (25), 67 (12), 81 (9), 59 (9), 41 (7), 102 (7), 79 (6), 100 (6).

212

Synthesis of 2-[5-(1-hydroxy-1-methylethyl)-2-methyltetrahydrothiophen-2-yl]-ethyl acetate 401

213

and 406. This five-step synthesis is shown in Figure 6:

214

6,7-Epoxyneral 246. Citral (1d, 100.0 g, 660 mmol) was dissolved in DCM (400 mL) and cooled to 0

215

°C. With stirring, a solution of m-chloroperbenzoic acid (136.0 g, 790 mmol) in DCM (200 mL) was

216

added dropwise to the reaction flask. After addition, the reaction mixture was stirred for an additional 2

217

h at 0 °C. The organic layer was washed with a 5% solution of NaHCO3 (2 x 100 mL) and brine (2 x

218

100 mL) and dried over MgSO4. After filtration and concentration, the crude of 246 (111.0 g) was

219

obtained and used as is for the next step.

220

S-[1-(3,3-dimethyloxiran-2-yl)-3-methyl-5-oxopentan-3-yl]-ethanethioate (2d). 246 (111.0 g, 660

221

mmol), thioacetic acid (60.0 g, 790 mmol), and piperidine (5.6 g, 70 mmol) were mixed and heated at 60

222

°C for 6 h under N2. The reaction mixture was dissolved in ether (500 mL) and washed with a 5%

223

solution of NaHCO3 (100 mL) and brine (100 mL), and dried over MgSO4. After filtration and

224

concentration, the crude of 2d (160.0 g) was obtained and used as is in the next step.

225

2-[5-(2-hydroxypropan-2-yl)-2-methyltetrahydrothiophen-2-yl]-acetaldehyde (3d). 2d (17.5 g, 70 10 ACS Paragon Plus Environment

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226

mmol) was dissolved in THF (200 mL) and cooled to 0 °C. With stirring, a solution of NaOH (2.9 g, 70

227

mmol) in water (10 mL) was added dropwise to the reaction flask and allowed to stir an additional 10

228

min at 0 °C. The mixture was added to ethyl acetate (200 mL), transferred to a separatory funnel, and

229

washed with a 10% solution of NH4Cl (100 mL) and brine (100 mL), and dried over MgSO4. After

230

filtration and concentration, 3d (10.2 g, 70%) was obtained by column chromatography (silica; ethyl

231

acetate/hexanes).

232

2-[5-(2-Hydroxypropan-2-yl)-5-methyltetrahydrothiophen-2-yl]-ethanol (4d). NaBH4 (3.0 g, 80 mmol)

233

was dissolved in ethanol (60 mL) and water (40 mL). The mixture was cooled down to 0 °C. With

234

stirring, the solution of 3d (16.0 g, 90 mmol) in ethanol (20 mL) was added dropwise to the reaction

235

flask. After addition, the reaction mixture was stirred for an additional 30 min at 0 °C. The mixture was

236

then added to ethyl acetate (200 mL), transferred to a separatory funnel, and washed with a 10% solution

237

of NH4Cl (100 mL) and brine (100 mL), and dried over MgSO4. After filtration and concentration, 4d

238

(10.7 g, 66%) was obtained by column chromatography (silica; ethyl acetate/hexanes).

239

2-[5-(1-hydroxy-1-methylethyl)-2-methyltetrahydrothiophen-2-yl]-ethyl acetate 401 and 406. 4d (3.0 g,

240

15 mmol) and triethylamine (1.5 g, 15 mmol) were dissolved in DCM (100 mL), and the mixture was

241

cooled to 0 °C. With stirring, a solution of acetyl chloride (1.2 g, 15 mmol) in DCM (10 mL) was added

242

dropwise to the reaction flask and stirred for an additional 10 min at 0 °C. The mixture was then

243

transferred to a separatory funnel, washed with a 10% solution of NH4Cl (100 mL), a 5% solution of

244

NaHCO3 (100 mL), and brine (100 mL), and dried over MgSO4. After filtration and concentration, 401

245

and 406 (3.4 g, 94%) was obtained by column chromatography (silica; ethyl acetate/hexanes). 1H NMR

246

(CDCL3, 500 MHz): 4.15-4.32 (m, 2H), 3.63-3.72 (m, 1H), 2.49 (br s, 1H), 1.98-2.12 (m, 7H), 1.79-1.96

247

(m, 2H), 1.41-1.44 (m, 3H), 1.18-1.24 (m, 6H). EI-MS: 246 (1, M+), 101 (100), 43 (82), 59 (35), 100

248

(28), 99 (25), 69 (22), 93 (16), 41 (16), 113 (14), 128 (13).

249

Synthesis of 2-(5-isopropylidene-2-methyltetrahydrothiophen-2-yl)-ethyl acetate 395. Compounds

250

401 and 406 (7.9 g, 32 mmol) were mixed with p-toluenesulfonic acid monohydrate (1.2 g, 6 mmol) in 11 ACS Paragon Plus Environment


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251

toluene (100 mL) and refluxed for 30 min (Figure 6). The mixture was then transferred to a separatory

252

funnel, washed with a 5% solution of NaHCO3 (100 mL) and brine (100 mL), and dried over MgSO4.

253

After filtration and concentration, 395 (4.1 g, 56%) was obtained by column chromatography (silica;

254

ethyl acetate/hexanes). 1H NMR (CDCL3, 400 MHz): 4.17-4.30 (m, 2H), 2.61-2.71 (m, 2H), 2.06 (s,

255

3H), 1.85-2.10 (m, 4H), 1.64-1.74 (m, 6H), 1.45 (s, 3H). EI-MS: 228 (74, M+), 43 (100), 141 (68), 85

256

(47), 41 (45), 67 (44), 59 (32), 79 (31), 99 (30), 125 (27), 107 (26).

257

The mass spectra for each of the synthesized VSCs and branched aliphatic aldehydes are detailed

258

in the Supporting Information.

259

CAS Registry Numbers for identified compounds. 2-(5-isopropyl-2-methyltetrahydrothiophen-2-yl)-

260

ethanol 304 and 307 (1612888-42-2).

261

Extraction of Lemon Peel. The peels from 100 lemons were zested over a microplane grater and

262

collected in 1 L jars. The peel (2.0 kg) was steeped in DCM (600 mL) for 24 h in a refrigerator (4 °C).

263

The solvent extract was passed through filter paper, dried over MgSO4, and concentrated using a

264

Zymark Turbovap (Biotage, Uppsala, Sweden) to 100 mL. It was then further reduced under a stream of

265

N2 to obtain a 25 mL concentrated extract.

266

Fractionation of Lemon Peel Extract. The peel extract (5.0 g) was diluted in hexane (5.0 g). The

267

diluted extract was fractionated using a Biotage Isolera Prime, which was equipped with a 40 x 150 mm

268

FLASH 40+M silica cartridge (Biotage). The first solvent gradient started with hexane:DCM at a ratio

269

of 100:0. When the ratio reached 0:100, a second gradient of DCM:MeOH began at 100:0 and increased

270

to 0:100. The flow rate was continuous at 25 mL/min and the collection mode was set to “collect all”. A

271

total of 105 fractions were collected. Based on gas chromatography (GC) and GC-MS analyses from a

272

selection of the individual fractions, the fractions were combined chronologically in groups of ten. The

273

resulting 11 fractions were each concentrated using a Zymark Turbovap to 15 mL and further reduced

274

under a stream of N2 to 1 mL.

275

Supercritical fluid extraction. A Spe-edTM Supercritical Fluid Extraction Prime (Applied Separations, 12 ACS Paragon Plus Environment

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276

Allentown, PA) instrument was used to extract the lemon peel oil. The chiller was turned on 12 h prior

277

to extraction and the system lines were pre-pressurized by liquid CO2 1 hr before the extraction. Freshly

278

zested lemon peels (20.0 g) were packed into a 50 mL high pressure cell. The dead volume in the cell

279

was filled with glass beads then capped with PTFE frits before securing the ends with threaded, steel end

280

caps. After connecting the sample cell to the unit, the cell temperature was set to 40 °C and pre-

281

pressurized with CO2. The compressed air was set to 120 psi and the sample was pressurized with

282

compressed air after the pre-pressurization process. The flow valve was opened to the metering valve

283

which then slowly opened to the recovery vial (placed in a dry ice/acetonitrile trap at -50 °C). The

284

metering valve was opened enough to maintain a sample cell flow of approximately 1-2 L/min. Once

285

stable flows were established, the peel was extracted for 45 min.

286

Gas Chromatography. The peel extract and its fractions were analyzed on apolar and polar phases

287

using an Agilent 7890A GC (Santa Clara, CA). The apolar capillary column had dimensions of 50 m x

288

320 µm x 0.52 mm (Restek RTX-1 F &F) and the polar capillary column was 50 m x 320 µm x 0.5 mm

289

(Varian CP-Wax 58 FFAP CB column). Samples were introduced to the GC using an autosampler at a

290

volume of 1 µL with a split ratio of 5:1. For the apolar column, the hydrogen carrier gas flow rate was

291

held constant at 2 mL/min, and the temperature program started at an initial temperature of 40 °C, then

292

increased 2 °C/min up to 310 °C with a 10 min hold at 310 °C. The temperature program for the polar

293

column had the same initial temperature and 2 °C/min ramp, but the final temperature only reached 250

294

°C with a 10 min hold at 250 °C. The GC was calibrated using a homologous series of C1-C18 ethyl

295

esters in order to generate index values for the observed peaks. The index values were calculated based

296

on previous work by van den Dool and Kratz,26 which takes into account the GC oven temperature

297

program and non-alkane based calibrants, specifically ethyl esters. While this is an industry standard, it

298

is commonly expected to report Kovats retention indices (RI), which are based off a calibration with of a

299

homologous series of n-alkanes. A linear relationship between the ethyl ester values and Kovats RI is

300

found when plotting the measured values for the homologous series of the ethyl esters versus the Kovats 13 ACS Paragon Plus Environment


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301

values. Based on this relationship, linear equations were derived for both polar and apolar phases, which

302

allowed for the calculated Kovats values reported herein.

303

Gas Chromatography-Olfactometry. The peel extract and its fractions were analyzed on the same

304

apolar column described above using an Agilent 6890A GC. All samples were introduced to the GC

305

inlet using an autosampler at 1 µL with a split ratio of 5:1. For all samples, the following parameters

306

remained constant. The hydrogen carrier gas flow rate was 2 mL/min, and the temperature program

307

started at an initial oven temperature of 40 °C, then increased 6 °C/min up to 80 °C, 4 °C/min up to 150

308

°C, 2 °C/min up to 200 °C, and finally 10 °C/min up to 310 °C with a 5 min hold. In addition, two

309

Olfactory Detection Ports (ODP, GERSTEL, Inc., Linthicum, MD) were equipped on the GC. The

310

effluent was split 4:4:1 (ODP:ODP:FID), and the ODP transfer lines were heated to 225 °C. Olfactory

311

comments were recorded using Dragon Naturally Speaking, Speech Recognition Software 12.0 (Nuance

312

Communications, Inc., Burlington, MA) in conjunction with the ODP software. The GC was calibrated

313

using a homologous series of C1-C18 ethyl esters in order to generate index values for the observed

314

peaks and then converted to Kovats values using the aforementioned equation.

315

Aroma Extract Dilution Analysis. To obtain dilutions of the original extract, the lemon peel extract

316

was diluted stepwise, 3-fold with DCM (1:3 by volume).27 Two experienced assessors simultaneously

317

conducted GC-O on these dilutions until no odorant could be detected. Each odorant was therefore

318

assigned a FD factor which represented the last dilution in which the odorant was detected.

319

Gas Chromatography-Chemiluminescence. The peel extract and its fractions were analyzed on an

320

Agilent 6890 GC equipped with an Antek 7090 Sulfur/Nitrogen chemiluminescence detector (PAC,

321

Houston, TX; CLSD) in the sulfur detection mode. All samples were introduced to the GC inlet using an

322

autosampler, at a volume of 1 µL in the splitless mode. The apolar, 50 m x 320 µm x 0.52 µm column

323

(Restek RTX-1 F &F) was split two ways to two detectors. The hydrogen carrier gas flow rate was 2

324

mL/min, and the temperature program had an initial oven temperature of 40 °C, then increased 2 °C/min

325

up to 270 °C with a 10 min hold at 270 °C. The FID and CLSD were calibrated using a homologous 14 ACS Paragon Plus Environment

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326

series of C1-C18 ethyl esters in order to generate index values for the observed peaks and then

327

converted to Kovats values.

328

Gas Chromatography-Mass Spectrometry. The chromatographic conditions were the same as

329

described for the GC analyses. All data was acquired using a Waters GCT-Premier orthogonal

330

acceleration time of flight mass spectrometer (Milford, MA) in electron ionization (EI) mode. The ion

331

source was operated at 150 oC with an electron energy of 70 eV and a trap current of 50 µA. The

332

temperature of the transfer line was 250 oC. Spectra were acquired between 27 and 400 Da in a time of

333

0.05 sec and a delay of 0.01 sec (approximately 20 spectra/sec). Exact mass spectra were obtained using

334

a single–point lock mass (m/z 218.9856 from perflurotri-n-butylamine) infused into the ion source

335

continuously during the run. Mass spectral library identification was achieved using in-house and

336

commercial libraries, including Wiley 8 and NIST. Standard relative retention data was used for

337

confirmation, which was obtained by calibrating the instrument with a homologous series of ethyl esters.

338

An Agilent 6890 GC, coupled to a Waters Quattro Micro triple quadrupole mass spectrometer

339

was used to develop a method operating in the multiple reaction monitoring (MRM) mode. The

340

instrument was equipped with a Varian CP-Wax 58 FFAP CB column (50 m x 320 µm x 0.20 µm) and a

341

Varian VF-1ms column (50 m x 320 µm x 0.40 µm). The oven ramp was set to an initial temperature of

342

75 °C with an increase of 3 °C/min up to 240 °C. The collision energy was set to 10 eV, and the

343

following transitions were monitored as the major mass intensities from the spectrum: m/z 110.0 to 95.0

344

and 95.0 to 67.0 were monitored for 6-methylnonanal 136; m/z 188.0 to 101.0 and 145.0 to 101.0 were

345

monitored for 2-(5-isopropyl-2-methyltetrahydrothiophen-2-yl)-ethanol 304 and 307.

346

Multidimensional Gas Chromatography-Mass Spectrometry. A Shimadzu GCMS-QP2010 system

347

(Shimadzu Corporation, Kyoto, Japan), that consisted of two GC2010 GCs, was equipped with a Deans

348

switch transfer device, an MS-QP2010 quadrupole mass spectrometer, and an AOC-20i autosampler.

349

The first GC, where the initial split/splitless injections take place, contained the apolar, 50 m x 320 µm x

350

0.50 µm column (Restek RTX-1 F &F), a transfer line to a FID, and a transfer line to the Deans switch. 15 ACS Paragon Plus Environment


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351

An advanced pressure control (APC) system is utilized to supply carrier gas at constant pressure, in this

352

case Helium, to the Deans switch. The Deans switch serves as a means to transfer effluent from the first

353

GC to the second GC, which is equipped with an orthogonal column phase in order to achieve advanced

354

second dimensional separation. The instrument parameters were as follows: the 300 °C inlet was held at

355

a constant pressure of 220 kPa, and samples were introduced to the inlet using an auto sampler at 1 µL

356

with a 10:1 split ratio. The first dimension oven temperature program started at an initial temperature of

357

40 °C then increased 2 °C/min up to 310 °C. The transfer line between the two GCs was 150 °C and the

358

APC, operating in the constant pressure mode, was held at 180 kPa. The second GC was equipped with

359

a Sol-Gel Wax polar, 60 m x 320 µm x 0.25 µm column (SGE Analytical Services). The oven was set to

360

an initial temperature of 30 °C and increased at a rate of 4 °C/min up to 260 °C. The end of the second

361

column was inserted into the mass spectrometer source which was operated under EI conditions. The

362

source temperature was 200 °C, with the interface temperature at 230 °C. The mass scan range was set

363

to scan from m/z 33 to m/z 250 with a scan speed of 800 u/sec, and the EI energy was set to 70 eV.

364

Nuclear Magnetic Resonance. NMR spectra were recorded at 26.8 °C in deuterated chloroform

365

(containing 0.05% v/v tetramethylsilane) on a Bruker Avance III 400 MHZ or a Bruker Avance 500

366

MHz spectrometer (Billerica, MA), with 5mm BBO probes. 1H chemical shifts are expressed as parts

367

per million (ppm) with residual chloroform (δ 7.26) or tetramethylsilane (δ 0.00) as references and are

368

reported as chemical shift (δΗ), relative integral, multiplicity (s = singlet, br = broad, d = doublet, t =

369

triplet, higher multiplicities as e.g. dd = doublet of doublets, m = multiplet); and coupling constants (J)

370

reported in Hz.

371

RESULTS AND DISCUSSION

372

Aroma Active Compounds in Lemon Peel Extract. Peel oil can be obtained commercially by cold-

373

pressed or distillation techniques, as well as in the laboratory by hand pressing or grating. For the grated

374

peel, different organic solvents are available to preferentially extract the volatile constituents from the

375

grated peel. The Lisbon lemon peels in this research were zested and extracted with DCM, an organic 16 ACS Paragon Plus Environment

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376

solvent with a high extraction capacity and affinity for a wide range of volatile constituents. Using paper

377

blotters, the aroma of the lemon peel extract was described as bright, aldehydic, and citrus-like with

378

subtle sulfurous notes. Identifying and characterizing the odor active compounds in lemon oil has been

379

completed in the past.28,29 To the best of our knowledge, this was the first attempt to perform AEDA and

380

assign FD factors to the aroma active compounds from laboratory extracted lemon peel oil.

381

In the lemon extract, twenty-seven odor-active regions were detected by GC-O with a FD factor

382

of at least three (Table 1). Four of the odor-active areas with the highest dilution factors included

383

limonene 80, linalool 106, neral 202, and geranial 216. The fifth area with an equally high dilution

384

factor had a “sulfurous, skunk-like” aroma. At this particular area, no peak appeared by FID or CLSD

385

runs. The concentration at which this presumed VSC was found in the lemon extract must therefore be

386

below the limit of detection for the sulfur chemiluminescence detector, which is 0.1 parts per million of

387

sulfur. With knowledge of the RI from the odor from two orthogonal columns by GC-O, prenyl

388

mercaptan was proposed as the source of the odor. This compound has been identified as a highly

389

odorous compound in other natural products, including beer,30 coffee,31 wine,32 durian,33 and most

390

recently virgin olive oil,34 but never in citrus fruits. This compound was subsequently spiked into the

391

lemon extract at a concentration high enough to produce a signal on the GC. The RI and odor descriptors

392

from the two GC-O runs on orthogonal columns of this spiked extract matched that of the original

393

extract. Although there is no GC-MS evidence to support this claim, prenyl mercaptan, which can be

394

formed from cysteine via free radical mechanisms,35 appears to be found in the lemon peel extract in the

395

parts per billion or parts per trillion range and significantly contributes to the aroma of the extract.

396

Relative flavor activity (RFA), which is a measure of the log of the FD factor divided by the

397

weight percent of the compound, can be a valuable calculation to understand which flavor components

398

contribute to the aroma of a sample.36,37 These values are not shown in Table 1 mainly because of the

399

difficulty calculating this value when compounds do not have a measured weight percentage. For

400

example, prenyl mercaptan and 3-mercapto-3,7-dimethyl-6-octenyl acetate 378 did not have measured 17 ACS Paragon Plus Environment


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401

weight percentages by GC, but would have two of the highest RFA values due to its FD factor and

402

extremely low concentration, which is quite remarkable and unique for lemons. A similar conclusion

403

could be made for 8-methylnonanal 143 (green, peely, citrus) and 8-methyldecanal 220 (green, woody,

404

cucumber) which were found at trace levels in the fractions and possess low odor thresholds.38

405

Composition of Volatile Compounds in the Lemon Peel Extract. Due to the complexity of natural

406

product extracts, fractionation has become a primary technique to circumvent peak coelution and

407

identify lower concentration compounds by selectively separating compound classes using gradient

408

elution. Specifically for lemons, fractionation on both the juice39 and the peel10 has led to novel findings.

409

In order to separate and enhance the trace volatiles from the complex volatile lemon peel matrix, and to

410

assist with properly linking GC-O data, the peel extract in this study was fractionated into its different

411

chemical classes. Further, mono-dimensional analysis has its limitations in citrus extracts as terpenes

412

and sesquiterpenes interfere with lower concentration, potentially odor active components. Therefore,

413

MDGC-MS was utilized for the analysis of the peel extract and its fractions. Unresolved peaks on the

414

primary column were heart-cut onto a secondary (orthogonal) column using the Deans switch.40 A

415

summary of the volatile compounds in the peel extract and fractions is listed in Table 2 in order of

416

elution on an apolar column. Known compounds were identified using RI from apolar and polar

417

columns, coupled with mass spectral data. Several unknown compounds were postulated, synthesized,

418

and confirmed in the lemon extract by comparing the RI of two orthogonal column phases and mass

419

spectral data. The major components of the peel extract included limonene (63.7%), β-pinene (14.5%),

420

and γ-terpinene (9.9%). The composition of the major chemical classes in the peel extract represented

421

94.8% hydrocarbons, 2.0% aldehydes, 1.6% alcohols, and 1.3% esters.

422

Aldehydes are known to play a key role in the aroma of citrus fruits, especially in lemons. The

423

medium polarity fractions from the lemon extract comprised a series of 2-methyl, 3-methyl, 4-methyl, 6-

424

methyl, and 8-methyl branched aliphatic aldehydes, many of which were synthesized and confirmed for

425

the first time in lemons, citrus fruits, or any other botanical species. Only the branched aldehydes 418 ACS Paragon Plus Environment

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426

methylnonanal 140 and 4-methylundecanal 274 have been previously identified in lemons.10 It should be

427

noted that 6-methylnonanal 136 required MRM for confirmation in one of the lemon fractions, where

428

the transitions of m/z 110.0 to 95.0 and 95.0 to 67.0 were monitored for the fraction, the standard

429

solution of 136, and the fraction spiked with the standard. The synthesis scheme for 136 is shown in

430

Figure 1. A similar mechanism was used for the synthesis of 6-methyldecanal 215 and the series of 4-

431

methyl branched aliphatic aldehydes.

432

Identification of Volatile Sulfur Compounds. As each of the fractions was analyzed, it was

433

increasingly evident that there were several unknown VSCs present. Formed through biochemical and

434

enzymatic pathways,41 as well as thermal processing,42 VSCs are rarely reported in citrus research

435

literature. The Lisbon lemons were purchased in early spring, the main harvest season for this lemon

436

variety in the state of California. An elemental specific analysis for VSCs in the peel extract was carried

437

out using a GC coupled to a CLSD in the sulfur mode only. Supercritical fluid extraction (SFE) of the

438

peel was used to compare and validate the chromatographic results from the CLSD trace of the lemon

439

peel DCM extract. Figure 2 shows the chromatograms obtained for both extraction techniques. Since the

440

two chromatograms mirrored one another, the peel extract that was steeped in DCM was chosen for

441

further research due to the large amount of extract, an important factor when carrying out multiple

442

analytical techniques. Three well-known sulfur compounds were identified in the fractions: sulfur

443

dioxide 1, 2-propionylthiophene 152, and methional 31. First identified in black tea,43 152 is new to

444

lemons and has been described in reaction mechanisms involving ascorbic acid and cysteine.44 The

445

medium polarity fractions led to the identification of 31, which is also new to lemons.

446

There were several novel VSCs identified in the peel extract (Figure 3), including one VSC that

447

was present at a high enough concentration in the extract to produce a mass spectral signal. Based on the

448

fragmentation pattern and accurate mass measurements of that peak, the structure was postulated as 3-

449

mercapto-3,7-dimethyl-6-octenyl acetate 378. This structure was synthesized (Figure 4) and confirmed

450

by NMR. During the sodium borohydride reduction to produce 378, it should be noted that a clean 19 ACS Paragon Plus Environment


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451

acetyl moiety migration occurred, as careful analysis of the crude did not yield the intermediate (4b).

452

The RI on two orthogonal columns and the mass spectrum for the synthesized structure supported the

453

presence of this compound in the peel extract. Even though the synthesized structure is highly unstable,

454

378 produced the strongest signal on the CLSD, as shown in Figure 2, and is considered stabilized by

455

the antioxidants and tocopherols in the matrix of the peel extract. This was confirmed experimentally as

456

the addition of tocopherols at the end of the synthesis of 378 produces a stable material in a shell-life

457

study (data not shown). The biochemical or enzymatic mechanism by which this VSC forms is still

458

under investigation. There is evidence that the amino acids cysteine and methionine, the precursors from

459

which VSCs could form, are present in lemons.45-48 In addition, hydrogen sulfide has been quantitated in

460

the headspace of fresh lemon juice.23 It was postulated that 378 could form from the reaction of

461

hydrogen sulfide and neryl acetate, which tends to comprise a larger percentage of the volatile

462

constituents in lemons compared to other citrus fruits.

463

In order to identify the other peaks on the CLSD chromatogram, the fractions were studied in-

464

depth by GC-MS and MDGC-MS. Two unknown peaks in two of the polar fractions appeared to be

465

isomers of each other with the same molecular weight and elemental formula as 378. With NMR and

466

mass spectral data from the synthesis and instability of 378 (Figure 4), there was a clear understanding

467

that these two unknown isomers were 2-(5-isopropyl-2-methyltetrahydrothiophen-2-yl)-ethyl acetate

468

373 and 375. The thiol group from 378 readily attacks the double bond and undergoes internal

469

cyclization to form 373 and 375. This internal cyclization occurs rapidly, as 378 is unstable even at

470

cooler temperatures.

471

Also in the polar fractions were what appeared to be two unknown isomeric VSCs that produced

472

a clear signal on a polar column. After further investigation from data on the mass intensities and the

473

elemental composition, the structure was proposed as 2-(5-isopropyl-2-methyltetrahydrothiophen-2-yl)-

474

ethanol 304 and 307 and synthesized (Figure 4). Using a Waters Quattro Micro triple quadrupole MS, a

475

method operating in the MRM mode was developed to identify this compound in the lemon extract 20 ACS Paragon Plus Environment

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476

using the synthesized structure. The collision energy was set to 10 eV, and the transitions, m/z 188.0 to

477

101.0 and 145.0 to 101.0, were monitored as the major mass intensities from the spectrum of 304 and

478

307 for the fraction, a standard solution of 304 and 307, and the fraction spiked with the standard.

479

Similar to the formation of 373 and 375, it was proposed that 304 and 307 could form in the lemon peel

480

from the internal cyclization from 3-mercapto-3,7-dimethyl-6-octenol (the reaction of hydrogen sulfide

481

and nerol; not observed) or from the hydrolysis of the corresponding esters 373 and 375 in acidic

482

conditions.

483

Interestingly, there was another sulfur peak with the same molecular weight and similar mass

484

spectral fragmentation as 304 and 307 that eluted earlier on both columns (see mass spectral data in

485

Supporting Information). From the analysis of the elemental composition, this sulfur peak had a

486

different molecular formula and double bond equivalency. The postulated structure, 2-(2-

487

methyltetrahydrothiophen-2-yl)-ethyl acetate 281, was synthesized as shown in Figure 5 and confirmed

488

as matching RI and mass spectral data for the peak in the lemon extract. The mechanism of formation

489

for 281 is very difficult to understand, as the earlier mentioned VSCs can partly be explained through

490

the internal cyclization of the citral-like backbone, but 281 appears to have derived through another

491

pathway.

492

The final VSCs identified in the polar fractions included 2-[5-(1-hydroxy-1-methylethyl)-2-

493

methyltetrahydrothiophen-2-yl]-ethyl

acetate

494

methyltetrahydrothiophen-2-yl)-ethyl acetate 395. Similar to 304 and 307, the synthesis of these

495

compounds involved an internal cyclization step. But, unlike 304 and 307 which used citral as the

496

starting material, 6,7-epoxyneral 246 was chosen as the starting material to generate the final tertiary

497

alcohol for 401 and 406 (Figure 6). In the lemon peel, however, the mechanism of formation is unknown

498

for these compounds, although it appears as if 395 could form from the dehydration of 401 and 406.

401

and

406

and

2-(5-isopropylidene-2-

499

As shown in Figures 4, 5, and 6, the lemon VSCs are a mixture of diastereomers. Unfortunately,

500

the authors cannot comment on the identity or quantity for each of the chiral compounds as it was 21 ACS Paragon Plus Environment


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501

outside the scope of the work.

502

Organoleptic Evaluation of Synthesized VSCs. The novel VSCs were synthesized to not only confirm

503

their presence in lemons but to gain a better understanding of the orthonasal and retronasal properties of

504

each compound. The orthonasal evaluations were completed on paper blotters and the retronasal

505

evaluations were completed in water (Table 3).

506

ACKNOWLEDGEMENT

507

The authors are grateful for the following members of the IFF team who assisted in all aspects of this

508

research: Susan Joseph, Stephen Toth, Sharon Brown, Dennis Swijter, Danielle Dinallo, Ubaideen

509

Hassan, Jerry Kowalczyk, Mauricio Poulsen, Kurt Nordman, Kelly Carroll, Dennis Kujawski, Jung-A

510

Kim, and Cynthia Vuich. We are also thankful for Dr. Russell Rouseff’s recommendation for analyzing

511

lemons as a potential source for VSCs.

512

ABBREVIATIONS USED

513

AEDA, aroma extract dilution analysis; CLSD, chemiluminescence detector, DCM, dichloromethane;

514

FD, flavor dilution; GC, gas chromatography; GC-MS, gas chromatography-mass spectrometry; GC-O,

515

gas

516

spectrometry; MRM, multiple reaction monitoring; ODP, olfactory detection port; RI, retention index;

517

RFA, relative flavor activity; SFE, supercritical fluid extraction; VSC, volatile sulfur compound.

chromatography-olfactometry;

MDGC-MS,

multidimensional

518 519 520 521 522 523 524 525 22 ACS Paragon Plus Environment

gas

chromatography-mass

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526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574


REFERENCES 1. Poore, H. D. Analyses and composition of California lemon and orange oils. Tech. Bull. 1932, 241, 1-30. 2. Bernhard, R. Analysis and composition of oil of lemon by gas-liquid chromatography. J. Chromatogr. 1960, 3, 471-476. 3. Ikeda, R. M; Rolle, L. A.; Vannier, S. H.; Stanley, W. L. Isolation and identification of aldehydes in cold-pressed lemon oil. J. Agric. Food Chem. 1962, 10 (2), 98-102. 4. Moshonas, M. G.; Shaw, P. E.; Veldhuis, M. K. Analysis of volatile constituents from Meyer lemon oil. J. Agric. Food Chem. 1972, 20 (4), 751-752. 5. Chamblee, T. S.; Clark Jr., B. C.; Brewster, G. B.; Radford, T.; Iacobucci, G. A. Quantitative analysis of the volatile constituents of lemon peel oil. Effects of silica gel chromatography on the composition of its hydrocarbon and oxygenate fractions. J. Agric. Food Chem. 1991, 39, 162-169. 6. Dellacassa, E.; Lorenzo, D.; Moyna, P.; Verzera, A.; Mondello, L.; Dugo, P. Uruguayan essential oils. Part VI. Composition of lemon oil. Flav. Frag. J. 1997, 12, 247-255. 7. Verzera, A.; Russo, C.; La Rosa, G.; Bonaccorsi, I.; Cotroneo, A. Influence of cultivar on lemon oil composition. J. Essent. Oil Res. 2001, 13, 343-347. 8. Lota, M.; de Serra, D.; Tomi, F.; Jacquemond, C.; Casanova, J. Volatile components of peel and leaf oils of lemon and lime species. J. Agric. Food Chem. 2002, 50, 796-805. 9. Dugo, P.; Ragonese, C.; Russo, M.; Sciarrone, D.; Santi, L.; Cotroneo, A.; Mondello, L. Sicilian lemon oil: composition of volatile and oxygen heterocyclic fractions and enantiomeric distribution of volatile components. J. Sep. Sci. 2010, 33, 3374-3385. 10. Naef, R.; Jaquier, A. New aldehydes and related alcohols in fresh lemon peel extract (Citrus limon L.). Flavour Frag. J. 2006, 21, 768-771. 11. Fischer, A.; Grab, W.; Schieberle, P. Characterisation of the most odour-active compounds in a peel oil extract from Pontianak oranges (Citrus nobilis var. Lour. Microcarpa Hassk.). Eur. Food Res. Technol. 2008, 227, 735-744. 12. Delort, E.; Jacquier, A. Novel terpenyl esters from Australian finger lime (Citrus australasica) peel extract. Flavour Frag. J. 2009, 24, 123-132. 13. Buettner, A.; Schieberle, P. Characterization of the most odor-active volatiles in fresh, handsqueezed juice of grapefruit (Citrus paradise Macfayden). J. Agric. Food Chem. 1999, 47, 5189-5193. 14. Buettner, A.; Schieberle, P. Stable isotope dilution assays for the quantification of odor-active thiols in hand-squeezed grapefruit juices (Citrus paradise Macfayden). In Frontiers of Flavor Science, 9; Schieberle, P., Engel, K.-H., Eds.; Deutsche Forschungsanstalt für Lebensmittelchemie: Freising, Germany, 2000; pp 132-134. 15. Lin J.; Rouseff, R. L. Characterization of aroma-impact compounds in cold-pressed grapefruit oil using time-intensity gc-olfactometry and gc-ms. Flavour Frag. J.. 2001, 16, 457-463. 16. Shaw, P. E.; Ammons, J. M.; Braman, R. S. Volatile sulfur compounds in fresh orange and grapefruit juices: identification, quantitation, and possible importance to juice flavor. J. Agric. Food Chem. 1980, 28, 778-781. 17. Demole, E.; Enggist, P.; Ohloff, G. 1-p-Menthene-8-thiol: a powerful flavor impact constituent of grapefruit juice (Citrus paradise Macfayden). Helv. Chim. Acta. 1982, 65, 1785-1794. 18. Hinterholzer, A.; Schieberle, P. Identification of the most odour-active volatiles in fresh, handextracted juice of Valencia late oranges by odour dilution techniques. Flavour Frag. J. 1998, 13, 49-55. 19. Rouseff, R. L.; Perez-Cacho, P.; Jabalpurwala, F. Historical review of citrus flavor research during the past 100 years. J. Agric. Food Chem. 2009, 57, 8115-8124. 20. Naef, R.; Velluz, A.; Meyer, A. P. Volatile constituents of blood and blond orange juices: a comparison. J. Essent. Oil Res. 1996, 8, 587-595. 21. Starkenmann, C.; Niclass, Y.; Escher, S. Volatile organic sulfur-containing constituents in Poncirus trifoliata (L.) Raf. (Rutaceae). J. Agric. Food Chem. 2007, 55, 4511-4517. 23 ACS Paragon Plus Environment


575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623

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22. Naef, R.; Velluz, A. Volatile constituents in extracts of mandarin and tangerine peel. J. Essent. Oil Res. 2001. 13 (3), 154-157. 23. Shaw, P. E.; Wilson, C. W. Volatile sulfides in headspace gases of fresh and processed citrus juices. J. Agric. Food Chem. 1982, 30, 685-688. 24. Allegrone, G.; Belliardo, F.; Cabella, P. Comparison of volatile concentrations in hand-squeezed juices of four different lemon varieties. J. Agric. Food Chem. 2006, 54, 1844-1848. 25. Bunce, R. A.; Peeples, C. J.; Jones, P. B. Tandem SN2-Michael reactions for the preparation of simple five- and six-membered-ring nitrogen and sulfur heterocycles. J. Org. Chem. 1992, 57, 17271733. 26. Van den Dool, H.; Kratz, P. D. A generalization of the retention index system including linear temperature programmed gas-liquid partition chromatography. J. Chromatogr. 1963, 11, 463-471. 27. Schieberle, P. New developments in methods for analysis of volatile flavor compounds and their precursors. In Characterization of Food: Emerging Methods; Gaonkar, A. G., Ed.; Elsevier Science: Amsterdam, The Netherlands, 1995; pp 403-431. 28. Drawert, F.; Christoph, N. Significance of the sniffing-technique for the determination of odour thresholds and detection of aroma impacts of trace volatiles. In Analysis of Volatiles.; Schreier, P., Ed.; Walter de Gruyter: Berlin, Germany, 1984; pp 269-291. 29. Schieberle, P.; Grosch, W. Identification of potent flavor compounds formed in an aqueous lemon oil/citric acid emulsion. J. Agric. Food Chem. 1988, 36, 797-800. 30. Kuroiwa, Y.; Naoki, H. Composition of sunstruck flavor substance of beer. Agri. Bio. Chem. 1961, 25, 257-258. 31. Holscher, W.; Vitzthum O. G.; Steinhart, H. Prenyl alcohol – source for odorants in roasted coffee. J. Agric. Food Chem. 1992, 40, 655-658. 32. Bailly, S.; Jerkovic, V.; Marchand-Brynaert, J.; Collin, S. Aroma extraction dilution analysis of Sauternes Wines. Key Role of polyfunctional thiols. J. Agric. Food Chem. 2006, 54, 7227-7234. 33. Li, J.; Schieberle, P.; Steinhaus, M. Characterization of the major odor-active compounds in Thai durian (Durio zibethinus L. ‘Monthong’) by aroma extract dilution analysis and headspace gas chromatography-olfactometry. J. Agric. Food Chem. 2012, 60, 11253-11262. 34. Vichi, S.; Cortes-Francisco, N.; Romero, A.; Caixach, J. Determination of volatile thiols in virgin olive oil by derivatisation and LC-HRMS, and relation with sensory attributes. Food Chem. 2014, 149, 313-318. 35. Kuroiwa, Y.; Hashimoto, N. Composition of sunstruck flavor substance and mechanism of its evolution. Proc. Am. Soc. Brew. Chem. 1961, 28-36. 36. Song, H. S.; Sawamura, M.; Ito, T.; Ido, A; Ukeda H. Quantitative determination and characteristic flavor of daidai (Citrus aurantium L. var. cyathifera Y. Tanaka) peel oil. Flavour Frag. J. 2000, 15, 323-328. 37. Choi, H.; Kondo, Y.; Sawamura, M. Characterization of the odor-active volatiles in Citrus Hyuganatsu (Citrus tamurana Hort. ex Tanaka). J. Agric. Food Chem. 2001, 49, 2404-2408. 38. Miyazawa, N.; Tomita, N.; Kurobayashi, Y.; Nakanishi, A.; Ohkubo, Y.; Maeda, T.; Fujita, A. Novel character impact compounds in yuzu (Citrus junos Sieb. ex Tanaka) peel oil. J. Agric. Food Chem. 2009, 57, 1990-1996. 39. Mussinan, C. J.; Mookherjee, B. D.; Malcolm, G. I. Isolation and identification of the volatile constituents of fresh lemon juice. In Essential Oils; Mookherjee, B. D.; Mussinan, C. J., Eds.; Allured Publishing Corp.: Wheaton, Illinois, 1981, pp 199-228. 40. Mondello, L.; Catalfamo, M.; Dugo, G. Multidimensional tandem capillary gas chromatography system for the analysis of real complex samples. Part I: Development of a fully automated tandem gas chromatography system. J. Chromatogr. Sci. 1998, 36, 201-209. 41. Starkenmann, C.; Troccaz, M.; Howell, K. The role of cysteine and cysteine-S conjugates as odour precursors in the flavor and fragrance industry. Flavour Frag. J. 2008, 23, 369-381. 24 ACS Paragon Plus Environment

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624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639


42. Perez-Cacho, P.; Mahattanatawee, K.; Smoot, J. M.; Rouseff, R. Identification of sulfur volatiles in canned orange juices lacking orange flavor. J. Agric. Food Chem. 2007, 55, 5761-5767. 43. Mick, W.; Schreier, P. Additional volatiles of black tea aroma. J. Agric. Food Chem. 1984, 32, 924-929. 44. Liu, Y; Yu, A. Effect of reaction temperature and time on aroma compounds generation from Maillard reaction of ascorbic acid and cysteine. Shipin Keji. 2011, 36, 262-267. 45. Miller, J. M.; Rockland, L. B. Determination of cysteine and glutathione in citrus juices by filter paper chromatography. Arch. Biochem. Biophys. 1952, 40, 416-423. 46. Lifshitz, A.; Stepak. Y. Detection of adulteration of fruit juice. I. Characterization of Israel lemon juice. J. AOAC. 1971, 54 (6), 1262-1265. 47. Vandercock, C. E.; Price, R. L. The application of amino acid composition to the characterization of citrus juice. J. Food Sci. 1972, 37 (3), 384-386. 48. Ortiz, J. M.; Garcia-Lidon, A.; Tadeo, J. L.; De Cordova, F.; Martin, B.; Estelles, A. Comparative study of physical and chemical characteristics of four lemon cultivars. J. Hort. Sci. 1986, 61 (2), 277-281.

640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 25 ACS Paragon Plus Environment


656

Figure captions

657

Figure 1. Synthesis of 6-methylnonanal 136.

658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692

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Figure 2. Comparison of the CLSD chromatograms by SFE (A) and DCM extraction (B). Peak identification from the DCM extraction (B) is as follows: (1) sulfur dioxide 1, (2) methional 31, (3) 2propionylthiophene 152, (4) 2-(2-methyltetrahydrothiophen-2-yl)-ethyl acetate 281, (5) 2-(5-isopropyl2-methyltetrahydrothiophen-2-yl)-ethanol 304 and 307, (6) 2-(5-isopropyl-2-methyltetrahydrothiophen2-yl)-ethyl acetate (isomer 1) 373, (7) 2-(5-isopropyl-2-methyltetrahydrothiophen-2-yl)-ethyl acetate (isomer 2) 375, (8) 3-mercapto-3,7-dimethyl-6-octenyl acetate 378, (9) 2-(5-isopropylidene-2methyltetrahydrothiophen-2-yl)-ethyl acetate 395, (10) 2-[5-(1-hydroxy-1-methylethyl)-2methyltetrahydrothiophen-2-yl]-ethyl acetate (isomer 1) 401, (11) 2-[5-(1-hydroxy-1-methylethyl)-2methyltetrahydrothiophen-2-yl]-ethyl acetate (isomer 2) 406. Figure 3. Chemical structures of synthesized VSCs. Figure 4. Synthesis of 2-(5-isopropyl-2-methyltetrahydrothiophen-2-yl)-ethanol 304 and 307, 3mercapto-3,7-dimethyl-6-octenyl acetate 378, 2-(5-isopropyl-2-methyltetrahydrothiophen-2-yl)-ethyl acetate 373 and 375. The asterisks in the final product of both lemon VSCs indicate there are a set of unresolved chiral centers from the diastereomeric mixtures. Figure 5. Synthesis of 2-(2-methyltetrahydrothiophen-2-yl)-ethyl acetate 281. The asterisk in the final product of 281 indicates there is an unresolved chiral center from the enantiomeric mixture. Figure 6. Synthesis of 2-(5-isopropylidene-2-methyltetrahydrothiophen-2-yl)-ethyl acetate 395 and 2[5-(1-hydroxy-1-methylethyl)-2-methyltetrahydrothiophen-2-yl]-ethyl acetate 401 and 406. The asterisks in the final product of both lemon VSCs indicate there are a set of unresolved chiral centers from the diastereomeric mixtures.


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Table 1. Aroma-active Compounds in California Lemon Peel Extract

RI (apol.) 773 813 839 932 983 988 1017 1040 1060 1066 1090 1136 1156 1162 1174 1188 1215 1219 1241 1247 1257 1286 1347 1360 1365 1504 1562

Odor descriptionᵃ green, apple sulfurous, skunk-like green, grass pine, green aldehydic, orange orange, zest, citrus minty, eucalyptus citrusy, lemon zest like lemon juice Plastic floral, sweet floral, waxy, herbal green, peely, citrus floral, fresh floral, metallic, citrus aldehydic, soapy floral, citrus fresh, peely, sweet floral, rose lemony, sweet green, woody, cucumber woody, eugenol-like citrus, soapy woody, pencil shavings citrus, soapy lemony, citrus Sulfury

Odorantᵇ

FD Factor (3ⁿ) 9 243 3 9 27 81 27 243 27 9 243 27 3 3 3 9 9 243 9 243 27 27 27 27 9 3 9

(Z)-3-hexenal prenyl mercaptanᶜ (Z)-3-hexenol α-pinene octanal myrcene 1,8-cineole limonene γ-terpinene p-cresol linalool citronellal 8-methylnonanal 4-terpinenol α-terpineol decanal nerol + citronellol neral geraniol geranial 8-methyldecanal 2-methoxy-4-vinylphenol neryl acetate unknown geranyl acetate β-bisabolene 3-mercapto-3,7-dimethyl-6-octenyl acetate a Odor perceived at the end of the ODP. bIdentified by matching odor quality of odorant to the mass spectrum and RI on an apolar column. cCoincidence of RI and odors on two capillary columns (apolar and polar) with that of pure reference standard.



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Table 2. Volatile Compounds Identified in the Extraction and Fractionation of Lisbon Lemon Peels (Citrus limon L. Burm. f., cv. Lisbon). RI RI c b (apol.) (pol.)d IDe Noᵃ Compound % 1 sulfur dioxide tr 441 1 534 2 acetaldehyde tr 476 1 1232 3 acetic acid tr 565 1 634 1 4 ethyl acetate tr 593 722 5 valeraldehyde tr(7) 664 1 1077 1 6 acetoin tr 669 1310 7 propionic acid tr(10) 693 1 734 1 8 ethyl propionate tr(7) 694 700 9 heptane tr 696 1 1001 1 10 2-methylbutanol tr(10) 724 769 11 butan-2-yl acetate tr(8) 740 1 1003 1 12 isoamyl alcohol tr 747 727 13 1-methyl-1,3-cyclohexadiene tr 752 1 796 1 14 isobutyl acetate tr(7) 754 852 15 3-hexanone tr(6-10) 762 1 859 1 16 2-hexanone tr(6-10) 765 935 17 (Z)-3-hexenal tr(7) 773 1 877 1 18 hexanal 0.02 778 983 19 3-hexanol tr(6-10) 785 1 1022 1 20 2-hexanol tr(7-11) 788 868 21 butyl acetate tr(6-8) 798 1 1240 1 22 furfural tr(8) 801 800 23 octane tr 801 1 1021 1 24 (E)-2-hexenal 0.01 826 1177 25 (Z)-3-hexenol 0.02 839 1 752 1 26 1,3,3-trimethylcyclohexene tr(8) 841 965 27 2-methylenehexanal tr(6) 842 1 1193 28 (E)-2-hexenol tr 850 1 1141 29 hexanol 0.01 853 1 920 30 2-methylbutyl acetate tr(7) 862 1 1237 1 31 methional tr(6) 865 974 32 2-heptanone tr(6-8) 868 1 1392 1 33 γ-butyrolactone tr(8) 874 949 34 heptanal tr 881 1 899 35 nonane tr 902 1 985 36 methyl hexanoate tr(7) 912 1 814 37 tricyclene 0.01 919 1 817 38 α-thujene 0.36 924 1 1300 39 benzaldehyde tr 928 1 28 ACS Paragon Plus Environment

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40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82


α-pinene 2-ethylhexanal α-fenchene 2-methylheptanal camphene 6-methylheptanal 4-octanone heptanol 2,3-octanedione 2,2,6-trimethyl-6-vinyltetrahydropyran 4-methylpentyl acetate hexanoic acid 6-methyl-5-hepten-2-one (Z)-5-octenal phenol sabinene 2-amylfuran β-pinene 2,3-dehydro-1,8-cineole octanal myrcene 2-methyl-5-isopropenyl-2-vinyltetrahydrofuran (isomer 1) (Z)-3-hexenyl acetate 2-methyl-5-isopropenyl-2-vinyltetrahydrofuran (isomer 2) hexyl acetate α-phellandrene 3-carene benzyl alcohol α-terpinene 2-methyl-(E)-2-heptenal γ-hexalactone p-cymene p-menth-1-ene 1,8-cineole β-phellandrene 2-ethylhexanol acetophenone 3-methylbenzaldehyde (E)-2-octenal 2,5,5-trimethyl-2-cyclohexenone limonene (Z)-β-ocimene melonal 29 ACS Paragon Plus Environment

1.56 tr(6) 0.01 tr(6,7) 0.05 tr(6,7) tr(7) tr tr(6) tr(6) tr(7) tr tr tr(7) tr 0.44 tr(6,7) 14.52 tr(9,10) 0.04 1.44 tr(9,10) tr tr(7-10) tr(7) 0.04 tr(4) tr 0.06 tr(7,8) tr 0.33 tr(9) tr(6-11) tr(3-7) tr tr(6-8) tr(6) tr(8) tr(8) 63.74 0.05 tr

932 935 938 943 943 945 952 960 960 962 962 965 969 971 971 973 974 977 978 983 988 991 994 995 998 999 1004 1008 1011 1014 1014 1015 1015 1017 1019 1022 1034 1036 1036 1037 1040 1041 1043

835 990 866 1009 873 1048 1024 1242 1121 924 1002 1616 1131 1136 1758 928 1037 925 973 1082 962 1010 1111 1043 1072 973 953 1630 983 1164 1477 1071 944 1012 1013 1277 1430 1393 1226 1243 1002 1035 1149

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1


83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125

dihydrotagetone 2-methyloctanal (E)-β-ocimene 4-methyloctanal (Z)-5-octenol 6-methyloctanal γ-terpinene octanol linalool oxide furan (isomer 1) (E)-sabinene hydrate p-cresol 4-methylhexyl acetate 2-nonanone fenchone linalool oxide furan (isomer 2) (Z)-4-nonenal 2,3-myrcene epoxide p-α-dimethylstyrene terpinolene rosefuran (Z)-sabinene hydrate nonanal unidentified linalool heptyl acetate (Z)-rose oxide perillene p-mentha-1,3,8-triene undecane (Z)-p-mentha-2,8-dien-1-ol (E)-4,8-dimethyl-1,3,7-nonatriene (Z)-p-menth-2-en-1-ol pinanol (isomer 1) 2-methyl-(E)-2-octenal campholenic aldehyde methyl octanoate octyl formate 3,5,5-trimethyl-2-cyclohexen-1,4-dione melonol (E)-p-mentha-2,8-dien-1-ol (Z)-limonene-1,2-oxide 4-acetyl-1-methyl-1-cyclohexene camphor

tr(3) tr(6) 0.06 tr(6,7) tr tr(6,7) 9.89 0.03 tr 0.08 tr(9) tr(7) tr(6) tr tr(10) tr(6) tr(8) tr 0.42 tr 0.06 0.11 tr 0.17 tr tr(8) tr tr(2-5) tr tr tr tr tr tr(7,8) tr tr tr(6) tr tr tr tr tr(8) 0.01 30

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1044 1045 1045 1046 1053 1054 1060 1063 1064 1065 1066 1068 1070 1072 1072 1075 1076 1077 1083 1085 1086 1088 1090 1090 1092 1093 1094 1099 1103 1105 1107 1109 1110 1112 1112 1114 1114 1115 1116 1118 1119 1120 1120

1105 1115 1050 1144 1401 1167 1049 1354 1232 1265 1836 1184 1192 1258 1237 1209 1233 1084 1194 1332 1190 1334 1174 1155 1212 1209 1097 1409 1111 1358 1339 1261 1270 1193 1231 1472 1440 1450 1243 1349 1299

1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

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126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168


alloocimene pinanol (isomer 2) (E)-p-menth-2-en-1-ol isopulegol (isomer 1) 4,8-terpinolene epoxide (E)-limonene-1,2-oxide citronellal isopulegol (isomer 2) (E)-2-nonenal pinocarvone 6-methylnonanal δ-terpineol 7-methyl-3-methylene-6-octenal 2-methylnonanal 4-methylnonanal 4-methylacetophenone rosefuran epoxide 8-methylnonanal pinocamphone 3,7-dimethyl-(E)-3,6-octadienal (E)-2-nonenol borneol 3,7-dimethyl-(Z)-3,6-octadienal nonanol 4-limonenol 4-terpinenol 2-propionylthiophene p-cymen-8-ol myrtenal dill ether octanoic acid 4,7-dimethyl-3-bicyclo[3.2.1]octen-6-one (Z)-4-decenal 2-decanone (E,Z)-2,4-nonadienal methyl chavicol α-terpineol butyl hexanoate (E)-dihydrocarvone 3,4-dimethylphenol 3-octenyl acetate methyl salicylate 3-decanone

tr(2) tr(10) tr tr(6-10) tr tr(7,8) 0.09 tr(6-9) tr(7,8) tr tr(6) tr(10) 0.01 tr tr tr(9) tr(8) tr tr tr(9) tr tr tr(7-9) 0.01 0.01 0.08 tr(7) tr tr(7,8) tr tr tr tr(6,7) tr(6-8) tr(7,8) tr(6) 0.32 tr(7) tr tr(8,9) tr(7) tr(7) tr(7) 31


1121 1121 1125 1128 1132 1135 1136 1140 1140 1142 1143 1144 1145 1147 1151 1152 1152 1156 1157 1158 1158 1159 1160 1160 1161 1162 1163 1165 1166 1167 1169 1170 1170 1171 1171 1173 1174 1176 1176 1177 1177 1177 1178

1184 1389 1424 1350 1256 1253 1270 1362 1331 1374 1246 1471 1306 1213 1254 1537 1382 1254 1305 1362 1500 1481 1335 1452 1480 1385 1627 1627 1402 1308 1820 1338 1295 1445 1446 1475 1218 1385 1959 1555 1263

1 1 1 1 1 1 1 1 1 1 2f 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1


169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211

hexyl butyrate (E)-1,2-dihydroperillaldehyde myrtenol 5-limonenol 8,9-limonene epoxide (isomer 1) propyl heptanoate safranal ethyl octanoate 8,9-limonene epoxide (isomer 2) (Z)-piperitol decanal 1-dodecene (Z)-5-octenyl acetate p-menth-1-en-9-al (isomer 1) p-menth-1-en-9-al (isomer 2) (E)-carveol octyl acetate (E)-piperitol 2,3-epoxygeranial (isomer 1) 2-hydroxy-1,8-cineole dodecane methyl nonanoate 2,3-epoxygeranial (isomer 2) (Z)-p-mentha-1(7),8-dien-2-ol (E)-p-mentha-1(7),8-dien-2-ol 2-methyl-6-methylene-2,7-octadienal ascaridole cuminaldehyde 4-vinylphenol citronellol nerol carvone 2-methyl-(E)-2-nonenal neral 4-methylnonanol carvotanacetone anisaldehyde hexyl 2-methylbutyrate piperitone unidentified isogeraniol isopiperitenone melonyl acetate

tr(7) tr(7,8) tr tr(7) tr tr(8) tr(7) tr(7) tr tr 0.07 tr(1) tr tr tr tr(10) 0.01 tr tr tr(10) tr(1) tr 0.01 tr(8) tr tr tr(8) tr(7) tr 0.05 0.21 tr(8,9) tr 0.60 tr(10) tr tr tr(6) 0.01 tr tr tr tr(7) 32


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1179 1179 1180 1183 1183 1183 1183 1184 1184 1185 1188 1192 1193 1197 1198 1198 1199 1201 1202 1202 1203 1203 1204 1204 1207 1208 1210 1211 1213 1214 1215 1216 1218 1219 1220 1223 1225 1226 1227 1233 1233 1237 1237

1217 1396 1619 1314 1223 1438 1234 1324 1466 1291 1051 1327 1414 1417 1611 1278 1524

1199 1294 1634 1644 1489 1545 2106 1545 1574 1510 1358 1460 1515 1455 1786 1229 1503 1565 1597 1349

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

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212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254


piperitone epoxide geraniol (E)-2-decenal 6-methyldecanal geranial 4-methyldecanal perilla aldehyde 2-methyldecanal 8-methyldecanal (E)-2-decenol indole decanol (Z)-1,2-dihydroperilla alcohol p-mentha-1,4-dien-7-al 3-undecanone (E)-1,2-dihydroperilla alcohol nonanoic acid 2-undecanone bornyl acetate (E,Z)-2,4-decadienal 10-limonenol perilla alcohol thymol ascaridole epoxide p-menth-1-en-9-ol (isomer 1) p-menth-1-en-9-ol (isomer 2) carvacrol (E,E)-2,4-decadienal 2-methoxy-4-vinylphenol 2,4,7-decatrienal undecanal 1-tridecene nonyl acetate methyl nerate 6,7-epoxyneral tridecane myrtenyl acetate methyl geranate methyl decanoate p-menthane-3,8-diol citronellic acid neric acid limonen-4-yl hydroperoxide

tr(8) 0.34 tr tr(6) 0.92 tr 0.03 tr(6) tr(6,7) tr tr 0.01 tr tr tr(5,6) tr 0.02 tr(5,6) 0.01 tr(8) 0.01 tr 0.01 tr tr tr 0.02 tr tr tr(8) 0.04 tr(1) 0.01 tr(7-9) tr(8) 0.01 tr 0.01 tr(6) tr tr tr tr(9) 33 ACS Paragon Plus Environment

1237 1241 1244 1245 1247 1248 1249 1250 1257 1257 1258 1261 1263 1265 1266 1267 1269 1270 1272 1272 1275 1277 1279 1281 1282 1282 1283 1284 1286 1289 1289 1291 1298 1300 1301 1304 1306 1307 1308 1309 1311 1314 1317

1495 1619 1427 1341 1509 1344 1552 1311 1374 1596 2171 1551 1675 1584 1260 1698 1928 1395 1381 1536 1755 1767 1938 1634 1723 1727 1963 1584 1946 1592 1397 1151 1380 1426 1726 1300 1476 1488 1396 1859 1991 2077

1 1 1 2 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1


255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297

(E)-carvyl acetate piperitenone limonen-1,2-diol linalyl propionate δ-elemene (isomer 1) γ-nonalactone p-menth-1-en-3-yl acetate δ-elemene (isomer 2) 6,7-epoxygeranial geranic acid citronellyl acetate eugenol α-cubebene 3-methylundecanal 2-methylundecanal (E)-2-undecenal (Z)-carvyl acetate neryl acetate decanoic acid 4-methylundecanal vanillin α-terpinyl acetate (Z)-5-dodecenal undecanol (Z)-3-hexenyl hexanoate geranyl acetate 2-(2-methyltetrahydrothiophen-2-yl)-ethyl acetate hexyl hexanoate α-copaene 4-dodecanone 2-dodecanone (E)-2-hexenyl hexanoate 6-dodecanone 8-hydroxycarvone ethyl decanoate methyl eugenol (Z)-6-dodecenal β-elemene methyl n-methylanthranilate 1-tetradecene dodecanal decyl acetate limonen-10-yl acetate 34 ACS Paragon Plus Environment

tr tr(8,9) tr tr(7) tr(2-4) tr(6) tr(7) 0.01 tr(8) tr(9,10) 0.05 tr(8) tr(1) tr(6) tr(6) tr(6-8) tr(6) 0.60 tr tr tr(10) 0.01 tr(6,7) tr tr(6) 0.58 tr(8) tr(6) tr tr(6) tr(5-7) tr(6) tr(6) tr tr tr(7,8) tr(7) 0.01 tr(6,7) tr(1) 0.01 0.01 0.01

Page 34 of 46

1318 1319 1321 1328 1330 1330 1331 1333 1335 1336 1338 1338 1344 1344 1345 1345 1346 1347 1351 1353 1358 1360 1363 1363 1365 1365 1365 1369 1372 1372 1374 1376 1377 1380 1382 1384 1386 1388 1388 1390 1390 1391 1392

1523 1696 2034 1409 1275 1788 1277 1790 2099 1455 1925 1259 1429 1415 1542 1555 1517 2019 1445 2250 1479 1529 1659 1447 1541 1674 1413 1291 1495 1460 2104 1434 1789 1390 1832 1244 1506 1484 1634

1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 2 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Page 35 of 46

298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338


nonyl propionate cuminyl acetate p-menth-1-en-9-yl acetate 4-methyl-5-(3-methylbut-2-en-1-yl)-furan-2(5h)-one tetradecane methyl undecanoate 2-(5-isopropyl-2-methyltetrahydrothiophen-2-yl)-ethanol (isomer 1) (Z)-α-bergamotene α-santalene 2-(5-isopropyl-2-methyltetrahydrothiophen-2-yl)-ethanol (isomer 2) β-caryophyllene perilla acetate citronellyl propionate 2-methyl-(E)-2-undecenal (E,Z)-2,6-dodecadienal geranyl acetone (E)-α-bergamotene undecanoic acid epi-β-santalene 3-methyldodecanal (E)-2-dodecenal α-humulene 2-methyldodecanal 4-methyldodecanal (E)-β-farnesene neryl propionate β-santalene geranyl propionate β-ionone-2,3-epoxide 5-hexyl-4-methyldihydro-2(3h)-furanone γ-muurolene β-ionone γ-curcumene ar-curcumene 3-tridecanone 1-pentadecene unidentified limonen-10-yl propionate 2-tridecanone γ-elemene valencene 35 ACS Paragon Plus Environment

tr(7) tr(7) tr tr 0.02 tr(6) tr(8)

1392 1396 1399 1402 1404 1409 1410

0.03 tr(1) tr(8)

1410 1413 1414

0.26 tr tr tr tr tr(7,8) 0.42 tr(10) 0.01 tr(6,7) tr 0.02 tr(6) tr(6) 0.02 0.02 0.02 tr tr tr tr(8) tr tr(1-3) 0.01 tr(5,7) tr 0.02 tr 0.01 tr(2) 0.09

1414 1418 1420 1427 1433 1434 1434 1441 1443 1443 1444 1446 1447 1449 1450 1452 1454 1455 1461 1464 1465 1469 1470 1472 1472 1474 1476 1479 1481 1484 1484

1435 1732 1614 1998 1401 1499 1929

1 1 1 1 1 1 2f

1370 1930

1 1 2f

1382 1681 1521 1569 1670 1641 1382 2121 1415 1537 1640 1460 1518 1546 1465 1581 1429 1610 1753 1867 1486 1706 1468 1562 1563 1356 1734 1593 1505

1 1 1 1 1 1 1 1 1 2 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1


339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379

α-selinene (Z)-α-bisabolene decyl propionate bicyclogermacrene myristicin (E,E)-α-farnesene ethyl undecanoate dihydroactinidiolide pentadecane sesquicineole α-muurolene undecyl acetate methyl dodecanoate γ-cadinene β-bisabolene calamenene tridecanal γ-bisabolene (isomer 1) δ-cadinene perilla propionate (Z)-9-dodecen-12-olide cubebol cadina-1,4-diene γ-bisabolene (isomer 2) 7-epi-α-selinene 2,6-dimethoxy-4-vinylphenol (E)-α-bisabolene hexyl benzoate γ-undecalactone neryl butyrate 2-methyltridecanal 4-methyltridecanal dodecanoic acid (E)-2-tridecenal 2-(5-isopropyl-2-methyltetrahydrothiophen-2-yl)-ethyl acetate (isomer 1) (E)-nerolidol 2-(5-isopropyl-2-methyltetrahydrothiophen-2-yl)-ethyl acetate (isomer 2) 4-tetradecenal (isomer 1) endo-1-bourbonanol 3-mercapto-3,7-dimethyl-6-octenyl acetate spathulenol 36 ACS Paragon Plus Environment

Page 36 of 46

tr(1) 0.07 tr 0.05 tr(7) tr(3) tr tr 0.06 tr(8) tr tr(7) tr(6) tr(8) 0.68 tr(5,8) tr 0.01 tr(1-9) tr(7) tr 0.01 tr(8) tr(1-3) tr 0.01 0.01 tr(6) tr(8) tr(7) tr(6) tr(6) tr(9,10) tr(7) tr(7,8)

1487 1487 1488 1489 1489 1489 1490 1493 1494 1495 1496 1496 1498 1500 1504 1504 1506 1508 1510 1510 1511 1514 1519 1520 1523 1531 1534 1537 1538 1539 1540 1543 1543 1545 1547

1513 434 1542 1514 2016 1543 1534 2037 1501 1538 1541 1574 1600 1549 1519 1601 1616

1560 2289 1564 1847 2005 1648 1621 1649 2232 1753 1822

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 2

tr tr(7,8)

1549 1553

1821 1841

1 2

tr(6,7) 0.02 tr tr

1559 1559 1562 1563

2111 1884 1889

1 1 2 1

1541 1772

Page 37 of 46

380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419


4-tetradecenal (isomer 2) (Z)-3-hexenyl octanoate β-caryophyllene oxide hexyl octanoate 5-dodecenyl acetate 5-heptyl-4-methyldihydro-2(3h)-furanone sesquisabinene hydrate 2-tetradecanone unidentified ethyl dodecanoate 1-hexadecene tetradecanal syringaldehyde dodecyl acetate hexadecane 2-(5-isopropylidene-2-methyltetrahydrothiophen-2-yl)-ethyl acetate isopropyl dodecanoate t-cadinol 2-methyl-(E)-2-tridecenal α-cadinol β-eudesmol 2-[5-(1-hydroxy-1-methylethyl)-2methyltetrahydrothiophen-2-yl]-ethyl acetate (isomer 1) α-eudesmol 2-isopropenyl-8,8a-dimethyloctahydro-4a(2h)-naphthol hydroxy-β-Santalene campherenol 2-[5-(1-hydroxy-1-methylethyl)-2methyltetrahydrothiophen-2-yl]-ethyl acetate (isomer 2) (E)-2-tetradecenal intermedeol β-bisabolol hexyl nonanoate α-bisabolol β-sinensal nootkatol 2-pentadecanone (E,Z)-farnesal pentadecanal methyl tetradecanoate oplopanone (E,E)-farnesal 37 ACS Paragon Plus Environment

tr(6) tr tr tr tr(7) tr(8) tr tr(7) 0.01 tr tr(1) 0.02 tr(10) tr tr(1) tr(7,8)

1564 1565 1566 1568 1570 1572 1575 1578 1578 1582 1588 1593 1594 1598 1600 1601

tr(6) tr(8) tr(7) tr(10) tr(11) tr(7,8)

1614 1617 1620 1621 1625 1630

1627 1936

tr(11) tr(7) tr(7-9) 0.05 tr(7)

1631 1632 1634 1635 1635

1964 1943 2020 2265

1 1 1 1 2

tr(7) 0.02 0.01 tr(6) 0.04 tr(7) tr tr(7) tr(8) 0.01 tr(6) tr(8) tr

1645 1648 1651 1663 1666 1666 1671 1680 1683 1696 1697 1697 1712

1858 1968 1920 1704 1980 1984 2188 1807 1994 1814 1804 2258 2044

1 1 1 1 1 1 1 1 1 1 1 1 1

1652 1734 1605 1976 1704 1634 1434 1708 2584 1683 1599 2004

1942 1870 2237

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 2


Page 38 of 46

1829 neryl hexanoate tr(6) 1713 1 2088 α-sinensal tr(7) 1718 1 1874 geranyl hexanoate tr(6) 1729 1 2429 tetradecanoic acid tr(9,10) 1734 1 β-bisabolenal tr 1744 1 1834 (Z)-3-hexenyl decanoate tr(6) 1744 1 methyl γ-tridecalactone tr(9) 1746 1 1800 octyl octanoate tr(6) 1748 1 2235 nootkatone 0.01 1763 1 1924 (Z)-9-hexadecenal tr(6) 1767 1 1834 ethyl tetradecanoate tr 1776 1 2210 6-isopropenyl-4,8a-dimethyl-4a,5,6,7,8,8a-hexahydro-2(1h)tr(9) 1793 1 naphthalenone 1914 0.02 1797 1 432 hexadecanal 1907 433 methyl pentadecanoate tr(6) 1798 1 2039 tr(7) 1813 1 434 (E,E)-farnesyl acetate 435 methyl γ-tetradecalactone tr(9) 1856 1 2014 436 heptadecanal tr(6) 1895 1 1995 437 methyl hexadecanoate tr(6) 1906 1 2751 438 citropten 0.12 1911 1 2745 439 hexadecanoic acid 0.02 1926 1 2129 440 (Z)-9-octadecenal tr(6) 1964 1 2129 441 (E)-9-octadecenal tr(6) 1969 1 2114 442 octadecanal tr(6) 1994 1 2253 443 methyl linoleate tr(6,7) 2041 1 2320 444 methyl linolenate tr(6,7) 2043 1 2195 445 methyl oleate tr(6,7) 2044 1 Total Adjusted PPT of Named Components 100.00 a b Compounds detected by order of elution on an apolar column. Relative percentage determined by FID on an apolar column with response factor adjustment; tr - trace compounds in the extract below 0.00% or identified in the fractions (1-11). cRI on an apolar column. dRI on polar column. e(1) tentative identification based on RI and/or EI mass spectral comparison with in-house, NIST and/or Wiley libraries; (2) identification based on RI and EI mass spectral comparison with synthesized standards. f Required MRM to confirm presence on polar column.

420 421 422 423 424 425 426 427 428 429 430 431


Page 39 of 46


Figure 1.



Figure 2.


Page 40 of 46

Page 41 of 46


Figure 3.



Figure 4.


Page 42 of 46

Page 43 of 46


Figure 5.



Figure 6.


Page 44 of 46

Page 45 of 46


Table 3. Orthonasal and Retronasal Evaluations of Novel VSCs.

ᵃOrthonasal evaluations were conducted using paper blotters at a 0.1% solution in ethanol; ᵇRetronasal evaluations were conducted at varying concentrations in water.



TOC Graphic


Page 46 of 46

Identification, Synthesis, and Characterization of Novel Sulfur

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