Research Article Received: 23 March 2015
Revised: 19 May 2015
Accepted article published: 28 May 2015
Published online in Wiley Online Library: 6 July 2015
(wileyonlinelibrary.com) DOI 10.1002/jsfa.7274
Analysis of volatiles from stored wheat and Rhyzopertha dominica (F.) with solid phase microextraction–gas chromatography mass spectrometry Yonghao Niu,a,b,c Lei Hua,a Giles Hardy,c Manjree Agarwalb,c and Yonglin Renb,c* Abstract BACKGROUND: Volatile organic compounds (VOCs) contribute significantly to food flavour and can be used as indicators of quality, age of storage, and hygiene condition of stored products. The VOCs in the headspace of three different samples – healthy wheat, Rhyzopertha dominica, and wheat with R. dominica – were analysed at 25∘ C by solid phase micro-extraction (SPME) coupled with gas chromatography–flame ionisation detection (GC-FID) and gas chromatography–mass spectrometry (GC-MS). All the experimental conditions were kept consistent except a polar column and a non-polar column were used to assess the differences in volatile fingerprints. RESULTS: A total of 114 volatiles were identified by both the polar and non-polar columns, of which 48 were specific to one of the three samples tested. The volatiles were mainly carbonyl chemical compounds such as aldehydes, ketones and alcohols. GC-MS results showed slightly more VOCs were identified from the polar column. The total number for the three samples was 43 from the polar column compared to 39 from the non-polar column. Conversely, 30 VOCs unique to a given sample were identified from the non-polar column compared to 18 from the polar column. CONCLUSION: The use of both polar and non-polar columns is essential to capture the full range of VOCs produced by the three specific sample types investigated. The data can form the basis of enquiry into the relationship between storage and grain quality, and insect infestation and grain quality by observing the impact that these circumstances have on the production of volatile organic compounds. © 2015 Society of Chemical Industry Keywords: wheat; Rhyzopertha dominica; solid phase microextraction (SPME); gas chromatography–mass spectrometry; volatile organic compounds
INTRODUCTION
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∗
Correspondence to: YongLin Ren, Murdoch University, School of Veterinary and Life Sciences, Murdoch, WA 6150, Australia. E-mail: [email protected]
a College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China b Australia Cooperative Research Centre for National Plant Biosecurity, LPO Box 5012, Bruce, ACT 2617, Australia c School of Veterinary and Life Sciences, Murdoch University, South Street, Murdoch, WA 6150, Australia
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Wheat is an essential food for human beings and how to maintain the quality of stored wheat has been a research focus for many years.1,2 Rhyzopertha dominica (F.) is one of the primary insect pests of stored wheat where it causes major physical and off-odour damage.3 Some grain volatiles contribute significantly to food flavour and can be used as indicators of the quality, age of storage and hygiene condition of stored products.4,5 At present, head-space solid phase micro-extraction (HS-SPME) coupled with gas chromatography–flame ionisation detection (GC-FID) and gas chromatography–mass spectrometry (GC-MS) are useful techniques for identifying volatiles in stored wheat, and might be used to ascertain if the grain is infested with insects or not.6,7 Several researchers have used HS-SPME coupled with GC-MS methods to analyse the volatile organic compounds (VOCs) of stored wheat and R. dominica. Most of these studies have chosen non-polar columns to separate VOCs7 – 10 and many
low-molecular-weight organic compounds emitted from stored grains have been identified.11 However, to date, there are no reports where polar columns have been used to separate volatiles from the headspace of stored grain. In this study, polar and non-polar columns are used for the first time to separate and compare VOCs from the following three sample types at 25∘ C: Wheat alone, R. dominica adults alone and wheat infested with R. dominica adults
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MATERIALS AND METHODS
A
Grain pre-treatment Freshly harvested (2011–2012) wheat (Australia Standard Wheat I) used for this investigation was procured from CBH (Co-operative Bulk Handling), Western Australia. The moisture content was 11.5% (w/w, HOH-Express 50, Electronic Moisture Meter; PFEUFFER, Kitzingen, Germany). The wheat sample was placed in sealed glass jars (4 L) and put in a freezer at −4∘ C for 1 week to ensure it was free of any live insects. The wheat was then stored at 4∘ C until use.
B
Insect cultures The insect species R. dominica (strain No. MUWRD 7) was obtained from the Stored Grain Research Laboratory, School of Veterinary and Life Sciences, Murdoch University, Perth, Australia. About 200 adult R. dominica were added to 400 g of whole wheat in 500-mL jars with meshed lids to obtain a mixed age insect population. The experimental insects were reared in the dark at 30∘ C and 60% relative humidity (RH), and incubated for 4–5 weeks until adults from the next generation emerged.
Figure 2. Chromatograms of R. dominica: (A) polar column, (B) non-polar column. (A) 1,3-Hydroxy-2-butanone; (B) 1, bromo-methane; 2, 3-methyl-2-butanol; 3, hexanal; and 4, 2-triophenecarboxylic acid, octyl ester.
A
B
Glassware and SPME fibres One hundred millilitre Erlenmeyer flasks (Quickfit, Cat. No FE 100/3; Fisher Scientific, Loughborough, UK) each equipped with a cone/screw-thread adapter (Code ST 5313; Crown Scientific, Wantirna South, Victoria, Australia) with a 7/16′′ blue septum (Cat. No. 6518; Grace Davison Discovery Sciences, Melbourne, Victoria, Australia) were used for sample preparation. The measured volume of each Erlenmeyer flask and inlet system was calculated from the weight of water required to fill the container. The SPME fibre used was 50/30 μm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS; Cat. No. 57348-U), from Analytical Sigma–Aldrich (Sydney, Australia), and it was conditioned prior to use in accordance with the manufacturer’s recommendations. Headspace extraction was through a 7/16′′ blue septum (Cat. No. 6518; Grace Davison Discovery Sciences). The fibre was inserted into the headspace of the flask containing samples at the end of the defined extraction time, the fibre was withdrawn from the headspace into the needle. The fibre holder was removed from the extraction flask and inserted into the injection port. The fibre was extended into a GC-PFPD inlet where sample components were desorbed. Each sample was replicated for three times and duplicate injections. A
B
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Figure 1. Chromatograms of wheat: (A) polar column, (B) non-polar column. (A) 1, Hexane; 2, acetone; 3, D-limonene; 4, 1-pentanol; and 5, 1-hexanol. (B) 1, Hexane; 2, hexanal; 3, 1-hexanol; 4, D-limonene; and 5, dodecamethyl-cyclohexasiloxane.
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Figure 3. Chromatograms of wheat with R. dominica adults: (A) polar column, (B) non-polar column. (A) 1, Hexane; 2, acetone; 3, D-limonene; 4, 3-methyl-1-butanol; 5, 1-pentanol; 6, 1-hexanol; and 7, 5-hydroxy-3,4,4trimethyl-(E)-2-hexenoic acid. (B) 1, 1-methylethyl-hydroperoxide; 2, hexane; 3, (S)-(+)-2-pentanol; 4, 1-hexanol; 5, decane; 6, D-limonene; 7, cyclopen-tanecarboxylic acid, pentyl ester; 8, 2-thiophenecarboxylic acid, octyl ester; and 9, thiophene-2-carboxylic acid, 3-pentyl ester.
Sample preparation Three different treatment samples were examined. These were: (1) 80 g wheat, (2) 100 R. dominica adults and (3) 80 g wheat with 100 R. dominica adults. Each sample was placed in a 100 mL Erlenmeyer flask and sealed with the cone/screw-thread adapter. The samples were then processed after conditioning for 24 h at 25 ± 2∘ C in a temperature controlled room.
Gas chromatography–flame ionisation detector An Agilent 6890 GC (serial# US00021731, USA) manufactured by Agilent Technology (Palo Alto, CA, USA) with a flame ionisation detector (FID) was used to analyse the volatile profiles extracted by HS-SPME. The columns used in this experiment were a Stabilwax® polar column (dimensions: 30 m × 0.25 mm × 0.25 μm film thickness, ZB-WAX, Cat. No. #10623; Torrance, CA, USA) and an Rxi®-5ms non-polar column (dimensions: 30 m × 0.25 mm × 0.25 μm film thickness, RESTEK, Cat. No. #13423; Bellefonte, PA, USA). The following GC conditions were used. Hydrogen was used as the carrier gas at a constant speed of 40 mL min−1 in the split-less mode. The GC inlet was operated at 250∘ C and FID temperature was 250∘ C. The oven temperature program used for optimising the GC condition was: 45∘ C for 5 min, increasing from 45∘ C to 250∘ C at 5∘ C min−1 and being held for 5 min at each increment with a total run of 51 min. Each sample was replicated for three times and duplicate injections.
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SPME-GCMS of volatiles from wheat and R. dominica
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Table 1. VOCs of wheat identified from GC-MS analysis using polar and non-polar columns RT VOC Hexane Bromo-methane 1-Methoxy-2-propanone Acetone 2-Butanone Isopropyl alcohol Cyclopentanol Dimethyl-silanediol Heptane Decane 3-Methyl-1-butanol Trichloromethane 2-Propenylidene-cyclobutene 1-Propanol Hexanal Undecane 3-Methyl-2-butanol Hexamethyl-cyclotrisiloxane 1-Butanol 1-Penten-3-ol D-Limonene Dodecane Decamethyl-cyclopentasiloxane 2-Pentyl-furan 1-Pentanol 2,3-Dimethyl-1-butanol Octamethyl-cyclotetrasiloxane 1-Hexanol Dodecamethyl-cyclohexasiloxane Nonanal 1-Heptanol Acetic acid 2-Ethyl-1-hexanol Tetradecamethyl-cycloheptasiloxane Tridecane Oxime-methoxy-phenyl-_ Tetradecane Hexanoic acid Pentadecane 2-Ethyl-hexanoic acid
a 1.27 ND ND 1.67 2.12 2.33 ND ND ND 3.07 ND 3.47 3.82 3.9 4.81 5.01 5.31 ND 6.89 7.38 8.13 8.42 8.79 9.41 10.23 11.64 ND 13.3 14.16 ND 16.07 16.12 16.97 18.6 ND 23.69 ND 25.26 ND 27.36
RI b 1.76 1.34 1.43 ND ND ND 2.39 2.49 2.58 11.86 3.09 ND ND 1.6 4.55 15.25 ND 5.37 ND ND 12.81 18.35 17.11 ND 3.73 ND 12 7.06 22.05 15.37 ND ND ND 26.46 21.21 8.56 23.87 ND 26.39 ND
a 977 ND ND 990 1004 1010 ND ND ND 1033 ND 1045 1056 1058 1086 1092 1102 ND 1150 1165 1189 1197 1209 1228 1253 1296 ND 1355 1386 ND 1455 1456 1487 1554 ND 1778 ND 1852 ND 1952
Tr b 722 710 713 ND ND ND 739 742 745 999 759 ND ND 717 799 1102 ND 822 ND ND 1028 1197 1159 ND 776 ND 1003 869 1330 1106 ND ND ND 1507 1297 910 1403 ND 1504 ND
a ++++++ ND ND +++ + ++ ND ND ND ++ ND + ++ + ++ + + ND + + ++++ + tr + ++++ + ND ++++++ + ND + + + + ND ++ ND + ND +
b ++++++ ++ +++ ND ND ND + +++ + ++ tr ND ND + ++ + ND ++ ND ND +++ ++ +++ ND +++ ND ++ +++++ + tr ND ND ND tr + ++++ + ND + ND
Rfs – – – – – – – – 14
– – – – – 13,14
– – – – – – – – 14
– – – 14,15
– 14 14,15
– – – – – – – – –
RT, retention time (min); RI, retention index; a, the data from the polar column; b, the data from the non-polar column. Rfs, previous reports of detection, references: 13–15. Trace level: tr, 100%; ND, means the compound was not detected. The percentages were calculated using the total ion current (TIC) for each compound relative to the TIC for hexane of 100%.
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was 70 eV and the scanning was performed from 35 to 500 atomic mass units. The volatiles were identified by comparison of the mass spectrum with the NIST08 mass spectra library. Statistical analysis The variations (standard deviation) of VOCs total GC peak areas between three replicates and the duplicate injections in comparison with average readings were analysed by Microsoft Excel 2007.
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Gas chromatography–mass spectrometry An Agilent 6890 GC (serial# US00035629, USA) coupled with an Agilent 5793 Network mass selective detector (MSD) together with an Agilent ChemStation (serial# US02480181, USA) was used. The temperatures of the GC inlet (at split-less mode), interface and MS source, were 250∘ C, 250∘ C and 230∘ C, respectively. Helium was used as the carrier gas at a constant airflow of 1.0 mL min−1 . Other aspects such as column and oven temperature were the same as the GC-FID conditions described above. The ionisation potential
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Table 2. VOCs of R. dominica adults identified from GC-MS analysis using polar and non-polar columns RT VOC Hexane Bromo-methane Acetone Hexamethyl-cyclotrisiloxane Dimethyl-silanediol 3-Methyl-2-butanol Acetic acid ethenyl ester Octamethyl-cyclotetrasiloxane 1-Pentanol Hexanal 2-Pentanol Decamethyl-cyclopentasiloxane 3-Hydroxy-2-butanone 2-Pentyl-furan 1-Hexanol Dodecamethyl-cyclohexasiloxane Nonanal Acetic acid DL-6-Methyl-5-hepten-2-ol Vanillin, tert-butyldimethylsilyl ether 2,5-bis((Trimethylsily)oxy)-benzaldehyde Decanal Tetradecamethyl-cycloheptasiloxane, (S-(R* ,R* ))-2,3-Butanediol Cyclopentanecarboxylic acid, pentyl ester 2-Triophenecarboxylic acid, octyl ester Oxime-methoxy-phenyl-_ Hexanoic acid Phenol
a
RI b
1.22 ND 1.66 1.78 ND ND 2.88 3.29 ND 4.78 6.29 8.74 11.22 ND 13.27 14.15 14.24 16.14 16.3 17.66 18.35 ND 18.59 19.23 ND ND 23.69 25.26 28.49
1.74 1.34 ND 5.37 2.52 2.56 ND 12 3.73 4.58 ND 17.12 ND 11.55 7.03 22.05 15.37 ND ND ND ND 18.5 26.46 ND 19.24 20.15 8.61 ND ND
Tr
a 976 ND 989 937 ND ND 1027 1040 ND 1085 1132 1207 1284 ND 1355 1386 1389 1457 1463 1514 1543 ND 1553 1580 ND ND 1778 1852 2006
b 721 710 ND 822 743 744 ND 1003 776 800 ND 1160 ND 990 868 1330 1106 ND ND ND ND 1202 1507 1230 1260 911 ND ND
a
b
++++++ ND +++ +++++ ND ND +++++ ++++ ND ++ +++ +++ ++++++ ND +++ ++ ++ ++ ++ ++ ++ ND + +++ ND ND ++++++ ++ +
++++++ + ND ++ +++ ++++ ND ++ +++ +++++++ ND + ND + ++ + ++ ND ND ND ND + + ND ++ +++++ ++++ ND ND
Rfs 7
– – – – – – – 7 7,14
– – – 7,14 14,15
– 14
– – – – 14
– – – – – – –
RT, retention time (min); RI, retention index; a, data from the polar column; b, data from the non-polar column. Rfs, previous reports of detection, references 7, 14, 15. Tr, trace level; tr, 100%; ND, means the compound was not detected. The percentages were calculated using the total ion current (TIC) for each compound relative to the TIC for hexane of 100%.
RESULTS AND DISCUSSION GC-FID analysis
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VOCs of wheat alone obtained from polar and non-polar columns Both the polar and non-polar columns separated similar important compounds of wheat, such as hexane, 1-hexanol and D-limonene (Fig. 1). However, acetone and 1-pentanol were only separated by the polar column and dodecamethyl-cyclohexasiloxane was only separated by the non-polar column. This is likely because acetone and 1-pentanol are polar compounds but dodecamethyl-cyclohexasiloxane is a non-polar compound. The results, therefore, conformed to the separation principle that polar columns retain polar compounds and non-polar columns retain non-polar compounds, which has also been described as ‘like dissolves like’.12 When peak number, peak height and peak area of the polar and non-polar columns were compared, the results showed that the data from the non-polar column produced more peaks and larger peaks than those from the polar column (Fig. 1).
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VOCs of adult R. dominica alone obtained from polar and non-polar columns The non-polar column detected the presence of more VOCs from R. dominica than the polar column (Fig. 2). These included 3-methyl-2-butanol, hexanal and 2-triophenecarboxylic acid, octyl ester. Bromo-methane was detected by both columns and it appears to be the first time that it has been detected from this species, though in low amounts. A comparison of the number of peaks, peak height and peak area between the polar and non-polar columns, indicated that the signal was stronger for the non-polar column (Fig. 2). VOCs of wheat with adult R. dominica obtained from polar and non-polar columns Several VOCs detected by both the polar and non-polar columns were the same compounds, such as hexane, D-limonene and 1-hexanol. However, there were also different compounds detected such as acetone, 3-methyl-1-butanol and 1-pentanol by the polar column, and 1-methylethyl-hydroperoxide, (S)-(+)-2-pentanol, cyclopentanecarboxylic acid, pentyl ester
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SPME-GCMS of volatiles from wheat and R. dominica
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Table 3. VOCs of wheat with adult R. dominica identified from GC-MS analysis using polar and non-polar columns RT VOC
a
Hexane Bromo-methane 1-Methylethyl-hydroperoxide Octane Acetone Hexamethyl-cyclotrisiloxane 2-Butanone Isopropyl alcohol Dimethyl-silanediol (S)-(+)-2-Pentanol 2-Pentanone Decane Trichloromethane Toluene Hexanal 3-Methyl-2-butanol 1-Butanol 1-Penten-3-ol D-Limonene Dodecane 3-Methyl-1-butanol 1-Pentanol 2-Pentyl-furan 2,3-Dimethyl-1-butanol 6-Methyl-5-hepten-2-one 1-Hexanol Cyclopentanecarboxylic acid, 2-penta-decyl ester Dodecamethyl-cyclohexasiloxane 2,3-Dimethyl-2-pentenoic acid Undecane 2-Ethyl-2,5-cyclohexadiene-1,4-dione 1-Octen-3-ol 5-Hydroxy-3,4,4-trimethyl-(E)-2-hexenoic acid 2-Ethyl-1-hexanol Decamethyl-cyclopentasiloxane Cyclopentanecarboxylic acid, pentyl ester 2-Thiophenecarboxylic acid, octyl ester Tridecane Thiophene-2-carboxylic acid, 3-pentyl ester Oxime-methoxy-phenyl-_ Tetradecane Thuiopsene Hexanoic acid Heptadecane 2-Ethyl-hexanoic acid
1.26 ND 1.43 1.58 1.66 1.79 2.11 2.31 ND ND 2.86 3.06 3.46 3.81 4.79 5.29 6.89 7.36 8.12 8.4 8.86 10.2 ND 11.6 12.7 13.3 ND 14.2 ND ND ND 16 16.1 17 ND ND ND ND ND 23.7 ND ND 25.3 ND 27.4
RI b 1.77 1.34 ND ND ND 5.37 ND ND 2.47 2.57 2.44 11.86 ND ND 4.55 ND ND ND 12.82 18.35 ND 3.74 11.55 ND 11.39 ND 13.86 ND 15.06 15.25 15.33 ND 17.77 ND 17.12 19.31 20.21 21.21 21.77 8.52 23.87 24.46 ND 26.39 16.12
Tr
a 977 ND 713 987 989 993 1003 1010 ND ND 1026 1033 1045 1055 1086 1101 1150 1165 1188 1197 1211 1253 ND 1296 1334 1355 ND 1386 ND ND ND 1450 1456 1486 ND ND ND ND ND 1779 ND ND 1852 ND 1952
b 722 710 ND ND ND 822 ND ND 742 744 741 999 ND ND 799 ND ND ND 1028 1197 ND 777 990 ND 986 ND 1060 ND 1096 1102 1105 ND 1180 ND 1160 1231 1262 1297 1319 909 1403 1427 ND 1504 1129
a ++++++ ND ++++ + +++ + + ++ ND ND ++ + + + ++ + + + +++ + + ++++ ND + + +++++ ND tr ND ND ND tr ++ + ND ND ND ND ND + ND ND + ND +
b ++++++ tr ND ND ND ++ ND ND ++ +++++ +++ ++ ND ND ++++ ND ND ND +++++ ++ ND +++++ + ND + ND + ND ++ + ++ ND + ND ++ ++++++ +++++++ + ++ ++ + tr ND + +
Rfs 7
– – – – – 7
– – – 7,14
– – – 7,13,14 7
– – – – – 7 7,14
– 7 7,14,15
– – – – – 7
– – – – – – – – – – – – –
RT, retention time (min); RI, retention index; a, data are from the polar column; b, data are from the non-polar column. Rfs: Previous reports of detection, references: 7, 13, 14, 15. Trace level: tr, 100%; ND, means the compound was not detected. The percentages were calculated using the total ion current (TIC) for each compound relative to the TIC for hexane of100%.
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GC-MS analysis Hexane was the VOC with the biggest peak that was detected by both the polar and non-polar columns and was also found in all of the three samples tested. Thus, for comparative purposes, the trace
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and 2-thiophenecarboxylic acid, octyl ester by the non-polar column (Fig. 3). Again, the results conformed to the separation principle and a comparison of peak numbers, peak heights and peak areas, showed that the non-polar column was more sensitive than the polar column.
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Figure 4. Total peak areas detected by polar and non-polar columns and the total shared same peak areas detected by both polar and non-polar columns from wheat alone, R. dominica adults alone and wheat with R. dominica adults with a variation less than 9% (SD) between three replicates and the duplicate injections. Bars represent standard deviations of the mean. Table 4. Quantities of VOCs from wheat alone, R. dominica adults alone and wheat with R. dominica adults detected by polar and non-polar columns
Sample
Type of GC column
Total GC peak area
Wheat
Polar Non-polar Polar Non-polar Polar Non-polar
431 903 220 524 492 085 205 622 304 497 116 481 507 952 749 766 464 356
R. dominica Wheat + R. dominica
Total shared same GC peak area*
Total no. of VOCs
335 054 757 418 938 377 113 160 703 363 657 499 308 220 005 284 729 277
28 25 21 18 28 28
Total no. of shared same VOCs 13 13 9 9 11 11
Total shared same GC peak area of total GC peak area (%) 77.58 79.88 55.03 73.15 60.68 37.15
* Shared same area refers to the total peak areas detected by both polar and non-polar columns. GC peak areas were measured.
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levels of all VOCs identified (Table 1, Table 2 and Table 3) have been determined relative to hexane.
Overall, 15 VOCs were detected by the polar column, while 12 were detected only by the non-polar column.
VOCs of wheat alone obtained from polar and non-polar columns In total, 40 VOCs from wheat were detected using the polar and non-polar columns, 13 of these were detected by both columns (Table 1). The total peak area of the non-polar column was bigger than that of the polar column (Fig. 4). The percentage of the total peak area of VOCs detected by the polar column that were also detected by the non-polar column was 77.58%, while the percentage of the total peak area of VOCs detected by the non-polar column that were also detected by the polar column was 79.88% (Table 4). This indicates that the polar and non-polar columns can both identify the most typical VOCs from wheat, such as hexane, hexanal, D-limonene 1-pentanol and 1-hexanol. However, some compounds like acetone, isopropyl alcohol and 2-propenylidene-cyclobutene were only identified by the polar column, while bromo-methane, 1-methoxy-2-propanone, dimethyl-silanediol, hexamethyl-cyclotrisiloxane and octamethylcyclotetrasiloxane were identified only by the non-polar column.
VOCs from R. dominica adults obtained from polar and non-polar columns Twenty-nine VOCs from R. dominica were detected using the polar and non-polar columns, with nine of these detected by both columns (Table 2). The total peak area of the non-polar column was obviously bigger than the polar column (Fig. 4). The percentage of the total peak area of VOCs detected by the polar column that were also detected by the non-polar column was 55.03%, while the percentage of the total peak area of VOCs detected by the non-polar column that were also detected by the polar column was 73.15% (Table 4). Eleven VOCs, of which 3-hydroxy-2-butanone and acetic acid ethenyl ester were the most abundant, were only detected by the polar column, while eight VOCs, of which 2-triophenecarboxylic acid, octyl ester and 3-methyl-2-butanol were the most abundant, were only detected by the non-polar column. In previous studies dominicalure-1 and dominicalure-2 were identified as aggregation pheromones of R. dominica.7,16 However,
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SPME-GCMS of volatiles from wheat and R. dominica these were not observed in the current study, which could possibly be due to the detection temperature used. Williams et al.16 collected VOCs of R. dominica at 28∘ C and Seitz and Ram7 collected at 80∘ C, whereas 25∘ C was used in the current study.
www.soci.org work is also required on the potential effects of such conditions on the capture of VOCs.
ACKNOWLEDGEMENT VOCs of wheat with R. dominica adults obtained from polar and non-polar columns Forty-five VOCs were detected from the wheat with R. dominica samples and 12 of these were detected by both columns (Table 3). The total peak area of the non-polar column was bigger than the polar column (Fig. 4). The percentage of the total peak area of VOCs detected by the polar column that were also detected by the non-polar column was 60.68%, while the percentage of the total peak area of VOCs detected by the non-polar column that were also detected by the polar column was 37.15% (Table 4). Sixteen VOCs, of which 1-hexanol and acetone were the most abundant, were only detected by the polar column, while 17 VOCS as well, of which four compounds were considerably abundant, were only detected by the non-polar column. These four VOCs, in descending quantity, were 2-thiophenecarboxylic acid, octyl ester; cyclopentanecarboxylic acid, pentyl ester; (S)-(+)-2-pentanol and 1-methylethyl-hydroperoxide. Seitz14 found several compounds from samples of wheat infested by R. dominica, but these were not detected in the present study. This again could be due to the fact that a different collection temperature (28∘ C) was used compared to the current study.
CONCLUSION The GC-FID results appeared to show that most of the compounds in the three different samples, wheat, R. dominica, and wheat with R. dominica were non-polar compounds due to the fact that more peaks and larger peaks were produced. This would indicate that the non-polar column is more sensitive and can detect more VOCs than the polar column. However, the GC-MS results showed that in two out of the three samples types, a slightly greater number of VOCs were identified from the polar column. The total number for the three samples was 43 from the polar column compared to 39 from the non-polar column. Conversely, a considerably greater number of VOCs unique to a given sample were identified from the non-polar column. Here the total number was 30 from the non-polar column compared to 18 from the polar column. This study was the first screening of a major food grain, wheat, and one of the most important storage pests, R. dominica, for volatile organic compounds using GC-FID and GC-MS coupled with both polar and non-polar columns at 25∘ C. A total of 114 compounds were detected of which 48 were specific to one or other of the three samples tested. This data base can form the basis of enquiry into the relationship between storage and grain quality, and insect infestation and grain quality. For example, it would be valuable to determine whether, or to what degree, VOCs vary as wheat ages or when infestations occur and as they increase. However, there are several variables that can impact the detection of VOCs, such as the equipment settings used, and the temperature and moisture content of the wheat. Therefore, further
We thank the Australian Government’s Cooperative Research Centre Program and the China Scholarship Council (CSC) for financial support. We would also like to acknowledge the support of staff from the Postharvest Biosecurity and Food Safety Laboratory (Murdoch University) for providing technical assistance.
REFERENCES 1 Evans P, Persaud KC, Mcneish AS, Sneath RW, Hobson N and Magan N, Evaluation of a radial base function neural network for the determination of wheat quality from electronic nose data. Sensor Actuat B: Chem 69:348–358 (2000). 2 Zhang HM and Wang J, Detection of age and insect damage incurred by wheat, with an electronic nose. J Stored Prod Res 43:489–495 (2007). 3 Singh CB, Jayas DS and Paliwal J, Detection of insect-damaged wheat kernels using near-infrared hyperspectral imaging. J Stored Prod Res 45:151–158 (2009). 4 Widjaja R, Craske JD and Wootton M, Changes in volatiles components of paddy, brown and white fragrant rice during storage. J Sci Food Agric 71:218–224 (1996). 5 Maria ABC, Caneppele C, Lazzari FA and Lazzari SMN, Correlation between the infestation level of Sitophilus zeamais Motschulsky, 1855 (Coleoptera, Curculionidae) and the quality factors of stored corn, Zea mays L. (Poaceae). Revista Brasileira de Entomologia 47:625–630 (2003). 6 Pavia DL, Gary M, George SK and Randall GE, Introduction to Organic Laboratory Techniques, 4th edition. Thomson Brooks/Cole, Pacific Grove, California, pp. 797–817 (2006). 7 Seitz LM and Ram MS, Metabolites of lesser grain borer in grains. J Agric Food Chem 52:898–908 (2004). 8 Perkowski J, Busko M and Chmielewski J, Content of trichodiene and analysis of fungal volatiles (electronic nose) in wheat and triticale grain naturally infected and inoculated with Fusarium culmorum. Int J Food Microbiol 75:600–625 (2008). 9 Piesik D, Weaverf DK and Runyon JB, Behavioural responses of wheat stem sawflies to wheat volatiles. Agric For Entomol 10:245–253 (2008). 10 Sumana B and Charlie S, Analysis of headspace compounds of distillers grains using SPME in conjunction with GC/MS and TGA. Cereal Sci 33:223–229 (2001). 11 Niinemets U, Loreto F and Reichstein M, Physiological and physico-chemical controls on foliar volatile organic compound emissions. Trends Plant Sci 9:180–186 (2004). 12 Risticevic S, Lord H, Górecki T, Catherine LA and Pawliszyn J, Protocol for solid-phase microextraction method development. Nat Protoc 5:122–139 (2009). 13 Maga JA, Cereal volatiles, A review. J Agric Food Chem 26:175–178 (1978). 14 Seitz LM, Volatile compounds in wheat cultivars from several locations in Kansas, in Food Flavors: Generation, Analysis and Process Influence, ed. by Charalambous G. Elsevier Science, Amsterdam, pp. 2183–2203 (1995). 15 Bu´sko M, Jelen´ H, Góral T, Chmielewski J, Stuper K, Szwajkowska-Michałek L, et al., Volatile metabolites in various cereal grains. Food Addit Contam A 27:1574–1581 (2010). 16 Williams HJ, Silverstein RM, Burkholder WE and Khorramshahi A, Dominicalure 1 and 2: Components of aggregation pheromone from male lesser grain borer Rhyzopertha dominica (F.). J Chem Ecol 7:760–782 (1981).
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© 2015 Society of Chemical Industry
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Revised: 19 May 2015
Accepted article published: 28 May 2015
Published online in Wiley Online Library: 6 July 2015
(wileyonlinelibrary.com) DOI 10.1002/jsfa.7274
Analysis of volatiles from stored wheat and Rhyzopertha dominica (F.) with solid phase microextraction–gas chromatography mass spectrometry Yonghao Niu,a,b,c Lei Hua,a Giles Hardy,c Manjree Agarwalb,c and Yonglin Renb,c* Abstract BACKGROUND: Volatile organic compounds (VOCs) contribute significantly to food flavour and can be used as indicators of quality, age of storage, and hygiene condition of stored products. The VOCs in the headspace of three different samples – healthy wheat, Rhyzopertha dominica, and wheat with R. dominica – were analysed at 25∘ C by solid phase micro-extraction (SPME) coupled with gas chromatography–flame ionisation detection (GC-FID) and gas chromatography–mass spectrometry (GC-MS). All the experimental conditions were kept consistent except a polar column and a non-polar column were used to assess the differences in volatile fingerprints. RESULTS: A total of 114 volatiles were identified by both the polar and non-polar columns, of which 48 were specific to one of the three samples tested. The volatiles were mainly carbonyl chemical compounds such as aldehydes, ketones and alcohols. GC-MS results showed slightly more VOCs were identified from the polar column. The total number for the three samples was 43 from the polar column compared to 39 from the non-polar column. Conversely, 30 VOCs unique to a given sample were identified from the non-polar column compared to 18 from the polar column. CONCLUSION: The use of both polar and non-polar columns is essential to capture the full range of VOCs produced by the three specific sample types investigated. The data can form the basis of enquiry into the relationship between storage and grain quality, and insect infestation and grain quality by observing the impact that these circumstances have on the production of volatile organic compounds. © 2015 Society of Chemical Industry Keywords: wheat; Rhyzopertha dominica; solid phase microextraction (SPME); gas chromatography–mass spectrometry; volatile organic compounds
INTRODUCTION
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∗
Correspondence to: YongLin Ren, Murdoch University, School of Veterinary and Life Sciences, Murdoch, WA 6150, Australia. E-mail: [email protected]
a College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China b Australia Cooperative Research Centre for National Plant Biosecurity, LPO Box 5012, Bruce, ACT 2617, Australia c School of Veterinary and Life Sciences, Murdoch University, South Street, Murdoch, WA 6150, Australia
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Wheat is an essential food for human beings and how to maintain the quality of stored wheat has been a research focus for many years.1,2 Rhyzopertha dominica (F.) is one of the primary insect pests of stored wheat where it causes major physical and off-odour damage.3 Some grain volatiles contribute significantly to food flavour and can be used as indicators of the quality, age of storage and hygiene condition of stored products.4,5 At present, head-space solid phase micro-extraction (HS-SPME) coupled with gas chromatography–flame ionisation detection (GC-FID) and gas chromatography–mass spectrometry (GC-MS) are useful techniques for identifying volatiles in stored wheat, and might be used to ascertain if the grain is infested with insects or not.6,7 Several researchers have used HS-SPME coupled with GC-MS methods to analyse the volatile organic compounds (VOCs) of stored wheat and R. dominica. Most of these studies have chosen non-polar columns to separate VOCs7 – 10 and many
low-molecular-weight organic compounds emitted from stored grains have been identified.11 However, to date, there are no reports where polar columns have been used to separate volatiles from the headspace of stored grain. In this study, polar and non-polar columns are used for the first time to separate and compare VOCs from the following three sample types at 25∘ C: Wheat alone, R. dominica adults alone and wheat infested with R. dominica adults
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MATERIALS AND METHODS
A
Grain pre-treatment Freshly harvested (2011–2012) wheat (Australia Standard Wheat I) used for this investigation was procured from CBH (Co-operative Bulk Handling), Western Australia. The moisture content was 11.5% (w/w, HOH-Express 50, Electronic Moisture Meter; PFEUFFER, Kitzingen, Germany). The wheat sample was placed in sealed glass jars (4 L) and put in a freezer at −4∘ C for 1 week to ensure it was free of any live insects. The wheat was then stored at 4∘ C until use.
B
Insect cultures The insect species R. dominica (strain No. MUWRD 7) was obtained from the Stored Grain Research Laboratory, School of Veterinary and Life Sciences, Murdoch University, Perth, Australia. About 200 adult R. dominica were added to 400 g of whole wheat in 500-mL jars with meshed lids to obtain a mixed age insect population. The experimental insects were reared in the dark at 30∘ C and 60% relative humidity (RH), and incubated for 4–5 weeks until adults from the next generation emerged.
Figure 2. Chromatograms of R. dominica: (A) polar column, (B) non-polar column. (A) 1,3-Hydroxy-2-butanone; (B) 1, bromo-methane; 2, 3-methyl-2-butanol; 3, hexanal; and 4, 2-triophenecarboxylic acid, octyl ester.
A
B
Glassware and SPME fibres One hundred millilitre Erlenmeyer flasks (Quickfit, Cat. No FE 100/3; Fisher Scientific, Loughborough, UK) each equipped with a cone/screw-thread adapter (Code ST 5313; Crown Scientific, Wantirna South, Victoria, Australia) with a 7/16′′ blue septum (Cat. No. 6518; Grace Davison Discovery Sciences, Melbourne, Victoria, Australia) were used for sample preparation. The measured volume of each Erlenmeyer flask and inlet system was calculated from the weight of water required to fill the container. The SPME fibre used was 50/30 μm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS; Cat. No. 57348-U), from Analytical Sigma–Aldrich (Sydney, Australia), and it was conditioned prior to use in accordance with the manufacturer’s recommendations. Headspace extraction was through a 7/16′′ blue septum (Cat. No. 6518; Grace Davison Discovery Sciences). The fibre was inserted into the headspace of the flask containing samples at the end of the defined extraction time, the fibre was withdrawn from the headspace into the needle. The fibre holder was removed from the extraction flask and inserted into the injection port. The fibre was extended into a GC-PFPD inlet where sample components were desorbed. Each sample was replicated for three times and duplicate injections. A
B
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Figure 1. Chromatograms of wheat: (A) polar column, (B) non-polar column. (A) 1, Hexane; 2, acetone; 3, D-limonene; 4, 1-pentanol; and 5, 1-hexanol. (B) 1, Hexane; 2, hexanal; 3, 1-hexanol; 4, D-limonene; and 5, dodecamethyl-cyclohexasiloxane.
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Figure 3. Chromatograms of wheat with R. dominica adults: (A) polar column, (B) non-polar column. (A) 1, Hexane; 2, acetone; 3, D-limonene; 4, 3-methyl-1-butanol; 5, 1-pentanol; 6, 1-hexanol; and 7, 5-hydroxy-3,4,4trimethyl-(E)-2-hexenoic acid. (B) 1, 1-methylethyl-hydroperoxide; 2, hexane; 3, (S)-(+)-2-pentanol; 4, 1-hexanol; 5, decane; 6, D-limonene; 7, cyclopen-tanecarboxylic acid, pentyl ester; 8, 2-thiophenecarboxylic acid, octyl ester; and 9, thiophene-2-carboxylic acid, 3-pentyl ester.
Sample preparation Three different treatment samples were examined. These were: (1) 80 g wheat, (2) 100 R. dominica adults and (3) 80 g wheat with 100 R. dominica adults. Each sample was placed in a 100 mL Erlenmeyer flask and sealed with the cone/screw-thread adapter. The samples were then processed after conditioning for 24 h at 25 ± 2∘ C in a temperature controlled room.
Gas chromatography–flame ionisation detector An Agilent 6890 GC (serial# US00021731, USA) manufactured by Agilent Technology (Palo Alto, CA, USA) with a flame ionisation detector (FID) was used to analyse the volatile profiles extracted by HS-SPME. The columns used in this experiment were a Stabilwax® polar column (dimensions: 30 m × 0.25 mm × 0.25 μm film thickness, ZB-WAX, Cat. No. #10623; Torrance, CA, USA) and an Rxi®-5ms non-polar column (dimensions: 30 m × 0.25 mm × 0.25 μm film thickness, RESTEK, Cat. No. #13423; Bellefonte, PA, USA). The following GC conditions were used. Hydrogen was used as the carrier gas at a constant speed of 40 mL min−1 in the split-less mode. The GC inlet was operated at 250∘ C and FID temperature was 250∘ C. The oven temperature program used for optimising the GC condition was: 45∘ C for 5 min, increasing from 45∘ C to 250∘ C at 5∘ C min−1 and being held for 5 min at each increment with a total run of 51 min. Each sample was replicated for three times and duplicate injections.
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SPME-GCMS of volatiles from wheat and R. dominica
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Table 1. VOCs of wheat identified from GC-MS analysis using polar and non-polar columns RT VOC Hexane Bromo-methane 1-Methoxy-2-propanone Acetone 2-Butanone Isopropyl alcohol Cyclopentanol Dimethyl-silanediol Heptane Decane 3-Methyl-1-butanol Trichloromethane 2-Propenylidene-cyclobutene 1-Propanol Hexanal Undecane 3-Methyl-2-butanol Hexamethyl-cyclotrisiloxane 1-Butanol 1-Penten-3-ol D-Limonene Dodecane Decamethyl-cyclopentasiloxane 2-Pentyl-furan 1-Pentanol 2,3-Dimethyl-1-butanol Octamethyl-cyclotetrasiloxane 1-Hexanol Dodecamethyl-cyclohexasiloxane Nonanal 1-Heptanol Acetic acid 2-Ethyl-1-hexanol Tetradecamethyl-cycloheptasiloxane Tridecane Oxime-methoxy-phenyl-_ Tetradecane Hexanoic acid Pentadecane 2-Ethyl-hexanoic acid
a 1.27 ND ND 1.67 2.12 2.33 ND ND ND 3.07 ND 3.47 3.82 3.9 4.81 5.01 5.31 ND 6.89 7.38 8.13 8.42 8.79 9.41 10.23 11.64 ND 13.3 14.16 ND 16.07 16.12 16.97 18.6 ND 23.69 ND 25.26 ND 27.36
RI b 1.76 1.34 1.43 ND ND ND 2.39 2.49 2.58 11.86 3.09 ND ND 1.6 4.55 15.25 ND 5.37 ND ND 12.81 18.35 17.11 ND 3.73 ND 12 7.06 22.05 15.37 ND ND ND 26.46 21.21 8.56 23.87 ND 26.39 ND
a 977 ND ND 990 1004 1010 ND ND ND 1033 ND 1045 1056 1058 1086 1092 1102 ND 1150 1165 1189 1197 1209 1228 1253 1296 ND 1355 1386 ND 1455 1456 1487 1554 ND 1778 ND 1852 ND 1952
Tr b 722 710 713 ND ND ND 739 742 745 999 759 ND ND 717 799 1102 ND 822 ND ND 1028 1197 1159 ND 776 ND 1003 869 1330 1106 ND ND ND 1507 1297 910 1403 ND 1504 ND
a ++++++ ND ND +++ + ++ ND ND ND ++ ND + ++ + ++ + + ND + + ++++ + tr + ++++ + ND ++++++ + ND + + + + ND ++ ND + ND +
b ++++++ ++ +++ ND ND ND + +++ + ++ tr ND ND + ++ + ND ++ ND ND +++ ++ +++ ND +++ ND ++ +++++ + tr ND ND ND tr + ++++ + ND + ND
Rfs – – – – – – – – 14
– – – – – 13,14
– – – – – – – – 14
– – – 14,15
– 14 14,15
– – – – – – – – –
RT, retention time (min); RI, retention index; a, the data from the polar column; b, the data from the non-polar column. Rfs, previous reports of detection, references: 13–15. Trace level: tr, 100%; ND, means the compound was not detected. The percentages were calculated using the total ion current (TIC) for each compound relative to the TIC for hexane of 100%.
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was 70 eV and the scanning was performed from 35 to 500 atomic mass units. The volatiles were identified by comparison of the mass spectrum with the NIST08 mass spectra library. Statistical analysis The variations (standard deviation) of VOCs total GC peak areas between three replicates and the duplicate injections in comparison with average readings were analysed by Microsoft Excel 2007.
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Gas chromatography–mass spectrometry An Agilent 6890 GC (serial# US00035629, USA) coupled with an Agilent 5793 Network mass selective detector (MSD) together with an Agilent ChemStation (serial# US02480181, USA) was used. The temperatures of the GC inlet (at split-less mode), interface and MS source, were 250∘ C, 250∘ C and 230∘ C, respectively. Helium was used as the carrier gas at a constant airflow of 1.0 mL min−1 . Other aspects such as column and oven temperature were the same as the GC-FID conditions described above. The ionisation potential
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Table 2. VOCs of R. dominica adults identified from GC-MS analysis using polar and non-polar columns RT VOC Hexane Bromo-methane Acetone Hexamethyl-cyclotrisiloxane Dimethyl-silanediol 3-Methyl-2-butanol Acetic acid ethenyl ester Octamethyl-cyclotetrasiloxane 1-Pentanol Hexanal 2-Pentanol Decamethyl-cyclopentasiloxane 3-Hydroxy-2-butanone 2-Pentyl-furan 1-Hexanol Dodecamethyl-cyclohexasiloxane Nonanal Acetic acid DL-6-Methyl-5-hepten-2-ol Vanillin, tert-butyldimethylsilyl ether 2,5-bis((Trimethylsily)oxy)-benzaldehyde Decanal Tetradecamethyl-cycloheptasiloxane, (S-(R* ,R* ))-2,3-Butanediol Cyclopentanecarboxylic acid, pentyl ester 2-Triophenecarboxylic acid, octyl ester Oxime-methoxy-phenyl-_ Hexanoic acid Phenol
a
RI b
1.22 ND 1.66 1.78 ND ND 2.88 3.29 ND 4.78 6.29 8.74 11.22 ND 13.27 14.15 14.24 16.14 16.3 17.66 18.35 ND 18.59 19.23 ND ND 23.69 25.26 28.49
1.74 1.34 ND 5.37 2.52 2.56 ND 12 3.73 4.58 ND 17.12 ND 11.55 7.03 22.05 15.37 ND ND ND ND 18.5 26.46 ND 19.24 20.15 8.61 ND ND
Tr
a 976 ND 989 937 ND ND 1027 1040 ND 1085 1132 1207 1284 ND 1355 1386 1389 1457 1463 1514 1543 ND 1553 1580 ND ND 1778 1852 2006
b 721 710 ND 822 743 744 ND 1003 776 800 ND 1160 ND 990 868 1330 1106 ND ND ND ND 1202 1507 1230 1260 911 ND ND
a
b
++++++ ND +++ +++++ ND ND +++++ ++++ ND ++ +++ +++ ++++++ ND +++ ++ ++ ++ ++ ++ ++ ND + +++ ND ND ++++++ ++ +
++++++ + ND ++ +++ ++++ ND ++ +++ +++++++ ND + ND + ++ + ++ ND ND ND ND + + ND ++ +++++ ++++ ND ND
Rfs 7
– – – – – – – 7 7,14
– – – 7,14 14,15
– 14
– – – – 14
– – – – – – –
RT, retention time (min); RI, retention index; a, data from the polar column; b, data from the non-polar column. Rfs, previous reports of detection, references 7, 14, 15. Tr, trace level; tr, 100%; ND, means the compound was not detected. The percentages were calculated using the total ion current (TIC) for each compound relative to the TIC for hexane of 100%.
RESULTS AND DISCUSSION GC-FID analysis
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VOCs of wheat alone obtained from polar and non-polar columns Both the polar and non-polar columns separated similar important compounds of wheat, such as hexane, 1-hexanol and D-limonene (Fig. 1). However, acetone and 1-pentanol were only separated by the polar column and dodecamethyl-cyclohexasiloxane was only separated by the non-polar column. This is likely because acetone and 1-pentanol are polar compounds but dodecamethyl-cyclohexasiloxane is a non-polar compound. The results, therefore, conformed to the separation principle that polar columns retain polar compounds and non-polar columns retain non-polar compounds, which has also been described as ‘like dissolves like’.12 When peak number, peak height and peak area of the polar and non-polar columns were compared, the results showed that the data from the non-polar column produced more peaks and larger peaks than those from the polar column (Fig. 1).
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VOCs of adult R. dominica alone obtained from polar and non-polar columns The non-polar column detected the presence of more VOCs from R. dominica than the polar column (Fig. 2). These included 3-methyl-2-butanol, hexanal and 2-triophenecarboxylic acid, octyl ester. Bromo-methane was detected by both columns and it appears to be the first time that it has been detected from this species, though in low amounts. A comparison of the number of peaks, peak height and peak area between the polar and non-polar columns, indicated that the signal was stronger for the non-polar column (Fig. 2). VOCs of wheat with adult R. dominica obtained from polar and non-polar columns Several VOCs detected by both the polar and non-polar columns were the same compounds, such as hexane, D-limonene and 1-hexanol. However, there were also different compounds detected such as acetone, 3-methyl-1-butanol and 1-pentanol by the polar column, and 1-methylethyl-hydroperoxide, (S)-(+)-2-pentanol, cyclopentanecarboxylic acid, pentyl ester
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SPME-GCMS of volatiles from wheat and R. dominica
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Table 3. VOCs of wheat with adult R. dominica identified from GC-MS analysis using polar and non-polar columns RT VOC
a
Hexane Bromo-methane 1-Methylethyl-hydroperoxide Octane Acetone Hexamethyl-cyclotrisiloxane 2-Butanone Isopropyl alcohol Dimethyl-silanediol (S)-(+)-2-Pentanol 2-Pentanone Decane Trichloromethane Toluene Hexanal 3-Methyl-2-butanol 1-Butanol 1-Penten-3-ol D-Limonene Dodecane 3-Methyl-1-butanol 1-Pentanol 2-Pentyl-furan 2,3-Dimethyl-1-butanol 6-Methyl-5-hepten-2-one 1-Hexanol Cyclopentanecarboxylic acid, 2-penta-decyl ester Dodecamethyl-cyclohexasiloxane 2,3-Dimethyl-2-pentenoic acid Undecane 2-Ethyl-2,5-cyclohexadiene-1,4-dione 1-Octen-3-ol 5-Hydroxy-3,4,4-trimethyl-(E)-2-hexenoic acid 2-Ethyl-1-hexanol Decamethyl-cyclopentasiloxane Cyclopentanecarboxylic acid, pentyl ester 2-Thiophenecarboxylic acid, octyl ester Tridecane Thiophene-2-carboxylic acid, 3-pentyl ester Oxime-methoxy-phenyl-_ Tetradecane Thuiopsene Hexanoic acid Heptadecane 2-Ethyl-hexanoic acid
1.26 ND 1.43 1.58 1.66 1.79 2.11 2.31 ND ND 2.86 3.06 3.46 3.81 4.79 5.29 6.89 7.36 8.12 8.4 8.86 10.2 ND 11.6 12.7 13.3 ND 14.2 ND ND ND 16 16.1 17 ND ND ND ND ND 23.7 ND ND 25.3 ND 27.4
RI b 1.77 1.34 ND ND ND 5.37 ND ND 2.47 2.57 2.44 11.86 ND ND 4.55 ND ND ND 12.82 18.35 ND 3.74 11.55 ND 11.39 ND 13.86 ND 15.06 15.25 15.33 ND 17.77 ND 17.12 19.31 20.21 21.21 21.77 8.52 23.87 24.46 ND 26.39 16.12
Tr
a 977 ND 713 987 989 993 1003 1010 ND ND 1026 1033 1045 1055 1086 1101 1150 1165 1188 1197 1211 1253 ND 1296 1334 1355 ND 1386 ND ND ND 1450 1456 1486 ND ND ND ND ND 1779 ND ND 1852 ND 1952
b 722 710 ND ND ND 822 ND ND 742 744 741 999 ND ND 799 ND ND ND 1028 1197 ND 777 990 ND 986 ND 1060 ND 1096 1102 1105 ND 1180 ND 1160 1231 1262 1297 1319 909 1403 1427 ND 1504 1129
a ++++++ ND ++++ + +++ + + ++ ND ND ++ + + + ++ + + + +++ + + ++++ ND + + +++++ ND tr ND ND ND tr ++ + ND ND ND ND ND + ND ND + ND +
b ++++++ tr ND ND ND ++ ND ND ++ +++++ +++ ++ ND ND ++++ ND ND ND +++++ ++ ND +++++ + ND + ND + ND ++ + ++ ND + ND ++ ++++++ +++++++ + ++ ++ + tr ND + +
Rfs 7
– – – – – 7
– – – 7,14
– – – 7,13,14 7
– – – – – 7 7,14
– 7 7,14,15
– – – – – 7
– – – – – – – – – – – – –
RT, retention time (min); RI, retention index; a, data are from the polar column; b, data are from the non-polar column. Rfs: Previous reports of detection, references: 7, 13, 14, 15. Trace level: tr, 100%; ND, means the compound was not detected. The percentages were calculated using the total ion current (TIC) for each compound relative to the TIC for hexane of100%.
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GC-MS analysis Hexane was the VOC with the biggest peak that was detected by both the polar and non-polar columns and was also found in all of the three samples tested. Thus, for comparative purposes, the trace
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and 2-thiophenecarboxylic acid, octyl ester by the non-polar column (Fig. 3). Again, the results conformed to the separation principle and a comparison of peak numbers, peak heights and peak areas, showed that the non-polar column was more sensitive than the polar column.
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Figure 4. Total peak areas detected by polar and non-polar columns and the total shared same peak areas detected by both polar and non-polar columns from wheat alone, R. dominica adults alone and wheat with R. dominica adults with a variation less than 9% (SD) between three replicates and the duplicate injections. Bars represent standard deviations of the mean. Table 4. Quantities of VOCs from wheat alone, R. dominica adults alone and wheat with R. dominica adults detected by polar and non-polar columns
Sample
Type of GC column
Total GC peak area
Wheat
Polar Non-polar Polar Non-polar Polar Non-polar
431 903 220 524 492 085 205 622 304 497 116 481 507 952 749 766 464 356
R. dominica Wheat + R. dominica
Total shared same GC peak area*
Total no. of VOCs
335 054 757 418 938 377 113 160 703 363 657 499 308 220 005 284 729 277
28 25 21 18 28 28
Total no. of shared same VOCs 13 13 9 9 11 11
Total shared same GC peak area of total GC peak area (%) 77.58 79.88 55.03 73.15 60.68 37.15
* Shared same area refers to the total peak areas detected by both polar and non-polar columns. GC peak areas were measured.
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levels of all VOCs identified (Table 1, Table 2 and Table 3) have been determined relative to hexane.
Overall, 15 VOCs were detected by the polar column, while 12 were detected only by the non-polar column.
VOCs of wheat alone obtained from polar and non-polar columns In total, 40 VOCs from wheat were detected using the polar and non-polar columns, 13 of these were detected by both columns (Table 1). The total peak area of the non-polar column was bigger than that of the polar column (Fig. 4). The percentage of the total peak area of VOCs detected by the polar column that were also detected by the non-polar column was 77.58%, while the percentage of the total peak area of VOCs detected by the non-polar column that were also detected by the polar column was 79.88% (Table 4). This indicates that the polar and non-polar columns can both identify the most typical VOCs from wheat, such as hexane, hexanal, D-limonene 1-pentanol and 1-hexanol. However, some compounds like acetone, isopropyl alcohol and 2-propenylidene-cyclobutene were only identified by the polar column, while bromo-methane, 1-methoxy-2-propanone, dimethyl-silanediol, hexamethyl-cyclotrisiloxane and octamethylcyclotetrasiloxane were identified only by the non-polar column.
VOCs from R. dominica adults obtained from polar and non-polar columns Twenty-nine VOCs from R. dominica were detected using the polar and non-polar columns, with nine of these detected by both columns (Table 2). The total peak area of the non-polar column was obviously bigger than the polar column (Fig. 4). The percentage of the total peak area of VOCs detected by the polar column that were also detected by the non-polar column was 55.03%, while the percentage of the total peak area of VOCs detected by the non-polar column that were also detected by the polar column was 73.15% (Table 4). Eleven VOCs, of which 3-hydroxy-2-butanone and acetic acid ethenyl ester were the most abundant, were only detected by the polar column, while eight VOCs, of which 2-triophenecarboxylic acid, octyl ester and 3-methyl-2-butanol were the most abundant, were only detected by the non-polar column. In previous studies dominicalure-1 and dominicalure-2 were identified as aggregation pheromones of R. dominica.7,16 However,
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SPME-GCMS of volatiles from wheat and R. dominica these were not observed in the current study, which could possibly be due to the detection temperature used. Williams et al.16 collected VOCs of R. dominica at 28∘ C and Seitz and Ram7 collected at 80∘ C, whereas 25∘ C was used in the current study.
www.soci.org work is also required on the potential effects of such conditions on the capture of VOCs.
ACKNOWLEDGEMENT VOCs of wheat with R. dominica adults obtained from polar and non-polar columns Forty-five VOCs were detected from the wheat with R. dominica samples and 12 of these were detected by both columns (Table 3). The total peak area of the non-polar column was bigger than the polar column (Fig. 4). The percentage of the total peak area of VOCs detected by the polar column that were also detected by the non-polar column was 60.68%, while the percentage of the total peak area of VOCs detected by the non-polar column that were also detected by the polar column was 37.15% (Table 4). Sixteen VOCs, of which 1-hexanol and acetone were the most abundant, were only detected by the polar column, while 17 VOCS as well, of which four compounds were considerably abundant, were only detected by the non-polar column. These four VOCs, in descending quantity, were 2-thiophenecarboxylic acid, octyl ester; cyclopentanecarboxylic acid, pentyl ester; (S)-(+)-2-pentanol and 1-methylethyl-hydroperoxide. Seitz14 found several compounds from samples of wheat infested by R. dominica, but these were not detected in the present study. This again could be due to the fact that a different collection temperature (28∘ C) was used compared to the current study.
CONCLUSION The GC-FID results appeared to show that most of the compounds in the three different samples, wheat, R. dominica, and wheat with R. dominica were non-polar compounds due to the fact that more peaks and larger peaks were produced. This would indicate that the non-polar column is more sensitive and can detect more VOCs than the polar column. However, the GC-MS results showed that in two out of the three samples types, a slightly greater number of VOCs were identified from the polar column. The total number for the three samples was 43 from the polar column compared to 39 from the non-polar column. Conversely, a considerably greater number of VOCs unique to a given sample were identified from the non-polar column. Here the total number was 30 from the non-polar column compared to 18 from the polar column. This study was the first screening of a major food grain, wheat, and one of the most important storage pests, R. dominica, for volatile organic compounds using GC-FID and GC-MS coupled with both polar and non-polar columns at 25∘ C. A total of 114 compounds were detected of which 48 were specific to one or other of the three samples tested. This data base can form the basis of enquiry into the relationship between storage and grain quality, and insect infestation and grain quality. For example, it would be valuable to determine whether, or to what degree, VOCs vary as wheat ages or when infestations occur and as they increase. However, there are several variables that can impact the detection of VOCs, such as the equipment settings used, and the temperature and moisture content of the wheat. Therefore, further
We thank the Australian Government’s Cooperative Research Centre Program and the China Scholarship Council (CSC) for financial support. We would also like to acknowledge the support of staff from the Postharvest Biosecurity and Food Safety Laboratory (Murdoch University) for providing technical assistance.
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