J. Agric. Food Chem. 2003, 51, 2115−2120
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Analysis of Terpenoids from Hemlock (Tsuga) Species by Solid-Phase Microextraction/Gas Chromatography/Ion-Trap Mass Spectrometry ANTHONY F. LAGALANTE*,†
AND
MICHAEL E. MONTGOMERY§
Worthington Scranton Campus, The Pennsylvania State University, 120 Ridgeview Drive, Dunmore, Pennsylvania 18512, and Forest Service, Northeastern Research Station, Forest Service, U.S. Department of Agriculture, 51 Millpond Road, Hamden, Connecticut 06514
A sampling method for determining the volatile terpenoid composition from single needles of seven Tsuga species was developed using headspace solid-phase microextraction (SPME). A reproducible sampling method for the volatile components was generated by examination of sample storage, method of needle cutting, and headspace sampling duration. Following SPME collection of the volatile compounds from the seven Tsuga species, gas chromatography/ion-trap mass spectrometry was used to identify 51 terpenoids present in the needle headspace. A semiquantitative method was devised to express individual terpenoid amounts as a percentage of all of the identified peaks in the chromatogram. The semiquantitative results permitted facile interspecies comparison using principal component analysis. Two components were able to account for 90% of the variance and were interpreted as a “species” component and a “resistance/susceptibility” component. Three interspecies groupings were evident from the principal component analysis: (1) Tsuga canadensis and Tsuga caroliniana; (2) Tsuga chinesnsis, Tsuga diversifolia, Tsuga heterophylla, and Tsuga sieboldii; and (3) Tsuga mertensiana. The finding that T. mertensiana was grouped alone and far removed from the other species adds to the morphological evidence that this species should be segregated from other Tsuga. KEYWORDS: Adelges tsugae; chemosystematics; terpenoids; GC-MS; hemlock; hemlock woolly adelgid; SPME; Tsuga canadensis; Tsuga caroliniana; Tsuga diversifolia; Tsuga chinensis; Tsuga heterophylla; Tsuga mertensiana; Tsuga sieboldii
INTRODUCTION
The genus Tsuga (hemlock trees) consists of nine species, two in eastern North America, two in western North America, and five in Asia (1). Phylogenetic relationships between species in the genus have been based on morphological and anatomical characters (2), geography (3), and molecular markers (4), but there is no general agreement on phylogeny. We wish to identify relationships between species in the context of resistance/ susceptibility of Tsuga species to the hemlock woolly adelgid (Adelges tsugae Annand). The Asian and western North American hemlock species are considered to be resistant to the hemlock woolly adelgid, and the eastern North American species are very susceptible, resulting in eventual tree death (5). Volatile terpenoids are abundant and diverse in conifers and play a complex, vital role in relationships between plants and insects. Signals for sexual reproduction (pheromones, kariomones), for defense against herbivores (allomones), or to attract * Corresponding author [telephone (570) 963-2564; e-mail afl1@ psu.edu]. † The Pennsylvania State University. § U.S. Department of Agriculture.
natural predators of herbivores (synomones) are conveyed through volatile terpenoids (6). Identifying these chemical signals and their function may suggest alternative methods to enhance resistance of plants to insect attacks. For instance, in Tsuga, the foliar terpenoids in Tsuga canadensis (L.) Carriere and Tsuga sieboldii Carriere were measured and related to the reproductive success of two scale insects, Fiorinia externa (Marlatt) and Nuculapsis tsuga Ferris (7). A wide variety of analytical methods are used to extract terpenoids from plant material (8, 9). These methods generally include a maceration or homogenization of the plant material to increase access to the essential oils in the resin canals of the plant. Techniques commonly used to extract the oils include steam distillation, Soxhlet extraction (10), and supercritical fluid extraction (11). Once isolated, the essential oil components are separated and identified using a suitable chromatographic method. Typically, gas chromatography-mass spectrometry (GC-MS) is chosen, largely due to the ability of GC-MS to identify the terpenoids through retention index matching and provide confirmation through comparison to library mass spectra.
10.1021/jf021028s CCC: $25.00 © 2003 American Chemical Society Published on Web 03/07/2003
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J. Agric. Food Chem., Vol. 51, No. 8, 2003
Lagalante and Montgomery
Table 1. Terpenoid Composition (Area Percent) from Individual Needles (n ) 3) in the Seven Species of Tsuga
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 a
tricyclene R-pinene camphene sabinene β-pinene myrcene R-phellandrene R-terpinene o-cymene limonene β-phellandrene cis-ocimene trans-ocimene γ-terpinene terpinolene linalool cis-p-menth-2-en-1-ol trans-p-menth-2-en-1-ol borneol ethyl octonoate trans-piperitol piperitone isobornyl acetate sabinyl acetate δ-elemene R-cubebene citronellyl acetate neryl acetate R-ylangene R-copaene geranyl acetate β-bourbonene β-elemene longifolene β-caryophyllene β-gurjunene Z-trans-R-bergamotene R-humulene γ-muurolene germacrene D β-selinene viridiflorene R-farnesene β-bisabolene cis-γ-bisabolene γ-cadinene δ-cadinene E-γ-bisabolene germacrene D-4-ol τ-cadinol R-cadinol
m/z
T. caroliniana
T. canadensis
T. chinensis
T. diversifolia
T. heterophylla
T. mertensiana
T. sieboldii
93 93 93 93 93 93 91 121 119 67 3 93 93 93 93 71 93 93 95 88 91 82 95 91 121 161 67 69 105 161 69 81 67 91 91 161 119 93 161 161 105 189 93 67 119 161 161 107 81 161 121
1.85 ± 0.20 10.07 ± 0.57 5.25 ± 0.36 0.41 ± 0.02 1.41 ± 0.10 8.26 ± 0.76 4.31 ± 0.29 0.27 ± 0.01 0.58 ± 0.06 0.85 ± 0.16 4.14 ± 0.07 3.62 ± 0.17
4.32 ± 0.37 13.19 ± 0.55 7.79 ± 0.76 0.15 ± 0.05 2.44 ± 0.06 1.65 ± 0.56 1.45 ± 0.58 * 1.63 ± 0.19 1.96 ± 0.18 3.06 ± 0.64 1.91 ± 0.37
1.93 ± 0.12 17.47 ± 0.67 5.37 ± 0.23
* 0.27 ± 0.05 0.16 ± 0.02 * * * 0.49 ± 0.09
0.18 ± 0.05 0.13 ± 0.01 * * * 2.99 ± 0.38
0.77 ± 0.06 18.74 ± 1.67 2.12 ± 0.15 1.46 ± 0.10 1.71 ± 0.09 0.62 ± 0.07 0.91 ± 0.14 * * 0.98 ± 0.20 1.75 ± 0.23 0.57 ± 0.03 2.00 ± 0.14 * *
3.24 ± 0.03 18.61 ± 0.46 8.39 ± 0.24 *b 1.77 ± 0.05 2.62 ± 0.46 0.52 ± 0.13 * 0.10 ± 0.03 1.79 ± 0.30 3.42 ± 0.35 1.00 ± 0.27 * 0.13 ± 0.02 0.17 ± 0.05 *
0.56 ± 0.09 26.62 ± 0.53 0.46 ± 0.07 0.40 ± 0.06 7.03 ± 0.48 1.54 ± 0.26 7.26 ± 0.16 * 0.51 ± 0.08 0.85 ± 0.06 19.85 ± 0.24 0.54 ± 0.05 0.62 ± 0.09 * 0.27 ± 0.04
2.03 ± 0.14 20.03 ± 2.35 4.84 ± 0.33 0.13 ± 0.02 4.47 ± 0.58 0.90 ± 0.14 1.51 ± 0.21 * * 0.63 ± 0.09 2.18 ± 0.30 0.21 ± 0.03 * * 0.12 ± 0.02
* 38.88 ± 1.57 * 0.81 ± 0.14 * 0.13 ± 0.13 * * 1.81 ± 0.11 * 1.77 ± 0.34 * 3.84 ± 0.60 0.23 ± 0.05 4.01 ± 0.12 0.11 ± 0.04 0.14 ± 0.01 0.27 ± 0.07 0.78 ± 0.09 0.31 ± 0.04 1.18 ± 0.02 2.28 ± 0.15 * * 0.15 ± 0.04 *
* 3.56 ± 0.18 42.86 ± 1.28 0.18 ± 0.03 0.12 ± 0.01 * 0.19 ± 0.04 * * * 1.37 ± 0.12 0.12 ± 0.01 * 3.26 ± 0.47 0.70 ± 0.11 0.66 ± 0.29 0.25 ± 0.07 0.48 ± 0.07 * * * 2.17 ± 0.53 3.23 ± 0.79 * *
*
2.07 ± 0.07 3.65 ± 0.38 2.22 ± 0.27 0.11 ± 0.01 0.41 ± 0.10 1.53 ± 0.17 7.21 ± 0.55 0.67 ± 0.10 * 0.28 ± 0.03 0.27 ± 0.03 * *
*
* * *
*
* * * *
9.52 ± 1.89 * * 0.83 ± 0.23 * * 0.56 ± 0.11 1.57 ± 0.29 * * * 13.32 ± 0.48 1.39 ± 0.24 10.79 ± 0.32 7.68 ± 1.37 4.58 ± 0.23 0.23 ± 0.01 * * * 5.56 ± 0.79 9.39 ± 1.36 * * *
18.98 ± 1.45 * 0.64 ± 0.01 1.23 ± 0.24 0.16 ± 0.01 1.09 ± 0.04 0.26 ± 0.05 * 0.31 ± 0.02 7.07 ± 0.07 0.31 ± 0.02 * 12.27 ± 0.10 1.25 ± 0.06 0.21 ± 0.03 0.29 ± 0.02 0.58 ± 0.04 *
* 28.40 ± 1.13 * 0.46 ± 0.03 0.49 ± 0.05 * 0.16 ± 0.01 0.69 ± 0.04 * * * 0.31 ± 0.02 6.09 ± 0.53 0.46 ± 0.03 12.32 ± 1.24 2.06 ± 0.08 0.46 ± 0.15 0.15 ± 0.01 0.29 ± 0.01 * *
4.66 ± 0.10 7.19 ± 0.27 *
2.20 ± 0.06 3.12 ± 0.06
*
*
3.24 ± 0.46 * * * * * 0.20 ± 0.02 1.11 ± 0.27 0.12 ± 0.01 0.33 ± 0.03 0.79 ± 0.06 0.27 ± 0.03 * 0.60 ± 0.05 0.61 ± 0.08 21.65 ± 0.98 * 0.10 ± 0.01 * 0.12 ± 0.01 0.05 1.30 ± 0.17 2.39 ± 0.51 *
21.37 ± 2.40 * * 1.25 ± 0.36 * 0.34 ± 0.04 2.12 ± 0.15 0.13 ± 0.04 0.23 ± 0.01 0.21 ± 0.02 6.01 ± 0.27 0.73 ± 0.09 0.32 ± 0.01 6.06 ± 0.30 2.38 ± 0.39 10.58 ± 0.20 0.49 ± 0.02 0.90 ± 0.04 0.21 ± 0.04 0.13 ± 0.01 2.76 ± 0.37 8.17 ± 0.13 * * *
m/z fragment values listed were used for single-ion quantification of a given compound. b Compound was present at
