Abstract
Solid-phase microextraction (SPME) was coupled to gas chromatography mass spectrometry (GC-MS) and a method optimized to quantitatively and qualitatively measure a large array of volatile metabolites in alfalfa glandular trichomes isolated from stems, trichome-free stems, and leaves as part of a non-targeted metabolomics approach. Major SPME extraction parameters optimized included SPME fiber composition, extraction temperature, and extraction time. The optimized SPME method provided the most chemically diverse coverage of alfalfa volatile and semi-volatile metabolites using a DVB/CAR/PDMS fiber, extraction temperature of 60 °C, and an extraction time of 20 min. Alfalfa SPME-GC-MS profiles were processed using automated peak deconvolution and identification (AMDIS) and quantitative data extraction software (MET-IDEA). A total of 87 trichome, 59 stem, and 99 leaf volatile metabolites were detected after background subtraction which removed contaminants present in ambient air and associated with the fibers and NaOH/EDTA buffer solution containing CaCl2. Thirty-seven volatile metabolites were detected in all samples, while 15 volatile metabolites were uniquely detected only in glandular trichomes, 9 only in stems, and 33 specifically in leaves as tissue specific volatile metabolites. Hierarchical cluster analysis (HCA) and principal component analysis (PCA) of glandular trichomes, stems, and leaves showed that the volatile metabolic profiles obtained from the optimized SPME-GC-MS method clearly differentiated the three tissues (glandular trichomes, stems, and leaves), and the biochemical basis for this differentiation is discussed. Although optimized using plant tissues, the method can be applied to other types of samples including fruits and other foods.
Highlights
Solid-phase microextraction (SPME) is a gas-phase, absorptive extraction method used for the isolation of volatile organic compounds with detection limits in the range of parts per billion by volume through static headspace sampling and gas chromatography mass spectrometry (GC-MS) analyses
An optimized SPME method wasplants developed the analysis of a broad range of volParameters investigated included fiber composition, extraction temperature, and extracatile metabolites from fresh alfalfa plants using a non-targeted metabolomics approach
These parameters were compared and optimized on the basis of both of the Parameters investigated included fiber composition, extraction temperature, and extracquantitative and parameters qualitative composition fromand the optimized large-scale on volatile metabolic profiling tion time. These were compared the basis of both of the associated with the various tested conditions, whereas many studies have considered only quantitative and qualitative composition from the large-scale volatile metabolic profiling the total amount of detected metabolites or the detected number of targeted metabolites associated with the various tested conditions, whereas many studies have considered only during determination of optimal SPME extraction parameters [18,19,20,22,23]
Summary
Solid-phase microextraction (SPME) is a gas-phase, absorptive extraction method used for the isolation of volatile organic compounds with detection limits in the range of parts per billion by volume through static headspace sampling and GC-MS analyses. SPME has been widely used for plant [1], fruit [2,3], beverage [4,5,6], and meat [7,8] volatile analyses because it is simple, automatable, highly sensitive, and rapid. Alfalfa (Medicago sativa L.) is a perennial flowering legume grown in many parts of the world that serves as a premium forage used to feed animals. Plant glandular trichomes are epidermal appendages with a stalk and secretary head (Figure 1).
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