Acquisition of Volatile Compounds by Gas Chromatography–Mass Spectrometry (GC-MS)

Abstract

Plants synthesize and emit a large range of volatile organic compounds (VOCs) that play important roles in their interactions with the environment, from attracting pollinators and seed dispersers to protectants such as repellants and pathogen inhibitors. As such, the development of techniques for headspace collection of volatiles in combination with gas chromatography–mass spectrometry (GC-MS) has an important impact on our understanding of the biosynthesis of plant VOCs. Furthermore, knowledge of the plant VOCs can be valuable in relation to plant breeding for improving fruit flavor or enhancing resistance to insects or pathogens. This chapter describes a reliable method for extracting volatile compounds by headspace solid-phase microextraction (HS-SPME), and separate and detect them by GC-MS.

1 Introduction

Metabolism in plant involves the conversion of high molecular weight precursors to smaller compounds that help to obtain viable seeds and to attract seed-dispersing species. Plants synthesize and emit a large variety of volatile organic compounds (VOCs). Physical properties of these compounds allow them to freely cross cellular membranes and be released into the surrounding environment [1]. These volatiles can be emitted from flowers, leaves, fruits, and roots into the atmosphere or soil, allowing the plant to interact with other organisms. Until now, more than 1700 volatiles have been identified from more than 90 plant families, which contain approximately 1% of all plant specialized metabolites currently known [2]. The significance of VOCs in plant physiology and ecology has been extensively investigated only in the past 15–20 years and has revealed roles for VOCs in the mutualistic interaction of plants with other organism, biotic and abiotic stress responses [3]. Probably the most well-known are volatiles emitted from floral tissues attracting pollinators [2]. Biosynthesis of VOCs depends on the availability of carbon, nitrogen, and sulfur, as well as energy provided by primary metabolism. According to the chemical structure, plant volatiles can be classed into hydrocarbons, alcohols, aldehydes, ketones, ethers, and esters. Based on their biosynthetic origin, plant volatiles are generally divided into several classes, including terpenoids, phenylpropanoid/benzenoid, fatty acid derivatives, and amino acid derivatives.

The increasing scientific interest in plant VOCs has led to the development of a variety of systems for the collection and analysis of volatiles [4,5,6]. All methods for the analysis of plant volatiles attempt to identify the authentic profile of volatile blends emitted by a plant. However, the choice of which system to use in a particular experiment for collection and analysis of plant volatiles dependents on the biological problem and plant material being investigated. Nowadays, VOCs analysis demands sensitive and time-efficient techniques or high-throughput profiling.

VOCs are usually identified and quantitated by extracting them from the airspace (headspace) surrounding aboveground plant parts and passing them through a gas chromatograph with a mass spectrometer detector (GC-MS). When conducting volatile profiling, several approaches are possible for sampling the headspace of plant tissue. Among them, solid-phase microextraction (SPME) has been demonstrated to be capable of isolating a high number of VOCs at detection limits in the ppb (parts per billion by volume). In SPME, volatile compounds are adsorbed on a stationary phase that is coated on a fused silica fiber. The fiber is inserted into the headspace above the sample, which has been allowed to equilibrate at 40–60 °C for 15–30 min, and is exposed for 5–30 min (see Note 1 ). The compounds adsorbed on the fiber are then thermally desorbed for 5 min in the GC injector port and then sent through the column and detector (see Note 2 ).

Numerous studies have used SPME extraction for the analysis of a broad range of volatile compounds in different matrices, such as whole plant, flower, fruit, air, soil and water samples [7,8,9,10]. In some applications, it is desirable to collect volatiles from parts of intact growing plants [9] (Fig. 1c) and others analysis requires homogenized samples in which is frequently added concentrated salt solution (NaCl or CaCl₂) to decrease the solubility of volatile compounds and force them into the headspace [10] (Fig. 1b).

Fig. 1

Strategies of plant volatile analysis. (a) Blank sample, H₂O or saturated CaCl₂. (b) Homogenized tissue in H₂O or saturated CaCl₂. (c) Intact plant tissue with 0.2 mL H₂O. (d) Blank sample for intact plant tissue

As shown in Fig. 2, the total ion current (TIC) chromatograms and thereby the metabolic profiles between the different extraction methods are highly variable.

Fig. 2

Total Ion Current (TIC) chromatogram of all samples from Fig. 1. (a) TIC chromatogram from samples in Fig. 1c (green) and Fig. 1d (light grey). (b) TIC chromatogram from samples in Fig. 1b (H₂O; light blue) and Fig. 1a (H₂O; light grey). (c) TIC chromatogram from samples in Fig. 1b (CaCl₂; dark blue) and Fig. 1a (CaCl₂; dark grey)

Some metabolites can be better isolated in in vivo tissues (Fig. 3a, b) and others could be only detected in homogenized tissues (Fig. 3c, d). Thus, it is vital to pay much attention to the extraction protocol design which depends on the target metabolite(s) or the biological questions to be answered. Also, a number of extra TIC peaks are often observed when homogenized samples is used, but not all observed peaks correspond to the detection of a real metabolite coming from the original tissue matrix. Indeed, it could be possible to detect environmental contaminations (Fig. 3e, f) or coming from CaCl₂ (Fig. 3g, h). Therefore, for each injection sequence it is a prerequisite to add sample blanks (see Note 3 ).

Fig. 3

Tentative annotation of metabolites on the basis of retention time (Table 1) and m/z value and MS fragmentation pattern with Golm Metabolome Database (GMD) and/or National Institute of Standards and Technology mass spectral library (NIST) . (Right Panel; b, d, f, h) Mass spectrum used for tentative metabolite annotated. (Left Panel; a, c, e, g) ion peak from each m/z (133, 128, 281, 182, respectively)

Here, we describe a method based on headspace solid-phase microextraction (HS-SPME) coupled to GC-MS to determinate volatile compounds in a variety of different matrixes (e.g., plant species/tissues). We exemplify it using intact in vivo or homogenized Arabidopsis flowers. An abbreviated scheme of sample preparation and analysis is shown in Fig. 4.

Fig. 4

Scheme of HS-SPME analysis of volatile from intact (I) or homogenized (II, H₂O and III, saturated CaCl₂ solution) plant tissue by GC-HS-SPME-MS

2 Materials

2.1 Sampling and Extraction

• 1. MilliQ water approx. 0.055 μS/cm.

• 2. Liquid nitrogen supply.

• 3. Oscillating ball mill MM200 (e.g., Retsch GmbH) or pestle and mortar.

• 4. Scalpel blades, spatula, tweezers, scissors.

• 5. Vortex.

• 6. Balance.

• 7. 2.0-mL microcentrifuge tubes (e.g., Eppendorf).

• 8. 0.2-mL glass vial (e.g., Sci-Vi Crimp Top Vial, Thermo Scientific).

• 9. 20 mL glass vial with screw neck and magnetic screw cap, septum silicone blue/PTFE white (e.g., Gerstel DHS vials).

• 10. StableFlex ™ SPME-fiber with 65 μm polydimethylsiloxane/divinylbenzene coating (e.g., Supelco 23Ga, autosampler, Pink) (see Note 4 ).

• 11. Pure standards of all the identified compounds (i.e., Sigma-Aldrich or OldChemin Ltd., Czech Republic).

• 12. Saturated NaCl or CaCl₂.

2.2 GC-HS-SPME-MS

• 1. Autosampler system, including SPME fiber cleaning and conditioning station and agitator (e.g., Gerstel MPS DHS 2×l Twister).

• 2. Gas chromatograph with electronic pressure control (Agilent 6890N).

• 3. Helium carrier gas.

• 4. Split/Splitless liner (e.g., Agilent liner, splitless, single taper).

• 5. Capillary column J&W DB-624, 60 m × 0.25 mm × 1.40 μm film thickness.

• 6. Electron impact ionization mass selective detector (EI/quadrupole MSD, Agilent 5975B).

3 Methods

3.1 Sample Preparation

3.1.1 Volatile Compound Analysis of Intact Plant Tissue (Fig. 4 I)

• 1. Fill 0.2-mL glass vials with MilliQ water (see Note 5 ).

• 2. Collect plant tissues and quickly put into a 0.2-mL glass vial containing water. Then, put it into a 20 mL vial and close it (Fig. 1c). Consider blank samples (Fig. 1a, d and Fig. 3).

• 3. Feed the autosampler and keep it at 15 °C.

3.1.2 Volatile Compound Analysis of Homogenized Tissue (Fig. 4 II and III) (See Note 6)

• 1. Collect the plant material and freeze immediately in liquid nitrogen.

• 2. Precool the steel cylinders and metal balls to grind the samples in liquid nitrogen. Alternatively, cool the pestle and mortar.

• 3. Quickly take out two samples and place them into independent steel cylinders together with a metal ball and cover the cylinders. Or use a pestle and mortar to grind the samples.

• 4. Fix cylinders in the mixer mill and mill at 20 Hz/s for 1 min. Or use a pestle and mortar until a very fine powder is obtained.

• 5. Quickly take out the cylinders and place back into liquid nitrogen.

• 6. Transfer the fine powder into a 2–0-mL microcentrifuge precooled tube and keep in liquid nitrogen.

• 7. Weigh the minimal amount of tissue necessary to obtain quantifiable data by GC-HS-SPME-MS (work quickly to get an exact fresh weight), and keep in liquid nitrogen or store at −80 °C until use.

• 8. The amount of plant material will vary with plant and tissue type (300 mg of flowers was used to get the data shown in this chapter).

• 9. Fill each 20 mL vials as follows: (a) Blank and sample vial for homogenized samples in water: 500 μL MilliQ water. (b) Blank and sample vials for homogenized samples in saturated CaCl₂: 500 μL of saturated CaCl₂.

• 10. Transfer the frozen material into 20 mL vial, close (Fig. 4 IIc and IIIc) and vortex it. Consider blank samples (Fig. 1a–d and Fig. 3 IIc and IIIc).

• 11. Feed the autosampler and keep it at 15 °C.

3.2 Data Acquisition

• 1. Incubate the vials from step 3 and/or 14 at 50 °C for 10 min with agitation at 250 rpm in autosampler.

• 2. Insert 24 mm the SPME holder into the vial and keep it for 20 min (extraction time) at 50 °C. Keep the agitation as described in step 1.

• 3. Inject sample in pulsed splitless mode, with the helium carrier gas flow set to 1 mL/min by using the autosampler.

• 4. Desorb the VOCs by injection of the fiber in the injection port at 250 °C for 1 min.

• 5. The flow rate is kept constant with electronic pressure control enabled.

• 6. The injection temperature is set to 250 °C.

• 7. Injection programs must include fiber cleaning and conditioning steps before and after each injection.

• 8. Helium flow goes through the fiber at 250 °C for 5 min.

• 9. Perform chromatography using a 60 m DB-624 capillary column. The temperature program should be isothermal for 2 min at 40 °C, followed by a 10 °C per min ramp to 260 °C, and holding at this temperature for 10 min. Cooling should be as rapid as the instrument specifications allow.

• 10. Set the transfer line temperature to 250 °C and match ion source conditions.

• 11. Set the ion source temperature to maximum instrument specifications, 250 °C.

• 12. The recorded mass range should be m/z 30–300 at 2 scan per s.

• 13. Proceed the remaining monitored chromatography time with a 360 s solvent delay with filaments turned off.

• 14. Manual mass defect should be set to 0, filament bias current should be -70 V, and detector voltage should be ∼1500–2000 V (see Note 7 ).

• 15. Transfer raw GC-MS profile chromatograms and analyze in comparison to internal standard (Table 1 and Fig. 4) (see Note 8 ).

Table 1 List of volatile compounds with retention time based on authentic standard or/and matching to GMD and NIST in our conditions

4 Notes

• 1. Solid-phase microextraction (SPME) fibers can be reused approximately 100 times.

• 2. SPME is an equilibrium extraction and desorption process, so precise control of sampling conditions is important. Recoveries may be enhanced by raising the temperature and time, but compounds may be displaced from the absorption sites and compounds that were not originally present in the sample may be formed.

• 3. To avoid carryover, a sample blank between 10 samples is recommended.

• 4. There are different types of fiber adsorbents available. By carefully selecting the polarity and thickness of the fiber coating, compounds of different polarity and volatility ranging from high-boiling or semivolatile to volatile compounds can be sampled.

• 5. In order to ensure the quality of the used water for the extraction, it is recommended to run a blank sample only with H₂O before mixing it with sample.

• 6. Once the sample has been frozen in liquid nitrogen it must not thaw out even slightly before analysis. Make sure therefore to keep samples at constant freezing temperature (in liquid nitrogen) to avoid degradation of VOCs and precool all components to be used (spatulas, vials, etc.) in liquid nitrogen before they come into contact with the sample.

• 7. In contrast to LECO’s MS, the detector voltage is automatically adjusted in Agilent’s MS.

• 8. For reporting chemical identification, authors should consider references [11,12,13,14,15].