Abstract
HS-SPME-GC-MS has been suggested as a fast and robust analytical platform for the product characterization of sesquiterpene synthases. The choice of fiber and injection temperature can have a significant effect on the observed product profile, due to the chemical rearrangements that can occur on the fiber material. Here we present a systematic study on the effects of fiber choice and injection port temperature on the observed sesquiterpenoid profile of four sesquiterpene synthases expressed in Nicotiana benthamiana. We found that the absorbent material PDMS was much less likely to support acid-induced rearrangement of sesquiterpenoids when compared to the adsorbent materials PDMS/DVB, PDMS/CAR, and PDMS/CAR/DVB. Furthermore, utilizing an injection port temperature at 160 °C almost eliminated the inherent thermal instability of germacrene sesquiterpenoids. Thus, for fast screening of sesquiterpene synthases, the results suggest that PDMS fibers and an injection temperature of 160 °C provide a fast and reproducible HS-SPME GC-MS method when using H2 as carrier gas.
Highlights
The development and accessibility of next-generation sequencing (NGS) technologies has made the generation of large amounts of genomic and transcriptomic data relatively straightforward
We provide examples of thermal and acid rearrangement on various SPME fibers and suggest solutions that can minimize these common problems associated with gas chromatography-mass spectroscopy (GC-mass spectroscopy (MS)) sesquiterpenoid analysis
Product release from fibers is correlated with the injection port temperature; decreasing the temperature from 250 °C will lead to lower levels of product detected by the gas chromatography (GC)-MS
Summary
The development and accessibility of next-generation sequencing (NGS) technologies has made the generation of large amounts of genomic and transcriptomic data relatively straightforward. It has since been shown that germacrenes are enzymatic end-products themselves, but that they are unstable and rearrange when exposed to high temperatures, acids, or reactive materials [7,9]. One such describe rearrangement is the heat-induced Cope rearrangement where the germacrene ring is transformed into the elemene structure (Figure 1b), but acid-assisted rearrangement has been described [9,12,13]. In the CAR/DVB/PDMS fiber material the primary extracting mechanism is adsorption to the porous material [20] Overall, in these materials, the diffusion is poor and the analytes are retained at the surface by adsorption to the stationary phase. We provide examples of thermal and acid rearrangement on various SPME fibers and suggest solutions that can minimize these common problems associated with GC-MS sesquiterpenoid analysis
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