Volatile apocarotenoid discovery and quantification in Arabidopsis thaliana: optimized sensitive analysis via HS-SPME-GC/MS

IntroductionIn the field of carotenoid metabolism researchers' focus has been directed recently toward the discovery and quantification of carotenoid cleavage products (i.e. apocarotenoids, excluding the well-studied carotenoid-derived hormones abscisic acid and strigolactones), due to their emerging roles as putative signaling molecules. Gas chromatography mass spectrometry (GC/MS) and sample preparation via headspace solid phase micro-extraction (HS-SPME) are widely used analytical techniques for broad untargeted metabolomics studies and until now, no optimized quantitative targeted HS-SPME-GC/MS method has been developed specifically for volatile apocarotenoids (VAs) in planta.ObjectivesOptimization and subsequent validation of the HS-SPME technique for extracting and quantifying volatile apocarotenoids in planta.MethodsFactors considered during method optimization were HS-SPME parameters; vial storage conditions; different adsorbent SPME fibre coating chemistries; plant tissue matrix effects; and fresh tissues to be analyzed.ResultsMean linear regression in planta calibration correlation coefficients (R-2) for VAs was 0.974. The resultant method mean limits of detection (LOD) and lower limits of quantification (LLOQ) for VAs using in planta standard additions were 0.3840.139 and 0.640 +/- 0.231 mu g/L, respectively. VAs remained stable at elevated SPME incubation temperatures, with no observable effects of thermal and photo-stereoisomerisation and oxidation. The bipolar 50/30 mu m divinylbenzene/carboxen on polydimethylsiloxane (PDMS/DVB/CAR) was identified as the optimal fibre for broad molecular weight range VA analysis.Conclusions p id=Par5 An optimized HS-SPME-GC/MS method for VA detection and quantification was validated in vitro and in planta: based on biological replicates and stringent QA/QC approaches, thereby providing robust detection and quantification of VAs across a broad range of Arabidopsis tissues, fifteen of which were identified for the first time in Arabidopsis.