Comprehensive chemical derivatization for gas chromatography–mass spectrometry-based multi-targeted profiling of the major phytohormones

In the present investigation we report selection of the N-methyl-N-(tert.-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) reagent as the most comprehensive derivatization protocol among 17 tested reactions covering trifluoroacetylation, pentafluorobenzylation, methylations, and trimethylsilylations. MTBSTFA allowed easy and robust tert.-butyldimethylsilyl derivatization of 1-aminocyclopropane-1-carboxylic acid, indole-3-acetic acid, (±)-jasmonic acid, salicylic acid, (±)-abscisic acid, meta-topolin, and trans-zeatin. Detection limits as analysed by selected ion monitoring quadrupole GC–MS were 0.2, 0.01, 1.0, 0.02, 0.3, 0.3, and 0.9 pmol of injected substance, respectively. Analysis of gibberellic acid A3, trans-zeatin riboside and (±)-abscisic acid-β-d-glucopyranosyl ester was best when coupled by splitting extracts and trimethysilylation. The MTBSTFA derivatization protocol was optimised, and validated. The preparation was insensitive to 2% residual water and to ≤1 day storage at room temperature. The final scheme was highly reproducible and successfully applied to extracts from ∼300 mg (fresh mass) of tobacco (Nicotiana tabacum) root and Arabidopsis thaliana seedling.