Comparative determination of some phytohormones in wild-type and genetically modified plants by gas chromatography–mass spectrometry and high-performance liquid chromatography–tandem mass spectrometry

Abstract

The analytical performances of two optimized analytical methodologies used for the determination of auxins, cytokinins, and abscisic acid in plant samples were critically compared. Phytohormones were extracted from Nicotiana glauca samples using a modified Bieleski solvent and determined both by gas chromatography–mass spectrometry (GC–MS), after derivatization with N, O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), and by high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) on the Bieleski extract without any further treatment. HPLC–MS/MS gave better results in terms of higher coefficients of determination of the calibration curves, higher and more reproducible recoveries, lower limits of detection, faster sample preparation, and higher sample throughput. Thus, two sets of N. glauca and N. langsdorffii samples, both wild-type and genetically modified by inserting the glucocorticoid receptor (GR) gene encoding for the rat glucocorticoid receptor, were first characterized by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and then analyzed by HPLC–MS/MS. Significant differences in the phytohormone content between the two sample sets were found and are very important in terms of understanding the mechanisms and effects on growth processes and the development of transgenic plants.