Čeština
EnglishAnalysis of gibberellins as free acids by ultra performance liquid chromatography–tandem mass spectrometry
Urbanová T., Tarkowská D., Novák O., Hedden P., Strnad M.
TALANTA 112: 85-94, 2013
Keywords: Gibberellins, Brassica napus, Arabidopsis thaliana, solid-phase extraction, ultra performance liquid chromatography, tandem mass spectrometry
Abstract: A robust, reliable and high-throughput method for extraction and purification of gibberellins (GAs), a group of tetracyclic diterpenoid carboxylic acids that include endogenous growth hormones, from plant material was developed. The procedure consists of two solid-phase extraction steps (Oasis MCX-HLB and Oasis MAX) and gives selective enrichment and efficient clean-up of these compounds from complex plant extracts. The method was tested with plant extracts of Brassica napus and Arabidopsis thaliana, from which total recovery of internal standards of about 72% was achieved. A rapid baseline chromatographic separation of 20non-derivatised GAs by ultra performance liquid chromatography is also presented where a reversed-phase chromatographic column AcquityCSH and a mobile phase consisting of methanol and aqueous 10mM-ammonium formate is used. This method enables sensitive and precise quantitation of GAs by MS/MS in multiple-reaction monitoring mode (MRM) by a standard isotope dilution method. Optimal conditions, including final flow rate, desolvation temperature, desolvation gas flow, capillary and cone voltage for effective ionisation in the electrospray ion source were found. All studied GAs were determined as free acids giving dominant quasi-molecular ions of [M–H]− with limits of detection ranging between 0.08 and 10 fmol and linear ranges over four orders of magnitude. Taking advantage of highly effective chromatographic separation of 20GAs and very sensitive mass spectrometric detection, the presented bioanalytical method serves as auseful tool for plant biologists studying the physiological roles of these hormones in plant development.
DOI: IEB authors: Ondřej Novák, Danuše Tarkowská
TALANTA 112: 85-94, 2013
Keywords: Gibberellins, Brassica napus, Arabidopsis thaliana, solid-phase extraction, ultra performance liquid chromatography, tandem mass spectrometry
Abstract: A robust, reliable and high-throughput method for extraction and purification of gibberellins (GAs), a group of tetracyclic diterpenoid carboxylic acids that include endogenous growth hormones, from plant material was developed. The procedure consists of two solid-phase extraction steps (Oasis MCX-HLB and Oasis MAX) and gives selective enrichment and efficient clean-up of these compounds from complex plant extracts. The method was tested with plant extracts of Brassica napus and Arabidopsis thaliana, from which total recovery of internal standards of about 72% was achieved. A rapid baseline chromatographic separation of 20non-derivatised GAs by ultra performance liquid chromatography is also presented where a reversed-phase chromatographic column AcquityCSH and a mobile phase consisting of methanol and aqueous 10mM-ammonium formate is used. This method enables sensitive and precise quantitation of GAs by MS/MS in multiple-reaction monitoring mode (MRM) by a standard isotope dilution method. Optimal conditions, including final flow rate, desolvation temperature, desolvation gas flow, capillary and cone voltage for effective ionisation in the electrospray ion source were found. All studied GAs were determined as free acids giving dominant quasi-molecular ions of [M–H]− with limits of detection ranging between 0.08 and 10 fmol and linear ranges over four orders of magnitude. Taking advantage of highly effective chromatographic separation of 20GAs and very sensitive mass spectrometric detection, the presented bioanalytical method serves as auseful tool for plant biologists studying the physiological roles of these hormones in plant development.
DOI: IEB authors: Ondřej Novák, Danuše Tarkowská