Analysis of gibberellins as free acids by ultra performance liquid chromatography-tandem mass spectrometry

0399103 - ÚEB 2014 RIV NL eng J - Journal Article
Urbanová, Terezie - Tarkowská, Danuše - Novák, Ondřej - Hedden, P. - Strnad, M.
Analysis of gibberellins as free acids by ultra performance liquid chromatography-tandem mass spectrometry.
Talanta. Roč. 112, č. 2013 (2013), s. 85-94. ISSN 0039-9140. E-ISSN 1873-3573
R&D Projects: GA AV ČR KAN200380801
Grant - others:GA MŠk(CZ) ED0007/01/01
Program: ED
Institutional research plan: CEZ:AV0Z50380511
Keywords : Gibberellins * Brassica napus * Arabidopsis thaliana
Subject RIV: EC - Immunology
Impact factor: 3.511, year: 2013

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 (R) MCX-HLB and Oasis (R) 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 20 non-derivatised GAs by ultra performance liquid chromatography is also presented where a reversed-phase chromatographic column Acquity CSH (R) and a mobile phase consisting of methanol and aqueous 10 mM-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 20 GAs and very sensitive mass spectrometric detection, the presented bioanalytical method serves as a useful tool for plant biologists studying the physiological roles of these hormones in plant development.
Permanent Link: http://hdl.handle.net/11104/0226484