Cytokinin profiling in plant tissues using ultra-performance liquid chromatography–electrospray tandem mass spectrometry

0322259 - ÚEB 2009 RIV GB eng J - Journal Article
Novák, Ondřej - Hauserová, Eva - Amakorová, Petra - Doležal, Karel - Strnad, Miroslav
Cytokinin profiling in plant tissues using ultra-performance liquid chromatography–electrospray tandem mass spectrometry.
[Cytokininová analýza v rostlinných tkáních pomocí ultra-účinné kapalinové chromatografie-elektrosprej tandemové hmotnostní spektrometrie.]
Phytochemistry. Roč. 69, č. 11 (2008), s. 2214-2224. ISSN 0031-9422. E-ISSN 1873-3700
R&D Projects: GA AV ČR KAN200380801
Institutional research plan: CEZ:AV0Z50380511
Keywords : Ultra-performance liquid chromatography (UPLC) * Tandem mass spectrometry (MS/MS) * Microextraction
Subject RIV: EC - Immunology
Impact factor: 2.946, year: 2008

We have developed a simple, high-throughput batch immunoextraction (IAE) micropurification procedure for extracting a wide range of naturally occurring cytokinins (bases, ribosides, O- and N-glucosides, and nucleotides) from plant tissues in solutions that are compatible with ultra-performance liquid chromatography (UPLC), thereby facilitating sensitive subsequent analysis. The UPLC system was coupled to a tandem quadrupole mass spectrometer (MS/MS) equipped with an electrospray interface (ESI). Small (mg) amounts of tissues were purified by solid-phase extraction (SPE) followed by an immunoaffinity clean-up step and two fast chromatographic separations of most cytokinin metabolites (bases, ribosides, and 9-glucosides in the first, O-glucosides and nucleotides in the second). Using UPLC, the runs were up to 4-fold faster than in standard cytokinin analyses, and both retention times and injection volumes were less variable (RSDs, 0.15–0.3% and 1.0–5.5%, respectively). In multiple reaction monitoring (MRM) mode, the detection limit for most of the cytokinins analyzed was close to 1 fmol (5–25 fmol for O-glucosides and nucleotides) and the linear range spanned at least five orders of magnitude. The extraction and purification method was optimized using poplar (Populus × canadensis Moench, cv Robusta) leaf samples, and the analytical accuracy was further validated using IAE-purified 10-day-old Arabidopsis thaliana plants spiked with 1 and 10 pmol of cytokinin derivatives. This approach can be used for rapid, sensitive qualitative and/or quantitative analysis of more than 50 natural cytokinins in minute amounts of plant tissues with high performance, robustness, and accuracy.

Byla vyvinuta jednoduchá imunoextrakční mikropurifikační metoda pro extrakci přirozeně se vyskytujících cytokininů kombinovaná s ultra-účinnou kapalinovou chromatografií spojenou s tandemovou hmotnostní spektrometrií. Nové extrakční a purifikační postupy byly optimalizovány na vzorcích topolového listí a analytická přesnost validována na 10-ti denních semenáčcích A. thaliana. Tento přístup může být použit pro rychlou, citlivou kvalitativní a/nebo kvantitativní analýzu víc než 50 přirozených cytokininů v mg množství rostlinných tkání.
Permanent Link: http://hdl.handle.net/11104/0170575