Analytical Methods For Determination Of Cytokinins Based On Hydrophilic Interaction Chromatography Coupled With Tandem Mass Spectrometry

Naturally occurring cytokinins (CKs) are a group of plant hormones whichregulate a wide variety of physiological and developmental processes in plants, suchas promotion of cell devision and differentiation, influence of shoot formation anddevelopment, control of apical dominace and leaf senescence. Accurate determinationof CKs in plant tissues is of great significance for in-depth understanding of theirbiological function. However, the complicated matrix of plant extracts, theextremely low concentration levels of CKs and the presence of enzymes, which canlead to the transformantion and degradation of CKs, make their quantificationbeseting with difficulties. Thus, a highly sensitive, selective and reliable method foridentification and quantification of CKs is particularly required. In recent reports,several technologies have been used in the fields of CKs analysis. However, todevelop simpler, quicker and more practical approaches are still the hotspots.In this dissertation, the author is devoted to the development of new samplepretreatment methods and separation technologies for CKs quantitative analysis.The major contents of this dissertation are described as follows:1. The development of CK analysis was reviewed. Emphasis is laid on thetechniques for sample preparation, separation and detection of CKs and theintroduction of new pretreatment methods and separation technologies in separationscience.2. A sensitive assay of CKs was developed using polymer monolithmicroextraction/hydrophilic interaction chromatography/electrospray ionization-tandem mass spectrometry (PMME/HILIC/ESI-MS/MS). The extraction wasperformed on a poly(2-acrylamido-2-methyl-1-propanesulfonic acid-co-ethylenedimethacrylate)(poly(AMPS-co-EDMA)) capillary monolith and the subsequentseparation was carried out on a Luna silica column in HILIC mode. PMME is a novelsolvent-free and convenient technique with small sample consumption, and HILICcan provide better separation with isocratic elution and higher sensitivity whencoupled with MS detector. Finally, the developed method was applied to the determination of CKs in various samples including Oryza sativa, Arabidopsis thalianaand oil seed rape and demonstrated to be an alternatively reliable and sensitive toolfor CKs studies.3. A simple and rapid one-step tandem solid phase extraction (SPE) method bycombining C₁₈and mixed mode SPE (silica@C8/SO₃H) was developed for thedetermination of CKs in plant samples. The tandem SPE offers a high recoverygreater than80%and an advantage for convenient analysis due to the compatibility ofthe extraction solvent (Bieleski solvent) for plant samples and the sampling solvent ofsilica@C8/SO₃H cartridge, which avoids the concentration of samples in a largevolume of modified Bieleski extraction solvent followed by a redissolving step andcan greatly reduce the time of sample preparation. This method can achieve rapid,efficient and high-throughput extraction toward CKs.4. Based on the third part, a rapid and highly sensitive analytical method for thedetermination of plant CKs was developed by employing tandem solid phaseextraction (C₁₈and silica@C8/SO₃H cartridges) and online trapping technique coupledwith HILIC-MS/MS (tandem SPE-online trapping-HILIC-MS/MS). Poly (methacrylicacid-ethylene glycol dimethacrylate)(poly(MAA-co-EGDMA) monolith and HILICwere used as the online trapping column and separation mode, respectively, whichimproved the detection sensitivity of CKs by at least one order of magnitudecompared with other previously reported methods. In this respect, only20mg of planttissue were required for simultaneous quantitative analysis of major CKs in plantsamples. Using this method we successfully determined the concentration of theendogenous CKs in three plant samples, which demonstrated the potential applicationof this method on the highly sensitive detection of CKs in small amount plant samplesfor the studies of plant metabolism and physiology.5. A2-acrylamido-2-methyl-1-propanesulfonic acid-co-ethylene glycoldimethacrylate magnetic copolymer (Fe₃O₄/SiO₂/poly(AMPS-co-EGDMA)) wasprepared and used as a magnetic solid phase extraction (MSPE) medium for recoveryof endogenous CKs from plant extracts. This magnetic porous polymer wasdemonstrated to have high extraction capacity toward CKs in plants due to its good specificity, large surface area and porous structure. The dispersion extraction mode ofMSPE can be directly used for analysis of large-volume sample solvents with traceCKs, and the magnetic property made the procedure simple and rapid. In addition,coupling with HILIC–MS/MS, a rapid, simple, and effective MSPE–HILIC–MS/MSanalytical method for the quantitative analysis of endogenous CKs in Oryza sativaroots was successfully established.