Analytical Methods For Determination Of Endogenous Brassinosteroids In Plant Tissue

Brassinosteroids (BRs) are a class of naturally occurring phytohormones with polyhydroxy steroid structure. They regulate general plant growth and many physiological processes, including cell elongation, vascular differentiation, seed germination, rhizogenesis and pollen tube developmen. BRs can also confer resistance to various abiotic and biotic stresses. However, the physiological functions of BRs has not been fully elucidated. And the investigations of BRs functions heavily rely on monitoring the temporal and spatial variation of BRs concentration. Therefore, the development of BR analytical method is of great importance.LC-MS is a powerful technique with high sensitivity and selectivity, thus has become a routine analytical platform of BRs. Despite of that, BR analysis is still challenged by the trace amount of endogenous BRs, complex plant matrix and the inherently low MS response of BRs. Moreover, from the perspective of the biologists, minimized amount of plant tissue, simple and fast sample preparation procedure are favorable. Therefore, the analytical chemists focused on the development of sample preparation technique so as to realized the fast analysis of BRs in micro amount of plant tissue with high sensitivity and great selectivity. In the recent reports, several technologies were employed for BRs analysis and were able to detect endogenous BRs. However, the sample preparation was tedious, and hormone purification was inefficient, which restricted the fast analysis of trace BRs.Based on the issues above, several new mechanisms (including QuEChERS, boronate affinity chromatography, hydrophilic chromatography, extraction/derivatization integration techniques) and new technologies (including magnetic solid phase extraction, polymer monolith microextraction, in-tube solid phase microextraction, in situ derivatization) were combined together to establish new sample preparation methods of BRs. In this dissertation, the author has developed several new assays of BRs with high sensitivity, great selectivity and high throughput.1. A modified QuEChERS method was introduced for the purification of BRs in plant extracts. By employing a graphite carbon black/primary secondary amine silica (GCB/PSA) double layered solid phase extraction column as the purification medium, hydrophilic interferents and hydrophobic matrix in plant tissue were simultaneously removed. Therefore, lower detection limits of BRs in real sample were obtained, and the quantification of endogenous BRs was achieved with1g leaves, or0.5g flowers.2. For the selective extraction of BRs, boronate affinity chromatography was introduced in the sample pretreatment process. Poly(3-acrylamidophenylboronic acid-co-ethylene dimethacrylate)(p(AAPBA-co-EDMA)) capillary monolith was used to selectively extract BRs from plant extract, and an selective assay of BRs was developed using PMME/LC-MS/MS. Compared to the GCB/PSA double layered SPE method, boronate affinity PMME exhibited better purification effect, which has removed most of the matrix interferents in plant extract.3. To obtain a high throughput pretreatment method, magnetic QuEChERS sorbents and magnetic boronate affinity sorbents were prepared, which were employed to purify and extract BRs in sequences. Based on the two kinds of sorbents, a rapid and selective analytical method for the determination of plant BRs was developed by a tandem MSPE method coupled with LC-MS/MS. By endowing QuEChERS method, boronate affinity chromatography with magnetic separation ability, the proposed method not only exhibited high selectivity, but also is fast and simple. Under the optimal conditions, sample preparation could be accomplished in1hour.4. To realize the automatic analysis of BRs, an in-tube SPME-UPLC-MS/MS system was constructed based on two valves-two pumps. Using C18PEEK column as trapping column and4-(dimethylamino)phenylboronic acid (4-DMAPBA) as derivatization reagent, an on-line trapping and in-situ derivatization assay of BRs were developed. BRs could be programmed to fulfill the procedures of injection, extraction, derivatization, LC-separation and MS detection on the system. The detection limits of BRs were improved more than one order of magnitude by the online trapping and in-situ derivatization techniques, thus endogenous BRs could be quantified in only300mg plant tissues. 5. In order to improve the sensitivity, selectivity and throughput of BRs analysis method, A novel strategy by combination of MSPE and in situ derivatization was developed for the determination of endogenous BRs in plant tissues. TiO2coated magnetic hollow mesoporous silica spheres were used as both microextraction sorbents and "microreactor" for the capture and derivatization of BRs in sequence. The extraction based on hydrophilic interaction removed most of hydrophobic interferents in plant extract, thus matrix effect was largely reduced. The extraction/derivatization integration technology greatly simplified sample preparation procedure, which could be accomplished within10min. In the meantime, MS response of BRs were significantly improved due to the derivatization with4-DMAPBA, which benefited the quantification of BRs with small amount of plant tissue (100mg fresh weigh in current study).