title: Ultrasensitive quantification of endogenous brassinosteroids in milligram fresh plant with a quaternary ammonium derivatization reagent by pipette-tip solid-phase extraction coupled with ultra-high-performance liquid chromatography tandem mass spectrometry.
abstract: Determination of endogenous brassinosteroids (BRs) in limited sample amount is vital to elucidating their tissue- and even local tissue-specific signaling pathway and physiological effects on plant growth and development. In this work, an ultra-sensitive quantification method was established for endogenous BRs in milligram fresh plant by using pipette-tip solid-phase extraction coupled with ultra-performance liquid chromatography tandem mass spectrometry (PT-SPE-UPLC-MS/MS), in which a quaternary ammonium phenyl boronic acid, 4-borono-N,N,N-trimethylbenzenaminium iodide (BTBA) was first developed for chemical derivatization of BRs. Due to the cationic quaternary ammonium group of BTBA, the ionization efficiencies of the BRs chelates with BTBA (BTBA-BRs) were enhanced by 1190-448785 times, which is the highest response enhancement factor among all derivatization reagents reported for BRs. In addition, PT-SPE packed with C18 sorbent was first used for purifying BRs from plant extracts, so the required sample amount was minimized, and recoveries higher than 91% were achieved. Under the optimized conditions, the minimal detectable amounts (MDA) of five target BRs were in the range of 27-94 amol, and the correlation coefficients (R(2)) were >0.9985 over four orders of magnitude. The relative recoveries of 75.8-104.9% were obtained with the intra- and inter-day relative standard deviations (RSDs) less than 18.7% and 19.6%, respectively. Finally, three BRs were successfully quantified in only 5mg fresh rice plant samples, and 24-epiBL can even be detected in only 0.5mg FW rice leaf segments. It is the first time that the BRs content in sub-milligram fresh plant sample has been quantified.
authors.affiliation: Key Laboratory of Separation Science for Analytical Chemistry of CAS, Dept. of Instrumentation & Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, PR China; University of Chinese Academy of Sciences, Beijing 100039, PR China.
authors.forename: Ting
authors.initials: T
authors.lastname: Deng
authors.affiliation: Key Laboratory of Separation Science for Analytical Chemistry of CAS, Dept. of Instrumentation & Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, PR China. Electronic address: dpwu@dicp.ac.cn.
authors.forename: Dapeng
authors.initials: D
authors.lastname: Wu
authors.affiliation: Key Laboratory of Separation Science for Analytical Chemistry of CAS, Dept. of Instrumentation & Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, PR China.
authors.forename: Chunfeng
authors.initials: C
authors.lastname: Duan
authors.affiliation: Key Laboratory of Separation Science for Analytical Chemistry of CAS, Dept. of Instrumentation & Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, PR China. Electronic address: guanyafeng@dicp.ac.cn.
authors.forename: Yafeng
authors.initials: Y
authors.lastname: Guan
mesh_terms: D001565:Benzoates / Q000737:chemistry; D060406:Brassinosteroids / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D007202:Indicators and Reagents; D057230:Limit of Detection; D012275:Oryza / Q000737:chemistry; D018515:Plant Leaves / Q000737:chemistry; D010944:Plants / Q000737:chemistry*; D000644:Quaternary Ammonium Compounds / Q000737:chemistry*; D015203:Reproducibility of Results; D052616:Solid Phase Extraction; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D001565:Benzoates; D060406:Brassinosteroids; D007202:Indicators and Reagents; D000644:Quaternary Ammonium Compounds; C006829:2-benzylthio-5-trifluoromethylbenzoic acid
keywords: Brassinosteriod; Derivatization; Pipette-tip solid-phase extraction; Rice leaf segments