title: Quantification of endogenous brassinosteroids in plant by on-line two-dimensional microscale solid phase extraction-on column derivatization coupled with high performance liquid chromatography-tandem mass spectrometry.
abstract: An on-line two-dimensional microscale solid phase extraction (2DμSPE)-on column derivatization (OCD)-high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) method was developed for quantification of brassinosteroids (BRs) in plant tissues. Five BRs with widest distribution in plant species and high bioactivity (24-epibrassinolide, 24-epicastasterone, 6-deoxo-24-epicastasterone, teasterone and typhastero) were selected as target analytes. 2DμSPE column packed sequentially with phenyl boronic acid silica sorbent (the first dimension) and C18 silica sorbent (the second dimension) was used to selectively extract and enrich BRs by 110-146 times. OCD was carried out on the second dimension of 2DμSPE column with m-aminophenylboronic acid (m-APBA) as a derivatization reagent, enhancing the sensitivity of MS/MS to BRs by 13-8437 times. It was also found that pre-trap of derivatization reagent on the C18 section of 2DμSPE column could increase reaction efficiency by 3-10 times. The whole process time of the on-line system was less than 30min. The detection limits of the method for five BRs were between 1.4 and 6.6pg with RSDs less than 10%. Endogeneous BRs in tomato leaves were analyzed by using this method. Owing to the high selectivity of this on-line 2DμSPE system, BRs in plant extracts could be quantified using matrix-free standard calibration method with relative recoveries in the range of 80-124%.
authors.affiliation: Department of Instrumentation and Analytical Chemistry, Key Laboratory of Separation Science for Analytical Chemistry of CAS, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Wu
authors.forename: Dapeng
authors.initials: D
authors.lastname: Wu
authors.forename: Zheng
authors.initials: Z
authors.lastname: Shen
authors.forename: Chunfeng
authors.initials: C
authors.lastname: Duan
authors.forename: Yafeng
authors.initials: Y
authors.lastname: Guan
mesh_terms: D060406:Brassinosteroids / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D018551:Solanum lycopersicum / Q000737:chemistry; D010936:Plant Extracts / Q000737:chemistry; D018515:Plant Leaves / Q000737:chemistry; D010944:Plants / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D060406:Brassinosteroids; D010936:Plant Extracts