title: Determination of endogenous brassinosteroids in plant tissues using solid-phase extraction with double layered cartridge followed by high-performance liquid chromatography-tandem mass spectrometry.
abstract: BACKGROUND
  Brassinosteroids (BRs) are a group of important phytohormones that play vital roles in plant growth, development and a series of physiological phenomena. In order to understand biosynthesis, degradation and metabolic pathways of BRs, a reliable analytical method of BRs with effective sample pre-treatment process is favourable.
  
  
  OBJECTIVE
  The development of a quick and effective method for the quantification of endogenous BRs in plant tissue with the aid of double layered solid-phase extraction (SPE) cartridges (graphite carbon black and primary secondary amine silica sorbent: GCB/PSA).
  
  
  METHODS
  The method involved an initial extraction of BRs with acetonitrile, a dehydration process with anhydrous MgSO4 and NaCl, a SPE purification process with a double layered cartridge, and a further clean-up step utilising liquid-liquid extraction (LLE). The purification process was mainly realised on the GCB/PSA cartridge. GCB could eliminate hydrophobic compounds, especially those containing a π system, and PSA was introduced to remove the polar interferences. Endogenous BRs were quantified by HPLC-ESI-MS/MS.
  
  
  RESULTS
  Good linearities were obtained in the range of 0.4-500 ng/mL (0.0124-15 ng), with the correlation coefficients above 0.9957. The relative recoveries of BRs of this method were in the range of 71.1-113.1%, with intra- and interday relative standard deviations (RSDs) less than 16.3%. With the proposed method, the requirement of plant tissue amount was minimised to 1 g fresh weight, which is the smallest amount reported so far, to our knowledge.
authors: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan, Hubei 430072, China.
authors: Jun
authors: J
authors: Ding
authors: Li-Jing
authors: LJ
authors: Mao
authors: Shao-Ting
authors: ST
authors: Wang
authors: Bi-Feng
authors: BF
authors: Yuan
authors: Yu-Qi
authors: YQ
authors: Feng
mesh_terms: D000097:Acetonitriles / Q000737:chemistry; D029688:Brassica napus / Q000737:chemistry; D060406:Brassinosteroids / Q000032:analysis* / Q000302:isolation & purification; D002021:Buffers; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D059625:Liquid-Liquid Extraction; D012275:Oryza / Q000737:chemistry; D015203:Reproducibility of Results; D052616:Solid Phase Extraction / Q000295:instrumentation / Q000379:methods*; D012997:Solvents / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000097:Acetonitriles; D060406:Brassinosteroids; D002021:Buffers; D012997:Solvents; C032159:acetonitrile