Protocol for Extraction and Isolation of Brassinosteroids from Plant Tissues
Brassinosteroids (BRs) are a class of steroid plant hormones that participate with other plant hormones in the regulation of numerous developmental processes, including root and shoot growth, vascular differentiation, fertility, and seed germination. A characteristic feature of all plant hormones, including BRs, is that their concentration is extremely low in plant tissues and, therefore, the methods dealing with their determination belong to ultra-trace analysis, for which very sensitive analytical tools are needed. The analysis of natural BRs is essential when their functions and roles in plant growth and development are to be elucidated. Here, we describe a reliable protocol for high-throughput extraction and purification of BRs. The procedure consists of two solid-phase extraction steps and provides selective enrichment and efficient cleanup of these compounds from complex plant extracts. The protocol is designed for sensitive liquid chromatography-tandem mass spectrometry-based method for simultaneous detection of 22 naturally occurring BRs, including their biosynthetic precursors and most of their biologically active metabolites, without need for derivatization.
Key wordsBrassinosteroids Plant hormones Solid-phase extraction Ultra-trace analysis Gas chromatography Liquid chromatography Mass spectrometry
The authors thank Sees-editing Ltd. for critical reading and editing of the manuscript. The work was supported by the Grant Agency of the Czech Republic (14-34792S) and by the Ministry of Education, Youth and Sport of the Czech Republic through National Program of Sustainability (LO 1204).
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