Determination of Trace Brassinosteroids by High Performance Liquid Chromatography

Since the discovery of brassinolide as a new plant growth hormonal steroid', a number of its related steroids (general term, brassinosteroids) have been found to occur in higher plants.2 Because of its remarkable biological activities and the very small amounts contained in plants2, microanalytical methods have been necessary for their screening and identification of brassinosteroids in plants. In our previous paper3, we reported that bismethaneboronates of brassinosteroids are useful derivatives for gas chromatography and gas chromatography-mass spectrometry. Our microanalytical method has been useful for the demonstration of the ubiquitous distribution of these hormonal steroids in the plant kingdom.4,5 However, high performance liquid chromatography (HPLC) analysis of brassinosteroids has not been intensively investigated, not withstanding the hope that more sensitive and selective analysis may be obtained by photometric or fluorimetric detection. The liquid chromatographic behavior of standard brassinosteroids was monitored with UV detector at 204 nm by Yokota et al.6 Because of the lack of the other UV active chromophore in brassinosteroids, derivatization is indispensable for their HPLC microanalysis. After employing a number of derivatization reagents, we have found that bisnaphthaleneboronate is a suitable derivative for brassinosteroids in HPLC analysis, due to the selectivity of 1-naphthaleneboronic acid for the two vicinal diol functional group of the steroids. In this paper, we report the HPLC microanalytical method for brassinosteroids as their bisnaphthaleneboronate derivatives and its application to the identification of brassinolide in the pollen of broad bean (Vicia faba L.) as a demonstration of the usefulness of our microanalytical method. Experimental