Analysis of positional isomers of hydroxylated aromatic cytokinins by micellar electrokinetic chromatography.

A micellar electrokinetic chromatography (MEKC) method was developed for the separation of six positional isomers of hydroxylated aromatic cytokinins (topolin and topolin riboside), including ortho-topolin, meta-topolin, para-topolin, ortho-topolin riboside, meta-topolin riboside, and para-topolin riboside. Optimum resolution and analysis time (ca. 20 min) for the six aromatic cytokinin standards were achieved with a running buffer at pH 8.0 consisting of 20 mM boric acid, 50 mM sodium dodecyl sulfate (SDS), and 20% v/v methanol. The method has good reproducibility and has been successfully applied to detect the presence of a putative ortho-topolin in coconut water extract sample purified using C(18) and mixed-mode solid-phase extraction (SPE) columns. Other advantages of this MEKC method are short analysis time, low solvent consumption, and separation of positional isomers which could be achieved by a simple aqueous buffer system without the use of expensive chromatographic columns. In addition, a high-performance liquid chromatography (HPLC) method with baseline separation of the six topolin and topolin riboside standards was developed for the confirmation of the endogenous ortho-topolin in coconut water sample. Finally, the presence of ortho-topolin was further confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) based on its characteristic fragmentation pattern.