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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 C18 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.