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

Abstract

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.

MEKC: An update focusing on practical aspects

“…Usually, a decrease in the BGE velocity is observed with increasing concentration of the organic modifier by effect of changes in the viscosity and dielectric constant of the separation electrolyte, and also in the micellar structure [78]. The most commonly used organic modifier in MEKC is ACN [79][80][81][82][83], which is especially effective in expanding the migration window; 2-propanol [84][85][86] and methanol [87][88][89] are also used, albeit to a lesser extent; and ethanol [90] and acetone [91] have also been used for this purpose in recent years. Despite the ability of organic modifiers to improve resolution in MEKC, they require the concentration of the organic solvent in the BGE to be rigorously controlled because too high concentrations can prevent the formation of micelles from monomers: it is generally accepted that micelles are not stable in mixtures of water and organic solvents containing more than 20-30% of the latter.…”

Separation of cytokinin isomers with a partial filling‐micellar electrokinetic chromatography‐mass spectrometry approach

“…As the optimized concentration of SDS for MEKC separation was 50 mM in our previous studies [12,13], thus the initial lowest ALS concentration was set at 50 mM in the current study. It is not too surprising that an increase in micelle concentration increased the separation resolution.…”

“…Furthermore, like any other CE technique, MEKC possesses many other advantages, such as simpler method development, minimal sample volume requirements, minimal organic waste, and separation could be achieved with simple and low cost capillaries instead of expensive chro- matographic columns. Our previous studies have shown that MEKC with UV detection could provide fast and efficient separations of structurally similar cytokinins, such as isomers [10][11][12][13]. Direct identification and structure elucidation of analytes after separation is most commonly achieved by coupling with MS [14,15].…”

Analyses of gibberellins in coconut (Cocos nucifera L.) water by partial filling‐micellar electrokinetic chromatography‐mass spectrometry with reversal of electroosmotic flow

“…MEKC has been employed for the separation of not only charged, but also neural compounds by means of its capacity to combine effects of partitioning between an aqueous and micellar phase, and electrophoretic mobility of sample components [13]. It has been reported that MEKC can compete with HPLC with regard to the separation resolution and separation time [14,15]. However, the direct coupling of MEKC to MS is limited by the use of nonvolatile surfactants.…”