On-line preconcentration and chiral separation of propiconazole by cyclodextrin-modified micellar electrokinetic chromatography

Abstract

A method for the chiral separation of propiconazole using cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) with hydroxypropyl-γ-cyclodextrin (HP-γ-CD) as chiral selector is reported. The use of a mixture of 30 mM HP-γ-CD, 50 mM SDS, methanol–acetonitrile 10%:5% (v/v) in 25 mM phosphate buffer solution was able to separate two enantiomeric pairs of propiconazole. Stacking- and sweeping-CD-MEKC under neutral pH (pH 7) and under acidic condition (pH 3.0) were used as two on-line preconcentration methods to increase detection sensitivity of propiconazole. Good repeatabilities in the migration time, peak area and peak height were obtained in terms of relative standard deviation (RSD). A sensitivity enhancement factor of 100-fold was achieved using sweeping-CD-MEKC at acidic pH. This is the first report on the separation of two pairs of propiconazole enantiomers and all the enantiomers of fenbuconazole and tebuconazole using sweeping-CD-MEKC. The limit of detection (S/N = 3) for the three triazole fungicides ranged from 0.09 to 0.1 μg/mL, which is well below the maximum residue limits (MRL) set by Codex Alimentarius Commission (CAC). Combination of solid-phase extraction (SPE) pretreatment and sweeping-CD-MEKC procedure was applied to the determination of selected triazole fungicides in grapes samples spiked at concentration 10–40 times lower than the MRL established by the CAC. The average recoveries of the selected fungicides in spiked grapes samples were good, ranging from 73% to 109% with RSD of 9–12% (n = 3).

Introduction

Separation of enantiomers has become one of the most important tasks of analytical chemistry especially in the field of pharmaceutical, clinical and agrochemical sciences, since it is well known that a pair of enantiomers can display different biological activities. Analytical methods used so far for the chiral separation include thin-layer chromatography [1], [2], gas chromatography [3], [4], high-performance liquid chromatography [5], [6], supercritical fluid chromatography [7], [8] and capillary electrochromatography [9]. However, a fast growing number of studies are reported on the use of capillary electrophoresis (CE) in chiral separation [10], [11], [12], [13]. Capillary electrophoresis has shown to be a powerful separation technique for enantiomers compared to conventional chromatographic techniques.

Micellar electrokinetic chromatography (MEKC) has become popular as a powerful technique for improving separation efficiency not only of neutral analytes but charged ones by using CE instrument without any alteration. The advantages of MEKC for enantiomeric separations are rapid method development, minimal use of expensive chiral reagents and provide high-separation efficiencies. Two approaches can be used to perform enantiomeric separation in MEKC, namely, the use of chiral surfactants and the addition of cyclodextrins (CDs) to the micellar solution [14]. However, CE suffers from poor concentration sensitivity when using UV detection because of the small injection volumes and narrow optical path length. Various alternatives have been developed such as the use of laser-induced fluorescence [15] and electrochemical detection [16]. In addition, off-line concentration technique such as solid-phase extraction (SPE) can be used as a clean-up method prior to CE [17], [18]. An attractive alternative is the use of on-line sample preconcentration techniques. This is a versatile and effective way of increasing concentration sensitivity. Two on-line sample preconcentration techniques, sample stacking and sweeping are known to be effective techniques for enhancement of the concentration sensitivity in MEKC [19], [20], [21].

Triazole fungicides represent the most important category of fungicides to date. They have excellent protective, curative and eradicant power towards a wide spectrum of crop diseases. Chirality is expected to play a crucial role in the bioactivities of triazole fungicides. The enantiomeric separations of triazole-type fungicides have been performed using CE procedures [22], [23], [24], [25]. Mixtures of uniconazole and diniconazole enantiomers were baseline resolved using 50 mM phosphate buffer (pH 6.5) containing 5 mM carboxymethyl-γ-CD and a temperature of 50 °C in less than 5 min [22]. Simultaneous chiral separation of triadimefon and triadimenol [23] and chiral separation of 14 triazole fungicides (including propiconazole, tebuconazole, without fenbuconazole) [24] were successfully achieved using sulfated-β-cyclodextrin-mediated CE under acidic conditions. However, the limit of detections (LODs) were not discussed. Cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) has also been employed to the enantiomeric and isomeric separation of seven commonly used pesticides (including propiconazole). However, only two diastereomers of propiconazole were baseline separated in this study [25]. It is therefore of interest to study the CD-MEKC method for the enantiomeric separation of two pairs of propiconazole enantiomers and the capabilities of two on-line preconcentration methods, i.e. stacking- and sweeping-CD-MEKC were evaluated. Sweeping-CD-MEKC at acidic pH was found to give the highest sensitivity (100-fold) and the method was applied to the chiral separation of propiconazole enantiomers followed by the chiral separation of two other triazole fungicides, i.e. fenbuconazole and tebuconazole (Fig. 1). This is the first ever reported separation of two pairs of propiconazole enantiomers and all the enantiomers of fenbuconazole and tebuconazole by CD-MEKC. The optimized on-line sample preconcentration-CD-MEKC procedure combined with SPE pretreatment was applied to the determination of selected triazole fungicides in grapes samples spiked at levels 10–40 times lower than the maximum residue limits (MRL) set by Codex Alimentarius Commission (CAC) [26].