Nanoemulsion supported microemulsion electrokinetic chromatography coupled with selected preconcentration techniques as an approach for analysis of highly hydrophobic compounds

Highlights

• Nanoemulsion sample matrix by cold dilution of microemulsion with water was developed.

• Sample matrix for the signal amplification of hydrophobic compounds was employed.

• The mechanism of signal amplification for hydrophobic compounds was described.

• Sensitivity enhancement factor for vitamin K₂-MK7 in NE matrix was determined.

Abstract

In this paper, an oil-in-water (O/W) nanoemulsion (NE) prepared by water cold dilution of an O/W microemulsion (ME) was introduced as a sample matrix in microemulsion electrokinetic capillary chromatography (MEEKC) for the highly hydrophobic compounds analysis. Several model compounds with log P _O/W values in the 4.1–10.9 range, from different chemical groups, including retinol, α-tocopherol, cholecalciferol, phylloquinone, menaquinone-7, dichlorodiphenyltrichloroethane, ivermectin have been tested. As a proof of the concept of NE formation, a dynamic light scattering technique was employed to determine the size distribution profile of NE particles. Moreover, due to relatively low conductivity of the NE matrix (50–100 times lower in comparison to the separation buffer) and a negative electric charge provided to hydrophobic compounds through NE dispersed phase, NE matrices have been combined with preconcentration techniques based on electrokinetic dosing, namely field amplified sample injection (FASI) and pressure assisted electrokinetic injection (PAEKI). The detection limits for vitamin K₁ and K₂-MK7 in the NE matrix in combination with FASI (NE–MEEKC–FASI) as well as PAEKI (NE–MEEKC–PAEKI) were up to 42.9 and 12.1 ng mL⁻¹, respectively. In comparison to standard hydrodynamic injection for microemulsion sample matrix NE–MEEKC–PAEKI grant 45-fold improvement in signal sensitivity. The study presents an innovative approach, as it enables the use of preconcentration techniques for highly hydrophobic compounds (log P _O/W > 4), which was not previously possible for implementation in the electromigration techniques. Likewise, the use of organic solvents has been reduced by using ME as a solvent for stock solutions and diluting with water prior to the analysis. The application to real samples was investigated using a dietary supplement containing vitamin K₂-MK7 obtained from the fermentation product of soybeans.

Introduction

Recently, nanoemulsions (NE) have gained interest in the field of pharmacy, cosmetics, food, and agrochemical industry as novel drug delivery systems that increase in the bioavailability of active agents [1]. The term "nanoemulsion" was first introduced by Nakajima et al. [2] and refers to the particle size of dispersed phase, which, according to various literature data, ranges from 20 up to 1000 nm. In comparison to microemulsions (ME), NEs are thermodynamically unstable and require lower concentrations of surfactant for their formation [3,4]. Taylor and Ottewill [5] presented the formation of NE by spontaneous emulsification through diluting an oil-in-water (O/W) ME for the first time, a process also termed a cold dilution, considered as a low energy method. As shown in Fig. 1, the molecular mechanism of this phenomenon involves the diffusion of co-surfactant molecules from the oil droplets into the water phase. In these conditions, the surfactant does not provide a low surface tension as a result of the decrease in concentration, the system is thermodynamically unstable, which appears in the formation of a nanoemulsion [3]. A further study on the dilution of MEs was conducted by Solè et al., which demonstrated that a single or multiple dilution of the O/W ME with water does not affect the final oil droplet size of the NE [6].

Microemulsion electrokinetic chromatography (MEEKC) is a well-known mode of electromigration technique [7], [8], [9], [10], in which the pseudostationary phase is the dispersed phase of the ME. Despite the higher resolution potential than capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) techniques [11], [12], [13], on-line preconcentration methods in combination with O/W MEEKC have not been studied so far during the analysis of highly hydrophobic compounds (log P _O/W > 4). The development of a sample matrix (SM) for high log P _O/W compounds is problematical due to the instability of the ME to changes in composition between the sample and the separation buffer zone; samples with a high concentration of organic solvents can lead to ME collapse and current disturbance. For this reason, separations of hydrophobic compounds by O/W MEEKC technique are based on the matrices prepared by dissolving the samples with a background electrolyte (BGE). This can lead to the inability of the application of such sample matrices in combination with preconcentration techniques [14], [15], [16]. Another advantage of proposed method over CZE and MEKC separations is that MEs have significantly higher solubility potential as they function as versatile solvents, therefore complex matrices consisting of both hydrophobic and hydrophilic compounds, separated under MEEKC conditions are common in the literature [17], [18], [19], [20]. The development of new methods for the separation highly hydrophobic compounds due to the composition of the sample matrix, and the possibility of combining them with on-line preconcentration techniques will advance the progress in the field of capillary electromigration methods.

Here, the development of sample matrix composition based on O/W NE prepared by cold dilution of O/W ME with water (Fig. 1) as a sample matrix for highly hydrophobic compounds has been presented. The aim of the study was to demonstrate that the NE matrix ensures the solubility of highly hydrophobic analytes, while providing them with a sufficient electric charge for the electrokinetic injection (EKI). Secondly, due to the low conductivity of NE matrices, the possibility of applying preconcentration methods based on EKI in an enhanced electric field for hydrophobic compounds was investigated. The prepared NEs were verified by dynamic light scattering technique to measure the particle size distribution, and the concentration sensitivity enhancement factor has been determined for menaquinone-7 (VK2) as exemplary sample.