On-line and off-line enrichment techniques combined with capillary electromigration separation methods in the analysis of highly hydrophilic analytes in biological and environmental samples

The current work investigates capillary electromigration separation methods as alternative strategies to the existing methodologies for the analysis of highly hydrophilic analytes. Combined with different on-line and off-line enrichment techniques, the applicability of the developed approaches is demonstrated in either biological fluids or environmental samples. Urinary nucleosides and α-aminocephalosporins are selected as model analytes for the current study. Nucleosides possess a cis-diol moiety that enables them to form negatively charged complexes with tetrahydroxyborate under alkaline pH conditions. From the effective electrophoretic mobility data, it is shown that the degree of complexation is close to one even at very low tetraborate concentration (2.5 mmol L-1). Insufficient resolution of the studied nucleosides using capillary zone electrophoresis (CZE) as a standing alone technique (in the presence of tetraborate buffer as a background electrolyte (BGE)) necessitates using alternative approaches which are: (i) another CE mode or (ii) buffer additives that can provide a different separation selectivity to permit the complete separation of the studied analytes. Following the first approach, a micellar electrokinetic chromatographic method (MEKC) is developed for the separation of urinary nucleosides in their ionic form using the ionic liquid-type surfactant 1-tetradecyl-3-methylimidazolium bromide (C14MImBr) as a cationic surfactant in the presence of tetrahydroxyborate. A complete separation of these hydrophilic metabolites is realized using a low concentration of C14MImBr (20 mmol L-1) in the BGE (5 mmol L-1 tetraborate, pH 9.38). Fundamental aspects underlying the separation of urinary nucleosides using C14MImBr are studied including the mode of interaction of these compounds with C14MImBr and regulation of the retention factors with respect to the oppositely charged PSP. It is proven that the negatively charged complexed nucleosides interact mainly with the C14MImBr micelles by electrostatic interaction, while hydrophobic interaction can be considered to be negligible. Moreover, it is demonstrated that the retention factors are increased with decreasing borate concentration and increasing pH of the BGE. Employing the conditions that maximize the interaction between the nucleosides and the C14MImBr micelles (quantified via the associated equilibrium constants), a fully optimized and validated MEKC method combined with different on-line enrichment techniques is successfully developed for the identification and quantification of nucleosides in urine samples. It is shown that “pseudostationary ion-exchanger” sweeping is the major contributor to the overall enrichment process. However, due to the low retention factors encountered for the nucleosides adenosine (Ado) and cytidine (Cyd), C14MImBr cannot be effectively employed for the sweeping of these analytes. In addition, these two nucleosides comigrate with urinary matrix constituents. As an alternative, SDS is investigated for the analysis of Ado and Cyd as positively charged species (under acidic pH conditions) together with “pseudostationary ion-exchanger” sweeping as on-line enrichment principle, which is applied successfully to the analysis of the two nucleosides in urine samples. Moreover, it is established that with a BGE containing the combination of an alkyl/aryl boronate and C14MImBr, the retention factors of all the studied nucleosides are significantly increased. The shift in the retention factors to higher values is attributed to the additional hydrophobic interaction sites introduced by the alkyl/aryl group of the boronate that forms a complex with the cis-diol group of the nucleoside. It is shown that these optimization strategies result in validated methods, which permit the successful analysis of the studied nucleosides in urine samples with limits of detection in the range of 0.1-0.2 mg L-1. Following the second approach, 2-hydroxypropyl-β-cyclodextrin (2-HP-β-CD) is used as buffer additive in the presence of tetraborate buffer, which permits the modification of the separation selectivity and enables a complete separation of the investigated nucleosides via CZE. Taking advantage of the high complex formation constant between the nucleosides and tetrahydroxyborate and employing 2-HP-β-CD as selectivity-tuning additive, a highly sensitive CZE method is developed based on a highly efficient on-line focusing procedure comprising three steps, which are dynamic pH junction, borate sweeping, and large volume sample stacking (LVSS). Limits of detection as low as 10-40 μg L−1 are achieved. The proposed method is validated according to ICH guidelines and is successfully applied to the analysis of the nucleosides under investigation in blank and spiked urine samples. The outcomes of the second approach are successfully transferred to the analysis of selected α-aminocephalosporins in surface water samples. Together with the use of a highly sensitive detection method such as laser-induced fluorescence (LIF) detection, CZE with LVSS-sweeping is applied successfully to the analysis of cefalexin and cefadroxil in spiked Lahn water samples reaching limits of detection as low as 5-8 ng L-1.