Further studies on the MEKC and MEEKC of UV-Absorbing compounds in human urine

Abstract

SummaryMicellar electrokinetic capillary chromatography (MEKC), a mode of capillary electrophoresis, can separate cations, anions and uncharged molecules simultaneously and with high resolution. So it is ideal for separating endogenous and exogenous analytes in complex biofluids, such as urine. Our previous system used 13-CD-enhanced SDS MEKC to aid resolution and could resolve a large numberof peaks from human urine in about 12 minutes.In this study we have investigated a number of alternative additives to enhance resolution even further. Various compounds have been claimed, in the literature, to improve MEKC e.g. mixed micelles, organic solvents, and urea. We investigated a number of such compounds as additives in ourstandard urine system butwithout much success. However some modified cyclodextrins were found to increase both resolution and the migration window. In particular sulphated 13-cyclodextrin gave excellent improvements in both resolution and selectivif,/. The optimised conditions (6.2 mM S-13-CD in 25 mM sodium tetraborate, pH 10, 75 mM SDS) resolved 70 components in a normal urine pool in < 15 minutes using < 10 nl of urine. Faster separations butwith some loss of resolution were obtained by injection from the detector end of the capillary (short-end injection).In addition we have investigated the use of microemulsion electrokinetic chromatography MEEKC but found that it offered no real advantages over MEKC for urine analysis.This optimised MEKC separation is simpler, faster and gives higher resolution than gradient RP-LC.

A comparative study of microemulsion electrokinetic capillary chromatography and micellar electrokinetic capillary chromatography for the analysis of UV‐absorbing compounds in human urine

“…120 mM SDS provided the best compromise between high resolution, reproducibility of separation and system stability. To conclude, the best MEKC separation of urinary solutes in terms of overall analysis time, efficiencies, resolution, and the number of peaks resolved was 30 mM aqueous disodium tetraborate, pH 10 and 120 mM SDS, conditions not dissimilar to those reported previously [36][37][38].…”

“…The majority of published applications have involved a microemulsion system consisting of octane/1-butanol/ SDS/borate buffer, pH 9.2 [8,23,45]. In our previous study [36] of urine analysis by MEEKC, we found that such microemulsion systems showed poor separation of components in human urine. In order to investigate and improve this separation, new microemulsions were studied.…”

“…The anodic and cathodic buffer vials were replaced every fifth run. In both the MEEKC and MEKC systems, urinary components were identified by comparison to previously published data [36][37][38], by spiking with authentic materials and spectral characterisation.…”

“…In a previous study [36], we found that a standard MEEKC system functioned poorly for complex neat biofluids such as urine. In this study, we compared optimised MEEKC and MEKC methods for the separation of UV-absorbing endogenous components in urine, a complex matrix.…”

Recent applications of microemulsion electrokinetic chromatography

“…Only two examples of urine analysis using MEEKC have been published [29,58] but both examples show very preliminary results and from these it is very difficult to judge the potential of the technique. In bioanalysis it is a problem that the detectability in CE techniques is less than that obtained in HPLC.…”