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

Micellar electrokinetic capillary chromatography (MEKC), a mode of capillary electrophores is, can separate cations, anions and uncharged molecules simultaneously and with high resolution. So ii is ideal for separating endogenous and exogenous analytes in complex biofluids, such as urine. Our previous system used beta -CD-enhanced SDS MEKC to aid resolution and could resolve a large number of 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 our standard urine system but without much success. However some modified cyclodextrins were found to increase both resolution and the migration window. In particular sulphated beta -cyclodextrin gave excellent improvements in both resolution and selectivity. The optimised conditions (6.2 mM S-beta -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 but with 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-IC.