ABSTRACT
In this preliminary study we have optimised micellar electrokinetic chromatography (MEKC, a form of capillary electrophoresis) to enable the chromatographic and spectral characteristics of human ouabainlike compound (OLC) to be investigated. Sera from fifty patients were combined to form a pool (100 ml) whilst urine (92.5 ml) was obtained from a normal healthy volunteer. Both samples were initially concentrated and partially purified by solid phase extraction before further purification by sequential HPLC separations. Final volumes for both extracts were 100 microl. MEKC was performed on a HP(3D) CE instrument with voltage set at 20 KV, capillary temperature at 20 degrees C, injection time 4 s, sample volume 10 nl, with detection by photodiode array. A compound was found in both serum and urine that had similar elution and spectral characteristics to authentic ouabain. We conclude that MEKC is potentially a useful tool for the analysis of human OLC.
Subject(s)
Chromatography, Micellar Electrokinetic Capillary , Digoxin , Hypertension/blood , Saponins/analysis , Saponins/blood , Cardenolides , Cardiotonic Agents , Chromatography, High Pressure Liquid , Humans , OuabainABSTRACT
In this study both native and chemically modified cyclodextrins (CDs) were investigated as buffer additives to improve the micellar electrokinetic capillary chromatography (MEKC) separation of endogenous bioanalytes in human urine. The following CDs were investigated: alpha, beta, gamma-CDs; hydroxypropyl-alpha-CD, hydroxypropyl-beta-CD, methylated beta-CD, sulphated beta-CD, sulphobutyl ether-beta-CD and hydroxypropyl-gamma-CD. The separations were compared to MEKC without additives. The best improvement in peak resolution and separation of urine components was observed with the sulphated beta-CD. A four-factor three-level full factorial design study was conducted on voltage, temperature, pH and sulphated beta-CD molarity. The optimum conditions were 25 mM sodium tetraborate, pH 9.5, 75 mM sodium dodecyl sulphate (SDS) and 6.25 mM sulphated beta-CD and were able to resolve 70 peaks from a urine pool in 12 min. These optimum conditions have been successfully applied to a number of clinical samples.
Subject(s)
Chromatography, Micellar Electrokinetic Capillary/methods , Cyclodextrins , Urine/chemistry , alpha-Cyclodextrins , beta-Cyclodextrins , gamma-Cyclodextrins , HumansABSTRACT
A micellar electrokinetic chromatographic method is described for the determination and quantitation of allantoin, an end-product of purine metabolism in mammals that is applicable to biofluids of different mammal species and man. The method was optimised following a study on the effect of pH and sample preparation procedure. Final conditions were 30 mM sodium tetraborate, pH 9.5, 75 mM sodium dodecyl sulphate, 20 kV and 20 degrees C. Allantoin was well resolved from endogenous compounds and could be determined in horse, dog, mouse and rabbit urine. No allantoin could be found in man. No complicated sample treatment was necessary, thus the developed method was rapid (< 5 min), sensitive (5 microM) and simple. Results from this work will permit the determination of allantoin in man as a measure of free radical generation reactions as well as its presence in the plasma and other biofluids with modification of the sample preparation procedures.
Subject(s)
Allantoin/urine , Chromatography, Liquid/methods , Electrophoresis, Capillary/methods , Animals , Dogs , Horses , Humans , Mice , Rabbits , Reproducibility of Results , Species SpecificitySubject(s)
Electrophoresis, Capillary/methods , Purine-Pyrimidine Metabolism, Inborn Errors/urine , Purines/metabolism , Urinalysis/methods , Allantoin/urine , Animals , Deoxyguanosine/urine , Guanosine/urine , Humans , Inosine/analogs & derivatives , Inosine/urine , Mammals , Purine-Nucleoside Phosphorylase/deficiency , Purine-Pyrimidine Metabolism, Inborn Errors/diagnosis , Reference Values , Spectrophotometry, UltravioletABSTRACT
A micellar electrokinetic capillary chromatography (MECC) method when compared to free solution capillary electrophoresis (CZE) was shown to offer improved selectivity and resolution for the separation of UV-absorbing components of human urine. Some of the factors affecting MECC separation e.g. methanol concentration, sodium dodecyl sulphate (SDS) concentration, beta-cyclodextrin (beta-CD) concentration, voltage, pH, temperature and electrolyte additives (urea, beta-CD and Brij 35) were optimised using chemometric techniques. Three-level three-factor (3(3)) factorial designs and simplex optimisation were used to achieve optimised conditions with the goal of obtaining the maximum number of peaks in the shortest possible analysis time. Using a TSP CE2000 instrument with detection from 195-300 nm and fitted with a 75 microns x 44 cm (37 cm effective length) fused silica capillary the final optimum conditions were found to be, an electrolyte consisting of 30 mM sodium tetraborate, pH 10, containing 75 mM SDS and 10 mM beta-CD, 15 degrees C, 20 kV, 4 s hydrodynamic injection of filtered urine. These conditions were capable of separating 70 peaks from a normal human urine pool in less than 12 min. The separation of components in urine using the optimised MECC was simpler, more reproducible, faster and gave better resolution than gradient reversed-phase high performance liquid chromatography.
