Validation and Further Optimisation of a Cyclodextrin-Modified Micellar Electrokinetic Capillary Chromatography Method for Urine Profiling

Abstract

Metabonomic studies require efficient and high-resolution analytical probes to monitor changes in the ‘metabolic fingerprint’. The advantageous characteristics of Capillary Electrophoresis, enabling highly efficient separations of diverse components present in minute sample volumes, may therefore prove a useful tool in biofluid analysis. This paper describes the optimisation and validation of a sulphated β-cyclodextrin-modified MECC method for urine profiling. Cyclodextrin substitution, experimental conditions including capillary length, injection mode and time, applied voltage, temperature and capillary pre-conditioning procedures were investigated and optimised. Precision, linearity, sensitivity and robustness of the method were assessed, as well as urine stability. The validated sulphated β-cyclodextrin-modified MECC method allows for the separation of over 80 urinary analytes in under 25 min, using a sodium borate/SDS/sulfated β-cyclodextrin (25/75/6.25 mM) electrolyte and an 18 kV applied voltage in a 40 cm (effective length), 50 μm i.d. fused silica capillary at 20 °C, pre-conditioned with HCl for 5 min and BGE for 1 min, and UV diode array detection (190–600 nm). Such methodology should prove invaluable in the rapid comparison of urine profiles and indication of metabolic disorders or abnormalities.