Analysis of Corticosteroids in Biofluids by Capillary Electrochromatography with Gradient Elution

Capillary electrochromatography (CEC) with gradient elution was used to separate mixtures of corticosteroids (adrenosterone, hydrocortisone, dexamethasone, fluocortolone) in extracts of equine urine and plasma. Urine samples were first purified using solid phase extraction. Two purification steps were necessary to prevent contamination of the CEC column. Plasma was purified using automated dialysis. A laboratory-built CEC interface, connected to a gradient HPLC system, delivered samples and mobile phase to the CEC column. CEC was performed in fused-silica capillaries of 50 μm i.d., 24 cm total length, and 16 cm effective length packed with Apex ODS, 3 μm particle size. The mobile phase was ammonium acetate (5 mM) in water/acetonitrile. Acetonitrile in the mobile phase was varied from 9 to 80% (v/v) using the gradient HPLC system. Detection was by UV absorbance at 240 nm. Samples, 10−250 μL, were injected into the mobile phase stream and loaded onto the CEC column under an applied field of 1.04 kV cm-1 and a CEC column head pressure of 12 bar. Mobile phase flow rate through the sampling interface was 100 μL min-1. The system was reproducible and could be left in unattended operation for long periods. After injection of 200 urine extracts, a broadening of peaks was observed but the CEC column was still serviceable.