Direct determination of valproate in serum by zone electrophoresis-isotachophoresis on a column-coupling chip

A feasibility study was performed using zone electrophoresis (ZE) coupled on-line with isotachophoresis (ITP) sample pretreatment on a poly(methyl methacrylate) column-coupling chip with integrated conductivity detection for direct determination of drugs in serum. Valproic acid (an antiepileptic drug), having a therapeutic range of 0.35-0.69 mmol/L (50-100 mg/L), was a test analyte while reference serum samples served as proteinaceous matrices. ITP provided in the ITP-ZE combination a multitask sample pretreatment: (1) separation of the analyte from the serum matrix and its concentration into a narrow ITP band, (2) removal of the matrix constituents migrating in the ITP stack from the separation compartment of the chip, (3) ITP stacking of the drug released on a continuous electrophoretic decomposition of the drug-protein complex. A high sample loadability, closely linked with the use of ITP in the first separation stage, made it possible to inject diluted serum samples with the aid of a 0.95 muL sample channel of the chip. Consequently, a 1-2 mumol/L concentration limit of quantitation for valproate from the response of the conductivity detector in the ZE stage of the combination was reached. The drug could be reliably determined in less than 10 minutes also in instances when its concentration in serum was below the lower value of the therapeutic range. 90-94% recoveries of valproate from serum samples were obtained in its direct ITP-ZE determination when the filtration of the diluted serum (a 0.45 mum pore size filter) was the only pre-column sample handling operation. No disturbances attributable to the precipitation of proteins from the loaded samples in the chip channels were detected.