Determination of free and total valproic acid in human plasma by capillary electrophoresis with contactless conductivity detection

Abstract

A new approach for the determination of free and total valproic acid in small samples of 140 µL human plasma based on capillary electrophoresis with contactless conductivity detection is proposed. A dispersive liquid–liquid microextraction technique was employed in order to remove biological matrices prior to instrumental analysis. The free valproic acid was determined by isolating free valproic acid from protein-bound valproic acid by ultrafiltration under centrifugation of 100 µL sample. The filtrate was acidified to turn valproic acid into its protonated neutral form and then extracted. The determination of total valproic acid was carried out by acidifying 40 µL untreated plasma to release the protein-bound valproic acid prior to extraction. A solution consisting of 10 mM histidine, 10 mM 3-(N-morpholino)propanesulfonic acid and 10 µM hexadecyltrimethylammonium bromide of pH 6.5 was used as background electrolyte for the electrophoretic separation. The method showed good linearity in the range of 0.4–300 µg/mL with a correlation coefficient of 0.9996. The limit of detection was 0.08 µg/mL, and the reproducibility of the peak area was excellent (RSD = 0.7–3.5%, n = 3, for the concentration range from 1 to 150 µg/mL). The results for the free and total valproic acid concentration in human plasma were found to be comparable to those obtained with a standard immunoassay. The corresponding correlation coefficients were 0.9847 for free and 0.9521 for total valproic acid.