Evaluation of diabetes-related short-chain organic acids in rat plasma by capillary electrophoresis.

Abstract

A capillary zone electrophoresis method was optimised to analyse low-molecular-mass organic acids for the purpose of monitoring diabetes in rat plasma. The method included acetoacetic, 2-hydroxybutyric, lactic and uric acids. A variation in the background electrolyte allowed us to measure pyruvic acid in the same sample. Conditions have been optimised for measuring a large number of plasma samples corresponding to control and diabetic rats. Samples were mixed with acetonitrile (1:1, v/v) to precipitate proteins, centrifuged, diluted and injected. Tropic acid was chosen as an adequate internal standard. Separation was developed with reversed voltage by using a column cartridge pre-treated with polyacrylamide. Two electrophoretic buffers were employed: 0.150MH3PO4 made up pH 6.20 with NaOH and 0.3mMCaCl2 for acetoacetic, hydroxybutyric, lactic and uric acids, and 200mM phosphate–10mM acetate pH 4.0 for pyruvic acid, both with direct detection at 200 nm. The method was validated for linearity, accuracy and precision and the limits of quantification were calculated. The method was successfully applied to analyse these organic acids in control and diabetic animals. Acetoacetic and hydroxybutyric acids were clearly increased in diabetic rats, meanwhile no statistically significant difference has been found with the other acids.