Determination of enkephalin peptides by nonaqueous capillary electrophoresis with electrochemical detection

Nonaqueous capillary electrophoresis with electrochemical detection (NACE-ED) was applied to the analysis of enkephalin peptides. The effect of different buffer compositions on the electrophoretic behavior of methionine enkephalin, leucine enkephalin, and D-Ala2]-leucine enkephalin was studied. Separation of the protonated and the deprotonated peptides was obtained using ACN/methanol-based electrolyte systems. The electrochemical behavior of the enkephalins was studied by the capillary batch injection analysis technique. NACE-ED yielded well-defined signals in the oxidation mode only for the negatively charged analytes. The optimized BGE for the counterelectroosmotic separation consisted of 10 mM ammonium acetate in ACN/methanol (3:1 v/v). Using a platinum microdisk electrode set to an actual potential of +0.65 V detection limits in the submicromolar range were observed which are about one order of magnitude lower compared to UV detection. Problems concerning EOF instability and electrode fouling caused by water and other neutral sample impurities transported by the EOF can be avoided in the EOF-inverted mode using poly(ethylene glycol)-coated capillaries and an actual working electrode potential of +1.0 V. For the quantification of the enkephalins D-Ala2]leucine enkephalin was used as internal standard. The practical utility for the determination of enkephalins in spiked plasma samples after SPE was demonstrated.