Determination of lactate dehydrogenase in human erythrocytes by capillary electrophoresis with electrochemical detection

Abstract

Capillary electrophoresis (CE) was employed to analyze lactate dehydrogenase (LDH) in human erythrocytes using an amperometric detector with a carbon fiber micro-disk bundle electrode. LDH activity was measured by determining the amount of NADH generated by LDH through a enzyme-catalyzed reaction between NAD + and lithium lactate. The factors influencing the enzyme-catalyzed reaction, separation and detection were examined and optimized. The following conditions were suitable for the determination of LDH: running buffer, 5.0×10 −2 mol/l Tris–HCl (pH 7.5); separation voltage, 20.0 kV; detection potential, 1.00 V (versus saturated calomel electrode (SCE)). The conditions of enzyme-catalyzed reaction were: reaction buffer, 5.0×10 −2 mol/l Tris–HCl (pH 9.3); substrates, 5.0×10 −2 mol/l lithium lactate and 5.0×10 −3 mol/l NAD +; reaction time, 10 min. The concentration limit of detection (LOD) of the method was 0.017 U/ml at a signal-to-noise (S/N) ratio of 3, which corresponded to 1.10×10 −10 mol/l, and the mass LOD was 2×10 −20 mol. The linear dynamic range was 0.039–4.65 U/ml for the injection voltage of 5.0 kV and injection time of 10 s. The relative standard deviation (R.S.D.) was 0.85% for the migration time and 1.8% for the electrophoretic peak area. The method was applied to determine LDH in human erythrocytes. The recovery of the method was between 98 and 101%.