Determination of free acetaldehyde in blood as the dinitrophenylhydrazone derivative by high-performance liquid chromatography

Abstract

A simple and sensitive method is proposed for the measurement of acetaldehyde in human blood. Venous blood samples were collected in EDTA Vacutainer tubes, and treated immediately with 0.6 M ice-cold perchloric acid in saline. After centrifugation at 4°C, the supernatants were treated with dinitrophenylhydrazine reagent. After addition of the internal standard (crotonaldehyde dinitrophenylhydrazone) and 3 M sodium acetate, the derivatives were extracted and analysed by high-performance liquid chromatography (HPLC) using an Ultrasphere ODS column. The compounds were separated using acetonitrile—water as the mobile phase and detected at 356 nm. A blank determination was carried out for each analysis and subtracted from the results. The specificity of the method was tested by UV and mass spectrometry and the purity of the derivatives by capillary gas chromatography. The recovery of blood acetaldehyde was 98%. Interference from ethanol was minimized by using the tripotassium salt of EDTA as an anticoagulant. The sensitivity of the method can be increased dramatically using microbore HPLC. The level of acetaldehyde was found to be 0.41 ± 0.13 μ M (mean ± S.D.) for eight fasting controls and 0.91 ± 0.73 μ M for fourteen alcoholics ( p<0.05). At 30 min after oral administration of ethanol (0.8 g/kg), the ethanol levels were 16.3 ± 2.8 and 17.7 ± 2.5 m M and the acetaldehyde levels were 1.67 ± 0.35 and 3.13 ± 2.43 μ M ( p<0.05) for the controls and alcoholics, respectively.