A Rapid Method for the Simultaneous Determination of Acetaldehyde and Ethanol in Blood using Gas Chromatography

Abstract

A simple, rapid, specific and reliable method for the simultaneous determination of blood levels of acetaldehyde (AcH) and ethanol (EtOH) by gas chromatography is described. Air samples equilibrated with blood were used rather than actual blood samples. A programmed temperature gas chromatograph with a hydrogen flame ionization attachment was used with a 1-mv. recorder. The operating procedures are described. Ethanol standards were prepared by diluting 1 g. of absolute ethanol to 50 ml. with distilled water. Volumetric dilutions of this solution with either distilled water or human blood provided EtOH standards of 2, 1, and 0.5 mg. per ml. Standard solutions of AcH were prepared by diluting 1 g. of AcH to 50 ml. with distilled water. Volumetric dilutions of this solution provided AcH standards of 20, 10 and 5 g. per ml. The reference standard solutions were equilibrated for 15 min. at 55 degrees C and injected periodically throughout the experiments. If endogenous EtOH was to be determined, a standard of 0.2 mg. per ml. was used and the attenuation changed to 1. The deproteinizing solutions used were 5% ZnSO4.6H2O and 0.3N Ba(OH)2. Standard and unknown samples were determined by identical procedures. Blood was obtained from rats either by direct cardiac puncture or by decapitation. No difference in levels was noted between these two methods. Degradation of AcH but not EtOH occurred in whole blood samples incubated at 37 or 55 degrees C. This was prevented by alkaline deproteinization of the samples but not by HgCl2 (1 times .0001 M). Following alkaline deproteinization, cold storage (5 degrees C) of samples was shown to be effective for 3 days in preserving initial AcH and EtOH levels. Solutions of lactic acid and pyruvic acid, equal to the normal blood concentrations of these metabolites, could not be detected. Solutions of acetone could be detected but its retention time lies half way between that of AcH and EtOH and its presence would not interfere with this method. The relative retention time of 6 volatile substances which have been encountered in human intoxication (acetaldehyde, propionaldehyde, acetone, methanol, isopropanol and n-propanol) were determined and no interference with AcH or EtOH determination could be demonstrated. The methods previously available for the determination of AcH are discussed and it is concluded that the gas chromatographic method described offers the rapidity, reliability and specificity that have been deficient in some older procedures.