An optimized method for the measurement of acetaldehyde by high-performance liquid chromatography.

THE LEVEL OF circulating acetaldehyde (AcH) after ethanol ingestion depends on the rate of its formation and elimination (Deitrich et al., 2007). Acetaldehyde is derived from the oxidation of ethanol by alcohol dehydrogenase, with lesser contributions from the microsomal ethanol oxidizing system and catalase. It is metabolized mainly by aldehyde dehydrogenase (ALDH) to acetate. The liver mitochondrial ALDH2 isozyme is responsible for almost all elimination of acetaldehyde in humans and rats. Low micromolar levels of acetaldehyde are detectable in tissues and biological fluids after ethanol consumption (Eriksson, 2007). There is great interest in measuring elevated acetaldehyde levels after ethanol consumption in: (i) Heavy drinkers (Eriksson, 1980; Lindros et al., 1980, Matthewson et al., 1986); (ii) Heterozygotes and homozygotes for the ALDH2*2 allele among Southeast Asians carrying a point mutation (G1510A) that causes a Glu487Lys substitution, with consequent decreased affinity for NAD+ and decreased activity of the liver enzyme (Yoshida et al., 1998), compared to ALDH2*1 carriers (Chen et al., 2009); (iii) Alcoholics treated with disulfiram (Antabuse) in aversive therapy for alcoholism (Barth and Malcolm, 2010) after ethanol consumption. Disulfiram is an FDA-approved small molecule that inhibits ALDH2 by irreversible binding of its metabolites to sulfhydryl groups in the active site of the enzyme (Lipsky et al., 2001); (iv) Screening for new drugs to provide aversion therapy treatment of alcoholics (for examples see Arolfo et al., 2009; Garver et al., 2001; Igoucheva et al., 2008); (v) Human alcohol-related diseases and experimental models in which acetaldehyde has been proposed as an etiologic agent (Quertemont and Didone, 2006; Preedy et al., 2007; Ren, 2007; Salaspuro, 2007; Seitz and Stickel, 2010). Several methods have been developed for the determination of acetaldehyde levels in biological samples. Measurement in this complex matrix is prone to errors introduced by the high volatility of acetaldehyde (boiling point 20°C), inherently low amounts (micromolar), residual amounts in common reagents and solvents (acetic acid, water, etc) and plastics that raise the background level, condensation reactions with all classes of macromolecules, and the presence of interfering substances (ethanol, proteins). Headspace gas chromatography (HS-GC) and reverse phase high-performance liquid chromatography (RP-HPLC) are the two main methods used for measurement of acetaldehyde. The HPLC method entails labeling with a derivatization reagent for stabilization and selective determination of acetaldehyde. Most derivatization protocols for acetaldehyde are based on reaction with hydrazines (Vogel et al., 2000). These reagents readily react with carbonyls in acidic media to form the respective hydrazones, which are typically separated by RP-HPLC and detected photometrically or fluorometrically. The eluting compounds are characterized by comparison of their retention time with those of internal and external standards and their spectra. The molecular weight and structure of the products can be further elucidated by mass spectrometry (Kempter et al., 1999; Peters et al., 2004; van Leeuwen et al., 2004; You et al., 2009). The most popular hydrazine for the derivatization of acetaldehyde has been 2,4-dinitrophenylhydrazine (DNPH) (Brady and Elsmie, 1926; references cited in Table 2). Table 2 Comparison of retention times of AcH-DNP and DNPH, separation interval between the two peaks, and length of different chromatographic methods. In this work, an optimized method for the determination of acetaldehyde in biological samples has been developed. Acetaldehyde in deproteinized samples was quantitatively derivatized with DNPH, and AcH-DNP was extracted. The separation of acetaldehyde was carried out using a RP C18 column. A new gradient system provided for increased HPLC resolving power, thus avoiding interferences from coelution with DNPH. The acetaldehyde peak was identified by its retention time against authentic AcH-DNP, and the concentration of acetaldehyde was calculated based on calibration curves. We have investigated the stability of derivatized acetaldehyde and the contribution of ethanol in the acetaldehyde measurements, and validated this method in hepatoma cell cultures capable of ethanol metabolism, and in rat blood and plasma samples.