Fast Quantification of Ethanol in Whole Blood Specimens by the Enzymatic Alcohol Dehydrogenase Method. Optimization by Experimental Design

Abstract

A sensitive, fast, simple, and high-throughput enzymatic method for the quantification of ethanol in whole blood (blood) on Hitachi 917 is presented. Alcohol dehydrogenase (ADH) oxidizes ethanol to acetaldehyde using the coenzyme nicotinamide adenine dinucleotide (NAD), which is concurrently reduced to form NADH. Method development was performed with the aid of factorial design, varying pH, and concentrations of NAD+ and ADH. The linear range increased and reaction end point decreased with increasing NAD+ concentration and pH. The method was linear in the concentration range 0.0024-0.4220 g/dL. The limits of detection and quantification were 0.0007 g/dL and 0.0024 g/dL, respectively. Relative standard deviations for the repeatability and within-laboratory reproducibility were in the ranges 0.7-5.7% and 1.6-8.9%, respectively. The correlation coefficient when compared with headspace gas chromatography-flame ionization detection methods was 0.9903. Analysis of authentic positive blood specimens gave results that were slightly lower than those of the reference method.

Estrogen receptor ERα plays a major role in ethanol-evoked myocardial oxidative stress and dysfunction in conscious female rats

“…Heparinized blood was centrifuged at 5000 rpm for 10 minutes and ethanol content was determined by the enzymatic method (Kristoffersen et al, 2005, El-Mas et al, 2008) with modification. Briefly, 150 μl of buffer (sodium pyrophosphate 74.5mM, semicarbazide hydrochloride 74 mM glycine 22.4 mM), 25 μl of β-Nicotinamide adenine dinucleotide (25.1mM), 25 μl of perchloric acid (2%) and 5 μl of sample or ethanol standard, was added in 96-well plate, respectively.…”

Impairment related to blood drug concentrations of zopiclone and zolpidem compared to alcohol in apprehended drivers

“…In addition, carisoprodol, meprobamate, methadone, carbamazepine and phenobarbital were routinely screened for from May 2001. The confirmation analyses were performed by LC-MS for zopiclone and zolpidem, and head-space GC analysis for alcohol (Kristoffersen et al, 2005). The cut-off value for zopiclone was 19 g/l, for zolpidem 15 g/l, and the cut-off value for alcohol was 0.04 g/dl (%).…”

Comparative analysis of hospital and forensic laboratory ethanol concentrations: A 15 month investigation of antemortem specimens

“…Oxidation of ethanol by this enzyme generates a stoichiometric amount of NADH, the rate of which is monitored spectrophotometrically at 340 nm. 4 …”

“…Enzymatic assays do not measure the concentration of ethanol directly, instead, they measure an absorbance change caused by the production of NADH which is then related back to the concentration of ethanol. 4 Unfortunately, some small molecules oxidized by their respective enzymes can also produce NADH, therefore possibly increasing the perceived concentration of ethanol. 5,6 Calibration differences between methods may also play a role in the variability of quantitation between different methods.…”