Fast quantification of ethanol in whole blood specimens by the enzymatic alcohol dehydrogenase method. Optimization by experimental design.
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.
Kristoffersen
Lena
L
Division of Forensic Toxicology and Drug Abuse, Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, N-0403 Oslo, Norway. lena.kristoffersen@fhi.no
Skuterud
Bjørn
B
Larssen
Bente R
BR
Skurtveit
Svetlana
S
Smith-Kielland
Anne
A
D000426:Alcohol Dehydrogenase / Q000378:metabolism*; D000431:Ethanol / Q000097:blood*; D005554:Forensic Medicine / Q000379:methods*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D015203:Reproducibility of Results; D012106:Research; D012107:Research Design; D015813:Substance Abuse Detection / Q000379:methods*
D000431:Ethanol; D000426:Alcohol Dehydrogenase