Quantifying ethanol by high performance liquid chromatography with precolumn enzymatic conversion and derivatization...

Area of Science:

• Analytical Chemistry
• Biochemistry

Background:

• Accurate quantification of blood ethanol is crucial for clinical diagnostics and forensic toxicology.
• Existing enzymatic methods provide a benchmark for ethanol measurement.

Purpose of the Study:

• To develop and validate a fluorimetric High-Performance Liquid Chromatography (HPLC) method for quantifying ethanol in blood.
• To assess the accuracy, precision, and clinical utility of the developed HPLC method.

Main Methods:

• Ethanol was quantified by measuring acetaldehyde increase post-reaction with alcohol dehydrogenase and nicotinamide adenine dinucleotide (ADH-NAD).
• A fluorimetric HPLC method was employed for detection and quantification.
• Method validation included assessing the limit of quantitation, accuracy against an enzymatic method, and intra-assay and inter-assay precision.

Main Results:

• The fluorimetric HPLC method demonstrated a limit of quantitation of 6 mg/dl.
• High correlation (r² = 0.993) was observed when comparing HPLC results to the ADH-NAD enzymatic method for blood samples.
• Intra-assay and inter-assay coefficients of variation were acceptable, indicating good precision.
• Correlation with the enzymatic method was also high (r² = 0.986) when analyzing volunteer blood ethanol concentrations over time.

Conclusions:

• The described fluorimetric HPLC method provides a reliable and accurate means for measuring blood ethanol concentrations.
• The method exhibits potential for widespread clinical application due to its performance characteristics.
• This HPLC technique offers a valuable alternative for blood alcohol analysis.