Determination of ethyl glucuronide in human hair by SPE and LC–MS/MS
Introduction
Alcoholism is one of the most frequent addictions and, therefore, is of particular interest in forensic and clinical medicine. The known enzymatic and hematological alcohol markers cannot be considered to be satisfying with regard to sensitivity and selectivity. Hair analysis proved to be an important diagnostic tool for the detection of chronic alcohol consumption. A series of different possibilities were presented by Pragst et al. [1], such as the determination of fatty acid ethyl esters and acetaldehyde adducts.
Ethyl glucuronide (EtG)—the phase II metabolite of ethanol—is a promising marker for alcohol abuse. The detection of alcohol consumption that has taken place several days, weeks or months earlier, via the determination of EtG in blood, urine or hair, represents a potential forensic tool as well as a possibility for monitoring alcohol consumption of patients being in treatment for alcoholism. Preliminary investigations with gas chromatography mass spectrometry (GC–MS) showed that EtG, although it is a hydrophilic compound, was detectable in hair of some alcoholics [2]. However, relatively high detection limits (2.2 ng/mg [3]) were obtained by GC–MS.
With protein-precipitation as the only sample pretreatment prior to derivatization, GC–MS methods with acetyl- [4] or trimethylsilyl (TMS) derivatives [5] have been used for urine and blood samples. Liquid chromatography (LC)–electrospray ionisation (ESI)–MS [6] and LC–ESI–MS/MS [7] methods have been used as well. For hair samples in particular, the analytical method has to show higher sensitivity than for the analysis of urine and serum. To overcome sensitivity and selectivity problems, a method was developed using a solid-phase extraction (SPE) procedure—which has been recently developed for GC/MS analysis for urine and serum [8]—followed by LC–ESI–MS/MS with enhanced sensitivity by post-column addition of organic solvent (acetonitrile).
Section snippets
Materials
EtG and D5-EtG (deuterium atoms located at the ethyl moiety) standards were obtained from Medichem (Stuttgart, Germany). SPE columns (ISOLUTE NH2 [aminopropyl] 1 g/6 ml) from Separtis (Grenzach-Wyhlen, Germany) were employed for SPE. The HPLC column (Synergy Polar-RP mm, 4 μm) with a guard column (Synergy Polar-RP mm) was obtained from Phenomenex (Aschaffenburg, Germany). HPLC-grade acetonitrile, formic acid and all solvents for SPE (analytical grade) were purchased from Merck
Validation
The calibration curve for EtG (25–2000 pg/mg) was obtained using weighted least-squares regression of the peak height ratio (analyte peak height/internal standard peak height) versus concentration. A weighting factor of 1/x2 was applied to the standard curve. Good linearity was observed for peak intensity within the specified EtG concentration range. The correlation coefficient of the calibration was 0.997. The mean absolute recovery at a concentration of 200 pg/mg EtG from hair was 96±2.3% (n
Conclusion
A new SPE–LC–MS/MS procedure for an improved determination of EtG in hair samples has been developed and validated. Further investigations concerning the incorporation of EtG in hair have to be performed, especially to address the question of the failure of EtG detection in hair of persons with known alcohol addiction. In addition, a higher number of samples with reliable information on the kind of alcohol consumption (amount, time period, etc.) are necessary to allow a detailed interpretation
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