Determination of ethyl glucuronide in human hair by SPE and LC–MS/MS

doi:10.1016/S0379-0738(02)00163-9

Forensic Science International

Volume 128, Issues 1–2, 14 August 2002, Pages 59-65

https://doi.org/10.1016/S0379-0738(02)00163-9 Get rights and content

Abstract

A method for the sensitive and selective determination of ethyl glucuronide (EtG) in hair has been developed using solid-phase extraction (SPE) and liquid chromatography–tandem mass spectrometry (LC–MS/MS). Washed and cut hair segments were extracted by ultrasonication (3 h, 50 °C) and the extracts were cleaned-up with aminopropyl SPE columns. LC–MS/MS analysis was performed using a polar-endcapped phenyl-hexyl-RP-phase with negative mode electrospray ionisation (ESI) using a triple quadrupole mass spectrometer (Sciex API 365) with a turboionspray source and post-column addition of acetonitrile for enhanced sensitivity. The MS/MS transitions monitored were m/z 221→75 for EtG and 226→75 for D₅-EtG as an internal standard. The method was selective and sensitive, with a detection limit of 51 pg/mg hair at a signal-to-noise ratio of 3:1. The mean recovery was 96%, with an intra- and inter-day precision of less than 11.7% at a concentration of 200 pg/mg. The linearity was assessed in the range of 25–2000 pg/mg hair, with a correlation coefficient of 0.997. The method was successfully applied to 97 human hair samples which were taken at autopsies from persons with known alcoholism or were obtained from alcoholics who were hospitalized for ethanol withdrawal, from social drinkers and from children having not consumed any alcohol. Although, approximately two-third of the alcoholics showed EtG concentrations in hair of higher than 51 pg/mg (up to >4000 pg/mg), in one-third the EtG concentration was below the detection limit. However, only in one of five hair samples of “social drinkers”, the EtG concentration was above the detection limit (51 pg/mg). No EtG has been detected in the hair of children. These investigations demonstrate that heavy alcohol consumption may be but not necessarily has to be detectable by EtG analysis in hair.

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 $150 mm ×2$ mm, 4 μm) with a guard column (Synergy Polar-RP $4 mm ×2$ 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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Determination of ethyl glucuronide in hair samples
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Ethyl glucuronide (EtG) is a minor, non-oxidative ethanol metabolite that can be detected in several matrices (e.g. blood, urine, hair, meconium) for variable periods of time. Quantification of EtG in hair (hEtG) has established itself, over recent years, as one of the most reliable biomarkers of long-term alcohol consumption habits, with the Society of Hair Testing (SoHT) offering cut-off values for assessment of both abstinence and heavy drinking (>60 g/day). Despite its high diagnostic performance, however, issues concerning inter- and intra-laboratory variability as well as data interpretation are still being investigated and represent the ultimate barrier to widespread acceptance of hEtG in the forensic context. The aim of this review is to summarize currently available analytical methods of hEtG testing, provide a framework to understand current hEtG cut-offs and their possible upcoming changes (in particular, a lower abstinence cut-off has been proposed for the 2019 revision of the SoHT consensus), and offer a schematic but exhaustive overview of the pitfalls in result reproducibility and interpretation that may limit applications of hEtG testing in the forensic context. Ultimately, the purpose of the authors is not to undermine the reliability of hEtG as an alcohol use marker, but rather to enhance it by promoting familiarization with all aspects related to it, from ethanol pharmacokinetics and EtG incorporation into hair, to sample preparation and analytical methods, to specific cases warranting close attention and additional tests for correct interpretation of hEtG results.
Novel zwitterionic HILIC stationary phase for the determination of ethyl glucuronide in human hair by LC-MS/MS
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Citation Excerpt :
The recommended cut-off levels for hEtG diagnostic purposes, suggested by the Society of Hair Testing (SoHT), are substantially two values: 30 pg/mg to distinguish from moderate and heavy alcohol consumption and below 7 pg/mg to pass the medical assessment of alcohol abstinence [4]. A small overview on analytical methods developed for hEtG determination published between years 2000 and 2017 is summarized in Tables 1a and 1b, where data on sample amount, details on analytical method and achieved sensitivity are reported for clarity [5–25,35,37–39]. Although GC–MS technique usually allows to reach lower LOQs also in presence of minor quantity of starting material, GC analysis requires a derivatization step, which contributes to increase complexity and renders the procedure time consuming.
Some recent studies have described a shift from traditional reversed-phase to more hydrophilic LC chemistry for EtG determination in hair (hEtG). The reason relies on the poor retention of C8– and C18-based columns for polar compounds, even in presence of great amount of aqueous phase. This work presents the development, validation and application of a new hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-LC-MS/MS) method based on a novel zwitterionic stationary phase for the analysis of hEtG. The linearity was assessed in the range of 5–100 pg/mg hair, with a correlation coefficient of >0.99. The method was selective and sensitive, with a limit of detection (LOD) and limit of quantitation (LOQ) of 1.4 pg/mg and 4.5 pg/mg in hair, respectively. Suitable diagnostic sensitivity was achieved without the introduction of a sample purification step, or a post column solvent addition. The method was successfully applied to real hair samples after full validation. This method, based on a separation at neutral conditions, confirmed the optimum retention and thus selectivity for weak acids in zwitterionic HILIC columns.
The influence of cleansing shampoos on ethyl glucuronide concentration in hair analyzed with an optimized and validated LC-MS/MS method
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Ethyl glucuronide (EtG) is widely used as a marker for assessment of alcohol consumption behavior. In this study the influence of special cleansing shampoos on ethyl glucuronide concentrations in hair was investigated. For that purpose an optimized LC–MS/MS method was developed using a Hypercarb™ porous graphitic carbon (PGC) column and validated according to the guidelines of the German Society of Toxicological and Forensic Chemistry (GTFCh). Twenty-five hair samples of persons with known alcohol consumption behavior were investigated (21 positive samples and 4 blank samples). The hair samples were divided into two strands of hair and were analyzed after treatment with one out of four cleansing shampoos and without shampoo treatment. EtG concentrations in hair did not show any significant differences after a single application of the different cleansing shampoos. EtG was still detectable in all the positive hair samples without significant concentration change. These results clearly demonstrated that a single application of the tested cleansing shampoos did not remove EtG from hair and therefore had no influence on EtG concentration in analytical hair analysis.
An evaluation of washing and extraction techniques in the analysis of ethyl glucuronide and fatty acid ethyl esters from hair samples
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Ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs) are alcohol metabolites measured in hair and are after a decade of research thought to be the best markers in hair to indicate alcoholism and abstinence Forensic Sci. Int. 218 (2012) 2. A great body of work concerning EtG and FAEEs detection in hair has been performed. However, no recent extensive comparison has been made concerning washing and extraction procedures. This work shows that the washing procedure of dichloromethane followed by a methanol rinse of the hair sample removes more than 16% of the FAEEs and 50% of the total EtG that is present in and on the hair. A review of ten washing protocols (where the removal is categorised: high, medium or low) showed that a relatively high percentage of FAEEs was removed and “medium” amount of EtG compared to the other washing protocols. This work shows promising results for the extraction of the FAEEs and the combined extraction of FAEEs and EtG by using 30 min of sonication with methanol. More FAEEs were recovered from hair with methanol than with any other extraction solvent including the commonly used dimethyl sulfoxide/heptane mixture. When the sonication time was increased a higher percentage of transesterification of the FAEEs was observed, the extraction was “dirtier” as solids and a colour change was observed whereas the extraction efficiency did not increase. Therefore, washing the hair sample with dichloromethane and methanol followed by an addition of 1 ml of methanol and sonication for 30 min to extract the FAEEs and EtG from hair is recommended for FAEEs as well as for the combined analysis of EtG and FAEEs. A linear calibration curve (r² > 0.99) was obtained for all analytes.
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Determination of ethyl glucuronide in human hair by SPE and LC–MS/MS

Abstract

Introduction

Section snippets

Materials

Validation

Conclusion

Forensic Sci. Int.

J. Chromatogr.

J. Chromatogr.

Forensic Sci. Int.

Determination of ethyl glucuronide in hair samples

Alcohol Alcohol.