Validation of a headspace solid-phase microextraction-GC-MS/MS for the determination of ethyl glucuronide in hair according to forensic guidelines

Labor Krone, Bad Salzuflen, Germany.
Forensic science international (Impact Factor: 2.12). 03/2010; 196(1-3):3-9. DOI: 10.1016/j.forsciint.2009.07.023
Source: PubMed

ABSTRACT The analysis of ethyl glucuronide (EtG) in hair is a powerful tool for chronic alcohol abuse control because of the typical wide detection window of the hair matrix and due to the possibility of segmentation, allowing evaluation of alcohol consumption in different periods. Additionally, EtG in hair is often the only diagnostic parameter of choice for alcohol abuse when other clinical parameters such as ALT, AST, gammaGT and CDT (asialotransferrin and disialotransferrin) are in the normal range and EtG in urine negative. In this paper, we describe the development, optimization and validation of a new method based on hair extraction with water, clean-up by solid phase extraction (SPE), derivatization with heptafluorobutyric anhydride and headspace solid-phase microextraction (HS-SPME) in combination with GC-MS/MS according to forensic guidelines. The assay linearity of EtG was confirmed over the range from 2.8 to 1000 pg/mg hair, with a coefficient of determination (r(2)) above 0.999. The LLOQ was 2.8 pg/mg and the LLOD was 0.6 pg/mg. An error profile calculated according to the "Guide to the Expression of Uncertainty in Measurement" (GUM) at 99% confidence intervals for the range 5-750 pg/mg hair did not exceed 10%. This range corresponds to more than 98% of the positive samples analysed.

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    ABSTRACT: The Society of Hair Testing (SoHT) has provided cutoffs for the quantification of ethyl glucuronide (EtG) in hair to indicate occasional or chronic/excessive alcohol consumption. Although several sensitive methods have been reported, past proficiency test results show a lack of reproducibility. An ultra-performance liquid chromatographic mass spectrometric method (LLOQ of 10 pg EtG/mg hair) has been validated according to the international guidelines, including the successful participation in five proficiency tests. This method was subsequently used to evaluate the impact of different grinding conditions (cut, weakly or extensively pulverized hair samples) on the final measured EtG concentration. Hair from alcohol consumers (n = 2) and commercially available quality control samples (QCs) (n = 2) was used. For the QCs, extensive pulverization led to a significantly higher amount of measured EtG. In the hair samples obtained from volunteers, cut or weakly pulverized hair resulted in EtG concentrations below the LLOQ, while the mean concentrations of 14 and 40 pg EtG/mg hair were determined after extensive pulverization. Differences in sample preparation could partially explain inter-laboratory variability. As the differences in results can lead to a different interpretation even when applying the SoHT cutoffs, it is of interest to standardize sample preparation techniques in the field of EtG hair testing.
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    ABSTRACT: Ethyl glucuronide (EtG), a minor metabolite of ethanol, accumulates in hair and is currently used as a long-term marker for the detection of chronic and excessive alcohol consumption. Sensitive methods are required to differentiate teetotalers from moderate drinkers according to the established cut-off (i.e., 7 pg/mg hair). The aim of this study was to develop a sensitive method using gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) operated in the negative ion chemical ionization (NICI) mode. The validated method was applied to hair samples from teetotalers, moderate and excessive alcohol consumers, and results were compared to a previously validated GC-NICI-MS method. The developed GC-NICI-MS/MS method showed linearity over a range from 2 to 400 pg/mg hair, with a limit of detection (LOD) of 0.05 pg/mg hair and a lower limit of quantification (LLOQ) of 0.2 pg/mg hair, compared to an LOD of 0.5 pg/mg hair and LLOQ of 1.5 pg/mg hair obtained with GC-NICI-MS. Furthermore, lower background noise was observed using GC-NICI-MS/MS. Comparison of results of hair samples (n = 58) obtained by GC-NICI-MS and GC-NICI-MS/MS showed no significant difference between both methods (paired-sample t-test, p > 0.05; mean CV = 1.0%). The differences between both methods were larger for EtG concentrations < 30 mg/pg hair (mean CV = 1.7%) than for EtG concentrations > 30 mg/pg hair (mean CV = 0.7%). This suggests a higher selectivity of GC-NICI-MS/MS at lower concentrations. In conclusion, by using GC-NICI-MS/MS, a higher analytical selectivity and an improved signal to noise ratio, can be achieved. Although GC-NICI-MS would not change the interpretation of the EtG concentrations, the present GC-NICI-MS/MS method should preferentially be used for the determination of EtG in hair, especially when differentiating between teetotalers and moderate drinkers according to the current cut-off (i.e., 7 pg/mg hair).
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