Development and Validation of a GC-EI-MS/MS Method for Ethyl Glucuronide Quantification in Human Hair

Abstract

Ethyl glucuronide (EtG) is a minor, non-oxidative ethanol metabolite detectable in several matrices for specific periods of time. In recent years, quantification of EtG in hair has been established as the most reliable biomarker for long-term alcohol consumption, with the Society of Hair Testing offering cut-off values for assessment of both abstinence and heavy drinking. Instrumental constrains and wide inter- and intra-laboratory variability represent the ultimate barriers to widespread acceptance of hair EtG determination in the forensic context. In this study, a new analytical method for hair EtG based on gas chromatographic (GC) separation, electron impact (EI) ionization, and tandem mass spectrometry (MS/MS) detection was developed and validated. At the same time, several parameters for sample pretreatment and instrumental analysis were optimized using real hair samples obtained from different drinking subjects. A full-factorial design-of-experiment approach included procedures for hair washing, pulverization, and extraction. Rigorous multi-step washing proved not to reduce the EtG content extracted in the subsequent sample incubation. Hair pulverization with a ball mill significantly improved the EtG extraction from the keratin matrix and allowed us to reduce the time needed for the subsequent extraction step, without affecting the extraction recovery. The hair extract was derivatized with N-methyl-N-(trimethylsilyl)-trifluoroacetamide. Upon electron impact ionization of the EtG-TMS derivative, triple quadrupole mass analyzers were operated in the selected reaction monitoring (SRM) mode using the fragment m/z 405 as the precursor ion (m/z 410 for the EtG-D5 internal standard), the transitions m/z 405 → 359 and m/z 410 → 359 for quantitation, and m/z 405 → 331 and m/z 405 → 287 for qualification/confirmation, all at 10 V collision energy. The final method was fully validated and then applied to 25 real hair samples. The calibration curve proved linear between 6 and 60 pg/mg. The limit of detection (LOD) was 4 pg/mg. Intra- and inter-assay precision and accuracy tests showed a variability and bias close to 15% or lower over the entire calibration range. The new method is routinely applied in the Italian State Police’s toxicology laboratory for hair analyses addressed to exclude excessive alcohol drinking and verify the psycho-physical requirements of the personnel.

Oxidative Stress in a Mother Consuming Alcohol during Pregnancy and in Her Newborn: A Case Report

“…The EtG analysis followed the protocol described by Mattia et al [ 37 ]. Briefly, 50 mg of hair samples was decontaminated by two consecutive washes in 5 mL of methanol and 5 mL of dichloromethane, manually shredded, and incubated overnight at 60 °C in water with EtG-D5 as the internal standard.…”

The Impact of Alcohol Consumption and Oral Microbiota on Upper Aerodigestive Tract Carcinomas: A Pilot Study

“…Certainly, serum CDT estimates alcohol consumption over a period of a maximum of 15 days before the sampling. On the other hand, the determination of EtG in the hair allows us to assess the “historical” consumption of alcohol [ 40 ], which is a metabolite that irreversibly accumulates in the keratin matrix. In this way, taking into account the average hair growth of 1 cm per month, it is possible to evaluate the habits connected to the consumption of alcoholic beverages more accurately [ 33 ].…”

“…Hair EtG analysis was performed according to the protocol described by Mattia et al (2022) [ 40 ]. Briefly, 50 mg of hair samples were decontaminated, manually shredded, incubated overnight at 60 °C in water with 15 μL of EtG-D5 100 mg/L solutions in methanol, obtained from Medichem (GmbH & Co., Steinenbronn, Germany) as internal standard, purified with solid phase extraction polymeric cartridges Strata X-A-33 μm obtained from Phenomenex Inc. (Torrance, CA, USA), air-dried, and reconstituted with ACN and N-Methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA) as derivatizing agent for injection in an Agilent Technologies 7890B gas chromatograph coupled to a 7000C tandem mass selective detector operating in EI ionization mode (Santa Clara, CA, USA).…”

High-resolution imaging in studies of alcohol effect on prenatal development

“…The direct metabolites of alcohol such as fatty acid ethyl esters (FAEE) in neonatal hair and meconium ( 86 – 88 ) and ethyl sulfate in maternal urine ( 86 , 89 ) are present as distinct biological biomarkers. There are also several indirect metabolites of ethanol such as ethyl glucuronide in neonatal meconium or maternal hair ( 90 – 92 ) and phosphatidyl ethanol in maternal blood ( 93 ), although these indirect markers are less specific and indicative of alcohol exposure ( 86 , 94 ). Still, no biomarker has been validated as a specific and sensitive diagnostic marker for PAE-induced toxic effects ( 86 , 95 ).…”