MSACL 2024 Abstract

Optimizated Sample Preparation for Low Level Determination of Alcohol Marker EtG from Hair Using UPLC-MS/MS Analysis.

Lee Williams, Zainab Khan, Thomas Smith, Adam Senior, Helen Lodder, Russell Parry, Geoff Davies, Lucy Lund, Alan Edgington, Claire Desbrow & Dan Menasco.
Biotage GB Limited, Cardiff. UK

Lee Williams (Presenter) Biotage GB Limited

Relevant Financial Disclosures (within past 24 months) Honorarium/Expenses BIOTAGE GB LTD Salary BIOTAGE GB LTD Conflict mitigation NOT REQUIRED.

Introduction
Hair analysis is growing in popularity due to the non-invasive nature of sample collection. Hair testing can provide advantages over other more widely used matrices, such as prolonged exposure and potential timelines. However, as a solid matrix sample preparation can often be lengthy and labour intensive. Minor ethanol metabolites such as ethyl glucuronide (EtG) and ethyl palmitate (EtPa) can be measured in hair as direct markers of alcohol consumption. As suggested by the Society of Hair Testing (SoHT) in 2019, EtG determination from hair is the preferred marker for the assessment of abstinence.

Objectives
This poster aims to demonstrate improved workflow and clean-up of hair matrix to allow low level EtG determination required for alcohol abstinence testing.

Methods
Hair samples (10 mg) were subjected to micro-pulverized extraction using the Biotage® Lysera bead mill homogenizer. Once in liquid form, sample extracts were further cleaned-up using polymer-based solid phase extraction (SPE) in 96-well plate format. Subsequent analysis was performed using a Waters ACQUITY Premier UPLC coupled to a Xevo TQ Absolute triple quadrupole mass spectrometer. Negative ions were acquired using electrospray ionization operated in the MRM mode.

Results
Initial method development focussed on investigation of LC mobile phases and column chemistry to achieve good chromatographic performance while providing adequate negative ion sensitivity. An HSS T3 column provided good reversed-phase chromatography albeit with acidic mobile phase composition, resulting in reduced negative ion sensitivity. HILIC chromatography yielded good sensitivity generally at the expense of compromised chromatographic peak shape.

Evaporative effects were investigated using a range of traditional SPE elution solvents. The results demonstrated between 15-25% analyte signal reduction depending on exact solvent combination.
Matrix homogenization was investigated after standard washing protocols. Both dry hair and pulverisation in solvent was evaluated. 10 mg of hair was extracted with up to 1 mL of H2O to effect efficient analyte solubilisation from the matrix.

Sample extraction and clean-up was performed using polymer-based mixed-mode strong and weak anion exchange SPE chemistries due to the carboxylic acid moiety on the analyte. Wash and elution optimization resulted in analyte recoveries greater than 80% for both chemistries. Suppression profiles were reduced resulting in matrix factors between 0.7-0.9, depending on exact protocol. Final workflow streamlining evaluated SPE bed reduction, minimum elution volumes and direct injection compared to evaporation/reconstitution protocols. Both approaches presented different advantages with respect to simplicity and time vs absolute sensitivity but ultimately achieved the required LOQs. Calibration curves were constructed using hair spiked between 1-100 pg/mg to cover the range of chronic excessive consumption and abstinence. Results demonstrated good linearity and coefficients of determination (r2) values greater than 0.99. LoQs were determined to be below the required SoHT guidelines for abstinence cases of equal to or below 5 pg/mg of hair.

Conclusion
This poster demonstrates optimized extraction and cleanup of hair matrix allowing low level alcohol marker determination in abstinence cases.