MSACL 2019 EU Abstract

Evaluation of Simplified Workflows for Hair Matrix Extraction Prior to UHPLC-MS/MS Analysis

Katie-Jo Teehan1, Lee Williams1, Rhys Jones1, Helen Lodder1, Alan Edgington1, Adam Senior1, Geoff Davies1, Steve Jordan1, Claire Desbrow1, Paul Roberts1.
Biotage GB Limited, Distribution Way, Dyffryn Business Park, Cardiff, CF82 7TS, UK.

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Alan Edgington (Presenter) Biotage GB Limited

Relevant Financial Disclosures (within past 24 months) Salary Biotage GB Limited Conflict mitigation NOT REQUIRED.

Introduction
Although not used routinely as per other matrices such as blood or urine, hair does have advantages in that the matrix can indicate prolonged drug exposure. This can provide valuable information with respect to therapeutic drug regimens or in abused drug abstinence cases.

Methodology
Hair samples of 10-20 mg were weighed and transferred to 2 mL reinforced tubes prior to the addition of 2.4 mm metal beads. Methanol (1 mL) was pipetted, either with or without pH modification and the samples were subjected to micro-pulverization using the Lysera bead disruption system. Methanolic extracts were cleaned-up using supported liquid extraction, ISOLUTE® SLE+ in 400 µL capacity 96-well plates or equivalent columns. Water-immiscible solvents: MTBE, EtOAc, hexane, DCM and DCM/IPA (95/5, v/v) were evaluated for cannabis and multi-panel drugs of abuse extraction. Final extracts were evaporated at 40 °C and reconstituted in respective mobile phases. UHPLC-MS/MS analysis was performed using a Shimadzu Nexera UHPLC system coupled to an 8060 triple quadrupole mass spectrometer.

Results
Due to substantially different limits of quantitation required between cannabis and other drug panels it was deemed necessary to separate analyses. Widely abused drugs comprising; amphetamine type, benzodiazepines and Z-drugs, cocaines, opiates and opioids, fentanyls and buprenorphines were treated separately to the cannabis panel comprising; cannabinol (CBN), cannabidiol (CBD), Tetrahydrocannabinol (THC), 11-Nor-9-carboxy-Δ⁹-tetrahydrocannabinol (THC-COOH), 11-Hydroxy-Δ⁹-tetrahydrocannabinol (THC-OH), Δ9-tetrahydrocannabinolic acid-A (THCAA).
Sample clean-up of the methanolic hair extract was performed using supported liquid extraction. A range of solvents were investigated from a recovery and suppression standpoint. In order to reach SoHT LoQs we compared methanolic extract evaporation and reconstitution prior to extraction with direct extraction of the methanol extract. Typical recoveries were greater than 60% or 80%, for the cannabinoid and extended drug panel respectively. Corresponding RSDs were below 10%. A range of extraction solvents were applicable depending on the exact panel required.

Calibration curves constructed between 0.1-200 pg/mg and 10-1000 pg/mg for the cannabinoid and extended drug panel respectively demonstrated good linearity and coefficients of determination (r2) values greater than 0.99 for all analytes. LoQs were below required SoHT quidelines for both screening and confirmation with THC-COOH performance achieving the low level 0.2 pg/mg levels set out.

Conclusion
This poster demonstrates a simplified workflow for the analysis of a range of drugs of abuse from hair matrix, from sample homogenization, through to extraction, cleanup, evaporation and finally quantitative analysis.