Poster Presentations for Kyle Dukes

Automated Extraction and Chromatographic Separation of Δ 8, 9, 10 THC Isomers from Whole Blood Kyle Dukes (Presenter) Biotage

Poster #37d View Map

This poster will be attended on Thursday at 14:30 for 1 hour 15 minutes in the Exhibit Hall.

INTRODUCTION:
Analysis of tetrahydrocannabinol (THC) and its isomers is crucial for understanding their pharmacological effects and legal implications of cannabis use, especially in clinical testing, forensic toxicology, and sport antidoping. THC, the primary psychoactive component of cannabis, exists in several isomeric forms, including Δ9, Δ8, and Δ10-THC, each with distinct pharmacokinetic and pharmacodynamic properties. Accurate identification and quantification of these isomers are essential for evaluating impairment, exposure, or intoxication. However, their analysis presents significant challenges due to their chemical similarities, which can result in co-elution during chromatographic separation and difficulty in distinguishing between isomers using traditional analytical methods.

OBJECTIVE:
This work will demonstrate extraction strategies for cannabigerol, Δ8, Δ9, and Δ10 THC, as well as the common metabolites 11-hydroxytetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH) from whole blood. In addition, two chromatographic methods of separating the isomers will be presented.

METHODS:
Δ9, Δ8, and Δ10-THC and the metabolites 11-hydroxytetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH) were spiked in whole blood samples concentrations ranging from 0.15-100ng/mL and extracted using ISOLUTE® SLE+ supported liquid extraction plates. Extraction performance was investigated by comparing recovery and matrix effects using MTBE, EtOAc, EtOAc followed by Hexane, and Hexane followed by EtOAc as an elution solvent. Chromatographic separation was investigated by comparing two different analytical column chemistries, biphenyl and fluorophenyl. Protocols were transferred to the Extrahera™ Classic automated sample preparation workstation and evaluated for linearity and precision. UHPLC-MS/MS analysis was performed using a Shimadzu Nexera X2 UHPLC coupled to a Sciex 5500 triple quad mass spectrometer. In addition, column chemistry and oven temperature were investigated to achieve optimal separation of the isomers

RESULTS:
Extraction performance was investigated by comparing recovery and matrix effects using the follow elution solvents: MTBE (750µLx2), EtOAc (750µLx2), 750 µL EtOAc followed by 750 µL Hexane, and 750 µL Hexane followed by 750 µL EtOAc. While these four elution protocols yielded similar matrix effects, EtOAc achieved the highest recoveries of analytes, 70-90% with minimal matrix effects (0.9-1.2). The optimized extraction procedure using ISOLUTE® SLE effectively removed more than 99% of the phospholipids present compared to the protein precipitation method. Final automated method performance and calibration curves demonstrated excellent linearity with coefficients of determination, r2 > 0.99 and excellent reproducibility with RSDs below 10% for all analytes.

CONCLUSION:
THC and its metabolites are effectively extracted by supported liquid extraction (SLE) due to favorable partitioning into the organic extraction solvent layer from the aqueous whole blood matrix. The SLE workflow is comparatively simple compared to a traditional SPE approach which requires additional conditioning, equilibration, and wash steps. Baseline separation of THC isomers can be challenging. Investigation of column chemistry showed that fluorophenyl chemistry achieved better resolution of isomers than biphenyl. Moreover, investigation of column oven temperature demonstrated that a lower oven temperature, 20◦C, is more effective in separating THC isomers and its metabolites.