Of recent, forensic toxicology laboratories have been grappling with the emergence of tetrahydrocannabinol isomers within biological specimens. Traditional methods for the identification and quantitation of cannabinoids only include the evaluation of Δ9-tetrahydrocannabinol (Δ9-THC) and its metabolites in biological matrices. Upon analysis of additional tetrahydrocannabinol isomers, laboratories often find co-elution or minimal separation between Δ9-THC, exo-THC, Δ8-THC, Δ10-THC, and Δ6a,10a-THC. These emerging isomers are commonly observed in the seized drug community, particularly in manufactured cannabis products (e.g., edibles, electronic cigarette cartridges). Trends within the seized drug community and legislative changes to include tetrahydrocannabinol isomers dictate the need for change within forensic toxicology. Traditional methods require adaptation to the ever-changing climate surrounding tetrahydrocannabinol. To accomplish this, additional method development with subsequent validation to meet ANSI/ASB Standard 036, Standard Practices for Method Validation in Forensic Toxicology, is often required.
A dual chromatographic column method was developed and optimized for the separation of tetrahydrocannabinol isomers. An Agilent Technologies 1290 Infinity liquid chromatograph was coupled independently to both a 6460 and 6470 quadrupole mass spectrometer for development and validation. Two independent chromatographic methods were developed using different analytical columns, mobile phase conditions, chromatographic gradients, and flow rates. The qualitative analytical method utilized an Agilent Technologies Poroshell 120 PFP 3.0 x 100 mm, 2.7 µm column held at 50°C. The quantitative analytical method used an Agilent Technologies Poroshell 120 EC-C18 3.0 x 50 mm, 2.7 µm column held at 50°C. The dual column methodology was used to enhance the separation of tetrahydrocannabinol isomers.
The sample preparation procedure consisted of a supported liquid extraction (SLE) using 0.5 mL of biological specimen. The biological specimen was acidified with 200 µL of formic acid in water prior to placement onto the SLE cartridge. Each specimen was allowed to incubate for 5 minutes prior to the addition of ethyl acetate (3.0 mL). After elution and collection, n-hexane (3.0 mL) was added to each cartridge. Samples were evaporated to dryness at approximately 50°C prior to reconstitution in 50 µL of methanol.
The optimized method was validated for quantitation of Δ9-THC, (±)-11-hydroxy-Δ9-THC (Δ9-OH-THC), (±)-11-nor-9-carboxy-Δ9-THC (Δ9-carboxy-THC), (-)-Δ8-tetrahydrocannabinol (Δ8-THC), and cannabidiol to meet ANSI/ASB Standard 036, Standard Practices for Method Validation in Forensic Toxicology. All other isomers were validated to meet qualitative identification criteria. During validation, samples were evaluated on both analytical methods to ensure congruence in results. The calibration range was 1/2/5 ng/mL to 100/200/500 ng/mL (Δ9-THC, Δ8-THC/Δ9-OH-THC, cannabidiol/Δ9-carboxy-THC). All compounds were within ±20% for bias and precision when evaluating pooled fortified samples of blank blood, antemortem blood, and postmortem blood. The estimated limit of detection and lower limit of quantitation were assessed for each matrix type within the validation. Significant ionization suppression (>25%) was noted for antemortem blood, postmortem blood, and urine. Further, interferences were evaluated including other isomers and derivatives of Δ9-THC.
An extensive validation was performed on the optimized SLE extraction with subsequent analytical analysis using liquid chromatography tandem mass spectrometry. The validated method optimizes chromatographic separation between tetrahydrocannabinols providing enhanced identification of these isobaric compounds.
Detailed Learning Objectives
- Attendees will be introduced to some of the analytical challenges associated with emerging tetrahydrocannabinol isomers in biological specimens.
- Attendees will be introduced to different analytical strategies for the chromatographic separation of tetrahydrocannabinol isomers.
- Attendees will be provided with a validated extraction procedure and analytical parameters that meet ANSI/ASB Standard 036, Standard Practices for Method Validation in Forensic Toxicology.