Cannabinoid Isomer Identification and Quantitation by UPLC-MS/MS Analysis in Forensic Urine Samples

In forensic toxicology, detecting use of cannabis by urine confirmation testing has been traditionally limited to    analysis of the major Δ⁹-tetrahydrocannabinol  (Δ⁹-THC) metabolite, 11-nor-9-carboxy-Δ⁹-tetrahydrocannabinol (Δ⁹-cTHC). However, in the United States legalization of hemp has led to increased production and sale of psychoactive cannabidiol (CBD) derivatives including Δ⁸-THC and Δ¹⁰-THC isomers. The increasing availability and growing use of these derivatives necessitates an expansion of cannabinoid confirmation test protocols. An isomer-selective definitive method is presented for the quantitative confirmation of Δ⁸-THC, Δ⁹-THC, Δ¹⁰-THC, Δ⁸-cTHC, Δ⁹-cTHC, and CBD by liquid chromatography/tandem mass spectrometry (UPLC-MS/MS).

Benefits

• Simple sample preparation
• Baseline separation between Δ⁸ and Δ⁹ isomers of THC and cTHC
• Accurate quantification of Δ⁸-THC, Δ⁹-THC, Δ¹⁰-THC, Δ⁸-cTHC, Δ⁹-cTHC, and CBD in urinary samples

Cannabis continues to be one of the widest used recreational drugs in the United States with over 50 million Americans (about 18%) reporting its use in 2021.¹ Detection of Δ⁹-THC use is typically accomplished by monitoring the major Δ⁹-THC excreted metabolite, Δ⁹-cTHC. Recently, however, formulations claiming to contain CBD derivatives such as Δ⁸ and Δ¹⁰ THC have gained in popularity, with recent studies demonstrating an increasing prevalence of these isomers in casework. The presence of isomers can interfere with the quantification of the traditional Δ⁹ isomers as they share MRM transitions and are often not fully resolved from each other by traditional methods. Therefore, an expanded scope of cannabinoid testing protocols is needed to both accurately quantify these isomers as well as the traditional Δ⁹ isomers. This application summarizes a rapid UPLC-MS/MS method for accurately identifying and quantifying Δ⁹-THC, Δ⁸-THC, Δ¹⁰-THC, Δ⁹-cTHC, Δ⁸-cTHC, and CBD. Baseline separation is achieved between isomers allowing accurate quantification and detection in authentic case samples.

Sample Preparation

All cannabinoid reference material and internal standards were from Millipore Sigma (Round Rock, TX) with the exception of Δ⁸-THC, which was from Cayman Chemical (Ann Arbor, MI). IMCSzyme glucuronidase and buffer were from Integrated Micro Chromatography Systems (Irmo, SC). 

Urine samples were prepared for testing by combining 20 µL of urine with 20 µL of internal standard mix (Δ⁹-THC-D3, Δ⁹-cTHC-D3, CBD-D3) and 20 µL of hydrolysis reagent. Samples were incubated for 1 hr at room temperature followed by the addition of 150 µL of 70% methanol containing 0.1% formic acid.

UPLC-MS/MS Analysis

Samples were analyzed using an ACQUITY™ UPLC™ I-Class (FTN) System interfaced with a Xevo™ TQD Mass Spectrometer. The UPLC system parameters are listed in Table 1 and chromatographic gradient in Table 2.

Table 3 describes the Xevo TQD Mass Spectrometer parameters used for all analyses and MRM parameters are shown in Table 4. All THC isomers and CBD were acquired under positive ESI and the cTHC isomers were acquired under negative ESI. Quantifier and qualifier MRM transitions were monitored for each analyte.

Data were acquired and analyzed using MassLynx™ Software (V4.1). Quantification was performed using TargetLynx™.

A more complete description and discussion of this assay validation, performance, and application to casework is detailed elsewhere.² 

Chromatography

Chromatographic resolution is a function of column selectivity and efficiency. To this end, multiple column chemistries were evaluated to achieve the separation between the isomers of THC and cTHC. Figure 1 shows the chromatography of Δ⁸-cTHC, Δ⁹-cTHC, CBD, Δ⁸-THC, Δ⁹-THC, and Δ¹⁰-THC. The chromatographic system provided near baseline separation of Δ⁸-cTHC and Δ⁹-cTHC as well as the Δ⁸, Δ⁹, and Δ¹⁰-THC isomers. The CORTECS™ C₁₈+ Column (p/n: 186007114) that was used has a solid core construction resulting in a narrow particle size distribution enabling maximum efficiency. This, combined with a small particle diameter and the column’s unique selectivity, resulted in excellent resolution in a time frame which is compatible with a routine confirmation workflow.

Quantitative Results

The analytical range for each analyte spanned from 10–1000 ng/mL with an LOD of 4 ng/mL. Calibration curves were linear with R² values >0.99 for all analytes. All calibration points were within 10% of target values. Representative calibration curves for Δ⁸-cTHC and Δ⁹-cTHC are shown in Figure 2. Precision and accuracy were evaluated both within (N=5) and between (N=6) analytical batches. These data are presented in Table 5. The coefficient of variation (%CV) of the LOD was under 17.3% for all compounds, meeting the detection and quantification criteria of <20%. Low and high controls were within 15% and assay bias for all QCs and the LOD was <±15%. Carryover was 0.03% for all analytes, meeting criteria. No interferences were seen with 12 analyte negative urines or in response to 102 drugs routinely tested for by NTC.

There were sample-to-sample variations in the values of the matrix effects for the individual analytes. However, these were paralleled by the internal standards so that these effects were well normalized and did not impact the sensitivity, accuracy, or precision of the method.

Method comparisons were performed using a previous method and 36 samples that were positive for Δ⁹-cTHC only, with no detected Δ⁸-cTHC. Regression analysis revealed a correlation with a slope of 1.004 and an R² value of 0.991 indicating excellent agreement between the two methods without any bias.

The method was applied to de-identified urine samples that had initially screened positive for cannabinoids by immunoassay. Δ⁹-cTHC was detected in >98% of cases. Δ⁸-cTHC was detected in 14% of samples. Parent drug was not detectable in any of the cases. This aligns well with the prevalence of Δ⁸-cTHC seen in vaping products in NY State.³

A method for the rapid and accurate determination of Δ⁸-THC, Δ⁹-THC, Δ¹⁰-THC, CBD, Δ⁸-cTHC, and Δ⁹-cTHC was developed, validated, and applied to authentic cannabinoid positive case samples. The high efficiency CORTECS C₁₈+ Column enabled the baseline separation of the individual cannabinoid isomers from each other, allowing the accurate quantification of Δ⁸-cTHC and Δ⁹-cTHC. Comparison to a previously validated method revealed excellent agreement for Δ⁹-cTHC. Application to case samples revealed significant concomitant use of Δ⁸-THC and Δ⁹-THC, with the prevalence of Δ⁸-THC matching closely with its prevalence in vaping products in NY State.

Acknowledgement

We would like to acknowledge the contributions of Thomas Rosano, Kiley Scholz and Michelle Wood from the National Toxicology Center, Center for Medical Science, Albany, NY and Jane Cooper of Forensic Testing Services, Mirfield, UK.

1. Substance Abuse and Mental Health Services Administration. (2022). Key Substance Use and Mental Health Indicators in the United States: Results from the 2021 National Survey on Drug Use and Health (HHS Publication No. PEP22-07-01-005, NSDUH Series H-57). Center for Behavioral Health Statistics and Quality, Substance Abuse and Mental Health Services Administration. https://www.samhsa.gov/data/report/2021-nsduh-annual-national-report (accessed Jan 2024).
2. Rosano TG, Cooper JA, Scholz KL and Wood M. Confirmation of Cannabinoids in Forensic Toxicology Casework by Isomer Selective UPLC-MS-MS Analysis in Urine. J. Anal. Toxicol. (2023) 47(8): 709–718.
   
3. Duffy BC, Li L, Lu S, et al. Chemotyping of Δ⁸-THC Containing E-Liquids Analyzed During the 2019–2020 New York State EVALI. J. Anal. Toxicol. (2022) 46: 743–749.