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Detection of Δ9-Tetrahydrocannabinol and Metabolites in the Meibomian Lipids of Tear Samples Through LC-MS/MS

Allen Mello and Sabra Botch-Jones with Boston University School of Medicine, Biomedical Forensic Sciences

Denise Valenti, OD with IMMAD: Impairment Measurement Marijuana and Driving

Jamie Foss with PerkinElmer, Inc.

INTRODUCTION

This research focused on the detection and quantitation of THC, 11-Hydroxy- THC (11-OH-THC), and 11-nor-carboxy-THC (THCOOH) as these analytes are produced in the metabolism of Δ9-THC. Meibomian fluid maintains a high lipid concentration and Fatty Acid Binding Protein 5 (FAPB5), a protein known to bind to cannabinoids. Due to the lipophilic nature of THC, tear fluid could be used as a less-invasive biological matrix to test for the presence of THC and its metabolites.

OBJECTIVES

There exist limitations with current methods of detection of Δ9-Tetrahydrocannabinol (THC) drug analyte in Driving Under the Influence of Drugs (DUID) cases. This research explores the use of meibomian tear fluid as a novel matrix to detect THC and its accompanying analytes.

METHODS

This project optimized a collection method for tear fluid that would be more suitable for the creation of a novel direct injection method. Collection was completed by BVI Weck-Cel Sterile Cellulose strips, measuring approximately 2 x 20 mm, and placed in Thompson eXtreme PVDF 0.2 mm, pre-slit, red cap, filter vials containing Quantisal buffer solution. All analysis and calibrations were completed with fortified matrix standards with concentrations ranging from 0.25 – 250 ng/mL. Method validation was consistent with the Academy Standards Board (ASB) Standards of Forensic Toxicology Standard 036, First Edition 2018.

Tear samples were collected from four participants according to Institutional Review Board (IRB) standards before and after administration of Marijuana. Samples were collected approximately 30 minutes post administration of THC. Samples and calibration standards were analyzed using Liquid Chromatography Tandem Mass Spectrometry (LC/MS-MS) with the QSight® 220 CR LC-MS/MS and using a Halo® C18 3.0×50 mm (2.7 µm) column.

RESULTS

Limit of Detection (LOD) and Quantitation (LOQ) for THC was calculated at 0.25 ng/mL. The LOD of THCOOH was detected at 0.25 ng/mL and LOQ was calculated at 1 ng/mL. The LOD of 11-OH-THC was detected at 2 ng/mL and was not quantitated.

The charted retention times vs analyte intensities were evaluated for the existence of peaks within the expected range. This expected range came from the calibration data as well as internal standard comparison. The goal was to observe peaks around the expected retention time for detection and to observe the area of those peaks to gather quantitative data. The data was then plotted on the calibration curves in order to perform quantitative analysis.

As can be seen in Figures 1 and 2 the procedure was sufficient in detection of THC and THCCOOH. Detection of 11-OH-THC was not sufficient. Data was referenced against blood samples taken at the same time as the tear samples (seen in Table 1).

Figure 1: Data Collected After Dose Patient 13 – THC

Figure 2: Data Collected After Dose Patient 13 – THCCOOH

Table 1: Blood Cannabinoid Concentration Results – Patient 13

CONCLUSION

Upon analysis of participant samples, it was determined that THC and metabolites could be detected and quantitated in tear fluid. However, it is noted that insufficient sample volume in collection is an issue that leads to poor quantitation and can readily be optimized in future research.

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