Development and Validation of a Liquid Chromatography High-Resolution Mass Spectrometry (LC-HRMS) Bioanalytical Method for Quantifying Cannabinoids in Whole Blood: Application for Determining Recent Cannabis Use

Abstract

Background Cannabis legalization is expanding rapidly throughout the United States, but there is no reliable means of establishing recent use. Objective To develop and validate a bioanalytical method for determination of Δ9-tetrahydrocannabinol (Δ9-THC), cannabinol, 11-hydroxy-Δ9-THC, 11-nor-9-carboxy-Δ9-THC, and 8β,11-dihydroxy-Δ9-THC in whole blood microsamples by liquid chromatography high-resolution mass spectrometry (LC-HRMS). Methods Cannabinoid extraction from whole blood was performed using a mixture of n-hexane/ethyl acetate (90:10, v/v). Chromatographic separation was performed with a C18 column using a binary mobile phase gradient of water and acetonitrile, each with 0.1% formic acid. Detection was performed by positive ion mode heated electrospray ionization with full scan MS on an Orbitrap mass spectrometer. A clinical study was performed in 30 subjects to identify recent cannabis use based on analysis of cannabinoids in blood samples up to 200 min post-smoking. Results Acceptable linearity of all calibration curves was observed (r2>0.99) for all analytes over a 1–100 ng/mL concentration range, with acceptable accuracy. Limit of detection (LOD) was 0.5 ng/mL. Accuracy and precision met acceptance criteria for all analytes. Repeatability (CV) was <5% at low (3 ng/mL) and high (90 ng/mL) concentrations. In the clinical study, the ratios between 11-nor-9-carboxy-Δ9-THC and Δ9-THC fell immediately after smoking and returned to near baseline levels by 200 min post-smoking, which is consistent with recent use. Conclusions and Highlights The developed LC-HRMS bioanalytical method is suitable for quantification of five key cannabinoids in whole capillary blood microsamples and can be used in conjunction with a test for determining recent cannabis use.

A Comprehensive Breath Test that Confirms Recent Use of Inhaled Cannabis within the Impairment Window

“…It is well known that cannabinoid pharmacokinetics differ depending on the route of administration. Because D 9 -THC metabolites cannot be detected in breath, the blood may contain critical information pertaining to recent use, including concentrations of glucuronide metabolites and changes in the ratios of the D 9 -THC metabolites such as 11-hydroxy-D 9 -THC and 11-nor-9-carboxy-D 9 -THC to D 9 -THC and to each other, as previously reported [14,17].…”

“…TraceFinder software performed all required analyses. The chromatographic conditions for the analysis of cannabinoids in exhaled breath were the same as previously described [14].…”

“…Extraction and analysis of Δ 9 -THC, its major metabolites, and other cannabinoids in whole blood was performed according to a validated method as previously described [14]. Brie y, 50 µL of each sample was mixed with 100 µL of high-purity water in a 1.5-mL microcentrifuge tube and spiked with 5.0 µL of IS solution.…”

“…All analytical data were collected and processed using TraceFinder version 4.1 software (Thermo Fisher Scienti c). The mass spectrometer and UHPLC system were con gured as previously described [14].…”

“…The twosample strategy makes detection of Δ 9 -THC and other cannabinoids possible in their distribution phases, which occur during the rst few hours after smoking, and for the evaluation of how cannabinoid levels in breath are changing with time relative to blood. Liquid chromatography high-resolution mass spectrometry (LC-HRMS) bioanalytical methods for the quanti cation Δ 9 -THC and other cannabinoids in microsamples of whole blood was developed and validated for this purpose [14].…”