2.3 | Sample Analysis
The cannabinoid concentrations were measured using a validated assay that was co-developed with Medlab Clinical and that was conducted by Agilex Biolabs (previously known as CPR pharma services), a National Association of Testing Authorities (NATA) accredited facility based in Adelaide, Australia. Agilex Biolabs co-developed and validated the assay method. Laboratory assessments included a two-dose levels approach analysis of pharmacokinetic data. Blood samples from all eligible participants were assayed for Δ9-THC, CBN, CBD, 11-hydroxy-THC (OH-THC) and 11-nor-9-carboxy-THC (COOH-THC). Determination of cannabinoids CBD, CBN, Δ9-THC, Δ9-THC metabolites 11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (COOH-THC) and their respective deuterated Internal Standards from 100 µL aliquots of human plasma was carried out using protein precipitation followed by solid phase extraction (using acidified acetonitrile) followed by Solid Phase Extraction procedure (using micro-elution plates). The analytes were separated by high performance liquid chromatography (HPLC) on a Phenomenex Kinetex Biphenyl column, and the eluates monitored by a QTRAP5500 tandem mass spectrometry (MS/MS) detector in negative MRM mode. The extract was then assayed against a calibration curve. The data were acquired an Analyst® (Sciex) system linked directly to the QTRAP5500 MS/MS detector and then processed in Watson LIMS™ (Thermo Scientific), where applicable. The detection range was from 0.100 to 10.0 ng mL−1 for CBD, CBN, Δ9-THC and OH-THC and from 1.00 to 100 ng mL−1 for COOH-THC, using 100 µL of plasma and has a run time of approximately 8 minutes per duplicate samples. Laboratory assessments of the cannabinoids
There was no significant interference at the retention time of the internal standard (IS), or at the retention time of the other analytes for each individual analyte injected at PS8 concentration without IS added (except for analyte CBN where the percentage interference was approximately 29.0% of the mean CBN Lower Level of Quantification response when Δ9-THC was injected at Upper Level of Quantification level of 10.0 ng mL−1). There were 5 repetitions performed on the same day. Coefficient of determination for curves run during validation had a linearity of >99%. Moreover, inter- and intra-assay precision had a mean % coefficient of variation of approximately 3% (n=18) and inter- and intra-assay accuracy had a mean % bias of approximately 3% (n=18). Furthermore, there were no significant effects observed for any of the analytes in six individual lots plus in lipemic and hemolysed sources.