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.