Methods exist for extraction of cannabinoids from oral fluid using the Quantisal device. However, the goal of this manuscript is to combine previous methods into a single extraction procedure without a hydrolysis step that would allow for the quantification of the following compounds by one method: THC, cannabidiol , cannabinol , cannabigerol , Δ9 -tetrahydrocannabinolic acid , tetrahydrocannabiverin , 11-hydroxy-Δ9 -THC , 11- nor-9-carboxy-Δ9 -THC , 11-nor-9-carboxy-Δ9 -THC-glucuronide , and Δ9 -THC-glucuronide . This assay will be useful for OF cannabinoid analysis in the establishment of a cannabinoid concentration associated with driving impairment.Oasis prime HLB 96-well extraction plates were purchased from Waters . Mass spectrometry grade methanol , acetonitrile , and formic acid were purchased from Fisher scientific . Blank synthetic OF matrix used to prepare calibrators and quality control specimens was purchased from Immunalysis . OF was collected with the Quantisal™ device also from Immunalysis. Participants refrained from food or drink for 10 min, then the absorptive cellulose pad was placed under their tongue until the indicator turned blue or 5 min had passed. The collection pad was then placed into the plastic collection device containing 3 mL of extraction/stabilization buffer. The extraction buffer is supplied with the Quantisal™ device. The capped tube was placed at room temperature for at least 4 h but not > 24 h. The pad was then removed from the stem using fisher brand standard serum filters and decanted into nunc 3 mL cryovials from Wheaton.The samples were then stored at 4 °C. Each sample was weighed in attempt to derive a short sample correction factor before being analyzed within 2 months of collection.
Positive QC standard solutions of 300, 60, and 12 ng/mL were prepared by parallel dilutions from a 1000 ng/mL stock in methanol made the same as described for the calibrator solution except using stock solutions from a different lot than the calibrators. Each solution was aliquoted into amber glass auto sampler vials,garden racks wholesale sealed with parafilm and stored at −20 °C. The solutions correspond to three levels of QC at 60, 12, and 2.4 ng/mL when processed in synthetic OF. The lower level of QC was chosen to reflect a low concentration that was closer to per se cut off values adapted by several states. QC results were reviewed according to an absolute criteria of ± 20% of target values.All calibrators and QCs were prepared by adding 50 μL of calibrator, 50 μL of working IS, followed by one mL of blank matrix to corresponding borosilicate tubes. Subject specimens were treated in a similar manner except methanol was substituted for the calibrator. Samples were then acidified with 400 μL of 4% phosphoric acid. Samples were vortexed briefly then contents were transferred to a well of a 96 well Oasis Prime HLB C18 SPE plate. Samples were forced through the wells using a positive pressure manifold on low pressure until all liquid was pushed through. This took approximately five minutes to drip through. Each well was washed with 500 μL of SPE wash buffer twice under low pressure. The pressure was switched to max flow for one minute following the second wash to push any excess liquid through the well. Compounds of interest were eluted into 750 μL glass inserts with three successive 100 μL aliquots of 98% ACN with 2% formic acid for a total of 300 μL eluant. Extracts were evaporated under nitrogen at 40 °C with gas flow set to 70 psi for 30 min. Dried extracts were reconstituted with 200 μL of 50% ACN containing 0.1% formic acid. Plates were covered with a silicone/PTFE treated, pre-slit cap mat and vortexed using the Fisher scientific Ana Multi-tube vortexer on speed setting of 4 for 5 min.
Plates were centrifuged at 1962 x g for 10 min in Sorvall legend XFR centrifuge and then transferred to the sample organizer for LC-MS/MS analysis.Chromatography was performed using a Waters Aquity i-class UPLC system equipped with sample organizer, binary solvent manager, auto sampler, and a column oven . Separation of analytes was achieved using a Waters 2.1 × 50 mm Acquity UPLC BEH C18 column packed with 1.7 μm sized particles. The analytical column was attached to a 2.1 mm × 5 mm ACQUITY UPLC BEH C18 VanGuard Pre-column packed with 1.7 μm particle size to prevent column degradation due to sample buildup. Guard columns were replaced after every 1000 injections. The auto sampler was set to 10 °C. The column heater was set to 40 °C. A full-loop 10 μL injection was made for each sample. Gradient elution was performed using a mobile phase A of 5 mM ammonium formate buffer with 0.1% formic acid and a mobile phase B of acetonitrile with 0.1% formic acid at a constant flow rate of 400 μL/min. The initial gradient conditions were 50% MPB, held for 30 s, and then linearly increased to 90% MPB over 3.5 min. The final MPB concentration was maintained for 15 s, before returning to initial conditions and holding for 45 s. The maximum pressure was set to 15,000 psi.The LC system was coupled to a Waters TQ-S-micro triple quadrupole mass spectrometer interfaced with an electrospray ionization probe. Negative ionization was used for THC-COOH-gluc. All other compounds used positive ionization. The mass spectrometry transition ions were collected using a scheduled multiple reaction monitoring mode with four separate time windows. The first time window was collected in negative ion mode from 1.00 to 1.50 min. The subsequent windows were collected in positive ion mode from 1.51 to 2.59 min for TW-2, 2.60 to 3.22 min for TW-3, and 3.23 to 4.20 min for TW-4. The selected precursor and product ions, collision energy, retention times and associated windows are displayed in Table 1. The source temperature was set to 550 °C for both modes.
The instrument was controlled with Masslynx V4.1 SCN945 SCN960 software and peaks were processed using TargetLynxs XS. A representative reconstructed chromatogram of all quantifier ions from a 20 ng/mL calibrator is displayed in Fig. 1.To demonstrate that the synthetic OF was a valid substitute for OF specimens from humans, OF from 10 drug free volunteers was collected and processed such that a one mL aliquot was fortified with low QC and IS, while another one mL aliquot was processed unaltered . The percent bias was calculated by dividing the difference between the averaged concentration of the low QC in human OF samples from the average concentration of low QC in blank matrix by the averaged concentration of low QC in blank matrix. A qualitative assessment of matrix interference was also performed by injecting each of the unspiked OF samples while simultaneously infusing a calibrator solution containing 10 ng/mL of each analyte. See Fig. 2 for total ion chromatograms of the blank OF samples. Potential drug interferences were assessed by generating 5 pools of 10 different drugs belonging to opiates, benzodiazepines, and other common drugs of abuse that could be present in a suspected DUI subject . Superphysiological concentrations of the pools of drugs were added to blank OF samples fortified with low QC. Recovery of the QC within ± 20% of expected concentration in the presence of super physiological concentrations of drug pools was required to demonstrate lack of interference.Auto-sampler stability was assessed by comparing the average area counts from the low QC to the area counts of an injection at 24 and 48 h post-extraction. Acceptable stability was set at ± 20% CV in area of a 1.25 ng/mL stock compared to the initial injection. Lack of carryover was established by injecting a blank matrix fortified with IS immediately after the highest calibrator and then comparing the area counts to the same blank matrix with IS injected prior to the calibration curve. The acceptable level of carryover was a set to < 20% increase in area counts of the blank matrix following reinjection after the highest calibrator.Proof of applicability is demonstrated by evaluating the concentrations of cannabinoids in three participants enrolled in an Institutional Review Board-approved study evaluating the effects of inhaled cannabis containing either placebo , 5.9% or 13.4% THC by weight. Inclusion criteria for participation were individuals had to be at a minimum an occasional user ,hydroponic racks abstain from marijuana use 48 h prior to testing, and have a valid drivers license. Oral fluid samples were collected upon arrival to the laboratory which was tested to demonstrate THC < 5 ng/mL using the Alere OF point of care instrument. Individuals whose OF screened negative on the Alere device then smoked a joint containing either placebo, 5.9%, or 13.4% THC. Oral fluid was then collected 15, 90, 210, and 280 min after smoking and processed as described above.
The complete study design and detailed methods will be published after the target enrollment of 180 subjects is complete.A qualitative matrix effect study was performed by infusing a 10 ng/ mL calibrator solution during an injection of an extracted oral fluid specimen from drug-free volunteers . There was no observable ion suppression or enhancement across any of the analytes peaks in the human oral fluid specimens . Quantitative assessment of matrix effects was performed by fortifying the human drug-free oral fluid specimens with low QC and calculating the percent recovery to expected values established in the inter-day validation . Acceptable critera were set as a percent difference < 20% from expected. No matrix effect exceeding this criteria was observed in the human oral fluid specimens when compared with the synthetic oral fluid used for calibrators and controls. Extraction efficiency was determined by comparing average peak areas of extracted blank matrix samples fortified with low, mid, or high QC divided by peak areas of blank matrix samples fortified post-extraction with QC. All analytes had less than a 9% difference in extraction efficiency between any level of QC with a range of efficiencies from 26.0–98.8% . Percent matrix bias were determined by comparing average peak areas of blank matrix samples fortified post-extraction with low, mid, or high QC divided by neat solutions of QC. The range of percent matrix bias was −37.6–23.7%. THCA-A observed the worst ion suppression followed by THC-V . THC-COOH observed the greatest ion enhancement due to matrix effects observed to be > 20% in the mid QC, whereas all other analytes had percent differences < 20%. The THC-COOH internal standard compensated for the matrix enhancement providing results within ± 20% of target values. Interferences were assessed from five pools of ten drugs in blank OF fortified with low QC. The percent bias for all cannabinoids ranged from −17.4–12.7% . Thus, no drugs that were tested caused any interference in calculating the low QC concentration.The areas for all the compounds were within ± 20% upon reinjection at 24 h in the auto-sampler. The 48 h injection of samples had a percent difference within 20% for CBN, CBD, THC, 11-OH-THC, THCCOOH, and CBG, but a percent difference < 28% for THC-gluc, THCCOOH-gluc, THC-V, and THCA-A. In the 48 h reinjections, the internal standards compensated for changes in area counts so quantitative results were within 20% of the initial values. Dilution integrity was acceptable within ± 20% of target concentrations for THC after diluting 1:10 with blank matrix. THC quantified within 3.1% of expected concentrations. There was no evidence of carry-over for any of the cannabinoids following injection of a sample containing 2000 ng/mL.Quantification of THC and related metabolites is part of a research project that aims to establish the concentration of cannabinoids associated with driving impairment following consumption of a low does , high dose , or placebo . Participants have their OF samples collected prior to and immediately after smoking one of the randomly assigned joints. The study is a double-blinded approach, thus the laboratory is blinded to which participants have smoked which kind of joint until the conclusion of the study. To demonstrate proof of applicability, the laboratory was unblinded to identify the first three participants in this study that smoked either the placebo, low or high dose joint. The purpose of including data from three subjects who smoked marijuana is to demonstrate the analytical method is capable of measuring these compounds in specimens obtained from human volunteers, and not to draw any conclusions regarding pharmacokinetics or determining severity of impairment. One participant from each group had their oral fluid samples assessed by this method with concentrations of each cannabinoid listed in Table 7.