Webinar Highlights: Quantifying Cannabinoids in Oral Fluid to Determine Driving Impairment Following Marijuana Consumption
Q&A from webinar on using an ACQUITY UPLC I-Class/Xevo TQ-S micro MS system for quantifying cannabinoids in oral fluid
The Waters forensics team recently sponsored a webinar discussion about the advantages of using oral fluid quantitative analysis for forensic toxicology and driving impairment. The webinar was given by Dr. Philip Sobolesky, Clinical Chemistry Fellow, University of California.
Below is a recap of questions asked at the end of the webinar, most of which weren’t answered live since time didn’t allow.
Is the extraction efficiency you presented for the HLB plate, or the Quantisal device? Are calibrators and controls also extracted as patient samples in the Quantisal device before SPE? Did you perform short- or long-term stability assessments for your calibrators?
The extraction efficiency I presented was for the HLB plate. I did some small experiments examining the % recovery of the analytes from the quantisal device and my results (~30% loss strictly due to the device) matched those of previously published values (M. Fabritius, C. Staub, P. Mangin, C. Giroud, Analysis of cannabinoids in oral fluid by liquid chromatography-tandem mass spectrometry, in: Forensic Toxicol., 2013: pp. 151–163. doi:10.1007/s11419-012-0168-z.).
So far we have a shown there is no significant effect (>20% change) in Cals and QC’s after 10 Thaw cycles from the freezer. Long-term stability studies are still ongoing with a validated 1-year stability for Calibrators and QC’s.
Different strains of marijuana contain different profiles of cannabinoids. How will this test avoid bias for high concentrations of say CBN, (a molecule that results from cannabinoid degradation via time heat and light)? If say CBN was used as an indicator, one could argue that this is not an accurate means of determining impairment in a time-dependent manner.
One could argue that CBN was not a good indicator due to high degradation, but we have not seen a patient yet that has a CBN level higher than the corresponding THC concentration. Therefore when our data is finalized most likely (>x) THC concentration will be associated with impairment and can further be confirmed by presence of (y) CBN concentration.
Additionally, were the results altered upon the introduction of food intake? (You mentioned there was a lunch break.) Does a high blood glucose level cause any problems in accuracy and precision?
So far we have not detected any abnormal results after lunch that could indicate dietary interference, however we are also not measuring blood glucose at any time point. So it is difficult to answer your question since no investigative studies were performed looking at glucose interferences on cannabinoid concentrations.
I had seen previous Waters application data where Acetonitrile (1 mL I think) was added to the Quantisal buffer. This seems to aid recoveries. Any comment on that, and how did you arrive at 1 mL of 4% phosphoric acid?
I arrived at adding 0.4 mL of 4% H3PO4 to 1 mL of OF/buffer due to previous validated studies using this amount. Also, adding the analytes in phosphoric acid is standard practice with Oasis Prime SPE, so I did not perform any optimization at this step. (M. Fabritius, C. Staub, P. Mangin, C. Giroud, Analysis of cannabinoids in oral fluid by liquid chromatography-tandem mass spectrometry, in: Forensic Toxicol., 2013: pp. 151–163. doi:10.1007/s11419-012-0168-z.).
THC-COOH concentrations in oral fluid are reported to be at the picogram/ml concentration. This seems to be independent of collector buffer. That is raw oral fluid and buffer based collectors are still giving very low THC-COOH concentrations. Would you agree?
I know the literature has reports of THC-COOH in Oral fluid at around 15-450 picograms/mL. I know that for this study we were unable to achieve a LOQ that low and did not believe that many labs would be able to achieve that LOQ either. So while some laboratories may be able to quantify down to 15 picograms/mL, that level of sensitivity requires additional processing and handling, resulting in lack of practicality.
Could you repeat what is being looked at in the study, other than blood, oral and breath? DRE, iPad distraction, what else?
The 10 cannabinoids were being quantified in oral fluid, 8 in blood and just THC in breath. Participants are assessed by the driving simulator, iPad based performance, and blinded police DRE exams. Obeying verbal commands is also assessed (i.e., after the third green light, turn left).
Was liquid extraction or dilute and shoot evaluated?
No – but these sample pre-treatment approaches have been tried in other laboratories . Due to the presence of stabilizers and surfactants in commercial oral fluid devices dilute and shoot will not work , very high ion suppression will result. Liquid liquid extraction is also not successful at reducing ion suppression when the oral fluid is collected in commercial devices.
What is the correlation between measured impairment and cannabinoid levels in the blood/saliva? Has your study looked at this yet or will that come later?
This will be done at the conclusion of this study, once we have 120 completed participants with completed data, we will be unblinded and then be able to assess what concentration of any of the cannabinoids measured are associated with impairment.
How do you ensure the uniformity of the cannabinoids concentrations in the joints you provided to the participants? What are the ranges of variations?
The joints are rolled by the pharmacist at UCSD Medical Center. The pharmacist is told by the statistician which type of marijuana to use (i.e., placebo, 5.9, or 13.4% THC) and then is given to the project coordinator, who delivers it to the patient. So only the statistician knows which participant is given what type of cannabis. The amount rolled in the joint is identical between the types.
How do you incorporate the cannabis using background for all the participants into the study? How are you going to interpret the results?
We are only enrolling chronic users (daily) and occasional users (>3x per week) into our study to reduce naïve user bias. At this time, I do not know how my collaborators will handle the differences in user background.
How is the spread of the drug in oral fluid samples? We collected two different samples in two different tubes and one tested positive and the other negative for THC, can you explain?
We have not collected enough data to really comment on the spread of drug in oral fluid samples but we have seen anywhere from 15 ng/mL – >4000 ng/mL of THC after smoking. Assuming you are talking about two different devices it is possible that the proprietary buffers could have contributed to your bias.
What would be the interpretation if there is one of the metabolites but not the parent drug in the oral fluid sample?
As of now, we do not have any speculations on potential sample interpretations, but with that said we have yet to have a sample that is THC negative with a metabolite positive in oral fluid. So it will likely be a very rare situation to have to speculate on what that might mean.
Why do you consider that THCA-A is so highly retained on the Hamilton Syringe?
I do not have a good answer as to why THCA-A is highly retained on the Hamilton Syringe. We chose not to investigate the cause but rather execute a solution (i.e., switch to disposable pipette tips).
With the Hamilton syringe carryover, did you try washing with other solvents like chloroform?
No, we did not try washing with chloroform or other nonpolar organic solvents.
There’s much that’s unknown about what causes strains to be (for example) ‘energizing’ versus ‘couch lock’. Those factors (e.g., terpene compliment) could certainly affect driving ability. How would you control for that variability?
Addressing this question is difficult since ingesting plant material takes a polypharmacy approach to medicine (i.e. multiple factors causing a response that is difficult to contribute to only one compound). However, the psychoactive component of cannabis is THC, so regardless of the other contributing factors, if THC ends up being associated with impairment in OF at a certain level then we hope that will hold true across different plant strains. This is a complex issue but future research on profiling plant strains is a topic that needs to be better regulated and controlled to tease out potential impairing effects.
What type of tasks were evaluated on the iPad?
The iPad task was to determine whether or not the subject was capable of selecting the bubble that was larger than all the others. The time taken to select the correct bubble will be assessed.
Our group measures retinal function/vision with acute use, but do not have the ability to determine biologics. We are finding in our limited data set, “plant” differences. Our previous research in AD and PD have shown that errors in tablet neuro tests often reflect vision dysfunction rather than cognitive. Your methods and ability to measure numerous cannabinoids will be important. Do you have means to determine non-cognitive functioning?
There are verbal commands that the subject are given to assess attention that would not be affected by vision dysfunction. This aspect of the project is overseen by my collaborators in the psychology department and would be better addressed to them as my experience is limited in that realm of research.
Older vision research (from 70s at Berkeley) found peak and dissipation curves similar to yours, i.e., peak around 10-15 minutes dissipated by 1.5 hours. These were functional test of retinal glare. Do any of the functional tests match these peaks and dissipation curves?
I am still blinded to any of the functional tests and my collaborators are blinded to any of the laboratory values until the completion of the study.
Hearing and vision are dysfunctional with acute marijuana use. Huestis reports tunneling — but no studies identified to date. We are finding tunneling. Reported at ARVO this year.
I am blinded to any of the functional assessments, simulators, and DRE exam outcomes.
So if NIH, limited in CBD then? What is max CBD can get?
Since we obtain our marijuana from the government, we are limited as to which strains and potency we are able to test. I do not know what the maximum amount of CBD is available from NIH.
Have you considered to use time shift method then look at the correlation between blood and OF? For example, if the compounds get absorbed after 15 minutes, use the 15 minutes blood with time-zero oral?
No we have not considered performing any time shift to our data at this time. Data analysis will occur at the conclusion of the study and there is a chance that the correlations will improve once we stratify the patients (i.e. impaired vs not impaired; or placebo vs Low vs High THC; etc.).
Why only deuterated IS?
Deuterated internal standards are commonly used for cannabinoid metabolites. 13C internal standards could be potentially used.
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