With the legalization of medical and recreational marijuana, there has been a rise in the use of cannabinoid products and thus, increased demand for techniques that can measure the levels of cannabinoids in the human body. Just as alcohol is measured using a breathalyzer, law enforcement officers also want to measure the levels of cannabinoids in the breath.
However, the concentration of cannabinoids in the breath is very low, so any detection method employed must be highly sensitive. One solution is a liquid chromatography-mass spectrometer-based assay to quantify cannabinoids in breath samples.
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Disadvantages of previous methods
After the use of marijuana, they can be detected in blood, urine, oral fluids, and breath. Studies show that the levels of marijuana in-breath can be as low as pg/mL. One of the most commonly used methods for detecting cannabinoids is liquid chromatography-tandem mass spectrometry (LC-MS/MS) using electrospray ionization.
To increase the sensitivity of LC-MS/MS, the analytes often have to be structurally modified (derivitization) to increase the efficiency of the method. The additional step of derivitization, including extraction, dry-down, reconstitution, etc. result in a lengthy period of sample preparation.
A novel derivatization method
Azo coupling refers to an electrophilic aromatic substitution reaction where a hydrogen atom in the aromatic ring is replaced by a diazonium ion, leading to the formation of an azo-derivative. The structure of this compound includes an aromatic compound and the diazonium that are bound using the azo group. This derivative can be further protonated to increase the efficiency of ionization.
The addition of this feature increases the sensitivity of the LC-MS/MS method. As this method is compatible with various samples, such as water, methanol, acetonitrile, etc. it can be performed in neat samples.
This method was used to analyze the presence of five cannabinoid products, including Δ9-tetrahydrocannabinol (THC, major cannabinoid), 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC, THC metabolite), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THCCOOH, THC metabolite), cannabinol (CBN, minor cannabinoid and THC degradation product), and cannabidiol (CBD) in the breath samples.
The study included cannabinoid users in various stages, from infrequent users (once a month) to frequent users (5 times a day). A total of 180 breath samples were collected in the breath capture module and eluted in ethanol. Samples were analyzed within three hours of the participants’ smoking cannabis. The derivatization assay can detect from pg/mL to sub-pg/mL levels of cannabinoids.
While THC and CBN were found in all breath samples, 11-OH-THC and THCCOOH were not detected. CBD was found in a subset of samples (53%). When the levels of CBN and THC were plotted against each other, a strong correlation was observed showing a potential relationship between the two in the breath of people who smoke cannabinoids. There was no interference between the five structurally similar cannabinoid compounds. Simultaneously, 180 blood samples were also analyzed for comparing the two methods and a good correlation was found.
Lasting impressions
This derivatization method can enhance the sensitivity of the LC-MS/MS method to analyses breath samples. This is also easier to use and faster compared to conventional LC-MS.MS analysis. Using this method, five cannabinoid compounds could be analyzed, whose levels were as low as 0.5pg/Ml to 2.5 pg/mL.
The ultrasensitivity of the method also helped to draw correlations between THC and CBN in human breath samples. One of the hypotheses is that CBN is converted into THC during cannabis combustion. The high sensitivity also helped to detect THC in samples collected within three hours of smoking.
Adding the steps of sample clean-up, this assay was also used to analyze the blood samples, showing that this method can be used in various types of biological samples. This method can be further applied to other aromatic compounds and thereby increasing the efficiency of the LC-MS/MS analysis.
Source
- Luo et al. (2019) Quantitation of Cannabinoids in Breath Samples Using a Novel Derivatization LC–MS/MS Assay with Ultra-High Sensitivity. Journal of Analytical Toxicology, 2019;1–9 (doi: 10.1093/jat/bkz023)
Further Reading