The goal of this work is to present a liquid chromatography/tandem mass spectrometry (LC-QqQ-MS/MS) method for detecting a variety of cannabis exposure and oxidative stress biomarkers in exhaled breath condensate (EBC) and oral fluid (OF) matrices.
Many states in the U.S. continue to legalize medicinal and recreational cannabis. As a result, there is a growing need in forensic toxicology to investigate cannabis biomarkers that can differentiate between recent/long-term use and licit/illicit consumption, be sampled with relative ease, and, ultimately, correlate with impairment. Furthermore, clinicians are interested in determining whether inhalation of recreational/medicinal cannabis can exacerbate or provide protection from oxidative respiratory stress following exposure.
EBC and OF are alternative, non-invasive matrices that hold promise for identification of cannabis exposure biomarkers. OF is currently being explored as a matrix for cannabis exposure analysis. However, there are currently no reports on the use of EBC for this purpose, although other drugs have been reported to be detected in this matrix. EBC is an aqueous specimen consisting of condensed water vapor, volatiles, and non-volatile components and has several potential advantages for cannabis biomarker detection, including ease of collection and the presence of a wide variety of volatile and non-volatile analytes, metabolites, and markers of respiratory stress.
In this study, selected cannabis and oxostress analytes were spiked into blank EBC and OF and analyzed by LC-QqQ-MS/MS. A total of 20 major and minor cannabinoids, acid precursors, and metabolites were targeted for analysis based on detection reported in human specimens, compounds identified in recreational and/or medicinal cannabis, and availability of standards. Seven internal standards were included for quantitation analysis. Five commonly monitored respiratory oxidative stress biomarkers were also selected for analysis: 8-isoprostane, 8-oxo-7,8-dihydroguanine, 8-oxo-7,8-dihydro-2’-deoxyguanosine, 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone, and 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanol. An Agilent 1290 UHPLC coupled to a 6470 LC-QqQ-MS/MS with positive and negative mode ESI was utilized for this work. Chromatographic separation was achieved using a Zorbax 120 EC-C18 column (3.0 x 100 mm, 1.8 μm, Agilent Technologies) and a step gradient elution with an aqueous mobile phase (A) of 5 mM ammonium formate with 0.1% formic acid in water and an organic mobile phase (B) of 0.1% formic acid in 75% acetonitrile and 25% methanol. ESI parameters were optimized to the mobile phase composition of choice, and dMRM parameters including MRM transitions, collision energy, and fragmentor voltage were optimized to effectively analyze both the cannabis and oxidative stress biomarkers in a single run time of 26 minutes.
The method was successful in identifying all analytes, including the difficult to separate delta-8-Tetrahydrocannabinol (8-THC), delta-9-Tetrahydrocannabinol (9-THC), cannabidiol, cannabichromene, and cannabicyclol. LOD and LOQ for the target cannabinoids and oxidative stress biomarkers ranged from 1 to 10 ng/mL and 7 to 18 ng/mL, and from 0.13 to 54 ng/mL and 0.38 to 164 ng/mL, respectively. Future studies will utilize the method to explore cannabis exposure and oxidative stress biomarkers in a human cohort of cannabis smokers with different exposure characteristics.