Determination of cannabinoids in human cerumen samples by use of UPLC-MS/MS as a potential biomarker for drug use

A quantitative analytical procedure was developed and validated by the use of Ultra- Performance Liquid Chromatography tandem Mass Spectrometry (UPLC-MS/MS) for the determination of Cannabidiol (CBD), Cannabinol (CBN), Δ9-Tetrahydrocannabinol (Δ9-THC), Cannabichromene (CBC), Cannabigerol (CBG) and 11-Nor- 9- Carboxy- Tetrahydrocannabinol (THC-COOH) in an unconventional biological matrix, cerumen. All the investigated calibration curves were characterized by high correlation values (R2 ≥ 0.9965). The LODs and LOQs ranged from 0.004 to 0.009 μg g-1 and 0.012-0.029 μg g-1, respectively. Intra-assay and inter-assay precision were found to be 0.6-2.5%, and 0.8-2.2%, respectively. All recovery values of cannabinoids, with the use of the optimum cotton swab, at low (0.008 μg g-1 of cerumen), medium (0.037 μg g-1of cerumen) and high (0.16 μg g-1 of cerumen) control levels, were estimated to be above 86%. The method developed here permitted the analysis of real cerumen samples obtained from fourteen cannabis users. In twelve out of fourteen cases, Δ9-THC was found to be positive, while in six cases, three major cannabinoids, CBN, CBG and Δ9-THC were quantified at concentrations 0.02-0.21 μg g-1, 0.01-0.24 μg g-1 and 0.01-4.86 μg g-1, respectively. Subject #8 has the highest amount of the detected substances in both left and right ear, with Δ9-THC at a concentration of 1.85 and 4.86 μg g-1, CBG 0.06 and 0.24 μg g-1, CBN 0.10 and 0.21 μg g-1, respectively. In addition, a detection window for the substances Δ9-Tetrahydrocannabinol, Cannabinol and Cannabigerol, in cerumen, was defined with success. In this case, Δ9-THC reached a maximum detection frame of up to fifteen days after smoking 0.5 g of marijuana cigarette. ANOVA-one-way analysis also indicated that the average earwax production of non-cannabis users differs significantly from the one of cannabis users (p = 0.048, <0.05). On the other hand, no significant difference was noticed between male and female users as the p value exceeded 0.05. In addition, no significant effect was observed on earwax production in regard to age, frequency and the last time of use (p > 0.05). These last three factors proved to have a significant impact on cannabinoids concentrations, since p values were less than 0.05.


Keywords:

ANOVA One-Way Factor Analysis; Cannabinoids; Cerumen; UPLC-MS/MS; Δ(9)-THC detection window.

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