Appearance of hexahydrocannabinols as recreational drugs and implications for cannabis drug testing - focus on HHC, HHC-P, HHC-O and HHC-H.

This study investigated the effects of hexahydrocannabinol (HHC) and other unclassified cannabinoids, which were recently introduced to the recreational drug market, on cannabis drug testing in urine and oral fluid samples. After the appearance of HHC in Sweden in 2022, the number of posts about HHC on an online drug discussion forum increased significantly in the spring of 2023, indicating increased interest and use. In parallel, the frequency of false positive screening tests for tetrahydrocannabinol (THC) in oral fluid, and for its carboxy metabolite (THC-COOH) in urine, rose from <2% to >10%. This suggested that HHC cross-reacted with the antibodies in the immunoassay screening, which was confirmed in spiking experiments with HHC, HHC-COOH, HHC acetate (HHC-O), hexahydrocannabihexol (HHC-H), hexahydrocannabiphorol (HHC-P), and THC-P. When HHC and HHC-P were classified as narcotics in Sweden on 11 July 2023, they disappeared from the online and street shops market and were replaced by other unregulated variants (e.g. HHC-O and THC-P). In urine samples submitted for routine cannabis drug testing, HHC-COOH concentrations up to 205 (mean 60, median 27) µg/L were observed. To conclude, cannabis drug testing cannot rely on results from immunoassay screening, as it cannot distinguish between different tetra- and hexahydrocannabinols, some being classified but others unregulated. The current trend for increased use of unregulated cannabinols will likely increase the proportion of positive cannabis screening results that need to be confirmed with mass spectrometric methods. However, the observed cross-reactivity also means a way to pick up use of new cannabinoids that otherwise risk going undetected.

Cannabinol (CBN) Cross-Reacts with Two Urine Immunoassays Designed to Detect Tetrahydrocannabinol (THC) Metabolite.

BACKGROUND: The psychoactive component of cannabis, tetrahydrocannabinol (THC), is one of many cannabinoids present in the plant. Since cannabinoids have extensive structural similarity, it is important to be aware of potential cross-reactivity with immunoassays designed to detect THC metabolite. This is especially important as cannabinoid products are increasingly marketed as legal supplements. The objective of this study was to assess the cross-reactivity of 2 commercial immunoassays designed to detect THC metabolite with 4 cannabinoids: cannabidiol, cannabinol, cannabichromene, and cannabigerol. METHODS: Deidentified residual patient urine samples that tested negative for THC metabolite on initial testing were pooled and fortified with the above compounds to detect cross-reactivity. We next tested a range of CBN concentrations to determine what concentration of CBN was required to trigger a positive immunoassay result. Finally, we tested whether CBN has an additive effect with THC in the immunoassay by adding CBN to 21 samples weakly positive for THC by a mass spectrometry method but negative by the EMIT II Plus immunoassay. RESULTS: Both the EMIT II Plus assay and the Microgenics MultiGent assay demonstrated cross-reactivity with CBN. For the EMIT II Plus assay, about 5-fold more CBN than THC metabolite was required to produce an assay signal equivalent to the cutoff concentration, and CBN displayed an additive effect with THC metabolite. For the Microgenics assay, 20-fold more CBN than THC metabolite was required to cross the cutoff concentration. CONCLUSIONS: These data may help guide the need for confirmatory testing when results of THC metabolite testing by immunoassay are inconsistent with expectations.

Confirmation of marijuana, cocaine, morphine, codeine, amphetamine, methamphetamine, phencyclidine by GC/MS in urine following immunoassay screening.

Rapid, reliable, sensitive, qualitative, and quantitative methods using small urine volumes (0.2-0.5 mL) were developed primarily for confirmation of marijuana, cocaine, benzoylecgonine, ecgonine methyl ester, morphine, codeine, amphetamine, methamphetamine, and phencyclidine. Using capillary gas chromatography/mass spectrometry (GC/MS) and selected ion monitoring (SIM), mass spectra were obtained for each analyte. Samples were prepared by hydrolysis where applicable, organic solvent extraction, and derivatization where necessary. Confirmation was achieved by comparing abundance of major ions and retention time of the total ion current (TIC) of an analyte with those of the appropriate analytical standard. Quantitation was achieved and calibration curves derived by obtaining the molecular ion ratios of that analyte/internal standard (IS) over a concentration range of 10-300 ng/mL (0.16-4.0 ng total injected into GC/MS). The overall extraction efficiency for these analytes ranged from 53% to 96%. Statistically significant cut-off values (p less than 0.01) were obtained for each analyte. The slope, y-intercept, and coefficient of determination (r2) were calculated for each analyte. All of the GC/MS methods were extensively tested against urine samples determined positive or negative by immunoassay (IA) and are now used in our laboratory.

Marihuana metabolites in urine of man. VII. Excretion patterns of acidic metabolites detected by sequential thin layer chromatography.

The 11-oic acid metabolites of delta-9-tetrahydrocannabinol (THC), cannabinol (CBN), and cannabidiol (CBD) can be identified presumptively in human urine using standard thin layer chromatographic procedures. Other acidic metabolites are also excreted but these are not yet identified. These acidic metabolites appear in urine soon after exposure but persist for at least 48 to 72 hours. Such long persistence does not permit their detection to be used for forensic purposes. Using only simple chemical methods, we have been able to identify presumptively the 11-oic acids of THC, CBN and CBD in the urine of persons taking these materials. A sequential thin-layer chromatography system we have devised provided the basis for this first report of such isolation without the use of labeled drug or mass spectrometric methods.