Abstract
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.
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.
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.
These data may help guide the need for confirmatory testing when results of THC metabolite testing by immunoassay are inconsistent with expectations.
Introduction
In addition to increasing rates of state legalization of cannabis for medicinal and/or recreational use, other cannabinoid-containing products, such as cannabidiol (CBD) oil, are becoming widely available in products claimed to exhibit anti-inflammatory, anti-anxiety, and antiseizure properties (1,). In 2018, the United States Food & Drug Administration approved Epidiolex, a CBD oral solution, to treat 2 rare forms of epilepsy in children, Dravet syndrome and Lennox-Gastaut syndrome (2,). Due to the structural similarity of cannabinoids, such as CBD, with tetrahydrocannabinol (THC), false positive drug screen results consequent to using alternative (non-THC) cannabinoid-containing products is of concern (3–5,). It is of particular importance to be aware of assay cross-reactivity when immunoassay results are not routinely confirmed by a definitive method (e.g., clinical drug testing). In this study, we evaluated the extent of cannabinoid cross-reactivity in 2 commercial immunoassays designed to detect THC metabolite. We selected 4 cannabinoid molecules, including CBD, for which there is scientific literature discussing their potential benefits in a variety of pathological conditions and for which pure standards were available (6, 7).
Materials and Methods
The EMIT II Plus Cannabinoids immunoassay (Siemens Healthcare Diagnostics), run on a Beckman AU5810 instrument and the Microgenics MultiGent Cannabinoids immunoassay (Microgenics Corporation) run on an Abbott Architect instrument were assessed in this study, according to instructions from the manufacturer. Information about the cross-reactivity of other cannabinoid compounds is not provided in the EMIT II package insert; data for CBD and cannabinol (CBN) are included in the Microgenics package insert. The EMIT II Plus assay is a homogeneous enzyme immunoassay with a positive cutoff of 20 ng/mL. Briefly, target analyte (11-nor-Δ9-THC-9-carboxylic acid) in the specimen competes with 11-nor-Δ9-THC-9-carboxylic acid labeled with glucose-6-phosphate dehydrogenase (G6PDH) enzyme in the reagent for binding sites on an antibody directed at the target analyte. G6PDH converts nicotinamide adenine dinucleotide (NAD) to NADH, which leads to a change in absorbance at 340 nm, and activity decreases when the 11-nor-Δ9-THC-9-carboxylic acid-G6PDH complex is bound to antibody. The assay is a two-point fixed assay. Single-point calibration using free THC metabolite establishes a comparator signal at the cutoff concentration. The Microgenics MultiGent Cannabinoid assay has a similar method principle to the EMIT II Plus assay, but has a positive cutoff of 50 ng/mL, employs 4 calibrators, and is assessed as a rate-up reaction.
Residual patient urine specimens with a negative result using the EMIT II Plus assay were deidentified and pooled for experimental use. The study was conducted under an “Exception Umbrella Protocol for the Use of Clinical Laboratory Data Extracts for Method Evaluations, Quality Assurance, and Research” approved by the University of Utah Institutional Review Board. All cannabinoid stocks (1 mg/mL in methanol) were obtained from Cerilliant Corporation, and stock dilutions were prepared in methanol and stored at −80 °C. The highest concentration of methanol in fortified samples was 2% (v/v), and this amount of methanol was added into the blank sample for each experiment. Given the tendency of THC and related compounds to adhere to plastic, glass pipets and glass storage vials were used in stock preparation and sample spiking. Plastic tubes were used during sample testing to keep the process consistent with actual patient testing.
We performed initial cross-reactivity screening in triplicate. Each sample was fortified with a methanol blank or 1000 ng/mL CBD, CBN, cannabichromene (CBC), or cannabigerol (CBG). Samples were tested immediately on the EMIT II Plus assay and after one day of refrigerated storage/transport on the Microgenics assay.
To assess the concentration of CBN required to elicit a positive response, we assayed samples fortified with a range of CBN concentrations. Three urine pools were prepared and aliquoted before being fortified with a methanol blank or CBN to a final concentration of 1000, 500, 250, 100, 50, 20, or 10 ng/mL depending on the assay. Samples were tested immediately by the EMIT II Plus assay and after 1 day of refrigerated storage/transport by the Microgenics assay.
To determine whether CBN has an additive effect to THC metabolite in the EMIT II Plus immunoassay, we selected 21 residual and deidentified urine samples that contained 5–10 ng/mL THC metabolite, based on analysis by a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method validated for clinical use. These samples were also tested by the EMIT II Plus assay to verify that signal measured below the 20 ng/mL cutoff concentration. We fortified each sample with 50 ng/mL CBN, repeated the EMIT II Plus assay, and compared the signal of the pre- and post-CBN addition samples. The concentration of 50 ng/mL CBN was chosen since that concentration by itself was determined to be insufficient to cause a positive result by the EMIT II Plus assay.
Results
The initial evaluation using samples fortified with 1000 ng/mL of various cannabinoid compounds revealed that both the EMIT II Plus assay and the Microgenics MultiGent assay showed cross-reactivity with CBN, but not the other compounds evaluated (Supplemental Fig. 1). This agreed with the information in the Microgenics package insert indicating that CBD did not cross-react with the assay. CBN-fortified samples demonstrate a linear relationship with the EMIT II Plus assay (milli-absorbance units, mAU) (Fig. 1) up to CBN concentrations of 250 ng/mL. A CBN concentration of at least 100 ng/mL was required to pass the signal corresponding to the assay cutoff, suggesting that CBN cross-reactivity is about five-fold lower than THC metabolite reactivity. For the Microgenics assay, a CBN concentration of at least 1000 ng/mL was required to produce a signal equivalent to the assay cutoff, suggesting that CBN cross-reactivity is about 20-fold lower than THC metabolite reactivity. This contrasts with the package insert, which indicates that CBN concentrations greater than 100 ng/mL will produce a signal equivalent to the cutoff. The package insert does not provide details on how the experiment was conducted, so potentially the sample storage or transportation that occurs within a normal patient-testing workflow introduced differences. Overall, the semiquantitative assay showed a linear relationship between the assay result and the added CBN concentration down to concentrations of 250 ng/mL (Supplemental Fig. 2). The limit of quantitation of the assay is 10 ng/mL, so we were unable to assess lower concentrations of CBN.
Individuals who are required to complete drug screens (e.g., as part of pain management programs) will benefit from the work presented here. The research explores cross-reactivity in immunoassays, which is an important factor to consider when interpreting drug screen results. Investigation of THC metabolite immunoassay cross-reactivity with other cannabinoid compounds has not been done with currently available commercial assays, or in the context of extensive marketing of cannabinoid-containing products. Therefore, this work addresses an important gap in the field.
Immunoassay results of samples fortified with various CBN concentrations. Results from each urine pool are shown in a different color. The horizontal orange line at 100 mAU signifies the cutoff between positive and negative results.
All of the 21 patient specimens containing 5–10 ng/mL THC metabolite and negative by initial immunoassay screen showed an appreciable increase in EMIT II Plus immunoassay signal when fortified with CBN (50 ng/mL). While CBN alone at concentrations of 50 ng/mL is insufficient to generate an immunoassay signal above the cutoff, 13 of the 21 specimens containing THC metabolite at 5–10 ng/mL crossed the cutoff threshold and would have been resulted as positive (Fig. 2). However, we saw significant variability in the magnitude of immunoassay result increase for each sample, ranging from 10–108 mAU with a median increase of 59 mAU. We compared the observed increase to the creatinine concentration of each sample and observed no correlation.
Difference in immunoassay results pre- and post-50 ng/mL CBN addition plotted against the average result.
Discussion
Immunoassay based drug screening to evaluate abstinence or to detect undisclosed drug use is an important aspect of many clinical programs. Confirmatory testing is not routine in many such settings. The propensity of some immunoassays to have significant false positive and false negative results is a major reason why confirmatory testing by definitive methods such as LC-MS/MS or gas chromatography-mass spectrometry is recommended, particularly when results are inconsistent with expectations (8). Even so, it is important for laboratorians and clinicians to be cognizant of potential cross-reactivity in order to guide optimal test utilization.
This study unsurprisingly illustrates that assays from different manufacturers will display different cross-reactivity profiles. Critically, identifying lack of cross-reactivity is equally as important as identifying compounds that exhibit cross-reactivity. With the increasing marketing of CBD containing products, there are concerns about whether CBD oil or other legal products will cause false positive results on THC metabolite immunoassays. This study allowed us to determine that pure CBD will not cause a false positive with 2 commercial immunoassays. However, one caveat that we note is that the purity of most cannabinoid products is not regulated, so THC may be a byproduct in CBD oil or other cannabinoid products. In addition, derivatives produced during metabolism may have different cross-reactivity.
The structure of CBN is quite similar to THC and its primary metabolites, so the cross-reactivity displayed is reasonable. CBN is an aromatized derivative of THC that is thought to exist in low concentrations in the cannabis plant itself, but appears to be produced mostly during aging and storage of cannabis products (6,). CBN is not consistently detected in the urine of marijuana users (9–11,); however, individuals may directly consume CBN products, which are marketed as sleep aids. Early studies suggested that CBN has similar pharmacokinetics to THC and CBD (12). Therefore, knowledge that CBN cross-reacts with the EMIT II Plus assay and the Microgenics assay is important interpretive information. We observed variability in the extent that CBN contributed to results for samples containing 5–10 ng/mL THC metabolite. Some samples may have already contained low concentrations of CBN, or other unknown interfering compounds, potentially explaining the variability of result increases seen following CBN addition.
One limitation of this study is that we did not test all the urine samples used in the pools by mass spectrometry individually; some samples may have had a low concentration of THC that would not be detected by the immunoassay. However, we did test the 3 pools fortified with 1000 ng/mL CBN from the second experiment by mass spectrometry and observed no quantifiable THC metabolite. This provides support to the fact that the urine pools were not contaminated with small amounts of THC. In addition, it illustrates that the mass spectrometry-based assay, as expected, does not have significant problems with interference due to CBN.
Nonstandard Abbreviations
THC, tetrahydrocannabinol; CBD, cannabidiol; CBN, cannabinol; CBC, cannabichromene; CBG, cannabigerol; G6PDH, glucose-6-phosphate dehydrogenase; NAD, nicotinamide adenine dinucleotide; LC-MS/MS, liquid chromatography-tandem mass spectrometry; mAU, milli-absorbance units.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved.
G.M. Kroner, statistical analysis; K.L. Johnson-Davis, administrative support; K. Doyle, administrative support; G.A. McMillin, administrative support.
Authors’ Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest: Employment or Leadership: G.M. Kroner, University of Utah; K.L. Johnson-Davis, University of Utah; K. Doyle, Intermountain Healthcare, University of Utah; G.A. McMillin, University of Utah/ARUP Laboratories. Consultant or Advisory Role: None declared. Stock Ownership: None declared. Honoraria: None declared. Research Funding: None declared. Expert Testimony: None declared. Patents: None declared.
Role of Sponsor: No sponsor was declared.
Acknowledgments
The authors wish to thank Meredith Ford, Jessica Kwon, Rachel Law, Triniti Jensen, Heather Reichman, and Jonathan Seiter for assistance with obtaining and assaying samples.
REFERENCES
FDA. FDA approves first drug comprised of an active ingredient derived from marijuana to treat rare, severe forms of epilepsy. https://www.fda.gov/news-events/press-announcements/fda-approves-first-drug-comprised-active-ingredient-derived-marijuana-treat-rare-severe-forms (Accessed June
List of abbreviations
- THC
tetrahydrocannabinol
- CBD
cannabidiol
- CBN
cannabinol
- CBC
cannabichromene
- CBG
cannabigerol
- G6PDH
glucose-6-phosphate dehydrogenase
- NAD
nicotinamide adenine dinucleotide
- LC-MS/MS
liquid chromatography-tandem mass spectrometry
- mAU
milli-absorbance units
