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Rapid screening tests for the detection of Δ⁹-tetrahydrocannabinol (THC) in fibre hemp

G. Grassi

Istituto Sperimentale per le Colture Industriali,
Via di Corticella 133, 40129 Bologna, Italy
e-mail: <ggrassi@bo.nettuno.it> http://www.inea.it/isci.html

Grassi, G. 1999. Rapid screening tests for the detection of Δ⁹-tetrahydrocannabinol (THC) in fibre hemp. Journal of the International Hemp Association 6(2): 63-66. A preliminary study was done to assess whether existing tests could be used for detection of Δ⁹-tetrahydrocannabinol (THC) in fibre hemp. A very simple test, derived from a human application, was used to verify whether THC could be evaluated in a plant extract and this method adopted to a field test application. Performance and practical use of this immunological test is described. An immunoassay (IA) for laboratory use, based on a competitive-enzyme linked immunosorbent assay (ELISA), is proposed as an alternative method to assess large numbers of samples in very short time intervals. The application of IA to hemp research and its use as a method of assay control for European Union policy application seems a very interesting opportunity as an alternative to traditional chemical analysis.

Introduction

The US Environmental Protection Agency writes in its immunoassay guidelines that: "Immunoassay can provide real-time field analysis of a wide variety of environmental parameters at a fraction of the cost and time for conventional full protocol laboratory analyses". The official EU method, which is part of the regulation N. 1164/89 of the European Commission, is gas-chromatography (GC) quantitative analysis to certify that a hemp variety has a particular THC content and, to date, this method is the only method recognized. However, the EU DG-6 commission is attempting to update this very old GC method and every country has been invited to submit suitable variations to make the new method practical, accurate and more simple than the previous one. The purpose of this report is to suggest alternative methods to reduce both the time and expense involved in THC evaluation of fibre hemp.

Immunoassays can provide real-time field analysis of cannabinoids for a preliminary evaluation, eliminating the need to use GC as the primary analytical method to examine field samples. The demand for ready-to-use versions of these tests, usable in simple laboratories, at home or in the field, with particular interest for general human sciences has greatly encouraged efforts to set up simple, fast and reliable tests. Starting from a urine dip strip to detect pregnancy, many devices have been developed. Many kits are now available for THC (natural THC-acid de-carboxylated) and its metabolites (11-hydroxy-Δ⁹-tetrahydrocannabinol and 11-nor-Δ⁹-tetra-hydrocannabinol-carboxylic acid) and some of them are very effective. "Contrast", "SureStep", "accuPINCH", "EZ-Screen" and "Frontline" are only a few of many brands of one-step methods which permit indication of THC metabolites in a few minutes. However, very often the other cannabinoids (cannabidiol and cannabinol) cross-react with the antibody used in these tests. We have tried several of these rapid tests, checked them for such cross-reactions and found that only with the "Frontline" kit was it possible to properly estimate the THC content of fibre hemp.

Screening assays are qualitative or semi-quantitative. The purpose of preliminary tests is to screen, economically and efficiently, large numbers of samples to identify those containing no drug or a drug below an established concentration (cut-off). This eliminates negative specimens from further consideration (usually a large proportion of the samples). Specimens giving a positive result with the screening method are confirmed with a reference method, i.e., GC or high performance liquid chromatography (HPLC) combined with mass spectrometry (MS).

When first applied, the enzyme-linked immunosorbent assay (ELISA) format was widely considered to be a qualitative method of limited sensitivity. However, starting with environmental chemistry, it was shown that ELISA could be reproducible, equaling or surpassing radioactive immunosorbent assay (RIA) in regard to sensitivity. Now this format has been adapted to field applications, which has caused the wide diffusion of ELISA to many applications.

ELISA for THC evaluation

This method is normally performed in 96-well microplates which are coated with an antibody (polyclonal or monoclonal). The competitive binding takes place between the antibody, the THC in the sample, and a constant concentration of tracer added to the sample. This tracer consists of THC conjugated with an enzyme (peroxidase or alkaline phosphatase) attached to the plate, which is detected by a specific substrate.

The reaction of substrate will be at maximum if THC is not present in the sample because only the tracer enzyme will stick to the antibody coating. On the other hand, the reaction will be very low if the THC concentration in the sample is very high because this will compete with the THC tracer and will prevent the enzyme from being blocked on the plate (Grassi et al. 1997). This method is quite simple and fast, and does not require very expensive equipment. There are no problems with waste of reagents and sensitivity is good (normally about 1 ng/ml). The result can be inspected with the naked eye, if only a positive or negative evaluation is required. Quantitation of THC concentrations in the sample can be done using data from standard concentrations in each plate in conjunction with a Scatchard plot or a simple computer program for comparison. Actually, many companies sell complete kits to detect THC or its metabolites that use competitive (C-) ELISA (e.g., Diagnosticx Inc., Diatech Diagnostic, Hycor Biomedical Inc., Imunotech Corporation, Neogen Corp.). Only some of these are specific for THC, while the majority detect its metabolites and show a low cross-reactivity towards natural THC acid or other cannabinoids in hemp extracts. We evaluated some commercial polyclonal and monoclonal antibodies and clone "THC-003" from Biogenesis Inc. was used in C-ELISA to test plant extracts. Its specificity for phytocannabinoids and THC metabolites in comparison with other antibodies is reported in Table 1. Although, it seems more specific for THC metabolites, it works well and it is the cheapest antibody available using purified IgG. A typical standard curve obtained with pure THC as a competitor and THC conjugated with peroxidase enzyme as a tracer is reported in Figure 1.

The minimum detectable concentration (MDC) of pure Δ⁹-THC was about 2 ng/ml, which is a dose much lower than that of a fibre hemp sample (0.3 % THC dry weight, 0.2% proposed the year 2001). This concentration corresponds to 3,000,000 ng/ml. We performed C-ELISA in our laboratory, using parallel GC analysis of the sample hemp extracts. The correlation coefficient was 0.95, which indicates that C-ELISA is suitable to test THC in hemp extracts. The samples tested with C-ELISA were diluted 100 times as compared with the sample injected in the GC column. To evaluate the THC content of hemp, the concentration should be included in the linear zone (range 13-280 ng/ml) of the curve (Grassi and Ranalli 1998).

The optimized test

"Frontline" is a semi-quantitative immunoassay screening test for the detection of THC. The immunoassay is performed with dry reagents on a chromatographic test strip using GLORIA (Gold Labeled Optical-read Rapid Immuno-Assay) technology. Using the "Frontline" test strips, the determination of THC in Cannabis is easy to perform without any sample pre-treatment or reagent handling. A test strip is dipped into the sample for 5 seconds and then removed and left in the air for chromatography. The result can be read after 2 min. by comparing the red reaction color against a comparison scale printed on the vial. The test strip results are in good agreement with other commonly used automated immunoassay methods. "Frontline" compared favorably with gas chromatography-mass spectrometry as a reference method, providing highly sensitive and specific data. The test strip consists of a carrier foil (support) with a number of fleece compartments screened-off by a cover foil. A colored pad, located towards the end of the test strip, displays the parameter evaluated.

When the test strip contacts the hemp extract the liquid level rises to an area between two marks on the cover foil. This area serves as a liquid reservoir for chromatography of the sample along the test strip. The attached fleece holds a gold-labeled monoclonal antibody conjugate specific to the THC to be detected. As the THC present in the sample passes through, it reacts with the conjugate, forming a red-colored antibody-analyte complex. In the next compartment of the test strip, the reaction mixture reaches the capture matrix with an analyte analog immobilized on the solid phase. The part of the antibody-gold conjugate that has not reacted with any analyte in the sample will be bound to the solid phase, whereas the gold antibody conjugates with a sufficient amount of analyte will pass to the detection area to give a positive signal. The intensity of reaction is strongly correlated with the concentration of analyte in the sample.

Results

Tables 2, 3 and 4 show the characteristics of the kit and the performance of the test. Data were obtained from the producer and were presented at a conference (Goerlach-Graw and Carstensen 1994), when this kit was first produced. We applied this method to evaluate hemp extracts obtained from leaf samples dissolved in methanol and the results were in close agreement with company data for human urine. Hemp analysis with the "Frontline" kit requires that the extraction of THC from plant samples must be quantified for a semi-quantitative result. Our procedure included the sampling of 10 leaf disks from different plants. The disks were obtained closing the cap of an Eppendorf tube provided with an appropriate safety lock, over the leaf sample, so that leaf disks were directly introduced into the tube. Methanol (1 ml) was then added and, to speed up the extraction, the tube was heated at 60-70? for 10 minutes. We evaluated the dry-weight of these hemp disks: it was about 28% of the mean weight of 10 fresh disks, which were 90 mg. The cut-off of the "Frontline" test is about 700 ng/ml of THC (i.e. 0.007%). Because the EU hemp subsidy limit may become 0.2% THC by dry weight (presently 0.3%), we calculated the amount of THC in our extract (representing 25 mg of leaf by d.w.) that corresponds to 0.2% as 50,000 ng. To find a concentration close to the cut-off of the test, the 1 ml of methanol extract, containing the THC, was then diluted 1:70 with a buffer (phosphate buffer 0.1 M pH 7.0) because concentrated methanol interferes with the immunological reaction. Three classes of THC concentrations were used and evaluation was in accordance with concentrations evaluated by GC analysis. Other and wider evaluations should be done, but for the moment, we have found the "Frontline" test a valuable method for a semi-quantitative estimate of the THC content of fibre hemp.

Conclusions

There are alternatives to GC analysis for THC eval-uation in hemp extracts. We have described two methods which probably could be improved, but they are already faster and cheaper than GC. The actual cost of GC equip-ment and all the laboratory accessories needed for THC analysis would be about 25,000 Euro. The price of the "Frontline" test, for a vial with 30 tests, is about 144 Euro.

C-ELISA is suitable for laboratory use when many samples have to be evaluated in a short period of time and the price for each test with commercial antibodies is less than one Euro. We also have a monoclonal antibody and the cost for one test in our laboratory is less than half a Euro.

In 1999, the EU countries will be free to use the method that they prefer to evaluate the THC content in hemp crops. Our institute is responsible for THC evaluation of hemp crops and we plan to perform preliminary open-field THC evaluations with the "Frontline" test and then confirm the test with GC analysis. We would then like to do a validation using the actual field samples and to test the various hemp crops under different Italian conditions.

There are many possible ways of speeding up THC evaluation in hemp crops. It is possible to save money if a preliminary test is used (immunological or colorimetric) to discriminate negative from positive samples. Only the limited amount of samples found positive for a high THC content with a preliminary test need be further evaluated with instrumental methods like gas-chromatography, and only might these cases become the object of legal control.

References

Goerlach-Graw, A. and C. Carsten 1994. TIAFT/SOFT Joint Congress, Oct. 31-Nov. 4, Tampa, FL, USA.

Grassi, G., A. Moschella and M. P. Fiorilli 1997. Evaluation and development of serological methods to detect THC in hemp. in Proceedings of the symposium: Bioresource Hemp , 27 February- 2 March, Frankfurt, Germany: 197-201.

Grassi, G. and P. Ranalli 1999. Detecting and monitoring of plant THC content: Innovative and conventional methods. in Ranalli, P. (ed.) Advances in Hemp Research. Haworth Press Inc., Binghamton, New York: 43-58.