Synthetic Cannabinoids

Often marketed as herbal incense or as a legal alternative to marijuana, synthetic cannabinoids grabbed headlines in recent years when hospital emergency departments experienced a sudden spike in overdoses and admissions for the designer drugs known as Spice and K2. The products, which are marketed to users looking for a drug similar to marijuana, consist of dried plant materials resembling potpourri that have been sprayed or laced with synthetic cannabinoids.

While Spice and K2 are the most widely known brands, hundreds of other brands are sold legally by Internet retailers, at convenience stores, and in tobacco shops for $8–20/g. Most are labeled with the warning ‘not for human consumption' to circumvent the U.S. Controlled Substances Act.

As these designer drugs continue to be sold and used, laboratories need to understand how to detect them. This article will describe the chemical origins of synthetic cannabinoids and their effects on users, legal status, and prevalence, as well as the limits of current testing methods.

Effects of Synthetic Cannabinoid

Synthetic cannabinoids have been compared with the psychoactive compound, Δ-9-tetrahydrocannabinol (THC), found in marijuana. On the molecular level, they are potent cannabinoid receptor agonists that also may have affinity for other types of receptors. Reported symptoms of toxicity include anxiety, agitation, paranoia, hallucinations, tachycardia, hypertension, excessive sweating, nausea, and vomiting.

Overdoses of synthetic cannabinoids can cause panic attacks and psychosis and lead to tragic results. In 2010, an Iowa teen smoked K2 with some friends, and then reportedly told them he was "going to hell" and went home, where he shot and killed himself. In another case, a 19-year-old in Illinois died when his car jumped a retaining wall at an estimated speed of 100 mph, flew 15 feet, and crashed into a house. About 90 minutes before the crash, he told his brother he had been smoking "legal potpourri." In both of these cases, the victims reportedly purchased the synthetic cannabinoids at a local shopping mall.

For people looking to get high, synthetic cannabinoids are readily available, fairly inexpensive, and in many cases, legal to purchase. Naive drug users also may incorrectly assume that a product sold at a convenience store and labeled as natural is safe to try. Furthermore, employees, such as transportation workers or military personnel who must undergo random drug testing, may be more likely to use synthetic cannabinoids because they are not detected by current drug screening programs.

Prevalence

Synthetic cannabinoids originally emerged in Europe in 2006, and by November 2008, the U.S. Drug Enforcement Agency's (DEA) forensic laboratory detected them in products in the U.S. The following year, the American Association of Poison Control Centers (AAPCC) reported 112 calls involving synthetic cannabinoids to poison control centers in 15 states. That number quickly soared. Within 9 months, 49 states plus the District of Columbia recorded 2,700 calls, and by 2011, the number rose to 6,549. In its latest report in October 2012, AAPCC logged an average of 580 calls per month for 2012.

Other signs of the growing popularity of synthetic cannabinoids are also evident. In 2010, DEA reported that 30–35% of specimens submitted by juvenile probation departments tested positive for synthetic cannabinoids, and according to the 2011 National Institutes of Drug Abuse (NIDA)-sponsored Monitoring the Future survey, 11% of high school seniors reported smoking synthetic marijuana in the past year, making it one of the most commonly abused drugs in this population—second only to marijuana. Furthermore, researchers recently reported that 4.5% of urine specimens collected from 5,956 U.S. athletes tested positive for synthetic cannabinoids, the highest of all drug classes detected (1). Synthetic cannabinoid use also has spiked among military personnel, and the Armed Forces are currently conducting a study to determine the prevalence of synthetic cannabinoid use within the military.

Classes and Structures of Synthetic Cannabinoids

There are three major categories of synthetic cannabinoids: classical cannabinoids, cyclohexylphenols, and aminoalkylindoles.

One well-known classical cannabinoid is the THC analogue HU-210. This chiral compound takes its name from Hebrew University where it was synthesized by Raphael Mechoulam in the 1980s. HU-210 is a schedule I controlled substance under the Controlled Substances Act. According to the U.S. Customs and Border Protection, it was discovered in January 2009 in herbal incense products in Wilmington, Ohio, where agents seized more than 100 lbs of the product. However, classical cannabinoids are difficult to synthesize and do not appear to be highly prevalent on the market.

Pfizer developed the second category of synthetic cannabinoids, cyclohexylphenols, as analgesics in the late 1970s. Dubbed CP for Charles Pfizer, CP-47,497 and its C8 homologue, cannabicyclohexanol, were among the first synthetic cannabinoids detected in herbal incense. In March 2011, DEA used its emergency scheduling authority to control these two compounds; however, they appear to have been replaced by new designer cannabinoids of the aminoalkylindole variety.

Aminoalkylindoles are currently the most prevalent synthetic cannabinoids. Included in this category are the JWH-018, JWH-073, and JWH-200 cannabinoids that DEA recently added to the class I schedule and other indole- and pyrrole-based analogues. Clemson University Professor J. W. Huffmann first developed the JWH series in the late 1990s. These cannabinoid analogues are synthesized in a simple two-step process, and undergraduate summer research students in his lab originally synthesized many of the original JWH analogues. A purification process also is necessary to achieve the final product. Recently, laboratories have detected phenylacetylindoles such as RCS-8, which stands for Research Chemical Suppliers, and benzoylindoles such as AM-694, named for Alexandros Makriyannis, in synthetic cannabinoid products.

Figure 1 Structures of Synthetic Cannabinoids Detected in U.S. Products as of September 2012 click for figure Other cannabinoids found in U.S. products, but not shown, include benzoylindole RCS-4, phenylacetylindole JWH-251, and napthoylindoles JWH-019, JWH-015, JWH-081, JWH-398, AM-1221, and WIN 55,212-2. * Indicates synthetic cannabinoids scheduled in March of 2011.

Legal Status

Following the DEA's March 2011 temporary emergency ban on the five synthetic cannabinoids described above, in July 2012, President Obama signed the Synthetic Drug Abuse Prevention Act of 2012 (S.3187) into law. The new law explicitly bans 15 synthetic cannabinoids in addition to 11 other synthetic designer drugs and increases the amount of time an analogue can be temporarily scheduled. At least 41 states also have legislative bans on synthetic cannabinoids.

But manufacturers of herbal incense products are financially motivated to stay one step ahead of such legislation. According to the Financial Times, assets for Psyche Deli, the original manufacturer of Spice in the U.K., grew by nearly 1300% from 2006 to 2007. In 2010, manufacturers in the U.S. claimed sales totaling more than $6,000/ day. Furthermore, in police testimony, one major manufacturer stated that if JWH-018 were banned, he would just switch and treat his dried plant products with another legal compound.

Recently, in fact, a new synthetic cannabinoid, AM-2201, began appearing in herbal incense products after the 2011 temporary scheduling of JWH-018. This compound is almost identical to JWH-018, except the terminal carbon of the alkyl chain has been changed to fluorine. Anecdotal reports from users posted on the Internet suggest AM-2201 is much more potent than JWH-018.

Pharmacokinetics

Hepatic CYP450 enzymes extensively metabolize the parent drugs of synthetic cannabinoids. For example, more than 20 metabolites of JWH-018 have been identified, including carboxylated, monohydroxylated, dihydroxylated, and trihydroxylated metabolites, that are excreted almost exclusively in urine as glucuronide conjugates (2–4).

Researchers have not detected the parent drugs in urine, and very limited data on detection time windows or expected concentrations of metabolites has been published. A study of one drug-naive individual showed the most abundant JWH-018 metabolite, JWH-018-N-pentanoic acid, was present in urine at approximately 0.1 ng/mL approximately 48 hours after a single use (5). Anecdotal evidence, however, suggests chronic users may produce positive urine for weeks after they stop using synthetic cannabinoids. In one study, researchers reported concentrations of JWH-018-N-pentanoic acid as high as 27,000 ng/mL in a urine specimen from an individual with an unknown smoking history (6).

The most comprehensive study of synthetic cannabinoids to date included 29 patients in Germany who presented to emergency departments after consuming the drugs (7). Among the patients, toxicity symptoms lasted 4–14 hours and serum concentrations of JWH-018 ranged from 0.38–13 ng/mL. Serum drug concentrations also varied depending on the specific synthetic cannabinoids the individual consumed. It is interesting to note that of the 29 patients, almost 40% had more than one synthetic cannabinoid in their serum. On the other hand, regular users of JWH-018 can have serum concentrations as high as 8 ng/mL without toxic symptoms, suggesting tolerance may develop.

In another study, researchers reported detecting JWH-018 in oral fluid specimens collected from two drug-naive individuals following a single smoking session (8). The concentration peaked 20 minutes after the individuals smoked the drug and remained detectable for 5–12 hours at ≤0.5 ng/mL.

Although pharmacokinetic data is beginning to accrue for scheduled analogues such as JWH-018, it remains unclear how this information will translate to modified analogues that manufacturers may produce in the future.

Methods for Detecting Synthetic Cannabinoids

Designing assays that detect synthetic cannabinoids is a rapidly moving target for laboratories. To avoid detection, illicit drug makers constantly change the structure of the synthetic cannabinoids used in the herbal incense market. In addition, because commercially available THC immunoassays do not cross-react with synthetic cannabinoids, laboratories usually develop their own mass spectrometry-based assays.

Recently, however, Randox introduced an ELISA test that uses polyclonal antibodies targeted toward different chemical moieties of the aminoalkylindole cannabinoids. Reported sensitivities for the assays are <1 ng/mL, and preliminary data from the manufacturer shows good cross reactivity with metabolites of 11 synthetic cannabinoids. Currently, there is no data on the assay's ability to detect metabolites of emerging synthetic cannabinoids; therefore, screening results generally need to be confirmed with mass spectrometric methods.

The best methods for detecting synthetic cannabinoids are liquid chromatography/ tandem mass spectrometry (LC-MS/MS) and gas chromatography/mass spectrometry (GC/MS). Protocols targeting JWH-018 and JWH-073 metabolites have been described in detail (3–6). Such targeted MS protocols are generally limited by the availability of reference standards, but vendors such as Cayman Chemical offer a wide variety of metabolite standards and deuterated internal standards.

For LC-MS/MS analysis, laboratories usually incubate urine specimens with glucuronidase and extract the sample by liquid-liquid or solid-phase extraction. A 10-minute chromatographic separation is necessary to separate isobaric cannabinoid metabolites and endogenous interferences. Testing saliva and serum is also possible using a modified extraction protocol followed by LC-MS/MS analysis that includes detecting parent synthetic cannabinoids.

The Challenges

As it stands today, laboratories must develop and validate their own methods for detecting synthetic cannabinoids. Furthermore, guidelines do not exist that clarify which metabolites should be measured or what cutoffs should be used, and there are no standardized quality control materials or proficiency tests.

This lack of guidance and standardization is keenly illustrated by the following example. In August 2011, the New York State (NYS) Department of Health identified 12 laboratories that test biological specimens for synthetic cannabinoids, primarily JWH-018 and JWH-073, for healthcare providers in the state (9). None of the laboratories tested for the same panel of metabolites, and the limits of quantitation varied >100-fold.

While much work still needs to be done to standardize methods for synthetic cannabinoids, cutoff values are a particularly good example of an area that would benefit from more data. One approach some laboratories have taken is to set the limit of detection as low as analytically possible. Considering the current lack of data, extremely low-level positives should be interpreted with caution. Does a urine concentration of 0.1 ng/mL indicate recent use or the slow release of cannabinoids from fat stores of a chronic user who is currently abstaining? Setting cutoffs too high is also detrimental since it can result in misclassifying too many positive specimens as negative. Similar issues exist for interpreting the lipid-soluble THC metabolite, 11-nor-9-carboxy-THC, but cutoffs for screening and confirmation are standardized and based on decades of data.

The bigger issue is keeping pace with the new synthetic cannabinoids that illicit drug makers produce. In 2010, researchers studying herbal products purchased from U.K.-based websites applied a high-resolution MS approach that identified previously unreported synthetic cannabinoids (10). In 2012, two independent groups in the U.S. used a similar approach to profile herbal mixtures they purchased from local stores and on the Internet. One group developed methods for more than 65 different designer drugs using available reference standards (11). The second group incorporated a "mass defect filter,"producing a method that does not require reference standards (12). Both groups identified previously undetected and unscheduled synthetic cannabinoids in recently purchased products, again illustrating the rapid evolution of this type of drug.

The Here and Now

Although synthetic cannabinoid testing will likely remain a moving target, developing accurate tests is an important need for laboratories. Having such tests will not only serve as a deterrent to drug makers and users, but it also will aid in diagnosing poisoned patients, monitoring compliance, and identifying patients at risk for drug abuse.

The question facing laboratories today, however, is how to detect these drugs. Laboratories that send out specimens from suspected users for testing should know which synthetic cannabinoids the reference laboratory tests for and what cutoff values are used. On the other hand, laboratories may want to consider developing in-house tests, which also requires keeping up with the constant influx of new synthetic cannabinoids.

Interpreting test results also remains challenging. Laboratorians and clinicians should keep in mind that only very limited pharmacokinetic data exist for just a few synthetic cannabinoids. Furthermore, time windows for detecting these drugs and their concentrations may vary depending on the frequency of drug use and particular flavor of synthetic cannabinoid consumed.

Until more studies are done, laboratorians would be well advised to pay close attention to the analytical methods used for detecting synthetic cannabinoids and to the interpretation of test results.

REFERENCES

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Bridgit O. Crews, PhD, is a clinical chemistry fellow at Washington University School of Medicine in St. Louis, Mo.
Email: [email protected]

Disclosure: The author has nothing to disclose.