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The manufacture, trafficking, and use of synthetic cannabinoids are of tremendous global concern. Synthetic cannabinoids are one of many types of new psychoactive substances (NPS), created from existing drugs, pharmaceuticals, or plant bases and modified to mimic the effects of traditional drugs such as marijuana or heroin. Because these drugs are newly developed and evolving quickly, little is known about their true effects or dosing limits. Most synthetic varieties tend to be more potent than their predecessors, turning inaccurate measurements into potentially lethal doses. In fact, synthetic cannabinoids are known to be stronger than cannabis due to active metabolites that stay in the brain longer, resulting in lasting and more serious effects, including severe withdrawal, psychotic episodes, convulsions, and life-threatening conditions for some individuals.
Due to the health risks and fatal consequences of synthetic cannabinoid use, law enforcement agencies have put legislation in place to halt the sale of cannabinoids and ban their use. However, with just small structural changes that push the molecules outside the scope of detection, new derivatives, isomers, and variants have been developed to keep the drugs in circulation. Attempts to regulate old and new compounds include the addition of numerous synthetic cannabinoids to the U.S. Drug Enforcement Administration’s Schedule I controlled substance list, containing drugs with the highest potential for abuse and psychotic effects. So many novel drug mixes are being introduced that it is essential for drug enforcement agencies to have the ability to detect both known and unknown molecules in trace amounts for forensic applications.
The challenge of detection
While known varieties of synthetic cannabinoids, such as K2 and Spice, or more recently Joker, Black Mamba, Kush, and Kronic, can be detected and resolved using basic techniques, new compounds and chemical mixtures remain undetectable. Synthetic cannabinoids have traditionally been tested using immunoassays and standard gas chromatography/mass spectrometry (GC/MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS). These methods are limited in their detection due to the use of known reference molecules to compare results and identify compounds. With a wide variety of novel variants constantly entering the drug market, these methods overlook any compounds not already identified.
Typical sample analysis can involve orthogonal techniques to extract and screen substances followed by a selective assay to confirm compound identity and potentially quantify concentrations, all of which takes time. Furthermore, many labs still use lengthy procedures on GC/MS or rely on orthogonal methods to confirm a known drug. If an unknown drug is suspected requiring additional testing or analysis through a contract testing lab, processing can take even longer. Time spent analyzing and confirming individual products delays potential legislation actions and allows the drugs more time on the market. Thus, there is a need for methods that allow quick and comprehensive identification of known and unknown compounds as they become available.
Cannabinoid analysis using high-resolution accurate-mass LC/MS
High-resolution accurate-mass (HRAM) LC/MS has become a method of choice for drug testing to routinely screen, confirm, and identify both known and unknown compounds. The technique provides a streamlined workflow for forensic screening and compound identification, solving the issue of testing for unknown drugs using untargeted detection. Detailed accurate-mass measurements and molecular isotopic pattern separation can differentiate between similar variants, while the sensitivity obtained using Thermo Scientific Orbitrap-based high-resolution technology allows for low limits of detection and quantitation.
Given the frequency of new compounds coming to market, the discovery and identification of novel analytes using untargeted screening has become an important aspect of synthetic cannabinoid testing. For example, work on the metabolism of cannabinoids JWH-018 and JWH-122 (Figure 1) has established multiple hydroxy metabolites occurring via substitution of a fluorine atom on the alkyl side chain or on the indole and naphthoyl moieties of the original compounds. Investigating full-scan HRAM MS data can uncover the presence of suspect compounds, and MS/MS data can confirm these as metabolites of known compounds. Identification and reporting of unknown metabolites in these types of cases would be extremely difficult without HRAM LC/MS.
Figure 1 – Structures of metabolites of JWH-018 and JWH-122Many variants, such as those described above, are chemically different with the same nominal mass, but have different accurate masses distinguishable by the selectivity of HRAM LC/MS. The regulated cannabinoid, JWH-122, for instance, can be modified to the unregulated compound MAM-2201 by adding a terminal fluorine onto its alkyl side-chain end. Mass accuracy of less than 1 ppm permits confident identification of differences in compounds like these. By generating richer, more complete information about each analyte, data can also be reviewed for missing metabolites and analyzed without additional runs.
Since synthetic cannabinoids can be much more potent than traditional drugs in much smaller amounts, sensitive detection methods such as mass spectrometry are needed to reveal very low levels of a drug and its analogs. High-resolution instrumentation can reduce signal contribution from interfering ions, allowing lower levels of quantitation. HRAM LC/MS has the high sensitivity to be able to detect these trace amounts, while being robust enough to identify multiple compounds in a sample, providing rapid, accurate, and conclusive results over an expanded scope of samples.
Once new compounds have been identified, reference library creation and maintenance is crucial to the development and validation of new screening procedures, and to reduce the time to update legislation. Historically, drug testing laboratories could create their own libraries or purchase one off-the-shelf. However, newer cloud-based reference libraries allow labs to share their discoveries with one another, helping to identify and regulate new drugs quickly. Newly discovered compounds can be uploaded, and libraries can be easily updated using these cloud-based applications, helping to drive timely law enforcement initiatives.
Determining geographic origin with isotope ratio mass spectrometry
An important aspect of synthetic cannabinoid identification is knowledge about its supply chain and production, yet this is a difficult task given that most illicit cannabinoids are likely manufactured in countless locations around the globe. Even though synthesis of common synthetic cannabinoids can easily be achieved with inexpensive equipment and attainable chemicals, details of production and sources of chemicals remain difficult to ascertain.
Using isotope ratio mass spectrometry (IRMS) to discover information about the geographical, chemical, and biological origins of substances helps determine the source of an organic substance. Instruments such as the Thermo Scientific Delta V isotope ratio mass spectrometer use mass spectrometry to measure the relative abundance of isotopes in a sample, detecting specific isotopic signatures of a product resulting from the geographical origin of the plant-derived material. The isotope method can also uncover isotopic variations resulting from unique production factors such as raw material used, synthesis methods, batch sizes, and manufacturer type. Learning about a drug’s history helps to establish links between precursors and synthetic drugs and enhance understanding of the synthetic cannabinoid market to gauge the impact of legal measures on drug supply.
When used to study drug samples, IRMS can determine the production source. Based on the obtained isotopic content, specific isotopes can help group drugs with common sources of manufacture or discriminate between different batches of drugs. These isotopic data allow labs to look into the origins of material to pinpoint a drug and its variants to potential distribution networks and ideally influence regulatory mandates.
Conclusion
Chemists are on the frontline in the war on drugs, identifying structures and their variants and developing screening procedures for tracking and regulation efforts. New compound identification and screening protocol improvements take time and resources, but advances in LC/MS technology and cloud-based systems provide a new level of efficiency for forensic labs. In addition, novel methods that allow traceability of drug samples and discovery of chemical origins can go a long way toward understanding the synthetic cannabinoid drug market and recommending adjustments to regulations.
Dominic Andrada is vertical marketing manager for forensics and sports antidoping, Thermo Fisher Scientific, 490 Lakeside Dr., Sunnyvale, CA 94085, U.S.A.; tel.: 408-203-8695; e-mail: [email protected]; www. thermofisher.com/forensics