Analysis of Synthetic Cannabinoids and Designer Drugs
By Thomas G. Brock, Ph.D.
Designer drugs are creating opportunities. Herbal blends advertised as "100% legal" are popular with students and young adults, although the ingredients aren't listed. That creates an opportunity for companies to develop ways to accurately detect synthetic cannabinoids (CBs) and designer drugs that might be mixed with flavorings and scents in plant material. There are opportunities for manufacturers of equipment to produce instruments that can detect parent compounds and metabolites in urine, saliva, serum, or hair. Also generating opportunities are those off-white powders that are found in foil packets, marketed as bath salts, plant food, or anything else that would not normally be for human consumption. These mystery mixtures might contain any of the usual controlled substances or their analogs, or they may have something quite different and unexpected. Most likely, they will contain a mixture of active compounds, as well as some filler or camouflaging compound. Several companies are developing devices to analyze these powders in the lab. Finally, an opportunity exists in the field, where bulk powders and liquids are being transported alongside legal goods. Point-and-shoot identification of designer drugs is needed there. Of course, these opportunities are generated by designer CBs, cathinones, and other compounds, currently being marketed to a curious public. In fact, the public is very accepting of pills, formulations, and herbal remedies that might alleviate any discomfort. Many have tried products to lose weight or increase their energy, knowing little about the ingredients. The phrase "100% legal" may suggest that the contents have been approved by authorities, which, in turn, indicates that the products are safe for consumption. Unfortunately, diverse stimulants, relaxants, entactogens, anxiolytics, and hallucinogens, which are commonly chemical isomers or analogs of known controlled substances, constitute the biological activity of many of these "100% legal" products currently available online, as well as at gas stations, head shops, and other commercial outlets. The magnitude of this problem is underscored by the number of users admitted to emergency rooms around the world.1,2
Synthetic Cannabinoids and Their Metabolites
It seemed like a good idea at the time: develop stable analogs of THC, the most potent ingredient in cannabis, that might be able to reduce pain or stimulate appetite without the psychoactive effects. Several laboratories accepted the challenge during the 1990s and 2000s, developing an array of structurally distinct compounds which avidly activate one or both of the CB receptors (see related article on page 4). Unfortunately, certain entrepreneurs had a different idea, to create synthetic marijuana by adding these synthetic CBs to dried plant material. Originally known as Spice or K2 and marketed as incense, these products contain varying mixes of synthetic CBs in uncharacterized concentrations. The use of these cannabimimetics translates, often, into hospital admissions due to cannabinoid toxicity and other adverse effects. Still, because the synthetic CBs are structurally distinct from THC, users who anticipate being tested for smoking weed have an added incentive: the synthetic CBs and their metabolites are not detected by marijuana tests.
Enter the Cayman . This assay detects urinary metabolites of many of the most popular synthetic CBs, including JWH 018, JWH 073, JWH 019, JWH 200, and AM2201. It has been validated with human urine samples and demonstrates a high degree of correlation with LC/MS analysis. This assay is designed as a rapid and inexpensive screening tool that generates a positive vs. negative answer (Figure 1). Samples testing positive in Cayman's assay should be confirmed by quantitative analysis, such as LC/MS.
Analysis at the Bench
The analysis of samples which might contain one or more forensic compounds can be performed using a variety of related techniques. Gas chromatography (GC) paired with mass spectroscopy (MS) has long been the gold standard for forensic analysis. In GC/MS, chemicals in a mixture are separated by GC, ionized, and then the mass to charge ratio (m/z) of each compound is determined by MS. The mass spectrum is then compared with a spectral library of known compounds for identification. While MS provides the m/z of a compound, tandem mass spectrometry (MS/MS) involves fragmenting the compound after initial MS and then determining the m/z of each piece, providing important information when parent compounds have identical mass. Different types of mass analyzers may be used, including quadrupole and time of flight (TOF), which differ in their sensitivity and selectivity. Tandem MS often combines sequential quadrupole devices (triple quadrupole, or QQQ) or quadrupole followed by TOF (QTOF). Other types of detectors may also follow GC (e.g., flame ionization detectors (FID)) or LC (e.g., photodiode array detectors (PDA, DAD)). Different equipment is suited to different goals in analysis. In some cases, analysis is targeted, seeking to specifically test whether a particular substance is present in a given sample. For this purpose, both the analytical hardware and the acquisition software should be optimized to avoid false positives. For this goal, Agilent's 6400 series Triple Quadrupole LC/MS systems with triggered MRM (tMRM) acquisition software would be an excellent match: this system produces quantitative data and a searchable library spectrum in a single injection in order to avoid false positive identification. Alternatively, the goal may be to identify a variety of compounds, some novel, in a complex mixture, as is often the case in designer drug preparations. In this case, the appropriate hardware must be combined with software that can scan an extensive library of compounds. Here, one might choose from Agilent's 6500 series Q-TOF LC/MS platforms which combine accurate mass analysis with the ability to retrospectively mine data for new compounds without reinjection. In addition, Agilent offers high quality accurate mass databases and libraries across all of its GC/MS and LC/MS instruments for thousands of compounds related to Forensic Toxicology. Cayman is actively synthesizing new and expected analytical reference standards, including synthetic CBs, cathinones, and others, to help develop these forensic libraries. Synthetic CBs and cathinones provide a unique challenge for MS analysis: many isomers have identical masses and cannot be distinguished by MS or MS/MS. One example would be flephedrone (4-fluoromethcathinone, 4-FMC) and 3-FMC, which are, respectively, para- and ortho-substituted isomers of a cathinone that may be found in bath salt-type pouders. The DiscovIR-GC™ from Spectra Analysis couples Fourier transform infrared spectroscopy (FTIR) with gas chromatography (Figure 2). The DiscovIR-GC™ provides a high resolution solid phase transmission spectrum for each component of a sample. Infrared spectroscopy can resolve ortho-, meta- and para-substituted isomers; even diastereomers can be resolved by infrared spectroscopy. FTIR can differentiate isomers based on spectral differences, so the DiscovIR-GC™ does not rely on retention time, a crucial capability when the mass spectra are identical and retention times are similar.
Please visit www.Spectra-Analysis.com for more information.
Not Outstanding in the Field
The curious can find designer drugs online, but where are they made? The answer is often some variation on ‘Clandestine labs in other countries, most likely China or India'. This means that the front line of defense is at the borders, where evaluation of imported powders and liquids for drugs must be performed rapidly and accurately, often in the field. One contemporary option is the portable Raman Spectrometer, a handheld device which offers point-and-shoot analysis.
Sir Chandrasekhara Venkata Raman was a Nobel Prize winning physicist from India. He discovered that, when light traverses a transparent material, some of the light that is deflected changes in wavelength (Raman scattering). In one current approach, laser light is directed at a sample and is scattered by specific molecules in the sample. A sensor then measures the intensity of light at each wavelength and converts it to a spectrum that fingerprints those molecules. Raman spectroscopy may be used in diverse applications, including profiling molecular components of cells and tissues (e.g., for cancer detection), studying static and changing chemical structure, and analyzing liquids for explosives. Raman spectroscopy is rapid and does not require processing or labeling of samples. Certain forms of Raman spectroscopy show low sensitivity to surface layers and can be used without opening packaging, including plastic bags, glass containers, and gel caps.
Portable or handheld Raman spectrometers are available in a variety of formats for forensic analysis in the field. Four devices were recently evaluated by the National Forensic Science Technology Center. It is worth noting that previous evaluations of portable GC-MS, near infrared (NIR) or FTIR devices revealed numerous drawbacks, most notably failure to identify compounds in samples accurately and reproducibly. The overall review of portable Raman spectrometers is somewhat favorable: testing is rapid and non-destructive, units are easy to operate, and very little sample preparation is required prior to analysis. However, accuracy remains a limitation, with only 50% accuracy being typical for mixtures of controlled substances (although the Thermo FirstDefender RM attained 70% accuracy). Reproducibility was less than 50% for all devices. Moreover, Raman spectrometry does not work well with trace amounts or with highly fluorescent or pigmented samples. In short, field testing remains an area for opportunity.
This brings us back to GC/MS. Companies have engineered instruments to be operated under field conditions, some with internal gas cylinders and vacuum pumps, capacities for rapid sample processing, and integrated software analysis. Torion Technologies has an extremely rugged and truly portable unit that combines GC with toroidal ion trap MS which offers both sample preparation (extraction) and sample injection in one device, its Solid Phase Micro Extraction (SPME) syringe. FLIR Systems offers several portable MS devices, including the Griffin 400 and 460 GC/MS models for mobile forensic investigations. These instruments also have SPME capacity and are MS/MS capable. Cayman Chemical is collaborating with FLIR Systems by providing analytical reference standards of known and anticipated designer drugs to produce a Mass Spectral Library that can be used with the Griffin GC/MS systems.
1. James,D., Adams,R.D., Spears,R., et al. Emerg. Med. J. 28(8), 686-689 (2011).
2. Schifano, F., Albanese, A., Fergus, S., et al. Psychoparmacol. (Berl). 214(3), 593-602 (2011).
Figure 1. The Cayman JWH Metabolite ELISA kit (top) selectively and sensitively detects several JWH metabolites, like JWH 018 N-pentanoic acid (bottom)
Figure 2. The DiscovIR-GC™ from Spectra Analysis differentiates isomers based on spectral differences
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