An effective and easy-to-replicate approach to the identification of synthetic cannabinoids in herbal incense blends by Gas Chromatography/Mass Spectrometry (GC/MS).

The rapid growth in popularity of synthetic cannabinoid use among teens and young adults is of serious concern in the U.S. today. Inadvertent overdosing can lead to severe short-term complications requiring emergency room visits. Complications include convulsions, anxiety attacks, elevated heart rate, increased blood pressure, vomiting, hallucinations, paranoia, and disorientation. Long-term health effects are unknown.

Synthetic cannabinoids are THC-mimics that are heavily advertised and readily available to consumers in convenience stores, gas stations, “head shops,” and over the Internet at an affordable price. They are typically formulated in botanical matrices and marketed for sale as “herbal incense” (Figure 1). The lack of homogeneity and variation in potency of these mixtures can lead to harmful dosing. Because they are not marketed for human consumption, there is no oversight by the U.S. Food and Drug Administration (FDA). As such, there is no control over their manufacture, raw material quality, potency, and thus overall safety. The large and growing number of chemical forms of synthetic cannabinoids has impeded their control by law enforcement. As soon as legislation is passed banning the use of a specific form, a new one is synthesized and introduced. Because the formulations are new and rapidly evolving, they are not detected in a routine urine drug screen.

Due to the severe health risks and public threat associated with their use, the U.S. Drug Enforcement Administration (DEA) exercised its emergency authority to control five specific synthetic cannabinoids for at least one year while it and the U.S. Department of Health and Human Services (DHHS) determine whether permanent control is warranted.1,2 Numerous states in the U.S. have banned specific forms of these chemicals.

Herbal incense blends containing synthetic cannabinoids are a growing problem for toxicology laboratories.

About Synthetic Cannabinoids
Synthetic cannabinoids were originally synthesized for medical research. They fall into the three structural types—THC Classical, JWH-018 Napthoylindoes, and CP47,497(C8) Non-Classical—shown in Figure 2. The DEA now controls the forms:

  • JWH-018
  • JWH-073
  • JWH-200
  • CP-47,497 (C7)
  • CP-47,497 (C8)

HU-210 is controlled under a previous DEA ruling. Over 20 uncontrolled forms remain, and the number is growing.

Figure 2: Synthetic cannabinoids fall into three distinct structural patterns.

Analytical Challenges
The rapid proliferation of synthetic cannabinoid analogs and homologs has resulted in several analytical challenges for forensic laboratories tasked with their identification. At the outset, the botanical matrix is surprisingly difficult to homogenize. Subsequent extraction requires a general approach because synthetic cannabinoids contain a variety of functional groups. However, a general approach extracts a large amount of matrix substances which in turn produce a complex chromatogram containing a substantial number of peaks. Herbal incense blends often contain a mixture of synthetic cannabinoids. Coeluting compounds and similar or overlapped mass spectra are common due to their structural similarities and isomeric forms. And because they can be extremely potent, synthetic cannabinoids can be present at trace levels relative to the matrix.

Further, reference material for use in positive identifications is difficult to obtain or does not exist. Until now, libraries of reference mass spectra and retention times were not commercially available to facilitate identifications. As such, laboratories had to make identifications through laborious manual interpretation of very similar, or worse yet, complex overlapped mass spectra.

In sum, laboratories are challenged to find trace-level cannabinoids in complex chromatographic data and to identify the subtle differences between cannabinoid species that yield very similar retention times and mass spectra.

Analytical Solution
To help laboratories overcome these emerging challenges, Agilent Technologies, Inc. collaborated with the Criminalistics Division of NMS Labs, an ASCLD certified independent forensic lab, to develop and validate an analytical method, as well as a supporting compendium and searchable mass spectral library of over 35 synthetic cannabinoids. The resulting validated method described here provides an effective and easy-to-replicate approach to the identification of synthetic cannabinoids in herbal incense blends by GC/MS.

Sample Preparation
Homogenization: The botanical material used as the carrier for synthetic cannabinoids, such as Damiana (Latin Name Tumera diffusa), is soft and light. These properties make it difficult to crush into a homogenous form for representative sampling. Various homogenization devices were tested including a mortar and pestle, an herb grinder, and electric grinders and mills. Surprisingly, none of these devices produced acceptable results. Instead, the most effective method is to grind approximately 500 mg of sample between two 5"x5" sheets of 100-grit sandpaper until a finely divided powder is obtained.

Extraction: The multiple functional groups associated with synthetic cannabinoids necessitate a generalized extraction approach: an acid/base combined extraction or a simple methanol incubation to solubilize the cannabinoids followed by centrifugation. Either approach will extract substantial amounts of matrix, producing a chromatogram with multiple peaks.

In the acid/base approach, an aliquot of homogenized sample (50–100 mg) is acidified by adding 1 mL of de-ionized water, followed by three drops of 10% hydrochloric acid. Next, 1 mL of solvent (95% methylene chloride/ 5% isopropanol v/v) is added and the sample is mixed. The sample is then centrifuged and the bottom solvent layer is retained and set aside. Two drops of concentrated ammonium hydroxide and 1mL of the solvent (95% methylene chloride/5% isopropanol v/v) are added to the remaining aqueous mixture (top layer). The sample is mixed and centrifuged again. The bottom solvent layer is removed, combined with the first bottom solvent layer collected and then mixed briefly. In most cases, the combined extract is now ready for GC/MS analysis.

Derivatization: Some synthetic cannabinoids contain multiple, active, polar functional groups such as phenols, alcohols, indoles, and ketones, which can make them much less amenable to GC/MS analysis (Figure 3, top). To enhance the chromatographic performance and sensitivity of the method, derivatization with BSTFA[N,o-Bis (Trimethylsilyl) trifluoroacetamide] with 1% TMCS (trimethylchlorosilane) can be used to “shield” these functional groups (Figure 3, bottom). Derivatization can also produce more unique ions for identification.

To derivatize, the extract is evaporated to dryness using a gentle stream of nitrogen. The extracts must be completely dry and free of residual water or alcohol which would neutralize the derivatizing agent. Next 50 uL of SELECTRA-SIL (BSTFA with 1% TMS) is added and the mixture is capped and incubated at 70°C for 30 minutes. After cooling, the derivatized mixture is ready for GC/MS analysis.

Figure 3:With the polar function groups de-activated, improved chromatographic peaks are achieved.

GC/MS Method
The GC/MS method was developed on an Agilent 6890 GC equipped with a fast oven and autoinjector with tray. The GC is coupled to an Agilent 5973 MSD. Table 1 lists the GC/MS method parameters used.

Table 1: Gas Chromatograph and Mass Spectrometer Conditions

Figure 4: Typical GC/MS results for an underivatized herbal incense sample.

Figure 5

Figure 5: These results reveal compounds with similar spectra and retention times.
Figure 5: These results reveal compounds with similar spectra and retention times. The peak at 12.746 minutes could be either JWH-015 (left) or JWH-073 (right).

Results and Discussion
Figure 4 shows the typical GC/MS results obtained for an underivatized herbal incense sample. Closer inspection reveals that many of the compounds detected have similar spectra and retention times. Figure 5 shows another example where the peak at 12.746 minutes could be either JWH-015 or JWH-073. Either choice is not definitive due to the overlapped mass spectra. As shown in Figure 6, when the spectrum is searched against the synthetic cannabinoid library using the standard NIST search algorithm, the best match is neither compound, but AM-694.

The results show that the data generated in a typical GC/MS analysis of an herbal incense blend can be difficult to interpret due to the presence of multiple related target analytes, including isomeric forms, which yield common fragment ions and close retention times. Even when a library is available, traditional peak-to-peak library searching may not correctly identify the specific forms present in the overlapped chromatographic peaks obtained. To overcome this challenge, Deconvolution Reporting Software (DRS) can be used. The DRS software automatically reviews the entire GC/MS full-scan data file, extracts the individual components as clean mass spectra grouping by ions with the same abundance versus time profile, searches the clean spectra against the target mass spectral library, and then generates a report. Using this approach, the DRS software substantially reduces the number of false positives and false negatives in complex samples, thereby providing a substantial time savings.

Figure 7 shows how the DRS software separates mass spectra of coeluting compounds to provide the correct search results. The peak due to AM-694 (blue trace) is overlapped with JWH-073 (red trace) which has significantly lower response. Once the mass spectra are deconvoluted, a correct match is readily made. It would be difficult to get an equally clean spectrum using conventional background subtraction. Figure 8 shows the DRS report which identifies the synthetic cannabinoids found in the chromatogram shown in Figure 4.

Figure 6: Spectrum searched against cannabinoid library using standard NIST search.

Figure 8: The DRS report of the synthetic cannabinoids found in the chromatogram.

Sidebar: Synthetic Cannabinoid Compendium and Library

Figure 9: Target peaks separated by multi-stage MS make cannabinoid identification fast and unequivocal.

GC/MS/MS Further Simplifies Data Interpretation
While the DRS uses a mathematical algorithm to rapidly deconvolute complex mass spectral data into its component parts to aid in identification, GC/MS/MS offers an alternative approach. In a GC/MS/MS instrument, the target analyte is selectively isolated from the matrix thereby yielding unique multiple reaction monitoring (MRM) transitions to make identifications. In this method, the mass spectrometer is set to monitor two analyte-specific MRM transitions for each target cannabinoid, producing only two peaks. If the two target ions are found along with their corresponding ion ratios, the cannabinoid is unequivocally confirmed. Figure 9 illustrates the potential of this method as an alternative for the identification of synthetic cannabinoids.


  1. Chemicals Used in “Spice” and “K2” Type Products Now Under Federal Control and Regulation. News Release. Public Affairs, U.S. Drug Enforcement Administration. March 1, 2010.
  2. Notice of Intent to Temporarily Control Five Synthetic Cannabinoids. Office of Diversion Control, U.S. Department of Justice, Drug Enforcement Administration, Federal Register Notices, Rules – 2011.
  3. Agilent Technologies, Inc. Identification of Synthetic Cannabinoids in Herbal Incense Blends by GC/MS, Application Compendium. P/N 5990-7967EN. April 2011.

Thomas J. Gluodenis Jr., Ph.D., is the Forensic & Toxicology Marketing Manager for Agilent Technologies, Inc.; 2850 Centerville Road,Wilmington DE 19808 USA;;