Download Identification of Synthetic Cannabinoids in Herbal Incense Blends

Identification of Synthetic Cannabinoids in Herbal
Incense Blends
By Thomas J. Gluodenis Jr., Ph.D. Article Posted: June 15, 2011
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.
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
 CP-47,497
(C7)
(C8)
HU-210 is controlled under a previous DEA ruling. Over 20 uncontrolled forms
remain, and the number is growing.
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 SELECTRASIL (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.
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.
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 AM694.
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 peakto-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.
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.
References
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.
http://www.justice.gov/dea/pubs/pressrel/pr030111.html
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.
http://www.deadiversion.usdoj.gov/fed_regs/rules/2011/fr0301.htm
3. Agilent Technologies, Inc. Identification of Synthetic Cannabinoids in
Herbal Incense Blends by GC/MS, Application Compendium. P/N 59907967EN. 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; [email protected]; www.agilent.com.