High time for synthetic cannabinoid analysis
19 Jun 2012 by Evoluted New Media
Herbal incense blends containing synthetic cannabinoids are being developed and introduced to the market at an alarming rate. Today’s forensic laboratories are challenged to confirm and quantify the controlled forms at trace levels in complex matrices with confidence. Here we discover that triple quadrupole GC/MS offers significant benefits over single quadrupole GC/MS for the analysis of synthetic cannabinoids in herbal incense blends
Over recent years, the use of synthetic cannabinomimetic compounds – also referred to as ‘legal highs’ or ‘herbal highs’ – has soared amongst teens and young adults. Originally developed for medical research purposes, such substances are now widely available in head shops and over the internet. In addition to their popularity in young-adult party scenes, herbal incense blends are also being used by much younger groups (i.e., school-aged), fuelled by the false sense of security of a ‘legal high’ and their ready availability.
Synthetic cannabinoids are usually formulated in botanical matrices and are marketed as herbal incense. All come with the label ‘not for human consumption’ and so they are exempt from health and safety regulations such as the US FDA (Food and Drug Administration). The lack of any control over the safety of these substances is a major concern, and the long-term health effects remain unknown. Known short-term complications include convulsions, anxiety attacks, elevated heart rate, increased blood pressure, vomiting, hallucinations, paranoia, and disorientation.
Furthermore, as there is no quality control process, large variations in potency exist across the mixtures. This means inadvertent overdoses are frequent.
Specific forms of synthetic cannabinoids have been banned in numerous countries including the US, but the constant evolution of new compounds makes the situation very difficult to control. By the time one compound is banned, new substances have been created and introduced to the market.
In 2011, the US DEA (Drug Enforcement Administration) placed five specific synthetic cannabinoids (JWH-018; JWH-073; JWH-200; CP-47,497 [C7]; and CP-47,497 [C8]) under control for at least a year while it and the US DHHS (Department of Health and Human Services) determined whether permanent control is warranted.1 In March 2012, they announced they propose to place all five into Schedule I of the Controlled Substances Act (CSA) (subject to a hearing).2 HU-210 is controlled under a previous DEA ruling. Over 20 uncontrolled forms remain and the number is growing.
Synthetic cannabinoids fall into three structural types. The first (Figure 1a) possesses a structural scaffold similar to that of tetradydrocannabinol. The second type (Figure 1b) is synthetic napthoylindole analogues, and the third type (Figure 1c) is phenylcyclohexyl moieties. A common motif inherent to most synthetic cannabinoids is a short aliphatic chain known to interact with the cannabinoid CB1 and CB2 receptors.
Although single quadrupole GC/MS is an effective approach for identifying and quantifying synthetic cannabinoids, it presents a number of analytical challenges. First of all, synthetic cannabinoids are present in a botanical matrix, which is surprisingly difficult to homogenise. 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 with a substantial number of peaks.
Furthermore, the blends often contain a mixture of synthetic cannabinoids which, due to their structural similarities and isomeric forms, co-elute producing overlapped mass spectra. Adding to the challenge, synthetic cannabinoids can be extremely potent and thus present at trace levels relative to the matrix. These difficulties result in complex data that are difficult to interpret without using post acquisition processing software (e.g., mass spectral deconvolution software).
Here we analyse a sample of herbal blends for the presence of synthetic cannabinoids to demonstrate the applicability of an alternative GC/MS/MS approach that offers enhanced selectivity and sensitivity, and that eliminates that need for mass spectral deconvolution.
Experimental
A total of 17 synthetic cannabinoids, as listed in Table 1, were selected to represent the structural diversity of synthetic cannabinoids present in popular herbal blends. The following herbal blends were analysed: EX 565, K2 Blondie, K2 Diamond, K3 XXX, K4 Purple Haze, Lunar Diamond, and Zombie.
The soft and light nature of the botanical materials used to carry synthetic cannabinoids makes it difficult to crush into a homogenous form for sampling. Therefore, we took ~500g of each sample and ground it between sandpaper (two 5 inch by 5 inch sheets; 100-grit) to obtain a fine powder.
As synthetic cannabinoids may have multiple functional groups, a generalised extraction process is necessary. We selected an acid/base combined extraction followed by centrifugation, as detailed in Figure 2.
Although not required in this analysis, some synthetic cannabinoids (e.g., HU-210) benefit from derivatisation with BSTFA (N, o-Bis, [trimethylsilyl] trifluoroacetamide) with 1% TMCS (trimethylchlorosilane) to cap the functional groups and to produce more intense ions for identification and quantification.
GC/MS/MS analysis
The GC/MS/MS analyses were performed on an Agilent 7000 Series Triple Quadrupole GC/MS system which couples the Agilent 7890A Gas Chromatograph with the Agilent 7000B Mass Spectrometer. The Gas Chromatograph was equipped with a HP-5MS UI column. Table 2 lists the run conditions. The mass spectrometer was operated in electron impact ionisation (EI) MS/MS mode using multiple reaction monitoring (MRM) for all analytes and reference standards. Table 3 lists the mass spectrometer operating conditions.
MRM transitions were developed empirically beginning with the collection of full-scan spectra from the reference standards, followed by product ion scanning to identify optimal precursor/product ion pairs for the analysis. Next, the collision cell energy was optimised to achieve the maximum ion intensity for each unique transition.
Results and discussion
The GC/MS/MS MRM method selectively isolates the target analyte from the matrix. The first quadrupole mass filter isolates a single precursor ion which is allowed to pass into the collision cell. In the collision cell, the precursor ion is fragmented by a collision gas and an applied electrical voltage; this process is known as CID (collision induced dissociation). CID fragments the precursor ion into specific and predictable product ions. The second quadrupole mass filter is set to pass only the specific product ions designated by the user. The most intense ion, the qualifier ion, is used for identification. The qualifier ion, when found in the correct abundance ratio with the quantifier ion, is used for confirmation. Even if an interfering ion is inadvertently allowed to pass through the first quadrupole into the collision cell, it is extremely unlikely that the interfering ion would yield the same product ions as the analyte precursor ion.
This MRM technique (made possible by GC/MS/MS) offers significantly improved selectivity and sensitivity over selected ion monitoring (SIM) with single quadrupole mass spectrometer for such trace level compounds in complex matrices.
All 17of the synthetic cannabinoids selected were found in the MRM total ion chromatogram (TIC) for 100ng/mL of the standard mixture, as shown in Figure 3. The high selectivity of GC/MS/MS negates chemical noise to give clean TIC.
Calibration curves were then constructed over the range 100-400ppb by spiking blank extracted matrix with known reference standards. Replicate injections were made at 100, 200, and 400ppb. The calibration curves for all analytes yielded an average correlation coefficient of linearity (r2) of 0.99 with standard deviations of 0.012.
The average RSD was 13%, 7%, and 6% at 100, 200, and 400ppb, respectively. Levels of quantification as determined by a signal to noise ratio ?10 were determined to range from 1-100ppb in the heavy botanical matrix.
Calibration curves for two synthetic compounds with very high activity, JWH-018 and JWH-073, showed the excellent linearity of the method (R2 = 0.996 for both).
Figure 4 shows the results for JWH-018 at 100ng/mL and represents typical chromatographic results. The shaded peak shows the quantifier ion transition (324 to 254 m/z). The trace shows the qualifier ion transition (341 to 167 m/z) is within the criteria (horizontal lines) established for the method.
JWH-073 and JWH-018 were detected in all of the blends analysed, with concentrations ranging from 50 to 150 ppb. Of interest, K2 Blondie contained JWH-073 and JWH-018 at concentrations extrapolated to be as great as 1,000-fold higher based on area counts alone. Each of the blends analysed contained at least two synthetic cannabinoids.
As demonstrated, triple quadrupole MS offers numerous benefits over single quadrupole MS for the analysis of synthetic cannabinoids in herbal incense. Triple quadrupole MS can:
- Negate matrix effects
- Improve signal-to-noise ratio
- Reduce false negatives and positives
- Lower detection limits
- Eliminate the need for additional post data-acquisition processing, such as mass spectral deconvolultion
Table 1: Analyte list Table 2: Gas Chromatograph Run Conditions (17 synthetic cannabinoids)
Table 3. Mass Spectrometer Operating Conditions
References
- Schedules of Controlled Substances: Temporary Placement of Five Synthetic Cannabinoids Into Schedule I. Office of Diversion Control, US Department of Justice, Drug Enforcement Administration, Federal Register Notices, Rules – 2011. http://www.deadiversion.usdoj.gov/fed_regs/rules/2011/fr0301.htm.
- Schedules of Controlled Substances: Placement of Five Synthetic Cannabinoids Into Schedule I. Office of Diversion Control, US Department of Justice, Drug Enforcement Administration, Federal Register Notices, Rules – 2012. http://www.deadiversion.usdoj.gov/fed_regs/rules/2012/fr0301_3.htm.
