Ling Huang, Ph.D.,
Assistant Professor of Chemistry
What Are “Spice” and “K2”?
“Is she breathing normal?” the operator asks.
“No, not so normal. More kind of shaking, convulsing, burning up,” the friend says as she hurries to Moore’s side, on the edge of panic.
“Demi, can you hear me?” she asks.
“Yes, she’s squeezing hands. … She can’t speak.”…
“Some form of … and then she smoked something. I didn’t really see. She’s been having some issues lately with some other stuff. So I don’t know what she’s been taking or not,” the friend says.
“She smoked something. It’s not marijuana. It’s similar to incense,” the friend says to the 911 operator. 
These were the responses Demi Moore’s friend gave to a 911 operator after she inhaled nitrous oxide and smoked herbal incense called “K2 Spice.” These herbal incenses originated in Germany, where some imaginative chemists read about Professor John W. Huffman’s (JWH) papers on the biological properties of the compounds his group had synthesized at Clemson University in the past 15 years. Most of those compounds bind to the same human cannabinoid receptors (CB1 and CB2) as Δ-9 tetrohydrocannabinol (THC), which is the same active ingredient in marijuana. Some of them mimic the effects of THC in mice; the others bind even more tightly than THC . These compounds were intended for the research and discovery of new painkillers or anesthetics. None have been tested on humans, and safe dosages are unknown. After reading literature, the “evil” drug producers in Europe started to synthesize the JWH-compounds in 2006, before dissolving them in organic solvents and spraying them on herbal base material. The end products were sold as “K2 Spice,” which saw instant popularity in Europe, Japan and later in the United States.
Since 2007, “Spice” herbal incense became a sensation among American youths. High school and college students can legally get the herbal incense cheaply and conveniently from local smoke shops and several popular online vendors such as eBay and amazon.com. One of my Hofstra students used “Spice” and saw a seizure in her friend who smoked one type of “Spice” product. A Google search of “herbal incense” or “herbal potpourri” generates numerous links to vendor websites. The spreading of the so-called “fake pot” went viral through YouTube videos and social networking sites such as Facebook. The video instructing others how to make these synthetic/ herbal blends can be found easily on YouTube. In one video, a man dissolved two types of synthetic cannabinoid powder in acetone purchased from Lowe’s prior to spraying them on damiana leaves, as seen in Figure 1C. He then used a spatula to stir the leaves in an attempt to evenly spread the mixture. After the solvent evaporated, he offered the dry leaves for sale. The packages are similar to the ones shown in Figures 1A and 1B. The online source of the powders was also provided in the video. The powders are packaged in plastic vials, as shown in Figure 1D, with simple paper labels. One vial (1g) often costs ~$25. One package of herb (1-3g) was sold for only several dollars.
High school and college
students can legally get the
herbal incense cheaply and
conveniently from local
smoke shops and several
popular online vendors such
as eBay and amazon.com.
What followed were lots of medical incidents like the Demi Moore’s story described above. The symptoms vary from seizure, bloodshot eyes, and anxiety attacks, to vomiting and psychotic episodes, resulting in increased emergency room visits . Multiple injuries and even death to teenagers and young adults have been reported in the news media. The hallucinogenic effects are vividly demonstrated and described by users in several YouTube videos. On March 1, 2011, the U.S. Drug Enforcement Administration (DEA) finally put five of the synthetic cannabinoids on the Schedule I controlled substance list. The five compounds include three JWHcompounds (JWH-018, JWH-073, and JWH-200), which are categorized as naphthoylindole cannabimimetic agents. The other two are close mimics or derivatives of THC, one synthesized at Pfizer (CP-497) and the other in one lab at Hebrew University (HU-210). There are, however, hundreds of JWH-series or similar analog compounds currently not banned. They are still being added to herbal products and sold legally everywhere. The actual identities and concentrations of cannabinoids are usually unknown to the consumers who buy the packages labeled with “not for human consumption” or “not for persons under 18 years old.”
Fortunately, law enforcement is working with Congress to create new laws to battle this drug problem. More than a handful of states have enacted a statewide ban on all “Spice” products. H.R. 1254, “Synthetic Drug Control Act,” passed the U.S. House of Representatives on December 8, 2011, attempting to curb the spreading of a wider range of synthetic cannabinoids and their isomers and analogs. Even if the law passes the Senate, there remain analytical challenges for law enforcement in quickly identifying and quantifying the amount of synthetic compounds in these herbal “Spices.” As discovered in our research, sometimes the powder distributors mislabeled their products, which consequently resulted in the wrong compounds being sprayed on the products. The compounds are often sprayed unevenly, with varying concentrations across one whole packet. The lack of high-quality analytical standard compounds hinders forensic chemists in their search for a perfect match between unknown and standard signals. We also found that multiple compounds (structures in Figure 2) are being sprayed on herbs. In one herb called “Sweet Leaf,” five synthetic cannabinoids were discovered. In observance of the DEA ban, many vendors are making newer generations of “Spice” products that do not contain controlled substances.
Isomers, or the compounds with the same molecular weights but different structures, are being made every day and sprayed on the herbs with various combinations, with fragrance added to entice consumers. Analogs, or compounds similar but bigger or smaller compared to the controlled substances, are still being made inexpensively overseas, shipped legally to the United States, and distributed by wholesalers. As demonstrated in multiple YouTube videos, there is virtually no quality control in the manufacturing of “Spice” products. In one video, one man is seen pouring the acetone solution containing JWH-compounds on a plastic bowl of leaves. The acetone solution partially dissolves the plastic bowl!
Good Chemists Strike Back!
Analytical chemists in a German pharmaceutical company first discovered the practice of mixing synthetic JWH-compounds with herbal base materials to make “Spice” . Liquid chromatography separation with mass spectrometry (LC-MS) detection was used to identify the synthetic compounds. The chromatographic methods separate compounds in a mixture based on the differential interaction between the analytes and the solid particles in the column. Less polar and more hydrophobic (oil-like) compounds will stick to the column longer than the more polar ones, and more hydrophilic (water-like) ones will elute from the column faster. In a mass spectrometer, a compound of interest, or analyte, is bombarded by electrons to form ion fragments that travel in a magnetic field with different speeds. The number of ion fragments that hit the detector will generate a histogram called mass spectrum, which can be used to identify compounds by using software to match them with standard spectra.
Besides MS, nuclear magnetic resonance (NMR) spectroscopy was used in Germany to identify the synthetic compounds in “Spice” after standard compounds were synthesized and cannabinoids were separated from herbal products and purified . NMR utilizes a pulsing radio frequency to disturb molecules in a strong magnetic field, producing a spectrum depicting hydrogen atoms in different local chemical environments. For example, the H-8″, H-4″ and H-5″ (the three hydrogens on the 10-member naphthyl ring on the left in Figure 2) of JWH-018 demonstrated three unique doublet peaks at 8.19, 7.95 and 7.89 ppm, respectively, on the NMR spectrum marked in a wide and red eclipse in Figure 3. Each standard cannabinoid possesses a unique set of molecular “fingerprints” in the form of an NMR spectrum or MS spectrum that can be used to make the identification.
Uchiyama et al, (in Japan) employed multiple spectroscopic and separation tools to detect and characterize synthetic cannabinoids in herbal blends . In order to obtain clean and clear mass spectra, NMR spectra, infrared and UV-visible absorbance spectra, the Japanese group took advantage of LC separation and thin layer chromatography (TLC), which are routinely used in forensic labs. The separation and purification steps from the dry herbs are cumbersome, laborintensive and time-consuming. In order to get a decent NMR signal, the herbs had to be crushed and ground before organic solvent extraction and repetitive separations on TLC to yield enough pure samples.
Using the same instrumental techniques, forensic scientists came up with new methods to detect the presence of synthetic cannabinoids and recently even the metabolites . In the past, many users preferred “Spice” because of its ability to emerge negative on the routine drug test, which did not include synthetic cannabinoid detection. So John Huffman’s comments on the metabolites are now, fortunately, incorrect. There have been efforts in developing a simple presumptive testing kit, like those used for other controlled substances. Besides the global banning trends on “Spice” cannabinoids, there are also initiatives to further study the addiction patterns of “Spice” and its adverse effects on the human body in the medical community, as more patients are seeking help with its physical and psychological effects.
“Weapons” for Rapid Detection
The Spice Accelerated Identification Team (SAIT) at the Chemistry Department of Hofstra University includes Adjunct Professor and retired NYPD detective Mercurio “Mark” Veltri, Pharm.D., several undergraduate students, and me. Dr. Veltri has devoted several years to monitoring this “legal pot” problem on the Internet and initiated this project with me. We are utilizing the state-of-the-art instrumentation in our department to tackle the analytical challenges faced by forensic communities in the detection and quantification of synthetic constituents in herbal incenses .
The actual identities and
cannabinoids are usually
unknown to the consumers
who buy the packages
labeled with “not for
human consumption” or
“not for persons under
18 years old.”
In order to improve the detection speed, we collaborated with Dr. Robert “Chip” Cody at JEOL Inc. Dr. Cody is the co-inventor of a technique called Direct Analysis in Real Time, or DART, which can ionize samples at room temperature for downstream mass spectrometry analysis. It is an extremely rapid screening method, as samples can be ionized with virtually no sample preparation steps. The results are obtained within seconds under atmospheric conditions with the ultra-fast speed from a time-of-flight mass spectrometer that separates ions with different mass-to-charge ratios based on their flight times. All of our samples (12 powder samples and 27 herbs) were analyzed on the instrument within a day. A thin capillary tube gently touched the powder, and the caught powder was instantly ionized at the nozzle (Figure 4A) where energized helium gas and water molecules were used to strip electrons away from the cannabinoids as well as to partially fragment them. The cannabinoid identity was determined in the mass spectrum (shown in Figure 4B), with the molecular ion peaks labeled.
The purchase and maintenance of our most expensive instrument, the 400 MHz JEOL NMR spectrometer, was generously supported by Hofstra’s Provost’s Office. This NMR was used by Hofstra student Michael A. Marino to track down the signals of synthetic cannabinoids, to make quick identifications to confirm the DART-MS results generated above. Mike was able to finish each scan within 2.5 hours and finish the analysis within 3 hours, including the run time. Mike performed NMR analysis on both powder samples and dried herbal extracts (Figure 3) that were prepared with a simple methanol rinse and a subsequent drying step. With the NMR, we could get absolute structural information to eliminate the misidentification sometimes associated with MS due to the same fragments from two isomers being produced in MS ionization and fragmentation. The NMR technique pairs well with MS.
The DART-MS and NMR would often catch one or two cannabinoid components with ease. Minor ingredients in “Spice,” however, are sometimes overlooked, due mainly to the arbitrary threshold setting in DART-MS analysis and the signal omission from Fourier Transform in NMR signal processing. High performance liquid chromatography (HPLC) serves as an extra safeguard to ensure the inclusion of all cannabinoids in the discovery, even for trace samples. We used a special column with particles coated with phenylhexyl, which greatly enhances the resolution in the separation of cannabinoids from herbal extracts, thanks to the selective interactions between the stationary phase and the aromatic groups (the rings in Figure 2) and the aliphatic chains (the zigzag lines in Figure 2). In 15 minutes, we were able to obtain the identities and quantities for all the cannabinoids in herbal incenses, as depicted in Figure 5.
With the combination of the three methods – DART-MS, NMR and HPLC – the Schedule I controlled substance JWH-018 was found in “Moon Spice” (Figures 1, 3 and 4), along with RCS-04, a still legal cannabinoid. The whole process took about 3 hours to complete, which will drastically enhance the speed for law enforcement and harm reduction efforts. The combinatory method can also be used to discover the identity of new designer drugs that are being made and sold legally to poorly educated consumers.
After testing 27 herbal products purchased from U.S. vendors, it was evident that AM-2201, JWH-122 and RCS-04 are the three most popular synthetic cannabinoids on the U.S. market today. The JWH-122 finding also echoes the discovered trends in Germany  and Japan . They all share close structural similarities (Figure 2) with the DEA-banned JWH-018 and JWH-073. AM-2201 has an extra fluorine at the distal end of the pentyl group, compared to JWH-018. JWH-122 has an extra methyl group on the naphthyl ring at the 4″ position. RCS-04 replaced the naphthyl ring in JWH-018 with a methoxylphenyl. Structurally analogous replacement compounds can be synthesized to circumvent the current ban, while still producing similar if not stronger effects. These replacements can then be easily and inexpensively obtained from overseas manufacturers who can promptly synthesize the analogs at a low cost and with little technical difficulty.
Where Is the Next High?
Besides the global
banning trends on “Spice”
cannabinoids, there are also
initiatives to further study
the addiction patterns of
“Spice” and its adverse
effects on the human body
in the medical community,
as more patients are seeking
help with its physical and
John W. Huffman’s group synthesized more than 470 JWH-series compounds, many of which bind to cannabinoid receptors CB1 and CB2 . To date, only two dozen or so have been exploited and used experimentally by drug users. There are also a handful or HU-compounds and AM-compounds that mimic the effects of THC, the active ingredient in cannabis. In the 1980s, Pfizer synthesized a class of CP-compounds that bind to CB1 and CB2. These compounds are also being abused and became controlled substances in several countries. The accelerated analytical techniques introduced in our work can be deployed to obtain quick identification and quantification, providing alternatives to other separation and spectroscopic methods such as TLC, LC-MS or GC-MS. Currently, SAIT is working on the careful analysis of the fragmentation MS spectra along with NMR confirmation with enhanced signals and better focus in order to provide concrete identification of newer cannabinoids in the future.
To reduce the harm, first the “Spice” users have to be educated on the devastating effects these products have on the human body. The herbal potpourri usually contains unknown amounts of unknown cannabinoids with varying concentrations, which increases the likelihood of overdose or misuse. Second, law enforcement agencies must act faster, perhaps using some of our methods, in the containment of the “Spice”-related crimes, and work to control or stop the distribution of powders. Third, forensic and medical communities need to develop better tools to assist in the identification and characterization of herbal incenses so that both users and law enforcement can better understand the seriousness of the situation and provide timely and effective remedies.