Abstract

Various “legal high” products were tested for synthetic cannabinoids and synthetic stimulants to qualitatively determine the active ingredient(s). Ultra-performance liquid chromatography with accurate mass time-of-flight mass spectrometry (UPLC–TOF) was used to monitor the non-biological specimens utilizing a customized panel of 65+ compounds comprised of synthetic cannabinoids, synthetic stimulants and other related drugs. Over the past year, the United States Drug Enforcement Agency has controlled five synthetic cannabinoid compounds (JWH-018, JWH-073, JWH-200, CP-47,497 and CP-47,497-C8) and three synthetic stimulant compounds (3,4-methylenedioxypyrovalerone, mephedrone and methylone) that were previously reported to be detected in these legal high products. Through our analyses of first and second generation products, it was shown that many of these banned substances are no longer used and have been replaced by other derivatives that are federally legal. Since enactment of the federal bans on synthetic cannabinoids and synthetic stimulants, 4.9% of the products analyzed at our facility contained at least one controlled substance. The remaining 95.1% of products contained only uncontrolled drugs. We demonstrate the UPLC–TOF methodology to be a powerful tool in the qualitative identification of these designer drugs, thus enabling a laboratory to keep current with the drugs that are being sold as these designer products.

Introduction

Herbal blend incense products and other legal high products, such as powders and pills, have been available for purchase on the Internet and in convenience stores, gas stations and smoke shops for at least the last three years in the United States. In late 2008 and early 2009, two reports were published regarding the detection of synthetic cannabinoid compounds in herbal blend products. A German laboratory detected CP-47,497-C8 (1) and a Japanese laboratory detected JWH-018 (2). Since that time, there have been multiple published reports in literature describing the detection of various cannabinoid and stimulant compounds in these types of products (2–14). Many of these products were marketed under names relating to “Spice” and “K2” as well as “bath salts” and “plant food” and were obtained by teenagers and adults as legal high drugs as an alternative to using the controlled or illegal amphetamines, cannabis and/or cocaine. None of the manufacturers of the products disclosed the presence of the drug or drugs detected and most were labeled with “not for human consumption” or a variant. The effects of the synthetic cannabinoids have been reported to include agitation, delusions, disorientation, hypertension, psychosis, sedation and tachycardia (15–17); the effects of the synthetic stimulants have been reported to include agitation, bizarre behavior, elevated body temperature, hallucinations, hypertension, increased alertness, restlessness, sweating, paranoia and tachycardia (18–23). On March 1, 2011, the United States Drug Enforcement Agency (DEA) moved to control five synthetic cannabinoid chemicals (JWH-018, JWH-073, JWH-200, CP-47,497 and CP-47,497-C8 homologue) and on September 7, 2011, the DEA also used its emergency scheduling powers to control three synthetic stimulants [3,4-methylenedioxypyrovalerone (MDPV), mephedrone and methylone]. Previous to the DEA controlling the substances, other countries, such as Germany, the Netherlands, Switzerland, Canada, France and Sweden, also moved to control various synthetic substances found in these types of products (24). At the present time, a vast majority of states in the United States of America have either passed legislation or have legislation pending controlling some of these substances.

Starting in December 2009, utilizing ultra-performance liquid chromatography (UPLC) and accurate mass time-of-flight mass spectrometry (TOF), we began monitoring these products and identifying which specific compounds were sprayed on them or used as the active ingredients. TOF mass spectrometry (MS) has been previously utilized in comprehensive drug screening procedures by Polettini et al. (25), Liotta et al. (26), Ojanperä et al. (27), ElSohly et al. (28) and Lee et al. (29). High resolution TOF mass spectrometry has also been utilized in the detection of these designer drugs by Hudson et al. (4). This type of methodology determines the mass-to-charge ratio of an ion by a simple time measurement. Ions are accelerated by an electrical field of a known strength through a vacuum region called a flight tube and when the ions reach a detector (at a known distance), a time of flight can be calculated. Theoretically, all ions should have the same kinetic energy, thus the velocity of the ion depends on its mass-to-charge ratio. Heavier ions travel slower and take a longer time to reach the detector, while lighter ions travel faster and take a shorter time to reach the detector.

Two time periods existed during this study—pre-federal ban and post-federal ban. For synthetic cannabinoids, the pre-federal ban time range was December 1, 2009 to March 1, 2010 and the post-federal ban time range was March 2, 2010 to April 1, 2012. For synthetic stimulants, the pre-federal ban time range was December 1, 2009 to September 7, 2011 and the post-federal ban time range is September 8, 2011 to April 1, 2012. We refer to the pre-ban products as “first generation” and the post-ban products as “second generation.” It is of significance that during the post-federal ban time range, specific legislation was passed in the state of Indiana and effective on July 1, 2011, which banned 25 compounds—19 synthetic cannabinoids and six synthetic stimulants. Derivative or analog language was not included in the law. The aim of this investigation was to analyze various products for synthetic cannabinoids and synthetic stimulants to determine the active ingredient(s). Our methods and findings are described in this paper.

Experimental

Chemicals, reagents and standards

4-Ethylmethcathinone (4-EMC), 4-methyl-a-pyrrolidinopropiophenone (MPPP), AM-251, AM-694, AM-1241, AM-2201, AM-2233, CP-47,497, CP-47,497-C8, HU-210, JWH-007, JWH-011, JWH-015, JWH-016, JWH-018, JWH-019, JWH-020, JWH-022, JWH-030, JWH-073, JWH-081, JWH-098, JWH-122, JWH-182, JWH-200, JWH-201, JWH-203, JWH-210, JWH-250, JWH-251, JWH-398, pentedrone, pentylone, RCS-4 and RCS-8 reference standards were obtained from Cayman Chemical Company (Ann Arbor, MI). 1-(3-Chlorophenyl)piperazine (mCPP), 3-trifluoromethylphenylpiperazine (TFMPP), MDPV, amphetamine, buphedrone, butylone, caffeine, cocaine, delta-9-tetrahydrocannabinol, ephedrine, ethylone, flephedrone, lidocaine, LSD, MDA, MDAI, MDEA, MDMA, mephedrone, methamphetamine, methcathinone, methylone, methedrone, phenylpropanolamine (PPA) and pseudoephedrine were obtained from Cerilliant (Round Rock, TX). 6-APB, 7-hydroxymitragynine, Alpha-PVP and mitragynine were obtained from Toronto Research Chemicals (Toronto, Ontario, Canada). 4-Fluoroamphetamine and phenazepam were obtained from Lipomed (Cambridge, MA). Acetonitrile (Optima grade) and methanol (Optima grade) were obtained from Fisher Scientific (Pittsburgh, PA). Water used for mobile phase and wash solvents was obtained from a Barnstead Nanopure Diamond Analytical Laboratory Water System (18.2 MΩ × cm). Concentrated formic acid (98%) was purchased from Sigma–Aldrich (St. Louis, MO). The gas used with the UPLC–MS system was high purity nitrogen. Specimen vials and caps were purchased from Micro-Liter Analytical Supplies (Suwanee, GA). All reference standards were diluted to a concentration of 10 µg/mL with methanol. The control specimens were prepared by diluting known amounts of reference standard with methanol. Mobile phase A [0.05% formic acid in deionized (DI) water] was prepared by adding 1 mL of concentrated formic acid to 2 L of DI water. Weak needle wash was prepared by adding 100 mL of acetonitrile and 10 mL of concentrated formic acid to 1.9 L of DI water. Strong needle wash was prepared by adding 500 mL of acetonitrile and 5 mL of concentrated formic acid to 500 mL of DI water.

Specimen collection

During the pre-federal ban time range, legal high products were acquired from local convenience stores and smoke shops or purchased over the Internet. Products were acquired during the post-federal ban time ranges from the same purchasing methods or from Indiana law enforcement agencies that had confiscated the materials from establishments selling the products. Specimens were either analyzed immediately after receipt by the laboratory or stored in ambient (25°C), dark, dry conditions until analysis.

Organic extraction procedure

A representative 50-mg portion of the legal high product was analyzed. The nonbiological material and 5 mL of acetonitrile–methanol (50:50) were added to a large glass tube. The mixture was sonicated in a water bath for 10 min and vortex-mixed for 2 min. After vortex mixing, specimens were diluted 1:50 with acetonitrile–DI water (20:80). The solution was mixed for 5 s and a portion of the solution was transferred to a plastic autosampler vial.

Instrumental analysis

Chromatographic separation was completed on a Waters (Milford, MA) Acquity UltraPerformance Liquid Chromatograph. The system consisted of a sample manager, solvent manager and single column manager. UPLC separation was performed by injecting 5 µL (partial loop) of specimen on a Waters Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.8 µm particle size) at a temperature of 60°C. Chromatographic separation was achieved by gradient elution at a flow rate of 0.5 mL/min. Mobile phases used during analysis were 0.05% formic acid in DI water (A) and acetonitrile (B). A summary of the inlet methods is shown in Table I. Two distinct gradients were needed due to the varied nature of the panel of drugs. Total run time for one specimen using the synthetic cannabinoid method was 12 min. Total run time for one specimen using the synthetic stimulant method was six min. After each injection, the system was washed with 1 mL of strong needle wash and 1 mL of weak needle wash.

Table I

Elution Gradients

Synthetic cannabinoids
 
Total time (min) % A % B 
Initial 58 42 
0.30 58 42 
11.0 97 
11.5 100 
11.6 58 42 
Synthetic stimulants 
 
Total time (min) % A % B 
 
Initial 96 
0.50 96 
4.50 44 56 
5.00 100 
5.50 100 
5.51 96 
Synthetic cannabinoids
 
Total time (min) % A % B 
Initial 58 42 
0.30 58 42 
11.0 97 
11.5 100 
11.6 58 42 
Synthetic stimulants 
 
Total time (min) % A % B 
 
Initial 96 
0.50 96 
4.50 44 56 
5.00 100 
5.50 100 
5.51 96 

Electrospray ionization mass spectrometry (ESI-MS) was performed on a Waters LCT Premier XE (TOF-MS) in W-optics positive ionization scan mode over a mass range 30–600 amu. A low voltage scan (cone voltage, 25 V; aperture voltage, 8 V) was used for precursor mass identification and a high voltage scan (cone voltage, 60 V; aperture voltage, 70 V) was used for in-source collision induced dissociation (CID) of the precursor mass producing fragments. Real time accurate mass data were acquired by reference to an independently sampled reference material or a lockspray (Leucine Enkephalin, [M + H]+ = 556.2771 amu). Capillary voltage was 3,000 V. Cone voltage was 25 V. Source temperature was 140°C and desolvation temperature was 500°C. The desolvation gas (nitrogen) and cone gas (nitrogen) were set to 900 and 25 L/min, respectively. An accurate precursor mass ion and at least one nominal mass fragment ion were monitored for the analytes of interest. The mass spectrometer was calibrated in positive ionization mode over a mass range 30–800 amu using sodium formate. Resolution was tuned to 12,000 FWHM.

The software used was MassLynx, version 4.1, in combination with the TargetLynx application manager. TargetLynx is designed for quantitative analysis, but a method was developed for qualitative analysis, which contained each analyte, its accurate mass, the fragment nominal masses and retention time, along with integration and peak detection parameters and tolerances.

Discussion

The initial list of analytes that was used in processing the acquired data from the LC–TOF consisted of 15 compounds and included all eventually DEA-controlled substances. As reference standards became available through certified vendors, it was recognized that it would be prudent to acquire what could be considered the more standard or central compounds, i.e., compounds that were being reported in literature at the time. Data were processed utilizing an ion mass window filter of ±0.025 mDa for the theoretical precursor accurate monoisotopic mass, ±0.5 mDa for the theoretical product nominal monoisotopic fragments and a retention time window filter of ±0.1 min. These filters were found to be sufficient for identification purposes. We did not take full advantage of the identification power of TOF-MS because we did not utilize the isotopic pattern and spacing abilities of this technology. Quality control specimens containing the analytes of interest were injected at various times throughout a batch of specimens to help determine whether there was retention time or mass accuracy drift throughout the run time of a batch of specimens. As new reference standards were acquired for the compounds, the full scan data of the TOF methodology was shown to be a powerful tool, because we were able to revisit raw data for previously-run specimens and reprocess the data without having to re-extract and re-inject or re-run specimens. Older data were able to be mined for the newer analytes of interest. A summary of the monitored analyte lists for both the synthetic cannabinoid method and the synthetic stimulant method is shown in Tables II–III. Every authentic specimen was analyzed on both synthetic cannabinoid and synthetic stimulant methods.

Table II

Analytes Monitored with the Synthetic Cannabinoid Method

Number Analyte Molecular formula Accurate mass (m/zNominal fragments (m/zRetention time (min) 
7-Hydroxymitragynine C23H30N2O5 415.2233 190, 268, 381, 397 0.4 
AM-251 C22H21Cl2IN4O 555.0215 454, 472 5.7 
AM-694 C20H19FINO 436.0574 230, 243 3.7 
AM-1241 C22H22IN3O3 504.0784 98, 275 0.7 
AM-2201 C24H22FNO 360.1764 155, 168, 232 4.3 
AM-2233 C22H23IN2O 459.0933 98, 112, 230, 362 0.6 
CP-47,497 C21H34O2 319.2637 121, 133, 148, 233 5.3 
CP-47,497-C8 C22H36O2 333.2794 121, 133, 148, 247 6.1 
Delta-9-THC C21H30O2 315.2324 123, 164, 193 6.8 
10 HU-210 C25H38O3 387.2899 201, 216, 301 6.4 
11 JWH-007 C25H25NO 356.2014 155, 168, 228 6.0 
12 JWH-011 C27H29NO 384.2327 155, 168, 286 7.0 
13 JWH-015 C23H21NO 328.1701 155, 168, 200 4.5 
14 JWH-016 C24H23NO 342.1858 155, 168, 214 5.3 
15 JWH-018 C24H23NO 342.1858 155, 168, 214 5.7 
16 JWH-019 C25H25NO 356.2014 155, 168, 228 6.4 
17 JWH-020 C26H27NO 370.2171 155, 168, 242 7.1 
18 JWH-022 C24H21NO 340.1701 155, 168, 212 5.2 
19 JWH-030 C20H21NO 292.1701 155, 168 4.4 
20 JWH-073 C23H21NO 328.1701 155, 168, 200 5.0 
21 JWH-081 C25H25NO2 372.1964 185, 198, 214 6.0 
22 JWH-098 C26H27NO2 386.2120 185, 198, 228 6.2 
23 JWH-122 C25H25NO 356.2014 169, 182, 214 6.2 
24 JWH-182 C27H29NO 384.2327 197, 214 7.5 
25 JWH-200 C25H24N2O2 385.1916 114, 155, 168 0.9 
26 JWH-201 C22H25NO2 336.1964 121, 135, 214 4.5 
27 JWH-203 C21H22ClNO 340.1468 125, 166, 214 5.4 
28 JWH-210 C26H27NO 370.2171 183, 214 6.8 
29 JWH-250 C22H25NO2 336.1964 121, 132, 214, 246 4.9 
30 JWH-251 C22H25NO 320.2014 105, 144, 214 5.3 
31 JWH-302 C22H25NO2 336.1964 121, 144, 162, 214 4.6 
32 JWH-398 C24H22ClNO 376.1468 189, 202 6.8 
33 Lidocaine C14H22N2O 235.1810 86 0.5 
34 Mitragynine C23H30N2O4 399.2284 159, 174 0.6 
35 MPPP C14H19NO 218.1545 91, 98, 132 0.5 
36 Phenazepam C15H10N2OBrCl 348.9743 179, 206, 242 1.4 
37 RCS-4 C21H23NO2 322.1807 135, 214 4.5 
38 RCS-8 C25H29NO2 376.2277 121, 132, 144, 254 6.4 
Number Analyte Molecular formula Accurate mass (m/zNominal fragments (m/zRetention time (min) 
7-Hydroxymitragynine C23H30N2O5 415.2233 190, 268, 381, 397 0.4 
AM-251 C22H21Cl2IN4O 555.0215 454, 472 5.7 
AM-694 C20H19FINO 436.0574 230, 243 3.7 
AM-1241 C22H22IN3O3 504.0784 98, 275 0.7 
AM-2201 C24H22FNO 360.1764 155, 168, 232 4.3 
AM-2233 C22H23IN2O 459.0933 98, 112, 230, 362 0.6 
CP-47,497 C21H34O2 319.2637 121, 133, 148, 233 5.3 
CP-47,497-C8 C22H36O2 333.2794 121, 133, 148, 247 6.1 
Delta-9-THC C21H30O2 315.2324 123, 164, 193 6.8 
10 HU-210 C25H38O3 387.2899 201, 216, 301 6.4 
11 JWH-007 C25H25NO 356.2014 155, 168, 228 6.0 
12 JWH-011 C27H29NO 384.2327 155, 168, 286 7.0 
13 JWH-015 C23H21NO 328.1701 155, 168, 200 4.5 
14 JWH-016 C24H23NO 342.1858 155, 168, 214 5.3 
15 JWH-018 C24H23NO 342.1858 155, 168, 214 5.7 
16 JWH-019 C25H25NO 356.2014 155, 168, 228 6.4 
17 JWH-020 C26H27NO 370.2171 155, 168, 242 7.1 
18 JWH-022 C24H21NO 340.1701 155, 168, 212 5.2 
19 JWH-030 C20H21NO 292.1701 155, 168 4.4 
20 JWH-073 C23H21NO 328.1701 155, 168, 200 5.0 
21 JWH-081 C25H25NO2 372.1964 185, 198, 214 6.0 
22 JWH-098 C26H27NO2 386.2120 185, 198, 228 6.2 
23 JWH-122 C25H25NO 356.2014 169, 182, 214 6.2 
24 JWH-182 C27H29NO 384.2327 197, 214 7.5 
25 JWH-200 C25H24N2O2 385.1916 114, 155, 168 0.9 
26 JWH-201 C22H25NO2 336.1964 121, 135, 214 4.5 
27 JWH-203 C21H22ClNO 340.1468 125, 166, 214 5.4 
28 JWH-210 C26H27NO 370.2171 183, 214 6.8 
29 JWH-250 C22H25NO2 336.1964 121, 132, 214, 246 4.9 
30 JWH-251 C22H25NO 320.2014 105, 144, 214 5.3 
31 JWH-302 C22H25NO2 336.1964 121, 144, 162, 214 4.6 
32 JWH-398 C24H22ClNO 376.1468 189, 202 6.8 
33 Lidocaine C14H22N2O 235.1810 86 0.5 
34 Mitragynine C23H30N2O4 399.2284 159, 174 0.6 
35 MPPP C14H19NO 218.1545 91, 98, 132 0.5 
36 Phenazepam C15H10N2OBrCl 348.9743 179, 206, 242 1.4 
37 RCS-4 C21H23NO2 322.1807 135, 214 4.5 
38 RCS-8 C25H29NO2 376.2277 121, 132, 144, 254 6.4 
Table III

Analytes Monitored with the Synthetic Stimulant Method

Number Analyte Molecular formula Accurate mass (m/zNominal fragments (m/zRetention time (min) 
4-Ethylmethcathinone C12H17NO 192.1388 144 2.6 
4-Fluoroamphetamine C9H12FN 154.1032 109, 124 1.9 
6-APB C11H13NO 176.1075 118, 131 2.2 
Alpha-PVP C15H21NO 232.1701 105, 118, 126 2.6 
Amphetamine C9H13N 136.1126 91, 106 1.7 
Buphedrone C11H15NO 178.1232 130 1.8 
Butylone C12H15NO3 222.1130 131, 174 2.1 
Caffeine C8H10N4O2 195.0882 123, 154 1.8 
Cocaine C17H21NO4 304.1549 82, 182 2.4 
10 Ephedrine C10H15NO 166.1232 91, 115, 132 1.5 
11 Ethylone C12H15NO3 222.1130 146, 174 1.9 
12 Flephedrone C10H12FNO 182.0981 148 1.8 
13 Lidocaine C14H22N2O 235.1810 86, 196 2.1 
14 LSD C20H25N3O 324.2076 180, 208 2.5 
15 mCPP C10H13ClN2 197.0846 118, 154 2.2 
16 MDA C10H13NO2 180.1025 118 1.7 
17 MDAI C10H11NO2 178.0868 103, 118, 144 1.5 
18 MDEA C12H17NO2 208.1338 118, 135 2.0 
19 MDMA C11H15NO2 194.1181 118, 135 1.8 
20 MDPV C16H21NO3 276.1600 126, 135, 149 2.7 
21 Mephedrone C11H15NO 178.1232 144 2.2 
22 Methamphetamine C10H15N 150.1283 91, 132 1.8 
23 Methcathinone C10H13NO 164.1075 118, 130 1.6 
24 Methedrone C11H15NO2 194.1181 118, 146 2.0 
25 Methylone C11H13NO3 208.0974 132, 160 1.7 
26 Pentedrone C12H17NO 192.1388 130, 144 2.4 
27 Pentylone C13H17NO3 236.1287 131, 188 2.5 
28 PPA C9H13NO 152.1075 91, 115 1.3 
29 Pseudoephedrine C10H15NO 166.1232 91, 115, 132 1.6 
30 TFMPP C11H15F3N2 231.1109 145, 188 2.6 
Number Analyte Molecular formula Accurate mass (m/zNominal fragments (m/zRetention time (min) 
4-Ethylmethcathinone C12H17NO 192.1388 144 2.6 
4-Fluoroamphetamine C9H12FN 154.1032 109, 124 1.9 
6-APB C11H13NO 176.1075 118, 131 2.2 
Alpha-PVP C15H21NO 232.1701 105, 118, 126 2.6 
Amphetamine C9H13N 136.1126 91, 106 1.7 
Buphedrone C11H15NO 178.1232 130 1.8 
Butylone C12H15NO3 222.1130 131, 174 2.1 
Caffeine C8H10N4O2 195.0882 123, 154 1.8 
Cocaine C17H21NO4 304.1549 82, 182 2.4 
10 Ephedrine C10H15NO 166.1232 91, 115, 132 1.5 
11 Ethylone C12H15NO3 222.1130 146, 174 1.9 
12 Flephedrone C10H12FNO 182.0981 148 1.8 
13 Lidocaine C14H22N2O 235.1810 86, 196 2.1 
14 LSD C20H25N3O 324.2076 180, 208 2.5 
15 mCPP C10H13ClN2 197.0846 118, 154 2.2 
16 MDA C10H13NO2 180.1025 118 1.7 
17 MDAI C10H11NO2 178.0868 103, 118, 144 1.5 
18 MDEA C12H17NO2 208.1338 118, 135 2.0 
19 MDMA C11H15NO2 194.1181 118, 135 1.8 
20 MDPV C16H21NO3 276.1600 126, 135, 149 2.7 
21 Mephedrone C11H15NO 178.1232 144 2.2 
22 Methamphetamine C10H15N 150.1283 91, 132 1.8 
23 Methcathinone C10H13NO 164.1075 118, 130 1.6 
24 Methedrone C11H15NO2 194.1181 118, 146 2.0 
25 Methylone C11H13NO3 208.0974 132, 160 1.7 
26 Pentedrone C12H17NO 192.1388 130, 144 2.4 
27 Pentylone C13H17NO3 236.1287 131, 188 2.5 
28 PPA C9H13NO 152.1075 91, 115 1.3 
29 Pseudoephedrine C10H15NO 166.1232 91, 115, 132 1.6 
30 TFMPP C11H15F3N2 231.1109 145, 188 2.6 
Table IV

Compounds Detected in Acquired Legal High Products

Product Name Time period acquired Form Compound(s) detected 
Black Mamba Pre-federal ban Dried plant material JWH-018 
Bliss Pre-federal ban Powder MDPV 
Eight Ballz Pre-federal ban Powder MDPV 
Ivory Wave Pre-federal ban Powder MDPV 
K2 Blonde Pre-federal ban Dried plant material JWH-018, JWH-073 
K2 Blue Pre-federal ban Dried plant material JWH-018 
K2 Citron Pre-federal ban Dried plant material JWH-018, JWH-073 
K2 Latte Pre-federal ban Dried plant material JWH-018 
K2 Mint Pre-federal ban Dried plant material JWH-018 
K2 Peach Pre-federal ban Dried plant material JWH-018 
K2 Pink Pre-federal ban Dried plant material JWH-018, JWH-073 
K2 Silver Pre-federal ban Dried plant material JWH-250 
K2 Standard Pre-federal ban Dried plant material JWH-018, JWH-073 
K2 Summit Pre-federal ban Dried plant material JWH-018, JWH-073 
K2 Summit Pre-federal ban Dried plant material JWH-018, JWH-073 
Tranquility Pre-federal ban Powder MDPV 
Tribal Warrior Pre-federal ban Dried plant material JWH-018, JWH-073 
7H Hydro Post-federal ban Dried plant material AM-2201 
7H Kush Post-federal ban Dried plant material AM-2201, JWH-122 
B2 Da Bomb Blueberry Post-federal ban Dried plant material AM-2201, JWH-073 
Baked Goods Post-federal ban Dried plant material AM-2201 
Bayou Blaster Post-federal ban Dried plant material AM-2201, AM-2233, JWH-210, MPPP 
Black Mamba Mango Post-federal ban Dried plant material AM-2201 
Black Rooster – Classic Post-federal ban Dried plant material AM-2201 
Cloud 9 Deversion 2.0 Post-federal ban Dried plant material AM-2201, JWH-019 
Cloud 9 Deversion 2.0 Post-federal ban Dried plant material JWH-250 
Cloud 9 Deversion 2.0 Post-federal ban Dried plant material JWH-122, JWH-250 
Cloud 9 Deversion 2.0 Post-federal ban Dried plant material AM-2201 
Cloud 9 Deversion 2.0 Post-federal ban Dried plant material AM-2201 
Cloud 9 Mad Hatter Post-federal ban Dried plant material AM-2201, JWH-019 
Cloud 9 Mad Hatter Post-federal ban Dried plant material AM-2201 
Cloud 9 Mad Hatter Post-federal ban Dried plant material AM-2201 
Cloud 9 Mad Hatter Post-federal ban Dried plant material AM-2201 
Cloud 9 Mad Hatter Post-federal ban Dried plant material AM-2201, JWH-019 
Cloud 9 Mad Hatter Post-federal ban Dried plant material AM-2201, JWH-019 
Cloud 9 Mad Hatter Post-federal ban Dried plant material AM-2201, JWH-019 
Colorado Post-federal ban Dried plant material AM-2201 
Crystal Clean Hookah Cleaner Post-federal ban Powder Alpha-PVP 
Dancing Monkey Groovy Grape Post-federal ban Dried plant material AM-2201, JWH-018, JWH-073 
Dank Potpourri Post-federal ban Dried plant material AM-2201, JWH-210 
Defiant Blue Post-federal ban Dried plant material AM-2201, JWH-073, JWH-122 
Demon Post-federal ban Dried plant material AM-2201, AM-2233, JWH-210, MPPP 
Demon Post-federal ban Dried plant material AM-2201, AM-2233, JWH-019, JWH-210, JWH-250 
Doves Ultra Post-federal ban Pills 6-APB 
Doves Ultra Post-federal ban Pills 6-APB 
Funky Monkey – Black Label Post-federal ban Dried plant material AM-2201, JWH-210, JWH-250, lidocaine, RCS-8 
HG2 Fire Post-federal ban Dried plant material JWH-122, JWH-250 
Jucci Intense Incense Post-federal ban Dried plant material AM-2201 
K2 XXX Chronic Post-federal ban Dried plant material JWH-122, JWH-203 
K4 Gold Post-federal ban Dried plant material JWH-250 
Karma Blueberry Post-federal ban Dried plant material AM-2201 
Karma Bubblegum Post-federal ban Dried plant material AM-2201, JWH-122, JWH-203 
Karma Mango Post-federal ban Dried plant material AM-2201, JWH-122, JWH-203 
Karma Original Post-federal ban Dried plant material AM-2201 
Karma Watermelon Post-federal ban Dried plant material AM-2201 
Kick-Ass Incense Post-federal ban Dried plant material AM-2201, MPPP 
Kottonmouf King Post-federal ban Dried plant material AM-2201, JWH-250 
Kush Pink Post-federal ban Dried plant material AM-2201, JWH-210 
M:20 Madness Potpourri Post-federal ban Dried plant material AM-2201 
Matrix - Green Post-federal ban Dried plant material JWH-122 
Matrix – Red Post-federal ban Dried plant material JWH-122 
Mexican Jumping Beans Post-federal ban Capsules Butylone 
Mexican Jumping Beans Post-federal ban Capsules Butylone 
Mexican Jumping Beans Post-federal ban Capsules Butylone 
Mind Candy Post-federal ban Pills 6-APB 
Mind Candy Post-federal ban Pills 6-APB 
Mr. Kush Post-federal ban Dried plant material JWH-122 
Mr. Kush: Blueberry Post-federal ban Dried plant material JWH-122 
Mr. Kush: Strawberry Post-federal ban Dried plant material JWH-122 
Mr. Nice Guy Post-federal ban Dried plant material AM-2201 
Mr. Nice Guy Post-federal ban Dried plant material AM-2201 
No More Mr. Nice Guy Post-federal ban Dried plant material JWH-122, JWH-250 
Nugz Bush Potpourri Post-federal ban Dried plant material AM-2201 
Purple Dragon Post-federal ban Dried plant material AM-2201 
Purple Haze Kryptonite Post-federal ban Dried plant material AM-2201, AM-2233 
Rosewood Pyara Post-federal ban Dried plant material JWH-122, JWH-250 
Scooby Snax Post-federal ban Dried plant material AM-2201, JWH-122, JWH-210 
Speed Rush Post-federal ban Pills Alpha-PVP 
Speed Rush Post-federal ban Pills Alpha-PVP 
Spike Max Post-federal ban Dried plant material JWH-250 
The Good Stuff Post-federal ban Dried plant material AM-2201 
Tranquility Post-federal ban Powder Methylone 
Ultra Zombie Matter Post-federal ban Dried plant material AM-2201, JWH-210, phenazepam 
Ultra Zombie Matter Acid Rain Post-federal ban Dried plant material AM-2201 
Urban Kaos – Blues Berries Post-federal ban Dried plant material AM-2201 
Urban Kaos – Blues Berries Post-federal ban Dried plant material AM-2201 
Urban Kaos - Hypnotic Post-federal ban Dried plant material AM-2201 
Urban Kaos – Pineapple Express Post-federal ban Dried plant material AM-2201 
Urban Kaos – Wicked Clown Post-federal ban Dried plant material AM-2201 
Urban Kaos – Wicked Clown Post-federal ban Dried plant material AM-2201 
Urban Kaos – Wicked Clown Post-federal ban Dried plant material AM-2201 
Urban Kaos – Wicked Clown Post-federal ban Dried plant material AM-2201 
Urban Kaos – Wicked Clown Post-federal ban Dried plant material AM-2201 
Urban Kaos – Whoop A**! Post-federal ban Dried plant material AM-2201 
Urban Kaos – XXX Post-federal ban Dried plant material AM-2201 
Vanilla Sky Post-federal ban Powder Alpha-PVP 
Zero Gravity Hypnotic Post-federal ban Dried plant material AM-2201, JWH-122, JWH-210 
Zero Gravity Watermelon Post-federal ban Dried plant material AM-2201, JWH-210, RCS-8 
Product Name Time period acquired Form Compound(s) detected 
Black Mamba Pre-federal ban Dried plant material JWH-018 
Bliss Pre-federal ban Powder MDPV 
Eight Ballz Pre-federal ban Powder MDPV 
Ivory Wave Pre-federal ban Powder MDPV 
K2 Blonde Pre-federal ban Dried plant material JWH-018, JWH-073 
K2 Blue Pre-federal ban Dried plant material JWH-018 
K2 Citron Pre-federal ban Dried plant material JWH-018, JWH-073 
K2 Latte Pre-federal ban Dried plant material JWH-018 
K2 Mint Pre-federal ban Dried plant material JWH-018 
K2 Peach Pre-federal ban Dried plant material JWH-018 
K2 Pink Pre-federal ban Dried plant material JWH-018, JWH-073 
K2 Silver Pre-federal ban Dried plant material JWH-250 
K2 Standard Pre-federal ban Dried plant material JWH-018, JWH-073 
K2 Summit Pre-federal ban Dried plant material JWH-018, JWH-073 
K2 Summit Pre-federal ban Dried plant material JWH-018, JWH-073 
Tranquility Pre-federal ban Powder MDPV 
Tribal Warrior Pre-federal ban Dried plant material JWH-018, JWH-073 
7H Hydro Post-federal ban Dried plant material AM-2201 
7H Kush Post-federal ban Dried plant material AM-2201, JWH-122 
B2 Da Bomb Blueberry Post-federal ban Dried plant material AM-2201, JWH-073 
Baked Goods Post-federal ban Dried plant material AM-2201 
Bayou Blaster Post-federal ban Dried plant material AM-2201, AM-2233, JWH-210, MPPP 
Black Mamba Mango Post-federal ban Dried plant material AM-2201 
Black Rooster – Classic Post-federal ban Dried plant material AM-2201 
Cloud 9 Deversion 2.0 Post-federal ban Dried plant material AM-2201, JWH-019 
Cloud 9 Deversion 2.0 Post-federal ban Dried plant material JWH-250 
Cloud 9 Deversion 2.0 Post-federal ban Dried plant material JWH-122, JWH-250 
Cloud 9 Deversion 2.0 Post-federal ban Dried plant material AM-2201 
Cloud 9 Deversion 2.0 Post-federal ban Dried plant material AM-2201 
Cloud 9 Mad Hatter Post-federal ban Dried plant material AM-2201, JWH-019 
Cloud 9 Mad Hatter Post-federal ban Dried plant material AM-2201 
Cloud 9 Mad Hatter Post-federal ban Dried plant material AM-2201 
Cloud 9 Mad Hatter Post-federal ban Dried plant material AM-2201 
Cloud 9 Mad Hatter Post-federal ban Dried plant material AM-2201, JWH-019 
Cloud 9 Mad Hatter Post-federal ban Dried plant material AM-2201, JWH-019 
Cloud 9 Mad Hatter Post-federal ban Dried plant material AM-2201, JWH-019 
Colorado Post-federal ban Dried plant material AM-2201 
Crystal Clean Hookah Cleaner Post-federal ban Powder Alpha-PVP 
Dancing Monkey Groovy Grape Post-federal ban Dried plant material AM-2201, JWH-018, JWH-073 
Dank Potpourri Post-federal ban Dried plant material AM-2201, JWH-210 
Defiant Blue Post-federal ban Dried plant material AM-2201, JWH-073, JWH-122 
Demon Post-federal ban Dried plant material AM-2201, AM-2233, JWH-210, MPPP 
Demon Post-federal ban Dried plant material AM-2201, AM-2233, JWH-019, JWH-210, JWH-250 
Doves Ultra Post-federal ban Pills 6-APB 
Doves Ultra Post-federal ban Pills 6-APB 
Funky Monkey – Black Label Post-federal ban Dried plant material AM-2201, JWH-210, JWH-250, lidocaine, RCS-8 
HG2 Fire Post-federal ban Dried plant material JWH-122, JWH-250 
Jucci Intense Incense Post-federal ban Dried plant material AM-2201 
K2 XXX Chronic Post-federal ban Dried plant material JWH-122, JWH-203 
K4 Gold Post-federal ban Dried plant material JWH-250 
Karma Blueberry Post-federal ban Dried plant material AM-2201 
Karma Bubblegum Post-federal ban Dried plant material AM-2201, JWH-122, JWH-203 
Karma Mango Post-federal ban Dried plant material AM-2201, JWH-122, JWH-203 
Karma Original Post-federal ban Dried plant material AM-2201 
Karma Watermelon Post-federal ban Dried plant material AM-2201 
Kick-Ass Incense Post-federal ban Dried plant material AM-2201, MPPP 
Kottonmouf King Post-federal ban Dried plant material AM-2201, JWH-250 
Kush Pink Post-federal ban Dried plant material AM-2201, JWH-210 
M:20 Madness Potpourri Post-federal ban Dried plant material AM-2201 
Matrix - Green Post-federal ban Dried plant material JWH-122 
Matrix – Red Post-federal ban Dried plant material JWH-122 
Mexican Jumping Beans Post-federal ban Capsules Butylone 
Mexican Jumping Beans Post-federal ban Capsules Butylone 
Mexican Jumping Beans Post-federal ban Capsules Butylone 
Mind Candy Post-federal ban Pills 6-APB 
Mind Candy Post-federal ban Pills 6-APB 
Mr. Kush Post-federal ban Dried plant material JWH-122 
Mr. Kush: Blueberry Post-federal ban Dried plant material JWH-122 
Mr. Kush: Strawberry Post-federal ban Dried plant material JWH-122 
Mr. Nice Guy Post-federal ban Dried plant material AM-2201 
Mr. Nice Guy Post-federal ban Dried plant material AM-2201 
No More Mr. Nice Guy Post-federal ban Dried plant material JWH-122, JWH-250 
Nugz Bush Potpourri Post-federal ban Dried plant material AM-2201 
Purple Dragon Post-federal ban Dried plant material AM-2201 
Purple Haze Kryptonite Post-federal ban Dried plant material AM-2201, AM-2233 
Rosewood Pyara Post-federal ban Dried plant material JWH-122, JWH-250 
Scooby Snax Post-federal ban Dried plant material AM-2201, JWH-122, JWH-210 
Speed Rush Post-federal ban Pills Alpha-PVP 
Speed Rush Post-federal ban Pills Alpha-PVP 
Spike Max Post-federal ban Dried plant material JWH-250 
The Good Stuff Post-federal ban Dried plant material AM-2201 
Tranquility Post-federal ban Powder Methylone 
Ultra Zombie Matter Post-federal ban Dried plant material AM-2201, JWH-210, phenazepam 
Ultra Zombie Matter Acid Rain Post-federal ban Dried plant material AM-2201 
Urban Kaos – Blues Berries Post-federal ban Dried plant material AM-2201 
Urban Kaos – Blues Berries Post-federal ban Dried plant material AM-2201 
Urban Kaos - Hypnotic Post-federal ban Dried plant material AM-2201 
Urban Kaos – Pineapple Express Post-federal ban Dried plant material AM-2201 
Urban Kaos – Wicked Clown Post-federal ban Dried plant material AM-2201 
Urban Kaos – Wicked Clown Post-federal ban Dried plant material AM-2201 
Urban Kaos – Wicked Clown Post-federal ban Dried plant material AM-2201 
Urban Kaos – Wicked Clown Post-federal ban Dried plant material AM-2201 
Urban Kaos – Wicked Clown Post-federal ban Dried plant material AM-2201 
Urban Kaos – Whoop A**! Post-federal ban Dried plant material AM-2201 
Urban Kaos – XXX Post-federal ban Dried plant material AM-2201 
Vanilla Sky Post-federal ban Powder Alpha-PVP 
Zero Gravity Hypnotic Post-federal ban Dried plant material AM-2201, JWH-122, JWH-210 
Zero Gravity Watermelon Post-federal ban Dried plant material AM-2201, JWH-210, RCS-8 

It was quite clear that during the pre-federal ban phase of testing that most (16 of 17 or 94.1%) of the first-generation products contained what would eventually become DEA-controlled substances. Of the five DEA-controlled synthetic cannabinoids, we detected only JWH-018 and JWH-073 and did not detect JWH-200, CP-47,497 or CP-47,497-C8. Of the three DEA-controlled synthetic stimulants, we detected MDPV.

In the post-federal ban time range, only four products (4.9% of the 81 products)—Dancing Monkey (Groovy Grape), B2 Da Bomb Blueberry, Defiant Blue, and Tranquility—contained a federally controlled substance. At the time of purchase of those products, JWH-018, JWH-073 and methylone were banned by both the federal government and also the state of Indiana. Table IV summarizes the analyses of all products during the two time ranges.

Our true focus in this investigation was the new federally uncontrolled substances detected in these so-called post-ban or second-generation highs. These included 6-APB, alpha-PVP, AM-2201, AM-2233, butylone, JWH-019, JWH-122, JWH-203, JWH-210, JWH-250, MPPP and RCS-8. Many of these compounds can be considered derivatives or analogs of the already controlled compounds. Figures 1–2 depict the analogous structural nature of many of these compounds; not all compounds are represented.

Figure 1.

The analogous nature of synthetic cannabinoids - not all monitored analytes represented.

Figure 1.

The analogous nature of synthetic cannabinoids - not all monitored analytes represented.

Figure 2.

The analogous nature of synthetic stimulants - not all monitored analytes represented.

Figure 2.

The analogous nature of synthetic stimulants - not all monitored analytes represented.

Federally uncontrolled compounds detected

A total of 12 different federally uncontrolled cannabinoid and/or stimulant compounds were detected in all of our analyses. All of the synthetic cannabinoid compounds were found as adulterants in “herbal blend,” “herbal incense” or “potpourri” products consisting of a dried plant material. All stimulant compounds were detected as adulterants in either powders or pills, with one exception, as a stimulant compound was found in three different dried plant material products.

Detected cannabinoids

AM-2201

The chemical name of AM-2201 is [1-(5-fluoropentyl)-1H-indol-3-yl]-1-naphthalenyl-methanone and it is a fluorinated derivative of JWH-018. It is a very potent synthetic cannabinoid with binding affinities (Ki) of 1.0 and 2.6 nm at the CB1 and CB2 receptors, respectively (30). AM-2201 was the most prevalent synthetic cannabinoid detected in products during the post-ban time frame, because it was detected in 70% of second generation products. These products include 7H Hydro, 7H Kush, B2 Da Bomb Blueberry, Baked Goods, Bayou Blaster, Black Mamba Mango, Black Rooster (Classic), Cloud 9 Deversion 2.0, Cloud 9 Mad Hatter, Colorado, Dancing Monkey Groovy Grape, Defiant Blue, Demon, Funky Monkey (Black Label), Jucci Intense Incense, Karma Blueberry, Karma Bubblegum, Karma Mango, Karma Original, Karma Watermelon, Kick-Ass Incense, Kottonmouf King, Kush Pink, M:20 Madness Potpourri, Mr. Nice Guy, Nugz Bush Potpourri, Purple Dragon, Purple Haze Kryptonite, Scooby Snax, The Good Stuff, Ultra Zombie Matter, Ultra Zombie Matter: Acid Rain, Urban Kaos (Blues Berries), Urban Kaos (Hypnotic), Urban Kaos (Pineapple Express), Urban Kaos (Wicked Clown), Urban Kaos (Whoop A**!), Urban Kaos (XXX), Zero Gravity Hypnotic and Zero Gravity Watermelon. AM-2201 was detected in products as the single active ingredient and alongside AM-2233, JWH-018, JWH-019, JWH-073, JWH-122, JWH-210, JWH-250, MPPP and/or RCS-8. Nakajima et al. have previously reported the detection of AM-2201 in various products obtained through the Internet (10). An extracted ion spectrum is shown in Figure 3.

Figure 3.

Extracted ion spectrum of AM-2201 detected in an authentic herbal blend product.

Figure 3.

Extracted ion spectrum of AM-2201 detected in an authentic herbal blend product.

AM-2233

The chemical name of AM-2233 is (2-iodophenyl)-[1-[[[(2R)-1-methyl-2-piperidinyl]methyl]-1H-indol-3-yl]-methanone. It is a novel potent cannabinoid compound that has not been reported in peer-reviewed literature as a compound detected in legal high products. This is the first published report of this cannabinoid found in an herbal blend product. AM-2233 is a full agonist at the CB1 receptor and has a Ki of 2.8 nm (31). It was detected in herbal blend products named Bayou Blaster, Demon and Purple Haze Kryptonite. It was detected alongside AM-2201 JWH-019, JWH-210, JWH-250 and/or MPPP. An extracted ion spectrum is shown in Figure 4.

Figure 4.

Extracted ion spectrum of AM-2233 detected in an authentic herbal blend product.

Figure 4.

Extracted ion spectrum of AM-2233 detected in an authentic herbal blend product.

JWH-019

The chemical name of JWH-019 is (1-hexyl-1H-indol-3-yl)-1-naphthalenyl-methanone; it is a 1-hexyl derivative of JWH-018 and a structural isomer of JWH-122. It has Ki values of 9.8 and 5.55 nm at the CB1 and CB2 receptors, respectively (29). Because they are isobaric compounds, chromatographic resolution between JWH-019 and JWH-122 is needed and detailed in Figure 5. At the time of detection, JWH-019 was controlled by the state of Indiana, but it was found to be a constituent of blends named Cloud 9 Deversion 2.0, Cloud 9 Mad Hatter and Demon. It was detected alongside AM-2201, AM-2233, JWH-210 and/or JWH-250. One other report has been published of the confirmed detection of this drug in legal high products. Nakajima et al. reported the confirmed presence of JWH-019 in multiple products obtained via the Internet (33). Also, Hudson et al. discussed detecting a compound that was either JWH-007 or JWH-019, but could not confirm the identity due to not knowing the chromatographic retention times of JWH-007 and JWH-019 (4). With the described method, JWH-007 elutes at 6.0 min, therefore, there is 0.4-min chromatographic resolution between the two isobaric compounds.

Figure 5.

Chromatographic resolution between isobaric compounds: JWH-019 and JWH-122.

Figure 5.

Chromatographic resolution between isobaric compounds: JWH-019 and JWH-122.

JWH-122

The chemical name of JWH-122 is (4-methyl-1-naphthalenyl)(1-pentyl-1H-indol-3-yl)-methanone; it is a 4-methyl derivative of JWH-018 and a structural isomer of JWH-019. It is a potent cannabinoid compound that exhibits Ki values of 0.69 and 1.2 nm at the CB1 and CB2 receptors, respectively (33–35). At the time of detection, JWH-122 was controlled by the state of Indiana, but it was found to be a constituent of several blends that were readily obtained, including 7H Kush, Cloud 9 Deversion 2.0, Defiant Blue, HG2 Fire, K2 XXX Chronic, Karma Bubblegum, Karma Mango, Matrix (Green), Matrix (Red), Mr. Kush, Mr. Kush (Blueberry), Mr. Kush (Strawberry), No More Mr. Nice Guy, Rosewood Pyara, Scooby Snax and Zero Gravity Hypnotic. It was detected as the single drug in the product or alongside AM-2201, JWH-073, JWH-203, JWH-210, JWH-250 and/or RCS-8. JWH-122 has been previously confirmed as an adulterant in herbal blend products by Ernst et al. (6). Hudson et al. detected a compound in an herbal blend that was determined to be either JWH-047 or JWH-122, but they could not confirm its true identity due to lack of known retention times (4).

JWH-203

The chemical name of JWH-203 is 2-(2-chlorophenyl)-1-(1-pentyl-1H-indol-3-yl)ethanone and it is a chlorophenylacetyl derivative of JWH-018. It has Ki values of 8.0 and 7.0 nm at the CB1 and CB2 receptors, respectively (36). This compound was only detected in three blend products named K2 XXX Chronic, Karma Bubblegum and Karma Mango. It was detected alongside AM-2201 and/or JWH-122. Previously published reports of the detection of this compound include Uchiyama et al. (38) and Nakajima et al. (39).

JWH-210

The chemical name of JWH-210 is (4-ethyl-1-naphthalenyl)(1-pentyl-1H-indol-3-yl)-methanone and it is a 4-ethyl derivative of JWH-018. It is a very potent synthetic cannabinoid with Ki values of 0.46 and 0.69 nm at the CB1 and CB2 receptors, respectively (35). JWH-210 was detected in products named Bayou Blaster, Dank Potpourri, Demon, Funky Monkey (Black Label), Kush Pink, Scooby Snax, Ultra Zombie Matter, Zero Gravity Hypnotic and Zero Gravity Watermelon. It was detected alongside AM-2201, AM-2233, JWH-019, JWH-122, JWH-250, MPPP and RCS-8. Nakajima et al. have previously identified JWH-210 in legal high products (33). An extracted ion spectrum is shown in Figure 6.

Figure 6.

Extracted ion spectrum of JWH-210 detected in an authentic herbal blend product.

Figure 6.

Extracted ion spectrum of JWH-210 detected in an authentic herbal blend product.

JWH-250

The chemical name of JWH-250 is 1-(1-pentyl-1H-indol-3-yl)-2-(2-methoxyphenyl)-ethanone and it is a 2-methoxyphenylacetyl derivative of JWH-018. It has Ki values of 11 and 33 nm at the CB1 and CB2 receptors, respectively (36). It was detected in one pre-federal ban product—K2 Silver—and multiple post-ban products—Cloud 9 Deversion 2.0, Demon, Funky Monkey (Black Label), HG2 Fire, K4 Gold, Kottonmouf King, No More Mr. Nice Guy, Rosewood Pyara and Spike Max. With the exception of the one pre-federal ban product, JWH-250 was detected during times when the compound was considered a banned substance in the state of Indiana. It was detected as the single active ingredient alongside AM-2201, AM-2233, JWH-019, JWH-122, JWH-210 and/or RCS-8. Uchiyama et al. (8) and Nakajima et al. (9) have previously identified JWH-250 as an adulterant in legal high drugs.

RCS-8

The chemical name of RCS-8 is 1-(1-(2-cyclohexylethyl)-1H-indol-3-yl)-2-(2-methoxyphenyl)ethanone and it is a cyclohexylethyl derivative of JWH-250. Activity at the cannabinoid receptors has not been reported in literature. It was only detected in the post-ban products named Funky Monkey (Black Label) and Zero Gravity Watermelon. At the time of detection, RCS-8 was a banned substance in Indiana. It was detected alongside AM-2201, JWH-210 and/or JWH-250. This is the first published report of the detection of this compound in an herbal incense blend. An extracted ion spectrum is shown in Figure 7.

Figure 7.

Extracted ion spectrum of RCS-8 detected in an authentic herbal blend product.

Figure 7.

Extracted ion spectrum of RCS-8 detected in an authentic herbal blend product.

Detected stimulants

6-APB

The chemical name of 6-APB is alpha-methyl-6-benzofuranethanamine. It is also commonly known as “benzo fury” because of the benzofuran ring that is contained within its structure. It is considered a substituted amphetamine drug and is most similar to 3,4-methylenedioxyamphetamine (MDA), but it is not scheduled in the United States. Toxicological data are not available for this drug because it has not been reported in literature. 6-APB was detected in products named Mind Candy and Doves Ultra, which were sold in a smoke shop as a package of two light-orange and light-red colored unmarked pills. This is the first published report of the detection of this compound in a legal high product. The isomeric compound, 5-APB, also exists, but at the time of publishing, we did not have access to a reference standard to verify retention time and whether any chromatographic resolution exists between 5-APB and 6-APB.

Alpha-PVP

Alpha-PVP is also known as alpha-pyrrolidinopentiophenone or desmethylpyrovalerone, a stimulant drug that was developed and patented in the 1960s. It is an derivative of MDPV; the only difference is the removal of the 3,4-methylenedioxy group. Toxicological data are not available for this drug because it has not been reported in literature. Alpha-PVP was detected in a product named Speed Rush, which was similar to the Mind Candy product, which was sold in a smoke shop as a package of two light-orange colored unmarked pills. It was also detected in a product named Crystal Clean Hookah and Pipe Cleaner, which was supplied as a tan-colored powder and sold in a gas station convenience store, and Vanilla Sky, a white-colored powder sold in a smoke-shop. This is the first published report of the detection of this compound in legal high products. An extracted ion spectrum is shown in Figure 8.

Figure 8.

Extracted ion spectrum of Alpha-PVP detected in an authentic legal high product.

Figure 8.

Extracted ion spectrum of Alpha-PVP detected in an authentic legal high product.

Butylone

Butylone's chemical name is Beta-keto-N-methylbenzodioxolylpropylamine. It is the beta ketone derivative of the designer drug MBDB (Eden) and also a derivative of the Schedule 1 drug methcathinone. Butylone was detected in a product named Mexican Jumping Beans, which was supplied as three capsules containing a tan-colored powdery substance. Butylone has been detected in previous legal high drugs by Brandt et al. (11) and Uchiyama et al. (37).

MPPP

MPPP's chemical name is 2-(pyrrolidin-1-yl)-1-(p-tolyl)propan-1-one. It is included in a group of compounds called pyrrolidinophenones. MPPP was detected in products named Bayou Blaster, Demon and Kick-Ass Incense, all of which were dried plant material sold as an herbal potpourri. Brandt et al. (40) and Kikura-Hanajiri et al. (41) have previously reported this drug's presence in legal high products.

Product anomalies

In four circumstances, we analyzed multiple packages of the same legal high product. The results show the true nature of these designer drugs and their clandestine roots, because the same product name with the same packaging (maintaining the same appearance) can contain distinctly different drugs and/or varied combinations of drugs. In two other cases, we detected unsuspected drugs, lidocaine and phenazepam, which are neither synthetic cannabinoids nor stimulants.

Herbal blend product #1

We analyzed five packages of the product named Cloud 9 Deversion 2.0. All four of these second-generation products were obtained at different locations at different times around Indiana. The first product contained the combination of AM-2201 and JWH-019. The second product contained only JWH-250. The third product contained the combination of JWH-122 and JWH-250. The fourth and fifth products contained only AM-2201.

Herbal blend product #2

We analyzed seven packages of the product named Cloud 9 Mad Hatter. The packages of second-generation material were obtained at different locations around Indiana. Three of the packages contained AM-2201 and JWH-019, while the other four packages contained only AM-2201.

Herbal blend product #3

We analyzed two packages of the product named Demon. The packages of material were obtained at the same location (convenience store) in Indiana. The first package of material contained AM-2201, AM-2233 and JWH-210. The second package of material contained AM-2201, AM-2233, JWH-019, JWH-210 and JWH-250.

Herbal blend product #4

The anesthetic and anti-arrhythmic drug lidocaine was detected as an adulterant in the herbal blend product Funky Monkey (Black Label). The use of caffeine, lidocaine or procaine in legal high products has been reported by Brandt et al. (11), but those products were powdery substances and not a dried plant material. Multiple replicates of this blend were analyzed to verify that the positive result was true and the LC–TOF results were confirmed by an multiple reaction monitoring-based liquid chromatography–tandem mass spectrometry (LC–MS-MS) methodology specific for lidocaine; no quantitation was performed. Lidocaine was not detected in any other product that we analyzed.

Herbal blend product #5

The potent benzodiazepine phenazepam was detected in the blend product Ultra Zombie Matter. Phenzepam is primarily used in Russia and other former Soviet republics; it is unscheduled in the United States (42). The presence of the drug was confirmed by a qualitative LC–MS-MS methodology specific for the drug. This is the first documented appearance of this drug in a legal high product. Phenazepam is legal to possess and consume in the United States.

“Bath salt” product #1

We analyzed two packages of the product named Tranquility. The first product was obtained during the pre-federal ban time frame and contained MDPV, while the second product was obtained during the post-federal ban time frame and contained methylone, which was one of the three federally banned stimulants. At the time of purchase, methylone was a banned substance in Indiana.

Conclusion

In our study of first-generation and second-generation products, it was shown that as compounds become banned or controlled, the manufacturers switch the active drug or drugs in the legal high product so that it stays legal and able to be sold. During the pre-ban phase of testing, JWH-018, JWH-073 and MDPV were quite common. After legislation and restrictions were put into place, other compounds, such as 6-APB, alpha-PVP, AM-2201, butylone, JWH-122, JWH-210 and JWH-250, became widespread and prevalent, while the use of the controlled compounds waned (but did not become totally extinct). It was also shown that even within the same product or packaging, different drugs or combinations of drugs may be present. To keep up with these moving targets, instrumentation and methodologies need to be adapted that allow a laboratory to acquire highly specific data in full scan mode. This method is suitable for use in the modern drug chemistry or forensic toxicology laboratory and allows for mining of the data and/or reanalysis of the data for the presence of new compounds without re-extraction or reinjection of the specimen. As these products evolve over time, modifications will be made to the methods in the future through the incorporation of additional compounds. Also, the use of isotopic patterns and spacing will be incorporated into the identification procedures.

Acknowledgments

The authors would like to thank Dr. Michael Evans for his support of the project as well as the Research and Development and Toxicology departments at AIT Laboratories, the Indiana State Police Crime Laboratory, Officer Ami Sunier of the Indiana State Excise Police, and the Wabash Police Department.

References

1
Auwarter
V.
Dresen
S.
Weinmann
W.
Muller
M.
Putz
M.
Ferreiros
N.
Spice and other herbal blends: Harmless incense or cannabinoid designer drugs?
Journal of Mass Spectrometry
 , 
2009
, vol. 
44
 (pg. 
832
-
837
)
2
Uchiyama
N.
Kikura-Hanajiri
R.
Kawahara
N.
Goda
Y.
Identification of a cannabimimetic indole as a designer drug in a herbal product
Forensic Toxicology
 , 
2009
, vol. 
27
 (pg. 
61
-
66
)
3
Lindigkeit
R.
Boehme
A.
Eiserloh
I.
Luebbecke
M.
Wiggermann
M.
Ernst
L.
, et al.  . 
Spice: A never ending story?
Forensic Science International
 , 
2009
, vol. 
191
 (pg. 
58
-
63
)
4
Hudson
S.
Ramsey
J.
King
L.
Timbers
S.
Maynard
S.
Dargan
P.I.
, et al.  . 
Use of high-resolution accurate mass spectrometry to detect reported and previously unreported cannabinomimetics in “herbal high” products
Journal of Analytical Toxicology
 , 
2010
, vol. 
34
 (pg. 
252
-
260
)
5
Uchiyama
N.
Kikura-Hanajiri
R.
Ogata
J.
Goda
Y.
Chemical analysis of synthetic cannabinoids as designer drugs in herbal products
Forensic Science International
 , 
2010
, vol. 
198
 (pg. 
31
-
38
)
6
Ernst
L.
Schiebel
H.-M.
Theuring
C.
Lindigkeit
R.
Beuerle
T.
Identification and characterization of JWH-122 used as new ingredient in “Spice-like” herbal incenses
Forensic Science International
 , 
2011
, vol. 
208
 (pg. 
e31
-
e35
)
7
Penn
H.J.
Langman
L.J.
Unold
D.
Shields
J.
Nichols
J.H.
Detection of synthetic cannabinoids in herbal incense products
Clinical Biochemistry
 , 
2011
, vol. 
44
 (pg. 
1163
-
1165
)
8
Uchiyama
N.
Kawamura
M.
Kikura-Hanajiri
R.
Goda
Y.
Identification and quantitation of two cannabimemetic phenylacetylindoles JWH-251 and JWH-250, and four cannabimimetic naphthoylindoles JWH-081, JWH-015, JWH-200, and JWH-073 as designer drugs in illegal products
Forensic Toxicology
 , 
2011
, vol. 
29
 (pg. 
25
-
37
)
9
Nakajima
J.
Takahashi
M.
Seto
T.
Suzuki
J.
Identification and quantitation of cannabimimetic compound JWH-250 as an adulterant in products obtained via the Internet
Forensic Toxicology
 , 
2011
, vol. 
29
 (pg. 
51
-
55
)
10
Nakajima
J.
Takahashi
M.
Nonaka
R.
Seto
T.
Suzuki
J.
Yoshida
M.
, et al.  . 
Identification and quantitation of a benzoylindole (2-methoxyphenyl)(1-pentyl-1H-indol-3-yl)methanone and a naphthoylindole 1-(5-fluoropentyl-1H-indol-3-yl)-(naphthalene-1-yl)methanone (AM-2201) found in illegal products obtained via the Internet and their cannabimimetic effects evaluated by in vitro (35S)GTPγS binding assays
Forensic Toxicology
 , 
2011
, vol. 
29
 (pg. 
132
-
141
)
11
Brandt
S.D.
Sumnall
H.R.
Measham
F.
Cole
J.
Analyses of second-generation ‘legal highs’ in the UK: Initial findings
Drug Testing and Analysis
 , 
2010
, vol. 
2
 (pg. 
377
-
382
)
12
Jankovics
P.
Varadi
A.
Tolgyesi
L.
Lohner
S.
Nemeth-Palotas
J.
Koszegi-Szalai
H.
Identification and characterization of the new designer drug 4-methylethcathinone (4-MEC) and elaboration of a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) screening method for seven different methcathinone analogs
Forensic Science International
 , 
2011
, vol. 
210
 (pg. 
213
-
220
)
13
Westphal
F.
Junge
T.
Girreser
U.
Greibl
W.
Doering
C.
Mass, NMR, and IR spectroscopic characterization of pentedrone and pentylone and identification of their isocathinone by-products
Forensic Science International
 , 
2012
, vol. 
217
 (pg. 
157
-
167
)
14
Maheux
C.R.
Copeland
C.R.
Chemical analysis of two new designer drugs: Buphedrone and pentedrone
Drug Testing and Analysis
 , 
2011
, vol. 
4
 (pg. 
17
-
23
)
15
Every-Palmer
S.
Warning: Legal synthetic cannabinoid-receptor agonists such as JWH-018 may precipitate psychosis in vulnerable individuals
Addiction
 , 
2010
, vol. 
105
 (pg. 
1859
-
1861
)
16
Zimmerman
U.S.
Winkelmann
P.R.
Pilhatsch
M.
Nees
J.A.
Spanagel
R.
Shulz
K.
Withdrawal phenomena and dependence syndrome after the consumption of “spice gold.”
Deutsches Ärzteblatt International
 , 
2009
, vol. 
106
 (pg. 
464
-
467
)
17
Simmons
J.
Cookman
L.
Kang
C.
Skinner
C.
Three cases of “spice” exposure
Clinical Toxicology
 , 
2011
, vol. 
49
 (pg. 
431
-
433
)
18
Spiller
H.A.
Ryan
M.L.
Weston
R.G.
Jansen
J.
Clinical experience with and analytical confirmation of “bath salts” and “legal highs” (synthetic cathinones) in the United States
Clinical Toxicology
 , 
2011
, vol. 
49
 (pg. 
499
-
505
)
19
Prosser
J.M.
Nelson
L.S.
The toxicology of bath salts: A review of synthetic cathinones
Journal of Medical Toxicology
 , 
2012
, vol. 
8
 (pg. 
33
-
42
)
20
Antonowicz
J.L.
Metzger
A.K.
Ramanujam
S.L.
Paranoid psychosis induced by consumption of methylenedioxypyrovalerone: Two cases
General Hospital Psychiatry
 , 
2011
, vol. 
33
 pg. 
640
 
21
Penders
T.M.
Gestring
R.
Hallucinatory delirium following use of MDPV: “Bath salts.”
General Hospital Psychiatry
 , 
2011
, vol. 
33
 (pg. 
525
-
526
)
22
Wood
D.M.
Davies
S.
Greene
S.L.
Button
J.
Holt
D.W.
Ramsey
J.
, et al.  . 
Case series of individuals with analytically confirmed acute mephedrone toxicity
Clinical Toxicology
 , 
2010
, vol. 
48
 (pg. 
924
-
927
)
23
Warrick
B.J.
Wilson
J.
Hedge
M.
Freeman
S.
Leonard
K.
Aaron
C.
Lethal serotonin syndrome after methylone and butylone ingestion
Journal of Medical Toxicology
 , 
2012
, vol. 
8
 (pg. 
65
-
68
)
24
Vardakou
I.
Pistos
C.
Spiliopoulou
Ch.
Spice drugs as a new trend: Mode of action, identification and legislation
Toxicology Letters
 , 
2010
, vol. 
197
 (pg. 
157
-
162
)
25
Polettini
A.
Gottardo
R.
Pascali
J.P.
Tagliaro
F.
Implementation and performance evaluation of a database of chemical formulas of pharmaco/toxicologically relevant compounds in biological samples using electrospray ionization-time-of-flight mass spectrometry
Analytical Chemistry
 , 
2008
, vol. 
80
 (pg. 
3050
-
3057
)
26
Liotta
E.
Gottardo
R.
Bertaso
A.
Polettini
A.
Screening for pharmaco-toxicologically relevant compounds in biosamples using high-resolution mass spectrometry: A metabolomic approach to the discrimination between isomers
Journal of Mass Spectrometry
 , 
2010
, vol. 
45
 (pg. 
261
-
271
)
27
Ojanperä
I.
Pelander
A.
Laks
S.
Gergov
M.
Vuori
E.
Witt
M.
Application of accurate mass measurement to urine drug screening
Journal of Analytical Toxicology
 , 
2005
, vol. 
29
 (pg. 
34
-
40
)
28
ElSohly
M.A.
Gul
W.
ElSohly
K.M.
Avula
B.
Khan
I.A.
LC-MS-(TOF) analysis method for benzodiazepines in urine samples from alleged drug-facilitated sexual assault victims
Journal of Analytical Toxicology
 , 
2006
, vol. 
30
 (pg. 
524
-
538
)
29
Lee
H.K.
Ho
C.S.
Iu
H.P.H.
Lai
P.S.J.
Shek
C.C.
Lo
Y.-C.
, et al.  . 
Development of a broad toxicological screening technique for urine using ultra-performance liquid chromatography and time-of-flight mass spectrometry
Analytical Chimica Acta
 , 
2009
, vol. 
649
 (pg. 
80
-
90
)
30
Markiyannis
A.
Deng
H.
Cannabimimetic indole derivatives
2005
 
United States Patent, US 7,241,799 B2
31
Deng
H.
Gifford
A.N.
Zvonok
A.M.
Cui
G.
Li
X.
Fan
P.
, et al.  . 
Potent cannabinergic indole analogues as radioiodinatable brain imaging agents for the CB1 cannabinoid receptor
Journal of Medical Chemistry
 , 
2005
, vol. 
48
 (pg. 
6386
-
6392
)
32
Aung
M.M.
Griffin
G.
Huffman
J.W.
Wu
M.-J.
Keel
C.
Yang
B.
, et al.  . 
Influence of the N-1 alkyl chain length of cannabimimetic indoles upon CB1 and CB2 receptor binding
Drug and Alcohol Dependence
 , 
2000
, vol. 
60
 (pg. 
133
-
140
)
33
Nakajima
J.
Takahashi
M.
Seto
T.
Kanai
C.
Suzuki
J.
Yoshida
M.
, et al.  . 
Identification and quantitation of two benzoylindoles AM-694 and (4-methoxyphenyl)(1-pentyl-1H-indol-3-yl)methanone, and three cannabimimetic naphthoylindoles JWH-210, JWH-122, and JWH-019 as adulterants in illegal products obtained via the Internet
Forensic Toxicology
 , 
2011
, vol. 
29
 (pg. 
96
-
110
)
34
Huffman
J.W.
Mabon
R.
Wu
M.J.
Lu
J.
Hart
R.
Hurst
D.P.
, et al.  . 
3-indolyl-1-naphthylmethanes: New cannabimimetic indoles provide evidence for aromatic stacking interactions with the CB(1) cannabinoid receptor
Bioorganic and Medicinal Chemistry
 , 
2003
, vol. 
11
 (pg. 
539
-
549
)
35
Huffman
J.W.
Zengin
G.
Wu
M.J.
Lu
J.
Hynd
G.
Bushell
K.
, et al.  . 
Structure-activity relationships for 1-alkyl-3-(1-naphthoyl)indoles at the cannabinoid CB(1) and CB(2) receptors: Steric and electronic effects of naphthoyl substituents. New highly selective CB(2) receptor agonists
Bioorganic and Medicinal Chemistry
 , 
2005
, vol. 
13
 (pg. 
89
-
112
)
36
Huffman
J.W.
Szklennik
P.V.
Almond
A.
Bushell
K.
Selley
D.E.
He
H.
, et al.  . 
1-pentyl-3-phenylacetylindoles, a new class of cannabimimetic indoles
Bioorganic and Medicinal Chemistry Letters
 , 
2005
, vol. 
15
 (pg. 
4110
-
4113
)
37
Uchiyama
N.
Kikura-Hanajiri
R.
Kawahara
N.
Goda
Y.
Analysis of designer drugs detected in the products purchased in fiscal year 2006
Yakugaku Zasshi
 , 
2008
, vol. 
128
 (pg. 
1499
-
1505
)
38
Uchiyama
N.
Kikura-Hanajiri
R.
Goda
Y.
Identification of a novel cannabimimetic phenylacetylindole, cannabipiperidiethanone, as a designer drug in a herbal product and its affinity for cannabinoid CB1 and CB2 receptors
Chemical and Pharmaceutical Bulletin (Tokyo)
 , 
2011
, vol. 
59
 (pg. 
1203
-
1205
)
39
Nakajima
J.
Takahashi
M.
Seto
T.
Yoshida
M.
Kanai
C.
Suzuki
J.
, et al.  . 
Identification and quantitation of two new naphthoylindole drugs-of-abuse, (1-(5-hydroxypentyl)-1H-indol-3-yl)(naphthalene-1-yl)methanone (AM-2202) and (1-(4-pentenyl)-1H-indol-3-yl)(naphthalene-1-yl)methanone, with other synthetic cannabinoids in unregulated “herbal” products circulated in the Tokyo area
Forensic Toxicology
 , 
2012
, vol. 
30
 (pg. 
33
-
44
)
40
Brandt
S.D.
Freeman
S.
Sumnall
H.R.
Measham
F.
Cole
J.
Analysis of NRG ‘legal highs’ in the UK: Identification and formation of novel cathinones
Drug Testing and Analysis
 , 
2011
, vol. 
9
 (pg. 
569
-
575
)
41
Kikura-Hanajiri
R.
Uchiyama
N.
Goda
Y.
Survey of current trends in the abuse of psychotropic substances and plants in Japan
Legal Medicine (Tokyo)
 , 
2011
, vol. 
13
 (pg. 
109
-
115
)
42
Bailey
K.
Richards-Waugh
L.
Clay
D.
Gebhardt
M.
Mahmoud
H.
Kraner
J.C.
Fatality involving the ingestion of Phenazepam and poppy seed tea
Journal of Analytical Toxicology
 , 
2010
, vol. 
34
 (pg. 
527
-
532
)