Am 2201 liquid

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Determination of 22 synthetic cannabinoids in human hair by liquid chromatography–tandem mass spectrometry

Journal of Chromatography B, 2012

Co-Authors: Melanie Hutter, Stefan Kneisel, Volker Auwärter, Merja A Neukamm

Abstract:

a b s t r a c t Herbal mixtures of the " Spice " -type contain a variety of synthetic cannabinoids. To prove the contact of a person with synthetic cannabinoids in a previous period of up to several months, hair testing is ideally suited. A rapid, simple and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay was developed to determine 22 synthetic cannabinoids in human hair. The synthetic cannabinoids JWH-007, JWH-015, JWH-018, JWH-019, JWH-020, JWH-073, JWH-081, JWH-122, JWH-200, JWH-203, JWH-210, JWH-250, JWH-251, JWH-398, AM-694, AM–2201, methanandamide, RCS-4, RCS-4 ortho iso-mer, RCS-8, WIN 48,098 and WIN 55,212-2 were extracted from 50 mg hair by 3-h ultrasonification in ethanol. The extracts were analysed on a triple-quadrupole linear ion trap mass-spectrometer in sched-uled multiple reaction monitoring mode (sMRM). The method was fully validated and proved to be accurate, precise, selective and specific with satisfactory linearity within the calibrated range and a lower limit of quantification of 0.5 pg/mg for 20 compounds. Authentic hair samples from chronic consumers showed the presence of two to six synthetic cannabinoids in the same segment. In the first segment, concentrations of up to 78 pg/mg JWH-081 were present. In segmented hair, the concentrations of most substances increased from the first (proximal) to the third segment. The highest concentration was ca. 1100 pg/mg JWH-081. The results of segmental hair analysis in chronic users suggest incorporation of the drugs in head hair via side-stream smoke condensation as a major route. In summary, the method can be used to prove the contact with herbal mixtures containing synthetic cannabinoids and thus contributes to an efficient abstinence control.

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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.

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 

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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 

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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/z) . Nominal fragments (m/z) . Retention 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/z) . Nominal fragments (m/z) . Retention 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 

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Table II

Analytes Monitored with the Synthetic Cannabinoid Method

Number . Analyte . Molecular formula . Accurate mass (m/z) . Nominal fragments (m/z) . Retention 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/z) . Nominal fragments (m/z) . Retention 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 

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Table III

Analytes Monitored with the Synthetic Stimulant Method

Number . Analyte . Molecular formula . Accurate mass (m/z) . Nominal fragments (m/z) . Retention 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/z) . Nominal fragments (m/z) . Retention 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 

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Table III

Analytes Monitored with the Synthetic Stimulant Method

Number . Analyte . Molecular formula . Accurate mass (m/z) . Nominal fragments (m/z) . Retention 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/z) . Nominal fragments (m/z) . Retention 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 

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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 

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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 

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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.

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.

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.

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.

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.

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.

Sours: https://academic.oup.com/jat/article/36/6/360/792298
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Effects of Bioisosteric Fluorine in Synthetic Cannabinoid Designer Drugs JWH-018, AM-2201, UR-144, XLR-11, PB-22, 5F-PB-22, APICA, and STS-135

†Department of Radiology, Stanford University School of Medicine, Stanford, California 94305, United States

‡School of Chemistry, The University of Sydney, Sydney, New South Wales 2006, Australia

§Faculty of Medicine and Health Sciences, Macquarie University, Sydney, New South Wales 2109, Australia

∥School of Psychology, The University of Sydney, Sydney, New South Wales 2006, Australia

⊥Center for Immersive and Simulation-based Learning, Stanford University School of Medicine, Stanford, California 94305, United States

#Department of Anaesthesia, Prince of Wales Hospital, Randwick, New South Wales 2031, Australia

∇Faculty of Pharmacy, The University of Sydney, Sydney, New South Wales 2006, Australia

¶School of Medical Sciences, The University of Auckland, Auckland 1142, New Zealand

◆Discipline of Medical Radiation Sciences, The University of Sydney, Sydney, New South Wales 2006, Australia

*E-mail: [email protected]

Sours: https://pubs.acs.org/doi/abs/10.1021/acschemneuro.5b00107
SCREWBACCO FREEBASE \u0026 SALT 35 MG REVIEW BY CREATIV LABZ . TAMBAH BOLD FLAVOURNYA

Detection of Synthetic Cannabinoids in Oral Fluid Using ELISA and LC–MS-MS

Abstract

Synthetic cannabinoids are often referred to as ‘Spice’ or K2 compounds. Detection of these compounds in oral fluid has, to date, been limited to chromatographic procedures such as liquid chromatography with tandem mass spectrometry detection. We report the first analytical immunoassay for the screening of some synthetic cannabinoids in oral fluid specimens collected with the QuantisalTM device. JWH-200 was chosen as the calibration standard, because parent compounds, not metabolites, are predominantly detected in oral fluid. The immunoassay is capable of detecting JWH-200, JWH-018, JWH-073, JWH-022, AM-2201, AM-2232 and AM-1220. The assay was validated according to accepted laboratory protocols and applied to 32 authentic oral fluid specimens previously analyzed using LC–MS-MS at an accredited laboratory. The assay is sensitive, with a cutoff concentration of 0.25 ng/mL, and has a wide working range from 0.1 to 5 ng/mL. Intra- and interday precision were determined to be <10%. The screening method was completely validated and characterized; critical aspects of the screening included the incorporation of a preincubation step that improves the sensitivity of the assay to allow relevant concentrations of synthetic compounds in oral fluid to be detected.

Introduction

Over the last few years, the emergence of synthetic cannabinoids, commonly known as ‘Spice’ or K2 compounds, has caused problems for analytical testing laboratories. The plethora of synthetic cannabinoids that continue to emerge have inundated toxicology laboratories and have the potential of not being detected by routine analytical drug screens, as most do not cross-react with cannabinoid immunoassays. Due to ease of chemical modification to the core naphthoyl indole ring structure, numerous derivatives are constantly being synthesized by chemists in order to circumvent existing drug enforcement laws, which are regularly revised. Chemical structures for the compounds detected by the assay, those included in the test profile and common structurally unrelated compounds, are shown in Figure 1. Liquid chromatography with tandem mass spectral detection (LC–MS-MS) appears to lend itself well to the analysis of synthetic cannabinoids in oral fluid. The first publication regarding this analysis included seven analytes (JWH-018; JWH-073; JWH-200; JWH-250; CP-47497, cannabicyclohexanol (CP-47497 C8) and HU-210) (1), but subsequent methodologies have expanded the detectability. Kneisel et al. (2) detected 28 substances with lower limits of quantification ranging from 0.15 to 3 ng/mL using LC–ESI MS–MS. When the method was applied to authentic specimens, 12% tested positive for at least one of the following synthetic cannabinoids: AM-694, AM-2201, JWH-018, JWH-019, JWH-081, JWH-122, JWH-203, JWH-210, JWH-250, JWH-307, MAM-2201 and RCS-4. Strano-Rossi et al. (3) incorporated the analysis of 24 compounds: benzylpiperazine, methylone, 5,6-methylenedioxy-2-aminoindane (MDAI), fenproporex, 4-fluoroamphetamine (4-FA), 4-methyl-N-ethylcathinone, 4-methylamphetamine, methylbenzodioxolylbutanamine, mephedrone, methylthioamphetamine, methylenedioxypyrovalerone (MDPV), mefenorex, nabilone, furfenorex, clobenzorex as well as the synthetic cannabinoids JWH-200, AM-694, JWH-250, JWH-073, JWH-018, JWH-019, JWH-122, HU-210 and CP-47497 into a published method, indicating that LC–MS-MS procedures have the flexibility to detect, in oral fluid, a wide range of new compounds.

The ease and rapidity of collection of oral fluid compared with other biological matrices makes it an attractive specimen for analysis (4) using immunoassay (5–7) and LC–MS procedures (8, 9). It is somewhat difficult to adulterate as the collection is observed and the presence of active drug is a reflection of recent drug intake. From a laboratory perspective, the addition of oral fluid to an already existing drug testing program is relatively straightforward and as technological improvements continue to occur, the sensitivity required for analysis is routinely achieved (10); however, analysts should be aware of potential pitfalls associated with some LC–MS procedures (11).

As a general rule, parent drugs are detected in oral fluid (12, 13) as opposed to metabolites, which tend to be in higher concentration in urine; therefore, the LC–MS-MS procedure described was not targeted at urinary metabolites. The requirement for LC–MS-MS analysis has, to date, centered around two issues: first, as previously stated, the routine cannabinoid immunoassays are not cross-reactive with the newer compounds, but secondly, the concentration of these compounds in oral fluid is lower than the levels detected in urine, so the sensitivity required for their routine detection in an immunochemical format has not been commercially available. Immunoassays are generally less expensive than GC–MS or LC–MS-MS methods, have higher throughput for faster turnaround time and have the benefit of cross-reaction with a wide range of structurally related compounds. Therefore, the aim of the project was to develop and validate an immunoassay sufficiently sensitive to detect some synthetic cannabinoids in oral fluid, and apply it to the analysis of authentic specimens previously paramethoxymethylamphetamine confirmed using LC–MS-MS. To achieve the necessary sensitivity, preincubation of the biological sample prior to addition of the enzyme conjugate was assessed as to the performance of the assay, because additional time allows for adequate reactivity of low concentration calibration standards, resulting in improved separation.

Materials and Methods

Collection devices, reagents and standards

Quantisal™ devices for the collection of oral fluid specimens were obtained from Immunalysis Corporation (Pomona, CA, USA). The devices contain a cotton collection pad, which is placed in the mouth and have been determined to collect 1 mL of oral fluid (±10%). After the volume adequacy indicator has turned blue, the pad is placed into transport buffer (3 mL), allowing a total specimen volume available for analysis of 4 mL (3 mL buffer + 1 mL oral fluid). Prepared controls and calibration standards accounted for a 1 : 4 dilution present in authentic collected specimens. The synthetic cannabinoid standard compounds JWH-018, JWH-073, JWH-081, JWH-210, JWH-250, AM-2201 and RCS-4 and their deuterated analogs JWH-018-D9, JWH-073-D7, JWH-210-D9, JWH-250-D5, JWH-081-D9, AM-2201-D5 and RCS-4-D9 were purchased from Cayman Chemicals (Ann Arbor, MI, USA).

Synthetic cannabinoid-specific polyclonal antibody was raised by Immunalysis Corporation, and all haptens and horseradish peroxidase (HRP)-labeled enzyme conjugates were synthesized at Immunalysis Corporation. The enzymes used in the process were bovine thyroglobulin (BTG) obtained from Sigma-Aldrich (Milwaukee, WI, USA), HRP obtained from BBI Enzymes (Madison, WI, USA) and bovine serum albumin (BSA) obtained from Proliant Biologicals (Boone, IA, USA). The substrate reagent 3,3′,5,5′-tetramethylbenzidine (TMB) used for the colorimetric reaction was obtained from Pierce (Rockford, IL, USA). All solvents and chemicals used were of ACS grade, obtained from Spectrum Chemicals (Gardena, CA, USA). For the immunoassay, the JWH-200 high calibrator at 100 ng/mL was prepared in oral fluid extraction buffer (Immunalysis Corporation). The calibrator was stored refrigerated at 4°C.

Enzyme-linked immunosorbent assay

The primary step in the development of the enzyme-linked immunosorbent assay (ELISA) method involved raising polyclonal antibodies, which were based on the immunochemical response of a selected animal species to a synthetic cannabinoid-specific antigen. Rabbits were immunized with an immunogen consisting of JWH-018 conjugated to BTG. JWH-018 was the primary target of interest and easier to chemically modify than JWH-200, so it was chosen as the precursor to the immunogen. The polyclonal serum was purified by affinity chromatography by elution through protein G columns, to obtain the purified IgG fraction and the IgG was then immobilized on 96-well polystyrene microtiter plates. Based on the immune response of the rabbits, the antibody produced showed the highest cross-reactivity to JWH-200, compared with the other parent synthetic cannabinoids, and was chosen for the oral fluid calibrator. Calibration standards were prepared by fortifying negative BSA buffer with JWH-200 at concentrations of 0.1, 0.25, 0.5, 1and 5 ng/mL. The calibration standards were diluted 1 + 3 with drug-free oral fluid extraction buffer.

Each calibrator and specimen (75 μL) was pipetted in duplicate into the wells on the ELISA plate. Before establishing the inclusion of a preincubation of the sample, a routine ELISA with no preincubation step was also assessed. The plate was allowed to preincubate at room temperature for 30 min in the dark. Without washing, the enzyme conjugate (50 μL) was added and the plate was incubated in the dark for 60 min. The plate was then washed six times with deionized water; then TMB substrate (100 μL) was added before allowing the plate to incubate a further 30 min. The reaction was stopped by adding stop solution (100 µL), and the plate absorbance was read at dual wavelengths of 450 and 650 nm. The 650-nm wavelength measures the background absorbance, which the plate reader then subtracts from the final absorbance. The intensity of the color produced is inversely proportional to the concentration of analyte in the sample.

Method validation: ELISA

Selectivity

Interference from related and non-related compounds was studied by spiking various compounds into synthetic negative oral fluid, dilution with Quantisal™ buffer and running them in the ELISA assay. Synthetic oral fluid was used as opposed to real saliva because of the quantity required to carry out the validation experiments. Cross-reactivity data with the compounds closely related in structure and pharmacological activity to JWH-200 were studied at the assay cutoff concentration of 0.25 ng/mL.

Imprecision

ELISA intra- and interday imprecision were performed in oral fluid extraction buffer. Synthetic saliva was fortified with 0.1, 0.25 and 0.5 ng/mL of JWH-200 and then diluted 1 : 4 with oral fluid extraction buffer. The assay was performed using eight replicates of each calibrator on the same day (n = 8) and eight replicates of each calibrator per day, over a 10-day period (n = 80). Imprecision was then calculated as coefficient of variation (CV%) from eight analyses carried out on the same day (intraday), and 80 analyses over 10 days (interday), the criteria for which was <10% CV.

Stability and shelf life of assay

An accelerated stability testing study was performed to determine the approximate shelf life of the ELISA kit. The JWH–HRP enzyme conjugate was held at 37°C for a period of 14 days and during that time assayed at 1- and 2-week intervals. The conjugate performance was compared with the refrigerated drug–enzyme conjugate, stored at 4°C during that same period. The dose–response curve was run at calibration levels of 0.1, 0.25 and 0.5 ng/mL in oral fluid extraction buffer over the 14-day period. This period of 14 days accelerated time testing represents the equivalent of at least 18 months of real-time stability at 4°C (14).

Liquid chromatography tandem mass spectrometry

A Shimadzu Prominence HPLC system connected to AB SCIEX 4000 QTrap mass spectrometer was used for LC–MS-MS analysis. The system was operated in electrospray ionization (ESI) mode with multiple reaction monitoring (MRM). Separation was performed on a Pinnacle DB biphenyl column (5 µm, 50 × 2.1 mm) at a flow rate of 0.5 mL/min. The mobile phases used for the analysis were water + 0.1% formic acid and 0.2% ammonium formate (A) and acetonitrile + 0.1% formic acid and 0.2% ammonium formate (B). The LC gradient started at 10% organic increasing to 90% organic preparation within 6 min, held at 90% organic for 1.5 min and then returned to the starting conditions.

Sample preparation

Five calibrators (0.25, 0.5, 1, 5 and 25 ng/mL), two positive controls (2 and 20 ng/mL) and one negative control prepared in synthetic negative saliva and prediluted 1 : 4 with extraction buffer to account for the dilution of authentic oral fluids in the collection device were included in each batch of specimens. An aliquot of the specimen (100 µL) was removed from the collection tube, diluted 1 : 1 with internal standard (IS; 5 ng/mL of mixed solution of all the seven deuterated standards prepared in dimethylformamide) and centrifuged (15,000 rpm; 2 min). Supernatant (100 µL) was then removed into sample vials and 20 µL of the prepared sample was injected into the LC system.

Data acquisition and analysis

Precursor [M−H]+ and product ions for the identification of JWH-018, JWH-073, JWH-250, JWH-081, JWH-210, RCS-4 and AM-2201 along with their corresponding energy values are summarized in Table I. Two ion transitions were monitored in the MRM mode for each analyte. The quantifier ion was chosen as the most abundant product ion, and the qualifier ion was chosen as the second most abundant product ion. One transition was monitored in the MRM mode for all the ISs. Calculations were performed automatically by the software by measuring the relative abundance of the unknown quantifier product ion and dividing by the relative abundance of the IS quantifier product ion. This ratio is compared with the standard curve of calibrator/IS abundance ratios to determine the concentration of the unknown.

Table I

Mass spectrometric conditions for fragmentation of synthetic cannabinoids

Compound . Q1 Mass . Q3 Mass . Declustering potential (V) . Collision energy (V) . 
AM-2201 1 360.0 155.1 96 37 
AM-2201 2 360.0 127.0 96 67 
AM-2201-D5 363.9 155.0 106 37 
JWH-018 1 342.0 155.1 86 37 
JWH-018 2 342.0 127.0 86 67 
JWH-018-D9 351.2 155.1 111 37 
JWH-073 1 328.0 155.1 71 35 
JWH-073 2 328.0 127.0 71 63 
JWH-073-D7 335.2 155.2 101 35 
JWH-081 1 371.9 185.1 111 37 
JWH-081 2 371.9 157.0 111 59 
JWH-081-D9 380.9 185.1 116 37 
JWH-210 1 369.9 183.1 116 35 
JWH-210 2 369.9 214.1 116 65 
JWH-210-D9 378.9 250.1 51 37 
JWH-250 1 336.2 120.9 86 29 
JWH-250 2 336.2 91.10 86 73 
JWH-250-D5 340.9 121.0 86 31 
RCS-4 1 321.9 135.0 106 33 
RCS-4 2 321.9 77.00 106 75 
RCS-4-D9 330.9 135.0 91 39 
Compound . Q1 Mass . Q3 Mass . Declustering potential (V) . Collision energy (V) . 
AM-2201 1 360.0 155.1 96 37 
AM-2201 2 360.0 127.0 96 67 
AM-2201-D5 363.9 155.0 106 37 
JWH-018 1 342.0 155.1 86 37 
JWH-018 2 342.0 127.0 86 67 
JWH-018-D9 351.2 155.1 111 37 
JWH-073 1 328.0 155.1 71 35 
JWH-073 2 328.0 127.0 71 63 
JWH-073-D7 335.2 155.2 101 35 
JWH-081 1 371.9 185.1 111 37 
JWH-081 2 371.9 157.0 111 59 
JWH-081-D9 380.9 185.1 116 37 
JWH-210 1 369.9 183.1 116 35 
JWH-210 2 369.9 214.1 116 65 
JWH-210-D9 378.9 250.1 51 37 
JWH-250 1 336.2 120.9 86 29 
JWH-250 2 336.2 91.10 86 73 
JWH-250-D5 340.9 121.0 86 31 
RCS-4 1 321.9 135.0 106 33 
RCS-4 2 321.9 77.00 106 75 
RCS-4-D9 330.9 135.0 91 39 

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Table I

Mass spectrometric conditions for fragmentation of synthetic cannabinoids

Compound . Q1 Mass . Q3 Mass . Declustering potential (V) . Collision energy (V) . 
AM-2201 1 360.0 155.1 96 37 
AM-2201 2 360.0 127.0 96 67 
AM-2201-D5 363.9 155.0 106 37 
JWH-018 1 342.0 155.1 86 37 
JWH-018 2 342.0 127.0 86 67 
JWH-018-D9 351.2 155.1 111 37 
JWH-073 1 328.0 155.1 71 35 
JWH-073 2 328.0 127.0 71 63 
JWH-073-D7 335.2 155.2 101 35 
JWH-081 1 371.9 185.1 111 37 
JWH-081 2 371.9 157.0 111 59 
JWH-081-D9 380.9 185.1 116 37 
JWH-210 1 369.9 183.1 116 35 
JWH-210 2 369.9 214.1 116 65 
JWH-210-D9 378.9 250.1 51 37 
JWH-250 1 336.2 120.9 86 29 
JWH-250 2 336.2 91.10 86 73 
JWH-250-D5 340.9 121.0 86 31 
RCS-4 1 321.9 135.0 106 33 
RCS-4 2 321.9 77.00 106 75 
RCS-4-D9 330.9 135.0 91 39 
Compound . Q1 Mass . Q3 Mass . Declustering potential (V) . Collision energy (V) . 
AM-2201 1 360.0 155.1 96 37 
AM-2201 2 360.0 127.0 96 67 
AM-2201-D5 363.9 155.0 106 37 
JWH-018 1 342.0 155.1 86 37 
JWH-018 2 342.0 127.0 86 67 
JWH-018-D9 351.2 155.1 111 37 
JWH-073 1 328.0 155.1 71 35 
JWH-073 2 328.0 127.0 71 63 
JWH-073-D7 335.2 155.2 101 35 
JWH-081 1 371.9 185.1 111 37 
JWH-081 2 371.9 157.0 111 59 
JWH-081-D9 380.9 185.1 116 37 
JWH-210 1 369.9 183.1 116 35 
JWH-210 2 369.9 214.1 116 65 
JWH-210-D9 378.9 250.1 51 37 
JWH-250 1 336.2 120.9 86 29 
JWH-250 2 336.2 91.10 86 73 
JWH-250-D5 340.9 121.0 86 31 
RCS-4 1 321.9 135.0 106 33 
RCS-4 2 321.9 77.00 106 75 
RCS-4-D9 330.9 135.0 91 39 

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Method validation: LC–MS-MS

Recovery from the collection pad

While the recovery of some synthetic cannabinoids (JWH-018, JWH-073, JWH-200, JWH-250, CP-47,497, CP-47,497 C8 and HU-210) from the Quantisal™ device has been previously reported (1), the recovery of a wider range of compounds was studied in this project at three different concentrations. Synthetic oral fluid samples spiked with JWH-018, JWH-081, JWH-073, JWH-250, JWH-210, RCS-4 and AM-2201 at concentrations of 5, 10 and 50 ng/mL (n = 15) were prepared. The collection pad was placed into the samples until 1 mL (±10%) had been collected, the volume adequacy indicator incorporated into the stem of the collector turned blue, then the pad was transferred to the Quantisal™ buffer, capped and stored overnight to simulate transportation to the laboratory. The following day, the tubes were vortexed, pads were separated and an aliquot of the specimen was analyzed as described. The amount recovered from the pad was compared with an absolute concentration (100%), where drug was added to the buffer and left overnight at room temperature without the pad, then subjected to extraction and analysis.

Sensitivity and linearity

The analytical sensitivity for the quantitative methods is expressed in terms of limit of detection (LOD) and lower limit of quantitation (LLOQ). To calculate the LOD and LLOQ for the assay, five replicates of 0.1, 0.25, 0.5 and 1 ng/mL of a mixed solution containing all the seven analytes were tested in the assay.

The LOD was determined as the minimum concentration where the analyte was detected with a signal-to-noise ratio of 10 : 1 and the ion ratios satisfied the ±20% criteria, but the average (AVG) of the five observed values was not within ±20% of the actual spiked concentration. The LLOQ was determined as the minimum concentration where the analyte was detected with a signal-to-noise ratio of 10 : 1, the ion ratios satisfied the ±20% criteria and the AVG of the five observed values was within ±20% of the actual spiked concentration with CVs of <10%.

Linearity studies were performed to determine the range of the assay expressed as LLOQ and upper limit of quantitation (ULOQ). This was carried out by analyzing successively increasing concentrations of the analyte/s. Five replicates of 5, 10, 25, 50, 100, 200 and 500 ng/mL of mixed solutions of all the seven analytes were tested, and the highest concentration for which the % agreement between the fortified value and measured value of an analyte was within ±20% and a CV of <10% between the five replicates was considered the ULOQ for that analyte.

Selectivity

The selectivity for the method was determined by testing for possible interference from specimen matrix (endogenous interference) as well as other spiked drugs (exogenous interference):

  • Endogenous interference: Negative sample matrix (oral fluid) from 20 volunteers was collected and each specimen was tested separately in the assay

  • Exogenous interference: Negative specimen matrix (oral fluid) was spiked at 2,500 ng/mL with the following structurally non-related drugs/compounds and analyzed: 7-aminoclonazepam; 7-aminoflunitrazepam; α-hydroxyalprazolam; α-hydroxytriazolam; amobarbital; amphetamine; benzylpiperazine; butabarbital; butalbital; butylone; carboxy-THC; cathinone; codeine; Δ9-THC; EDDP; ephedrine; ethylone; hydrocodone; hydromorphone; ketamine; lorazepam; meta-Chlorophenylpiperazine (mCPP); MDA; MDMA; methylenedioxypyrovalerone (MDPV); mephedrone; methamphetamine; methcathinone; methedrone; methylone; methylphenidate; morphine; nicotine; nordiazepam; oxazepam; oxycodone; phencyclidine; pentedrone; phenobarbital; phentermine; paramethoxyamphetamine (PMA); paramethoxymethylamphetamine (PMMA); pseudoephedrine; secobarbital; TFMPP.

Imprecision

Intra- and interday imprecision were performed in negative oral fluid spiked with a mixed solution of all the seven analytes at concentrations of 0.5, 10 and 25 ng/mL of neat oral fluid.

Intraday imprecision was calculated as CV% from 15 analyses carried out on the same day (n= 15).
formula
Interday imprecision was calculated from 15 analyses per day, over 5 days (n= 75).
formula

Matrix effect

The matrix effect is the suppression or enhancement of ionization of analytes by the presence of matrix components in biological samples. This is usually encountered in case of ESI techniques in LC–MS-MS analysis. The quantitative measurement of matrix effect provides useful information in the validation of mass spectrometry-based assays. The matrix effect can be termed matrix factor (MF) and defined as the ratio of the peak response in the presence of matrix ions to the analyte peak response in the absence of matrix ions.
formula
IS-normalized MF is the MF of an analyte divided by the MF for IS. An IS-normalized MF can also be obtained by substituting peak response with a peak response ratio (analyte/IS) in the above equation for MF. To predict the variability of matrix effects in samples from individual subjects, MF (or IS-normalized MF) for a minimum of 10 individual lots of matrix (oral fluid) was determined as follows:
formula
Either MF or absolute MF can be used to evaluate the matrix effect. IS-normalized MF is used in this case. Where both the IS and analyte are suppressed or enhanced to the same degree, the overall effect is mitigated.
  • Ten blank matrices (Quantisal collection device buffer) and 10 real matrices (drug-free oral fluid collected from individuals in the lab) were spiked with 2.5 ng/mL of JWH-018, JWH-073, JWH-081, JWH-210, JWH-250, AM-2201 and RCS-4 and analyzed using the assay.

  • Ten blank matrices (Quantisal collection device buffer) and 10 real matrices (drug-free oral fluid collected from individuals in the lab) were also spiked with the ISs: JWH-018-D9, JWH-073-D7, JWH-081-D9, JWH-210-D9, JWH-250-D5, AM-2201-D5 and RCS-4-D9 and analyzed using the assay.

  • Observed peak area values for all ISs with and without matrix were recorded.

  • AVG, standard deviation (STD) and coefficient of variation (CV%) for the values of 10 specimen matrices were calculated. Acceptance criteria for the MF were CVs of <10% between the 10 MF values.

  • The absolute MF was calculated using the equation below with CVs of <10% as the acceptance for absolute MF.

Stability of the synthetic cannabinoids in oral fluid

The stability of the synthetic cannabinoids in Quantisal™ buffer-oral fluid was determined at a concentration of 10 ng/mL. Spiked standards were maintained at room temperature, 4°C and −20°C for 8 weeks; aliquots were removed and analyzed at various time intervals: 1, 2, 4, 7, 15, 28 days, 6 and 8 weeks.

Authentic samples

Thirty-two authentic oral fluid specimens collected during routine testing for probation and parole were analyzed using the validated LC–MS-MS, and then assessed using the newly developed ELISA assay in order to determine the utility of the kit.

Results and discussion

Enzyme-linked immunosorbent assay

Dose–response curve

The method utilizes competitive binding between the enzyme conjugate and free analyte in the sample for a fixed amount of antibody-binding sites, proportional to their concentration in the mixture. The dose–response curve for JWH-200 was prepared in oral fluid extraction buffer, at the levels described earlier. B0 is the absorbance of the negative calibrator and B represents the absorbance of the individual calibrator levels. The percentage ratio of individual calibrator to negative calibrator (B/B0) was calculated for each calibrator level and plotted against the drug concentration (ng/mL). Figure 2 shows the dose–response curves plotted for oral fluid fortified with JWH-200 with and without a preincubation stage. The B/B0 values are inversely proportional to the concentration of drug in the sample, the reason being that the higher the drug concentration the less drug–enzyme conjugate binds to the antibody, thereby producing a lower absorbance value. As a general rule, the point at which the dose–response curve shows the greatest difference between calibration points (most linear) is selected as the cutoff concentration; in this case, 0.25 ng/mL consistently produced a B/B0 percentage of 60% when preincubation is included. Without preincubation, adequate separation is not achieved until concentrations of 1 ng/mL and above (Figure 2).

Selectivity

Table II summarizes the cross-reactivity data with the compounds closely related in structure and pharmacological activity to JWH-200, at the assay cutoff concentration of 0.25 ng/mL. None of the nonstructurally related compounds was found to interfere with the detection of synthetic cannabinoids in the assay. Matrix effects from synthetic cannabinoid-free authentic oral fluid specimens were also studied to determine their effects on the assay. No false-positive results were observed due to the matrix, when LC–MS-MS confirmed synthetic cannabinoid-free oral fluid specimens were screened with the assay.

Table II

Cross-reactivity with structurally and pharmacologically related compounds and metabolites

Compound . Concentration of drug added (ng/mL) . Equivalent response to JWH-200 at 0.25 ng/mL . Calculated cross-reactivity (%) . 
JWH-200 0.25 0.25 100 
JWH-018 0.14 14 
JWH-022 0.35 0.10 29 
JWH-073 0.2 0.13 65 
JWH-122 0.09 
JWH-081 50 0.16 0.32 
JWH-201 400 0.07 0.02 
JWH-210 100 0.27 0.27 
JWH-250 400 0.10 0.03 
JWH-015 1.2 0.14 12 
JWH-007 0.13 
JWH-019 0.8 0.07 
JWH-398 0.08 
AM-1220 0.1 0.07 70 
AM-2201 0.2 0.09 45 
AM-2232 0.09 
AM-2233 0.11 
XLR-11 10,000 3.4 0.03 
UR-144 10,000 Not detected 
JWH-018 N-pentanoic acid 0.09 
JWH-073 N-butanoic acid 0.1 0.16 160 
JWH-018 N-(5-hydroxypentyl) 0.08 0.11 138 
JWH-018 4-hydroxyindole 0.19 
JWH-018 5-hydroxyindole 20 0.92 
JWH-018 N-(5-hydroxypentyl)-β-d-glucuronide 0.1 0.13 130 
JWH-073 N-(4-hydroxybutyl) 0.06 0.10 167 
JWH-073 6-hydroxyindole 0.2 0.09 45 
JWH-122 N-(4-hydroxypentyl) 0.8 0.18 22 
AM-2201 N-(4-hydroxypentyl) 0.01 0.12 1,200 
AM-2201 6-hydroxyindole 0.4 0.22 55 
3-(1-Naphthoyl)-1H-indole 1.2 0.16 13 
Cannabipiperidiethanone 400 0.12 0.03 
Compound . Concentration of drug added (ng/mL) . Equivalent response to JWH-200 at 0.25 ng/mL . Calculated cross-reactivity (%) . 
JWH-200 0.25 0.25 100 
JWH-018 0.14 14 
JWH-022 0.35 0.10 29 
JWH-073 0.2 0.13 65 
JWH-122 0.09 
JWH-081 50 0.16 0.32 
JWH-201 400 0.07 0.02 
JWH-210 100 0.27 0.27 
JWH-250 400 0.10 0.03 
JWH-015 1.2 0.14 12 
JWH-007 0.13 
JWH-019 0.8 0.07 
JWH-398 0.08 
AM-1220 0.1 0.07 70 
AM-2201 0.2 0.09 45 
AM-2232 0.09 
AM-2233 0.11 
XLR-11 10,000 3.4 0.03 
UR-144 10,000 Not detected 
JWH-018 N-pentanoic acid 0.09 
JWH-073 N-butanoic acid 0.1 0.16 160 
JWH-018 N-(5-hydroxypentyl) 0.08 0.11 138 
JWH-018 4-hydroxyindole 0.19 
JWH-018 5-hydroxyindole 20 0.92 
JWH-018 N-(5-hydroxypentyl)-β-d-glucuronide 0.1 0.13 130 
JWH-073 N-(4-hydroxybutyl) 0.06 0.10 167 
JWH-073 6-hydroxyindole 0.2 0.09 45 
JWH-122 N-(4-hydroxypentyl) 0.8 0.18 22 
AM-2201 N-(4-hydroxypentyl) 0.01 0.12 1,200 
AM-2201 6-hydroxyindole 0.4 0.22 55 
3-(1-Naphthoyl)-1H-indole 1.2 0.16 13 
Cannabipiperidiethanone 400 0.12 0.03 

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Table II

Cross-reactivity with structurally and pharmacologically related compounds and metabolites

Compound . Concentration of drug added (ng/mL) . Equivalent response to JWH-200 at 0.25 ng/mL . Calculated cross-reactivity (%) . 
JWH-200 0.25 0.25 100 
JWH-018 0.14 14 
JWH-022 0.35 0.10 29 
JWH-073 0.2 0.13 65 
JWH-122 0.09 
JWH-081 50 0.16 0.32 
JWH-201 400 0.07 0.02 
JWH-210 100 0.27 0.27 
JWH-250 400 0.10 0.03 
JWH-015 1.2 0.14 12 
JWH-007 0.13 
JWH-019 0.8 0.07 
JWH-398 0.08 
AM-1220 0.1 0.07 70 
AM-2201 0.2 0.09 45 
AM-2232 0.09 
AM-2233 0.11 
XLR-11 10,000 3.4 0.03 
UR-144 10,000 Not detected 
JWH-018 N-pentanoic acid 0.09 
JWH-073 N-butanoic acid 0.1 0.16 160 
JWH-018 N-(5-hydroxypentyl) 0.08 0.11 138 
JWH-018 4-hydroxyindole 0.19 
JWH-018 5-hydroxyindole 20 0.92 
JWH-018 N-(5-hydroxypentyl)-β-d-glucuronide 0.1 0.13 130 
JWH-073 N-(4-hydroxybutyl) 0.06 0.10 167 
JWH-073 6-hydroxyindole 0.2 0.09 45 
JWH-122 N-(4-hydroxypentyl) 0.8 0.18 22 
AM-2201 N-(4-hydroxypentyl) 0.01 0.12 1,200 
AM-2201 6-hydroxyindole 0.4 0.22 55 
3-(1-Naphthoyl)-1H-indole 1.2 0.16 13 
Cannabipiperidiethanone 400 0.12 0.03 
Compound . Concentration of drug added (ng/mL) . Equivalent response to JWH-200 at 0.25 ng/mL . Calculated cross-reactivity (%) . 
JWH-200 0.25 0.25 100 
JWH-018 0.14 14 
JWH-022 0.35 0.10 29 
JWH-073 0.2 0.13 65 
JWH-122 0.09 
JWH-081 50 0.16 0.32 
JWH-201 400 0.07 0.02 
JWH-210 100 0.27 0.27 
JWH-250 400 0.10 0.03 
JWH-015 1.2 0.14 12 
JWH-007 0.13 
JWH-019 0.8 0.07 
JWH-398 0.08 
AM-1220 0.1 0.07 70 
AM-2201 0.2 0.09 45 
AM-2232 0.09 
AM-2233 0.11 
XLR-11 10,000 3.4 0.03 
UR-144 10,000 Not detected 
JWH-018 N-pentanoic acid 0.09 
JWH-073 N-butanoic acid 0.1 0.16 160 
JWH-018 N-(5-hydroxypentyl) 0.08 0.11 138 
JWH-018 4-hydroxyindole 0.19 
JWH-018 5-hydroxyindole 20 0.92 
JWH-018 N-(5-hydroxypentyl)-β-d-glucuronide 0.1 0.13 130 
JWH-073 N-(4-hydroxybutyl) 0.06 0.10 167 
JWH-073 6-hydroxyindole 0.2 0.09 45 
JWH-122 N-(4-hydroxypentyl) 0.8 0.18 22 
AM-2201 N-(4-hydroxypentyl) 0.01 0.12 1,200 
AM-2201 6-hydroxyindole 0.4 0.22 55 
3-(1-Naphthoyl)-1H-indole 1.2 0.16 13 
Cannabipiperidiethanone 400 0.12 0.03 

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Imprecision

Both intra- and interday imprecision were found to be <7% at all concentrations (Table III).

Table III

Intra- and interday imprecision for ELISA immunoassay

Drug concentration (ng/mL) . Mean absorbance . CV% . 
Intraday precision (n = 8) 
 0 3.42 1.91 
 0.1 2.71 3.17 
 0.25 2.07 6.07 
 0.5 1.53 5.52 
Interday precision (n = 80) 
 0 3.37 2.75 
 0.1 2.70 6.65 
 0.25 2.22 4.65 
 0.5 1.79 6.98 
Drug concentration (ng/mL) . Mean absorbance . CV% . 
Intraday precision (n = 8) 
 0 3.42 1.91 
 0.1 2.71 3.17 
 0.25 2.07 6.07 
 0.5 1.53 5.52 
Interday precision (n = 80) 
 0 3.37 2.75 
 0.1 2.70 6.65 
 0.25 2.22 4.65 
 0.5 1.79 6.98 

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Table III

Intra- and interday imprecision for ELISA immunoassay

Drug concentration (ng/mL) . Mean absorbance . CV% . 
Intraday precision (n = 8) 
 0 3.42 1.91 
 0.1 2.71 3.17 
 0.25 2.07 6.07 
 0.5 1.53 5.52 
Interday precision (n = 80) 
 0 3.37 2.75 
 0.1 2.70 6.65 
 0.25 2.22 4.65 
 0.5 1.79 6.98 
Drug concentration (ng/mL) . Mean absorbance . CV% . 
Intraday precision (n = 8) 
 0 3.42 1.91 
 0.1 2.71 3.17 
 0.25 2.07 6.07 
 0.5 1.53 5.52 
Interday precision (n = 80) 
 0 3.37 2.75 
 0.1 2.70 6.65 
 0.25 2.22 4.65 
 0.5 1.79 6.98 

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Stability and shelf life of assay

The assay was found to show very similar dose responses over the entire 2-week accelerated time study. The dose response on Day 1 of the study, at concentrations of 0.1 and 0.25 ng/mL, corresponded to 83 and 64% B/B0, respectively; Day 7 (4°C): 83 and 66%; (37°C): 81 and 64%, respectively; Day 14 (4°C): 81 and 64%; (37°C): 80 and 62%, respectively.

Liquid chromatography tandem mass spectrometry

Method validation

Recovery from the collection pad

The recovery of the synthetic cannabinoids from the collection device was not concentration dependent. The lowest recovery for any analyte was 68% for JWH-018 (Figure 3).

Sensitivity, linearity and reportable range

The LOD and LLOQ were determined to be 0.1 and 0.25 ng/mL, respectively, for all analytes; the ULOQ was 50 ng/mL for JWH-018, JWH-073, JWH-250, JWH-210 and 100 ng/mL for JWH-081, RCS-4 and AM-2201.

Selectivity

None of the negative specimen matrices produced a positive response on the assay, and none of the spiked substances showed a peak/response at the expected retention time of any of the analytes included in the assay at or above the LOD of the specific analyte.

Imprecision

Both intra- and interday imprecision for all the seven analytes were found to be <10%.

Matrix effects

The results for the absolute MF calculations are summarized in Tables IV and V. The AVG absolute MF for all analytes was close to 1 with CVs of <8.1% between the 10 individual matrices indicating no significant matrix effect in the assay. An MF of 1 signifies no matrix effect; a value of <1 suggests ionization suppression and can result in diminished sensitivity. An MF of >1 may be due to ionization enhancement and can also be caused by analyte loss in the absence of matrix during analysis. Stable isotope-labeled IS minimizes the influence of matrix effects most effectively since the matrix effects observed for stable isotope-labeled IS are generally similar to those observed for the matching analyte. An IS-normalized MF of ∼1 is not necessary for a reliable assay; however, highly variable MF in individual subjects may cause a lack of reproducibility.

Table IV

Matrix factors in LC–MS-MS

JWH-018 . JWH-073 . JWH-250 . JWH-210 . JWH-081 . AM-2201 . RCS-4 . 
Average MF (n = 10) 0.99 1.12 1.02 1.06 1.06 0.98 1.05 
STD 0.06 0.03 0.07 0.06 0.03 0.04 0.03 
CV% 3.0 
JWH-018 . JWH-073 . JWH-250 . JWH-210 . JWH-081 . AM-2201 . RCS-4 . 
Average MF (n = 10) 0.99 1.12 1.02 1.06 1.06 0.98 1.05 
STD 0.06 0.03 0.07 0.06 0.03 0.04 0.03 
CV% 3.0 

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Table IV

Matrix factors in LC–MS-MS

JWH-018 . JWH-073 . JWH-250 . JWH-210 . JWH-081 . AM-2201 . RCS-4 . 
Average MF (n = 10) 0.99 1.12 1.02 1.06 1.06 0.98 1.05 
STD 0.06 0.03 0.07 0.06 0.03 0.04 0.03 
CV% 3.0 
JWH-018 . JWH-073 . JWH-250 . JWH-210 . JWH-081 . AM-2201 . RCS-4 . 
Average MF (n = 10) 0.99 1.12 1.02 1.06 1.06 0.98 1.05 
STD 0.06 0.03 0.07 0.06 0.03 0.04 0.03 
CV% 3.0 

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Table V

Internal standard-normalized matrix factors in LC–MS-MS

JWH-018/JWH-018-D9 . JWH-073/JWH-073-D7 . JWH-250/JWH-250-D5 . JWH-210/JWH-210-D9 . JWH-081/JWH-081-D9 . AM-2201/AM-2201-D5 . RCS-4/RCS-4-D9 . 
Absolute MF (IS normalized, n = 10) 0.97 1.04 1.07 1.04 1.06 1.02 0.94 
STD 0.07 0.08 0.06 0.04 0.03 0.08 0.06 
CV% 7.2 7.5 5.9 4.1 3.0 8.1 6.4 
JWH-018/JWH-018-D9 . JWH-073/JWH-073-D7 . JWH-250/JWH-250-D5 . JWH-210/JWH-210-D9 . JWH-081/JWH-081-D9 . AM-2201/AM-2201-D5 . RCS-4/RCS-4-D9 . 
Absolute MF (IS normalized, n = 10) 0.97 1.04 1.07 1.04 1.06 1.02 0.94 
STD 0.07 0.08 0.06 0.04 0.03 0.08 0.06 
CV% 7.2 7.5 5.9 4.1 3.0 8.1 6.4 

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Table V

Internal standard-normalized matrix factors in LC–MS-MS

JWH-018/JWH-018-D9 . JWH-073/JWH-073-D7 . JWH-250/JWH-250-D5 . JWH-210/JWH-210-D9 . JWH-081/JWH-081-D9 . AM-2201/AM-2201-D5 . RCS-4/RCS-4-D9 . 
Absolute MF (IS normalized, n = 10) 0.97 1.04 1.07 1.04 1.06 1.02 0.94 
STD 0.07 0.08 0.06 0.04 0.03 0.08 0.06 
CV% 7.2 7.5 5.9 4.1 3.0 8.1 6.4 
JWH-018/JWH-018-D9 . JWH-073/JWH-073-D7 . JWH-250/JWH-250-D5 . JWH-210/JWH-210-D9 . JWH-081/JWH-081-D9 . AM-2201/AM-2201-D5 . RCS-4/RCS-4-D9 . 
Absolute MF (IS normalized, n = 10) 0.97 1.04 1.07 1.04 1.06 1.02 0.94 
STD 0.07 0.08 0.06 0.04 0.03 0.08 0.06 
CV% 7.2 7.5 5.9 4.1 3.0 8.1 6.4 

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Stability of the synthetic cannabinoids in oral fluid

The stability of the various synthetic cannabinoids followed the same trend. At room temperature, <40% of the original concentration remained after 7 days; and after 6 weeks <20% remained. At 4°C, 50–60% of the original concentration remained after 7 days and after 8 weeks 25–35% was remaining. When stored frozen at −20°C, 50–60% remained after 7 days, but by 8 weeks 30–40% was still intact. For long-term storage, the specimens should be frozen.

Authentic specimens

Thirty-two authentic oral fluid specimens collected using the Quantisal™ device were obtained from Redwood Toxicology Laboratory and analyzed using the newly developed ELISA. The results are presented in Table VI. These specimens were confirmed by LC–MS-MS and shown to contain AM-2201 (0.25–80.5 ng/mL), along with either JWH-018 (0.36–12.5 ng/mL) or JWH-210 (0.36–27.7 ng/mL). None of the specimens was positive for JWH-073, JWH-081, JWH-250 or RCS-4. Five specimens (15%) were negative using the ELISA assay at a cutoff concentration of 0.25 ng/mL. Specimen #2 was confirmed to contain JWH-210, which does not cross-react with the assay; the other four specimens (13, 16, 19 and 20) all were confirmed to have AM-2201 present at concentrations of <0.5 ng/mL. As the cross-reactivity of the kit to AM-2201 is 43%, these specimens were just below the equivalent cutoff level. Between the LC–MS-MS assay and the ELISA analysis, the samples were stored at −20°C. Stability of these drugs in oral fluid could, therefore, be a contributing factor to any discrepancies, because the LC–MS-MS assay and ELISA analysis were not concurrently performed on the specimens. One specimen (#21) contained 0.32 ng/mL of AM-2201, which should have caused the ELISA to be negative; however, it screened positively indicating the possible presence of other synthetic cannabinoids that do not show significant cross-reactivity with the ELISA kit but may cause a synergistic effect at higher concentrations. For this reason, the LC–MS-MS profile is currently being extended to include JWH-203, and JWH-122 as well as the newer synthetic cannabinoids such as 1-pentyl-1H-indol-3-yl (2,2,3,3-tetramethylcyclopropyl) methanone (UR-144) and 1-(5-fluoropentyl)-1H-indol-3-yl (2,2,3,3-tetramethylcyclopropyl) methanone (XLR-11). The total ion chromatogram of specimen #30, which was confirmed to contain AM-2201, JWH-018 and JWH-210, is shown in Figure 4. There are numerous synthetic cannabinoids available, not all of which would be detected by a single ELISA screen. ELISA is still, however, effective as a cost-lowering screening method when used in conjunction with LC–MS-MS analysis for confirmation.

Table VI

Results from authentic specimens

Specimen # . ELISA result . LC–MS-MS concentration (ng/mL)
AM-2201 . JWH-018 . JWH-210 . 
Positive 0.66 
Negative 0.36 
Negative 
Positive 13.9 
Positive 7.20 0.36 
Positive 11.0 0.57 
Negative 
Positive 80.5 3.51 
Negative 
10 Positive 2.80 
11 Borderline positive 0.40 
12 Positive 4.13 
13 Negative 0.28 
14 Negative 
15 Negative 
16 Negative 0.36 
17 Borderline positive 0.43 
18 Negative 
19 Negative 0.33 
20 Negative 0.25 
21 Positive 0.32 
22 Positive 1.22 
23 Positive 0.64 0.51 
24 Borderline positive 0.27 
25 Positive 0.25 
26 Positive 12.5 
27 Borderline positive 0.47 
28 Positive 4.20 
29 Positive 2.70 
30 Positive 63.7 0.59 27.7 
31 Positive 2.00 1.00 
32 Positive 1.37 
Specimen # . ELISA result . LC–MS-MS concentration (ng/mL)
AM-2201 . JWH-018 . JWH-210 . 
Positive 0.66 
Negative 0.36 
Negative 
Positive 13.9 
Positive 7.20 0.36 
Positive 11.0 0.57 
Negative 
Positive 80.5 3.51 
Negative 
10 Positive 2.80 
11 Borderline positive 0.40 
12 Positive 4.13 
13 Negative 0.28 
14 Negative 
15 Negative 
16 Negative 0.36 
17 Borderline positive 0.43 
18 Negative 
19 Negative 0.33 
20 Negative 0.25 
21 Positive 0.32 
22 Positive 1.22 
23 Positive 0.64 0.51 
24 Borderline positive 0.27 
25 Positive 0.25 
26 Positive 12.5 
27 Borderline positive 0.47 
28 Positive 4.20 
29 Positive 2.70 
30 Positive 63.7 0.59 27.7 
31 Positive 2.00 1.00 
32 Positive 1.37 

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Table VI

Results from authentic specimens

Specimen # . ELISA result . LC–MS-MS concentration (ng/mL)
AM-2201 . JWH-018 . JWH-210 . 
Positive 0.66 
Negative 0.36 
Negative 
Positive 13.9 
Positive 7.20 0.36 
Positive 11.0 0.57 
Negative 
Positive 80.5 3.51 
Negative 
10 Positive 2.80 
11 Borderline positive 0.40 
12 Positive 4.13 
13 Negative 0.28 
14 Negative 
15 Negative 
16 Negative 0.36 
17 Borderline positive 0.43 
18 Negative 
19 Negative 0.33 
20 Negative 0.25 
21 Positive 0.32 
22 Positive 1.22 
23 Positive 0.64 0.51 
24 Borderline positive 0.27 
25 Positive 0.25 
26 Positive 12.5 
27 Borderline positive 0.47 
28 Positive 4.20 
29 Positive 2.70 
30 Positive 63.7 0.59 27.7 
31 Positive 2.00 1.00 
32 Positive 1.37 
Specimen # . ELISA result . LC–MS-MS concentration (ng/mL)
AM-2201 . JWH-018 . JWH-210 . 
Positive 0.66 
Negative 0.36 
Negative 
Positive 13.9 
Positive 7.20 0.36 
Positive 11.0 0.57 
Negative 
Positive 80.5 3.51 
Negative 
10 Positive 2.80 
11 Borderline positive 0.40 
12 Positive 4.13 
13 Negative 0.28 
14 Negative 
15 Negative 
16 Negative 0.36 
17 Borderline positive 0.43 
18 Negative 
19 Negative 0.33 
20 Negative 0.25 
21 Positive 0.32 
22 Positive 1.22 
23 Positive 0.64 0.51 
24 Borderline positive 0.27 
25 Positive 0.25 
26 Positive 12.5 
27 Borderline positive 0.47 
28 Positive 4.20 
29 Positive 2.70 
30 Positive 63.7 0.59 27.7 
31 Positive 2.00 1.00 
32 Positive 1.37 

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The absorbance (B) of the low control (0.1 ng/mL) divided by the absorbance of the drug-free matrix (B0) expressed as a percentage is ∼78–80% (Figure 2), which is considered to be too high to use as a strict cutoff and, therefore, 0.25 ng/mL was recommended (B/B0 = 60–62%). Using the low control in the ELISA assay (concentration 0.1 ng/mL) as a decision point would have caused all the specimens to be confirmed.

Advantages and drawbacks

The immunoassay is predominantly targeted at the JWH-structured compounds and shows limited cross-reactivity to the newer synthetic cannabinoids such as XLR-11. The ability to identify these synthetic compounds continues to be difficult as more compounds are detected in herbal products on a regular basis (15, 16). A recent report from Japan discussed the detection in illegal products of a new class of designer drugs such as URB-754 (6-methyl-2-[(4-methylphenyl) amino]-1-benzoxazin-4-one) which inhibit an endocannabinoid-deactivating enzyme (17). Due to the varied structures of the synthetic cannabinoids, a single immunoassay or one LC–MS-MS procedure will not be adequate for the detection of all possible drugs in the class. As compounds are legally prohibited, other structurally related compounds tend to appear; however, a recent resurgence in JWH-018 in Germany has been reported (18).

Summary

The development and validation of an immunoassay for the determination of ‘Spice’ compounds in oral fluid is reported for the first time. While immunoassays for synthetic cannabinoids in urine are commercially available, to date, there are none for oral fluid. The inclusion of a preincubation step is critical to the improved sensitivity of the assay in order to detect low concentrations present in the oral fluid of synthetic cannabinoid users. The procedure is applicable to the analysis of specimens collected using the Quantisal™ device for the presence of synthetic cannabinoids. Over 84% of confirmed oral fluid specimens were identified by the ELISA assay using 0.25 ng/mL as a cutoff concentration.

Conflict of Interest

Warren C. Rodrigues, Philip Catbagan, Guohong Wang and Christine Moore are employed by Immunalysis Corporation, which manufactures the Quantisal™ oral fluid collection device and the ELISA assay described here. Sumandeep Rana is an employee of Redwood Toxicology Laboratory, a facility that offers commercial analysis of oral fluid specimens for synthetic cannabinoids.

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Liquid am 2201

Pharmacodynamic Effects, Pharmacokinetics, and Metabolism of the Synthetic Cannabinoid AM-2201 in Male Rats

Novel synthetic cannabinoids are appearing in recreational drug markets worldwide. Pharmacological characterization of these new drugs is needed to inform clinicians, toxicologists, and policy makers who monitor public health. [1-(5-Fluoropentyl)-1H-indol-3-yl](1-naphthyl)methanone (AM-2201) is an abused synthetic cannabinoid that was initially created as a research tool for investigating the endocannabinoid system. Here we measured the pharmacodynamic effects of AM-2201 in rats, and simultaneously determined plasma pharmacokinetics for the parent drug and its metabolites. Male Sprague-Dawley rats were fitted with surgically implanted temperature transponders and indwelling jugular catheters under pentobarbital anesthesia. One week later, rats received subcutaneous injection of AM-2201 (0.1, 0.3, and 1.0 mg/kg) or its vehicle, and serial blood specimens were withdrawn via catheters. Core temperatures and catalepsy were measured just prior to each blood withdrawal, and plasma was assayed for drug and metabolites using liquid chromatography-tandem mass spectrometry. We found that AM-2201 produced dose-related hypothermia and catalepsy that peaked at 2 hours and lasted up to 8 hours. AM-2201 plasma concentrations rose linearly with increasing dose and ranged from 0.14 to 67.9 µg/l. Concentrations of three metabolites, AM-2201 N-(4-hydroxypentyl) (≤0.17 µg/l), naphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH-018) N-(5-hydroxypentyl) (≤1.14 µg/l), and JWH-018 N-pentanoic acid (≤0.88 µg/l) were detectable but much lower. Peak AM-2201, JWH-018 N-(5-hydroxypentyl), and JWH-018 N-pentanoic acid concentrations occurred at 1.3, 2.4, and 6.5 hours, respectively. Concentrations of AM-2201, JWH-018 N-(5-hydroxypentyl), and JWH-018 N-pentanoic acid were negatively correlated with body temperature, but, given the low concentrations of metabolites detected, AM-2201 is likely the major contributor to pharmacodynamic effects under our experimental conditions.

Sours: https://pubmed.ncbi.nlm.nih.gov/30266766/
LEBIH ENAK 3MG atau 6MG NYA SIH? BANDINGIN LANGSUNG SI CROFFLEBOY

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