Abstract
Synthetic cannabinoids emerged on the designer drug market in recent years due to their ability to produce cannabis-like effects without the risk of detection by traditional drug testing techniques such as immunoassay and gas chromatography–mass spectrometry. As government agencies work to schedule existing synthetic cannabinoids, new, unregulated and structurally diverse compounds continue to be developed and sold. Synthetic cannabinoids undergo extensive metabolic conversion. Consequently, both blood and urine specimens may play an important role in the forensic analysis of synthetic cannabinoids. It has been observed that structurally similar synthetic cannabinoids follow common metabolic pathways, which often produce metabolites with similar metabolic transformations. Presented are two validated quantitative methods for extracting and identifying 15 parent synthetic cannabinoids in blood, 17 synthetic cannabinoid metabolites in urine and the qualitative identification of 2 additional parent compounds. The linear range for most synthetic cannabinoid compounds monitored was 0.1–10 ng/mL with the limit of detection between 0.01 and 0.5 ng/mL. Selectivity, specificity, accuracy, precision, recovery and matrix effect were also examined and determined to be acceptable for each compound. The validated methods were used to analyze a compilation of synthetic cannabinoid investigative cases where both blood and urine specimens were submitted. The study suggests a strong correlation between the metabolites detected in urine and the parent compounds found in blood.
Introduction
Synthetic cannabinoids were first brought to the public's attention in late 2008 when Auwärter et al. (1) reported results from an investigation into herbal incense products marketed at headshops and over the Internet that were described as reporting cannabis-like effects. After collecting blood and urine samples from the individuals consuming these products, it was determined that not only naturally occurring herbs but cannabinomimetic drugs were present in these products. These compounds had gone undetected using routine immunoassay and gas chromatography–mass spectrometry screening techniques indicating that these analyses would likely be ineffective for this group of compounds. This report also provided the first indications of the complex and confounding identification issue forensic drug testing laboratories would soon be confronting. Subsequently, an expanding array of synthetic cannabinoids has emerged on the market targeting individuals looking for the cannabis-like effects without the risk of detection.
Synthetic cannabinoids were originally developed to investigate the structure–activity relationships of CB1 and CB2 receptors in the endocannabinoid system and aid in the treatment of symptoms associated with a number of diseases (2–5). CB1 receptors, located primarily in the central nervous system, mediate the physiological and psychotropic effects; while CB2 receptors, located primarily in the immune system, mediate activity against neuropathic and inflammatory pain (2, 3, 6). Many of the synthetic cannabinoids bind to these receptors with greater affinity than Δ9-tetrahydrocannabinol (THC). Since this discovery, the pharmaceutical research community has manufactured hundreds of structurally diverse synthetic cannabinoid analogs with varying degrees of selectivity to the two receptor subtypes (2–5). These compounds are also being manufactured by clandestine laboratories for the sole purpose of illicit consumption and pose a significant challenge to the law enforcement, medical and forensic toxicology communities.
JWH 018, CP47,497, CP47,497-C8 homolog and oleamide were the first cannabinoids where human use was reported (1). This led German authorities to schedule these compounds in an effort to combat potential abuse. However, it was discovered that these recently scheduled compounds were quickly replaced by JWH 073 (7, 8) followed by JWH 015, JWH 081, JWH 122, JWH 200, JWH 250 and JWH 398 (8–10). It soon became apparent that as government agencies scheduled specific compounds or even classes of compounds, manufacturers would release a new synthetic cannabinoid containing minor structural modifications (7, 8, 11–14). The new structural variants commonly differ only by the addition of a halogen or modification of part or all of the aliphatic side chain. Additional modifications have included the tetramethylcyclopropyl (TMCP) ring seen in XLR11 and UR-144, the admantyl group seen in AKB48, 2NE1 and STS-135 and the 8-hydroxyquiniline group seen in PB-22 and BB-22. Furthermore, chemists continue to develop new cannabinoids, such as AB-PINACA, which was first described in the scientific literature in 2013 (15). Despite being marketed and sold as the same product, two identical packages may contain an entirely different spectrum of synthetic cannabinoids or unsuspected additives such as O-desmethyltramadol (8), phenazepam and lidocaine (12). In Japan, pharmacologically distinct designer drugs such as cathinones and tryptamines have also been detected in synthetic cannabinoid products (16, 17).
The constant evolution of synthetic cannabinoids available to the public makes it nearly impossible for analytical laboratories to detect and identify the myriad of emerging compounds. After a new compound enters the market, it often takes months for these laboratories to develop the tools and methodology to test for the compound. This process is made more difficult by the delayed availability of certified reference standards. Additionally, validation procedures and comprehensive metabolic studies need to be completed to identify the most appropriate metabolites for forensic analysis. Compounds may go undetected and significant numbers of forensic samples may be reported as negative before the laboratory realizes a new compound is being used.
Due to the extensive metabolism of synthetic cannabinoids, the parent compounds are rarely seen in the urine (18–25). Workplace testing, anti-doping and military programs which monitor urine samples only may miss synthetic cannabinoid positive samples unless they are monitoring metabolites. Dresen et al (10) reported early on the importance of developing analytical methodologies to detect parent compounds in the blood but also recognized that it is imperative to correlate blood samples to their major urinary metabolites.
Numerous in vitro and in vivo publications have shown the major metabolic pathways to be mono-hydroxylation of the N-alkyl side chain, the naphthyl, indole and adamantyl moieties and/or further oxidation to the carboxylic acid on the alkyl chain (18–29). In addition to these metabolic modifications, halogenated compounds, such as AM694, AM2201 and XLR11, will undergo an enzymatic dehalogenation (22, 23, 26). Urinary excretion profiles also indicate that synthetic cannabinoids undergo phase 2 metabolism and are primarily excreted as glucuronides (18, 19, 23–28). Use of these common metabolic schemes may be used to predict the metabolic products of structurally similar compounds. For example, AM2201 is the fluorinated analog of JWH 018. One route of metabolism of AM2201 is defluorination, causing the eventual production of hydroxyl and carboxyl metabolites that are structurally identical to metabolites of JWH 018 (23). Consequently, the detection of JWH 018 metabolites may indicate use of AM2201, JWH 018 or both making the interpretation of these metabolic results challenging.
While urine is the preferred matrix to indicate past exposure of ingested substances, there are advantages to analyzing whole blood. Forensically, analyzing blood increases the probability of identifying the ingested parent compound. Additionally, only blood concentrations allow an estimate to be made concerning levels of impairment based on pharmacological activity. At present, there are few studies reporting effective dose–response relationships for synthetic cannabinoids. Teske et al (29) published an early quantitative procedure for JWH 018 in serum after conducting a self-administration experiment. Two additional quantitative methods for serum followed, establishing analytical procedures for 8 and 27 synthetic cannabinoids (10, 30), respectively. These methods, developed and validated in the same laboratory, demonstrated the rapidly changing profile of available compounds observed in Germany as synthetic cannabinoids underwent scheduling. The first publication concerned with whole blood presented a method for the identification and quantification of JWH 018, JWH 073 and JWH 250 (31). Shanks et al. (32) published a quantitation method for JWH 018 and JWH 073 involving postmortem casework, Ammann et al. (33) published a validation method for 25 parent compounds and Kronstrand et al. (34) published a quantitation method for 29 parent compounds along with related toxicological casework.
The following paper presents validated procedures for identifying and quantifying 15 parent synthetic cannabinoids in blood along with their corresponding urine metabolites using liquid chromatography tandem mass spectrometry (LC–MS-MS). The validated procedures were applied to a compilation of blood and urine results from specimens collected over a 4-year period.
Experimental
Chemicals and reagents
All organic solvents were high-performance liquid chromatography (HPLC) grade or better and were purchased from Fisher Scientific (Pittsburgh, PA). Hydrochloric acid was also purchased from Fisher Scientific. Formic acid was purchased from Aldrich (Milwaukee, WI). Ammonium bicarbonate, β-glucuronidase from E. coli, sodium bicarbonate, sodium carbonate, sodium phosphate monobasic and dibasic were purchased from Sigma-Aldrich Chemicals (St. Louis, MO). AKB48, AM2201, BB-22, JWH 018, JWH 073, JWH 081, JWH 122, JWH 200, JWH 210, JWH 250, MAM2201, PB-22, RCS-4, STS-135, UR-144, XLR11, 2NE1, AM2201-d5, JWH 018-d9, JWH 073-d7, JWH 081-d9, JWH 122-d9, JWH 200-d5, JWH 210-d9, JWH 250-d5, PB-22-d9, RCS-4-d9, UR-144-d5, XLR11-d5, AM2201 N-(4-hydroxypentyl) metabolite (AM2201 N-OH), JWH 018 N-(5-hydroxypentyl) metabolite (JWH 018 N-OH), JWH-018 N-pentanoic acid metabolite (JWH 018 N-COOH), JWH 073 N-(4-hydroxybutyl) metabolite (JWH 073 N-OH), JWH 073 N-butanoic acid metabolite (JWH 073 N-COOH), JWH-081 N-(5-hydroxypentyl) metabolite (JWH 081 N-OH), JWH 122 N-(5-hydroxypentyl) metabolite (JWH 122 N-OH), JWH 210 N-(5-hydroxypentyl) metabolite (JWH 210 N-OH), JWH 210 N-pentanoic acid metabolite (JWH 210 N-COOH), JWH 250 N-(5-hydroxypentyl) metabolite (JWH 250 N-OH), JWH-250 N-pentanoic acid metabolite (JWH 250 N-COOH), RCS-4 N-pentanoic acid metabolite (RCS-4 N-COOH), MAM2201 N-(4-hydroxypentyl) metabolite (MAM2201 N-OH), MAM2201 N-pentanoic acid metabolite (MAM2201 N-COOH), UR-144 N-(5-hydroxypentyl) metabolite (UR-144 N-OH), UR-144 N-pentanoic acid metabolite (UR-144 N-COOH), and XLR11 N-(4-hydroxypentyl) metabolite (XLR11 N-OH), AM2201 N-(4-hydroxypentyl) metabolite-d5 (AM2201 N-OH-d5) and JWH 073 N-butanoic acid metabolite-d5 (JWH 073 N-COOH-d5) were purchased from Cayman Chemical (Ann Arbor, MI). The common names, structures and chemical formulas of the synthetic cannabinoid parent and metabolite compounds investigated in this study are displayed in Figures 1 and 2, respectively.
Figure 1.
Synthetic cannabinoid parent structures and chemical formulas.
Figure 2.
Synthetic cannabinoid metabolite structures and chemical formulas.
Preparation of standards and controls
Stock standards of the synthetic cannabinoids were prepared at target concentrations of 0.010, 0.10 or 1.0 mg/mL in acetonitrile (ACN) or methanol (MeOH) and stored at ≤−20°C. Stock internal standards (ISTDs) were made at a concentration of 0.10 mg/mL in MeOH and stored at ≤−20°C. Working solutions of the synthetic cannabinoid parent and metabolite compounds were prepared by serial dilution with ACN and MeOH, respectively, at concentrations of 100, 10 and 1.0 ng/mL. The ISTD working solution was prepared at a concentration of 10 ng/mL for the parent compounds and 100 ng/mL for the metabolites.
Calibrators were prepared in 1.0 mL of certified drug-free blood or 2.0 mL of certified drug-free urine at concentrations of 0.10, 0.25, 0.50, 1.0, 5.0 and 10 ng/mL. Positive blood (0.75, 4.0 and 7.5 ng/mL) and urine controls (0.25, 1.0 and 10.0 ng/mL) were prepared from different preparations of the stock standard sources. Calibrators, positive controls and a negative control were analyzed with each batch of samples.
Parent blood extraction
To 1 mL of samples, calibrators and controls, 100 µL of working ISTD solution was added for a final concentration of 1.0 ng/mL. Five-hundred microliters of 0.5 M sodium carbonate buffer, pH 9.3, were added and vortexed followed by 1.5 mL of 99:1 hexane–ethyl acetate. Samples were mixed for 20 min and centrifuged at 3,500 rpm for 10 min. The upper organic layer was immediately transferred to clean conical tubes and evaporated at 40°C under nitrogen. Samples were then reconstituted in 50 µL of mobile phase (50:50 0.1% formic acid in deionized (DI) H2O: 0.1% formic acid in ACN), vortexed and transferred to labeled autosampler vials.
Instrumental analysis for blood
The LC–MS-MS analysis of the parent synthetic cannabinoids was performed using a Waters Acquity Ultra Performance Liquid Chromatograph (UPLC) coupled to a Xevo tandem quadrupole detector (TQD) equipped with an electrospray ionization (ESI) source (Waters, Milford, MA). LC components consisted of a thermostatted column compartment, Waters Acquity ultra-high performance autosampler and UPLC quaternary pump. Masslynx and Targetlynx software were used for data acquisition and analysis, respectively.
Chromatographic separation was performed on a Waters Acquity UPLC T3 C18 column (2.1 × 100 mm, 1.8 µm). The column compartment was maintained at 45°C, and the injection volume was set at 5 µL. A gradient elution was performed with 0.1% formic acid in DI H2O (mobile phase A) and 0.1% formic acid in ACN (mobile phase B) at a constant flow rate of 0.5 mL/min. Gradient conditions were as follows: 42% B hold for 0.3 min after injection, increased to 56% B at 2.25 min, 70% B at 2.75 min, 88% B at 8.50 min and 95% B at 8.70 min, hold for 0.6 min, ramp to 20% B at 9.6 min, hold for 0.9 min and re-equilibrate at 42% B for 0.9 min, for a total run time of 11.5 min. A typical total ion chromatogram (TIC) for all parent cannabinoids and ISTD is displayed in Figure 3.
Figure 3.
TIC of 0.5 ng/mL calibrator. JWH 200-d5 (1), JWH 200 (2), AM2201 (3), RCS4-d9 (4), RCS4 (5), PB-22-d9 (6), PB-22 (7), MAM2201 (8), JWH 250-d5 (9), JWH 250 (10), JWH 073-d7 (11), JWH 073 (12), STS-135 (13), XLR11-d5 (14), XLR11 (15), BB-22 (16), JWH 018-d9 (17), JWH 018 (18), JWH 081-d9 (19), JWH 081 (20), JWH 122-d9 (21), JWH 122 (22), 2NE1 (23), UR144-d5 (24), UR144 (25), JWH 210-d9 (26), JWH 210 (27) and AKB48 (28).
The MS was operated in positive ESI mode, and the analysis was operated in multiple reactions monitoring (MRM) acquisition mode. Two MRM transitions were monitored for each compound and ISTD. Source dependent parameters were as follows: desolvation temperature, 450°C; desolvation gas flow, 850 L/h; cone gas flow, 10 L/h; source temperature, 150°C; cone voltage (CV), 27 V; capillary voltage, 2.5 kV and extractor voltage, 3.0 V.
The compound-dependent parameters for the MS–MS were determined by a combined mobile phase and standard infusion. The infusion pump delivered both the 1,000 ng/mL standard solution and mobile phase at initial conditions with a constant flow (10 µL/min) directly into the source. The optimizer auto-optimization program determined the optimal CV and collision energy (CE) for each MRM transition. Table I lists the compound-dependent parameters, retention times and MRM transitions monitored for the synthetic cannabinoid parent compounds.
Table I.
Synthetic Cannabinoid Parent Optimal Compound Dependent Parameters
| Compound | RT (min) | MRM transition (m/z) | Compound dependent parameters |
|
|---|---|---|---|---|
| CV (V) | CE (eV) | |||
| JWH 200-d5 | 1.02 | 390.2/155.0 390.2/127.0 |
54 54 |
22 52 |
| JWH 200 | 1.03 | 385.2/155.0 385.2/127.0 |
48 48 |
22 50 |
| RCS4-d9 | 4.85 | 331.2/135.0 331.2/107.0 |
52 52 |
24 44 |
| RCS4 | 4.88 | 322.1/135.0 322.1/107.0 |
52 52 |
24 42 |
| PB-22-d9 | 4.96 | 368.2/223.2 368.2/144.9 |
28 28 |
12 40 |
| PB-22 | 4.99 | 359.2/214.1 359.2/144.9 |
30 30 |
16 40 |
| JWH 250-d5 | 5.09 | 341.2/121.0 341.2/91.0 |
48 48 |
20 44 |
| JWH 250 | 5.12 | 336.2/121.0 336.2/91.0 |
48 48 |
20 44 |
| STS-135 | 5.27 | 383.3/135.0 383.3/107.0 |
62 62 |
30 44 |
| JWH 073-d7 | 5.23 | 335.2/155.0 335.2/127.0 |
52 52 |
24 46 |
| AM2201 | 4.69 | 360.2/155.0 360.2/127.0 |
56 56 |
26 52 |
| MAM2201 | 5.10 | 374.2/169.0 374.2/141.0 |
60 60 |
24 46 |
| JWH 073 | 5.26 | 328.2/154.9 328.2/127.0 |
64 64 |
24 46 |
| XLR11-d5 | 5.40 | 335.2/125.0 335.2/236.9 |
56 56 |
22 26 |
| XLR11 | 5.43 | 330.2/125.0 330.2/232.1 |
56 56 |
22 24 |
| JWH 018-d9 | 5.84 | 351.2/155.0 351.2/127.0 |
56 56 |
26 46 |
| BB-22 | 5.67 | 385.2/240.1 385.2/143.9 |
32 32 |
14 40 |
| JWH 018 | 5.88 | 342.2/155.0 342.2/127.0 |
52 52 |
24 44 |
| JWH 081-d9 | 6.08 | 381.2/185.0 381.2/157.0 |
56 56 |
28 44 |
| JWH 081 | 6.13 | 372.2/185.0 372.2/157.0 |
58 58 |
26 42 |
| JWH 122-d9 | 6.43 | 365.2/169.0 365.2/141.0 |
56 56 |
26 42 |
| JWH 122 | 6.48 | 356.2/169.0 356.2/141.0 |
52 52 |
24 40 |
| 2NE1 | 6.55 | 365.2/135.1 365.2/107.0 |
50 50 |
28 46 |
| UR144-d5 | 6.83 | 317.2/125.0 317.2/218.8 |
56 56 |
22 24 |
| UR144 | 6.88 | 312.2/125.0 312.2/214.1 |
50 50 |
22 24 |
| JWH 210-d9 | 7.09 | 379.2/183.0 379.2/223.1 |
60 60 |
28 26 |
| JWH 210 | 7.14 | 370.2/183.0 370.2/214.1 |
54 54 |
24 24 |
| AKB48 | 8.29 | 366.2/135.1 366.2/107.1 |
38 38 |
22 48 |
RT, retention time; CV, cone voltage; CE, collision energy.
Metabolite urine extraction
Twenty microliters of working ISTD solution was added to 2 mL of the sample, calibrators and controls for a final concentration of 1.0 ng/mL. Prior to extraction, the urine samples were hydrolyzed using 1 mL of 0.5 M phosphate buffer (pH 6.8), 1,250 units of β-glucuronidase (20 µL of Escherichia coli solution) and incubated at 55°C for 20 min. After the samples cooled to room temperature, 200 µL of HCl and 5 mL of chlorobutane were added, mixed for 20 min and centrifuged at 3,500 rpm for 5 min. The upper organic layer was transferred to clean conical tubes and evaporated at 55°C under nitrogen. Samples were reconstituted with 50 µL of mobile phase (50:50 10 mM ammonium formate–20% MeOH in ACN with 0.1% formic acid), vortexed and transferred to labeled autosampler vials.
Instrumental analysis for urine metabolites
The LC–MS-MS analysis of the synthetic cannabinoid metabolites was performed using a Shimadzu MPX series liquid chromatograph coupled with an AB SCIEX 3,200 QTRAP LC–MS-MS (Foster City, CA) equipped with a Turbo V™ source. The LC components consisted of a vacuum degasser, binary pump, LEAP PAL HTS-xt automated liquid sampler and thermostatted column compartment. Analyst 1.5.2 software was used for data acquisition and analysis.
Chromatographic separation was performed on a Phenomenex Gemini C18 analytical column (150 × 4.6 mm ID × 3.0 µm; Torrance, CA). The column compartment was maintained at 40°C, and the injection volume was set at 10 µL. A gradient elution was performed with 10 mM ammonium formate, pH 4.5 (mobile phase A) and 20% MeOH in ACN with 0.1% formic acid (mobile phase B) at a constant flow of 0.80 mL/min. Gradient conditions were as follows: starting conditions 75% B, increased to 95% B at 5.0 min, hold for 2.5 min, ramp to 75% B over 0.25 min, hold for 0.25 min and re-equilibrate at 75% B for 4 min, for a total run time of 12 min. Baseline resolution was achieved but not needed for most compounds due to the differences in molecular weights. However, baseline resolution was critical for the accurate quantitation of JWH 073 N-COOH and JWH 018 N-OH which have the same MRM transitions and elute at approximately the same retention time (Figure 4).
Figure 4.
TIC of 1.0 ng/mL calibrator. RCS4 N-COOH (1), JWH 250 N-COOH (2), JWH 250 N-OH (3), JWH 073 N-COOH-d5 (4), JWH 073 N-COOH (5), JWH 073 N-OH (6), JWH 018 N-COOH (7), AM2201 N-OH-d5 (8), AM2201 N-OH (9), JWH 018 N-OH (10), XLR11 N-OH (11), MAM2201 N-COOH (12), MAM2201 N-OH (13), JWH 081 N-OH (14), JWH 122 N-OH (15), UR144 N-COOH (16), JWH 210 N-COOH (17), UR144 N-OH (18) and JWH 210 N-OH (19).
The MS was operated in positive ESI mode. The analysis of the synthetic cannabinoids was operated in MRM acquisition mode. Two MRM transitions were monitored for each compound as well as the ISTDs. The source dependent parameters for the analysis were as follows: GS1 gas (nebulizer) was set to 60 psi, GS2 gas (drying) was set to 70 psi, CUR (curtain gas) was set to 15 psi, nebulizer current was set to 4 V, CAD (collision cell) was set to medium, IS (ion spray voltage) was set to 4,500 V and the source temperature was set at 450°C.
The compound dependent parameters for the MS–MS analysis of synthetic cannabinoid metabolites were determined by direct infusion. An integrated infusion pump delivered a 1,000 ng/mL standard solution at a constant flow (10 µL/min) directly into the ESI source. The auto-optimization process determined the optimal parameters for each MRM transition. The following parameters were optimized during the process: declustering potential (DP), entrance potential (EP), collision cell entrance potential (CEP), CE and collision cell exit potential (CXP). The retention times, compound dependent parameters and MRM transitions monitored for the synthetic cannabinoid metabolites are listed in Table II.
Table II.
Synthetic Cannabinoid Metabolite Optimal Compound Dependent Parameters
| Compound | RT (min) |
MRM Transition (m/z) |
Compound dependent parameters |
||||
|---|---|---|---|---|---|---|---|
| DP (V) |
EP (V) |
CEP (V) |
CE (V) |
CXP (V) |
|||
| JWH 073 N-COOH-d5 | 3.17 | 363.2/155.2 363.2/127.2 |
51 51 |
7.5 7.5 |
22 22 |
31 65 |
4 4 |
| RCS4 N-COOH | 2.87 | 352.1/135.1 352.1/92.1 |
56 56 |
9.5 9.5 |
19 16 |
31 87 |
4 4 |
| JWH 250 N-COOH | 2.95 | 366.0/121.1 366.0/130.2 |
56 56 |
4.5 4.5 |
19 19 |
29 51 |
4 4 |
| JWH 250 N-OH | 3.08 | 352.1/121.0 352.1/130.1 |
56 56 |
4.5 4.5 |
19 19 |
29 49 |
4 4 |
| JWH 073 N-COOH | 3.19 | 358.2/155.2 358.2/127.1 |
51 51 |
9.5 9.5 |
24 24 |
33 61 |
4 4 |
| JWH 073 N-OH | 3.28 | 344.2/155.2 344.2/127.1 |
61 61 |
8 8 |
22 22 |
35 65 |
4 4 |
| JWH 018 N-COOH | 3.32 | 372.2/155.2 372.2/127.1 |
51 51 |
4.5 4.5 |
26 26 |
33 69 |
4 4 |
| JWH 018 N-OH | 3.50 | 358.2/155.2 358.2/127.2 |
61 61 |
10.5 10.5 |
26 26 |
33 71 |
4 4 |
| JWH 081 N-OH | 3.68 | 388.1/185.2 388.1/157.2 |
66 66 |
10.5 10.5 |
20 20 |
35 57 |
4 4 |
| JWH 210 N-COOH | 3.96 | 400.1/183.2 400.1/155.1 |
61 61 |
10.5 10.5 |
20 20 |
35 59 |
4 4 |
| JWH 210 N-OH | 4.20 | 386.1/183.1 386.1/155.2 |
81 81 |
10 10 |
20 20 |
33 55 |
4 4 |
| AM2201 N-OH-d5 | 3.33 | 381.2/155.1 381.2/127.0 |
66 66 |
10 10 |
24 24 |
31 69 |
4 4 |
| AM2201 N-OH | 3.35 | 376.1/155.1 376.1/127.2 |
66 66 |
10.5 10.5 |
20 20 |
35 79 |
4 4 |
| XLR11 N-OH | 3.59 | 346.2/248.2 346.2/144.0 |
56 56 |
7.5 7.5 |
18 18 |
29 45 |
4 4 |
| MAM2201 N-COOH | 3.62 | 386.3/169.2 386.3/141.2 |
51 51 |
11 11 |
22 22 |
35 59 |
4 4 |
| MAM2201 N-OH | 3.66 | 390.3/169.2 390.3/141.2 |
33 63 |
9.5 9.5 |
18 18 |
33 63 |
4 4 |
| JWH 122 N-OH | 3.83 | 372.2/169.2 372.2/141.2 |
61 61 |
10.5 10.5 |
20 20 |
33 61 |
4 4 |
| UR144 N-COOH | 3.89 | 342.2/125.0 342.2/244.3 |
51 51 |
9 9 |
36 36 |
29 35 |
4 4 |
| UR144 N-OH | 4.11 | 328.2/125.0 328.2/244.1 |
41 41 |
10 10 |
30 30 |
29 45 |
4 4 |
RT, retention time; DP, declustering potential; EP, entrance potential; CEP, collision cell entrance potential; CE, collision cell; CXP, collision cell exit potential.
Identification of the synthetic cannabinoids in both the blood and urine specimens was based on the MRM transition ratios being within ±20% of the average MRM transition ratio and ±3% of the relative retention time generated from all six calibrators.
Method validation
The following parameters were evaluated for the validation of synthetic cannabinoids: selectivity, specificity, linearity, limit of quantitation (LOQ), limit of detection (LOD), upper limit of linearity (ULOL), within- and between-day precision, accuracy, carryover, extraction recovery and ion suppression effects.
Selectivity of the method was examined to determine if the matrices would produce any significant extraneous peaks that may interfere with the analysis at the retention time of the analytes of interest. Selectivity was assessed by extracting and analyzing different and separate sources of negative matrices with ISTD. Specificity was evaluated by analyzing negative samples fortified with 202 structurally similar or commonly encountered compounds at 1,000 ng/mL for possible interference with identification or quantitation of the analytes of interest.
Linearity was determined by analyzing a multipoint calibration curve over a minimum of 10 days. The linear relationship was evaluated by calculating the line of regression of the six point curve(s) using the least squares method. The correlation of determination (R2) of the calibration curve was required to be 0.980 or better. Additionally, each calibrator was back calculated against the generated curve and compared with the theoretical concentrations to ensure the concentration was within ±20% of the theoretical fortified concentration. The LOD was defined as the lowest concentration for which the MRM transition ratios are within ±20% of the average MRM transition ratio and ±3% of the relative retention time but had no defined relationship to the theoretical fortified concentration. The LOQ and ULOL were defined as the lowest and highest concentrations, respectively, that met all the above criteria and were within ±20% of the theoretical fortified concentration.
Within-day precision was evaluated by analyzing multiple aliquots of each pooled control in the same analytical extraction. Between-day precision was evaluated by analyzing one aliquot of each pooled control over a minimum of 10 separate extractions. The acceptable values of precision, expressed as the coefficient of variance (CV), for each within- and between-day assay must be ≤15%. Accuracy, defined as the percent difference (% diff) between the average calculated concentration and the theoretical fortified concentration, was also evaluated during each extraction. Each pooled control had to be within ±20% of the theoretical fortified concentration in order to meet acceptability criteria.
Carryover was defined as the inadvertent transfer of analyte from one sample to the subsequent sample during instrumental analysis. For the parent method, carryover was evaluated with blood controls fortified at 50, 250 and 500 ng/mL; while the metabolite method was evaluated with urine controls fortified at 100, 500 and 1,000 ng/mL. Blank samples were injected between each of the fortified controls to look for any sign of residual analytes.
Recovery and matrix effect were examined at both a low and high concentration within the linear range. Three sets of samples were generated to evaluate these parameters. Set 1 consisted of analytes of interest and ISTD fortified into solvent (unextracted). Set 2 contained analytes of interest and ISTD fortified into blank matrix and extracted as mentioned above (pre-extracted). Set 3 consisted of analytes of interest and ISTD fortified after the extraction was completed but prior to dry down (post-extracted). Extraction recovery, which measured the amount of analyte lost during the extraction procedure, was calculated by dividing the pre-extracted response by the response from the post-extracted. Matrix effect was evaluated by comparing post-extracted response to the unextracted response. Matrix effect values over 100% indicate ion enhancement and values <100% indicate ion suppression.
Results and discussion
Method validation
Method selectivity was determined by evaluating the potential interferences from six different sources of drug-free negative blood and 10 sources of drug-free negative urine. Specificity was evaluated by analyzing negative blood and urine samples fortified with 202 structurally similar and commonly encountered compounds. Analysis identified no peaks within the retention time windows corresponding to the transitions of either synthetic cannabinoid parent or metabolite compounds. These results suggest that endogenous components found in blood and urine specimens and/or commonly encountered compounds should have no effect or interfering impact on the LC–MS-MS analysis of the synthetic cannabinoids of interest.
Linearity for both methods was assessed with a six-point curve (0.10, 0.25, 0.50, 1.0, 5.0 and 10 ng/mL) ranging from 0.10 to 10 ng/mL. The linearity was determined to be acceptable with 1/x weighting for the blood method; while no weighting was required for the urine method. The LOQ for both methods was administratively set at 0.10 ng/mL. This was the lowest concentration that met all quantitation acceptance criteria for each parent compound and all but three of the metabolites. The LOQ for JWH 250 N-OH and RCS-4 N-COOH is 0.25 ng/mL; while the LOQ for JWH 250 N-COOH is 0.50 ng/mL. The LOD, defined as the lowest concentration that meets all identification criteria, was 0.05 ng/mL. We experienced some compound dependent variability when determining the LOD. Compounds displaying this variability are presented in Table III along with the experimentally determined LOQ and ULOL.
Table III.
Linearity and Experimentally Determined LOD, LOQ and ULOL for Blood (A) and Urine (B) Specimens
| Linearity (ng/mL) |
LOD (ng/mL) |
LOQ (ng/mL) |
ULOL (ng/mL) |
|
|---|---|---|---|---|
| (A) Blood | ||||
| JWH 200 | 0.10–10 | 0.025 | 0.05 | 50 |
| RCS4 | 0.10–10 | 0.05 | 0.05 | 50 |
| PB-22 | 0.10–10 | 0.025 | 0.10 | 40 |
| JWH 250 | 0.10–10 | 0.025 | 0.025 | 50 |
| STS-135 | 0.10–10 | 0.05 | 0.05 | 50 |
| AM2201 | 0.10–10 | 0.05 | 0.10 | 50 |
| MAM2201 | 0.10–10 | 0.05 | 0.10 | 40 |
| JWH 073 | 0.10–10 | 0.05 | 0.10 | 30 |
| XLR11 | 0.10–10 | 0.05 | 0.10 | 50 |
| BB-22 | 0.10–10 | 0.05 | 0.05 | 50 |
| JWH 018 | 0.10–10 | 0.025 | 0.05 | 30 |
| JWH 081 | 0.10–10 | 0.025 | 0.05 | 50 |
| JWH 122 | 0.10–10 | 0.05 | 0.05 | 10 |
| 2NE1 | 0.10–10 | 0.05 | 0.10 | 10 |
| UR144 | 0.10–10 | 0.05 | 0.05 | 10 |
| JWH 210 | 0.10–10 | 0.10 | 0.10 | 50 |
| AKB48 | 0.10–10 | 0.025 | 0.05 | 10 |
| (B) Urine | ||||
| RCS 4 N-COOH | 0.25–10 | 0.10 | 0.25 | 100 |
| JWH 250 N-COOH | 0.50–10 | 0.50 | 0.50 | 50 |
| JWH 250 N-OH | 0.25–10 | 0.25 | 0.25 | 50 |
| JWH 073 N-COOH | 0.10–10 | 0.05 | 0.10 | 50 |
| JWH 073 N-OH | 0.10–10 | 0.10 | 0.10 | 50 |
| JWH 018 N-COOH | 0.10–10 | 0.05 | 0.10 | 50 |
| JWH 018 N-OH | 0.10–10 | 0.05 | 0.10 | 50 |
| JWH 081 N-OH | 0.10–10 | 0.05 | 0.10 | 50 |
| JWH 210 N-COOH | 0.10–10 | 0.025 | 0.10 | 50 |
| JWH 210 N-OH | 0.10–10 | 0.05 | 0.10 | 50 |
| AM2201 N-OH | 0.10–10 | 0.05 | 0.10 | 50 |
| XLR11 N-OH | 0.10–10 | 0.05 | 0.10 | 50 |
| MAM2201 N-COOH | 0.10–10 | 0.01 | 0.10 | 50 |
| MAM2201 N-OH | 0.10–10 | 0.01 | 0.10 | 50 |
| JWH 122 N-OH | 0.10–10 | 0.05 | 0.10 | 50 |
| UR144 N-COOH | 0.10–10 | 0.05 | 0.10 | 50 |
| UR144 N-OH | 0.10–10 | 0.05 | 0.10 | 50 |
LOD, limit of detection; LOQ, limit of quantitation; ULOL, upper limit of linearity.
Within- and between-day precision and accuracy data were evaluated at low, middle and high concentrations (blood: 0.75, 4.0, 7.5 ng/mL; urine: 0.25, 1.0, 10.0 ng/mL). Within-day precision for both methods was below 15% with a maximum CV of 9.9% for the blood method and 9.6% for the urine method. Between-day precision for the parent blood method was <15.0%, with the exception of JWH 122 and 2NE1. These two compounds were outside the acceptable CV range and are therefore considered qualitative only. Between-day precision for the urine method was <12% for all metabolites. Within- and between-day accuracies were <18% for the blood method and 15% for the urine method. Precision and accuracy data are displayed in Table IV.
Table IV.
Precision and Accuracy of the Blood (A) and Urine (B) Assays
| Precision (% CV) |
Accuracy (% difference) |
|||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Within run (n = 5) |
Between day (n = 11) |
Within run (n = 5) |
Between day (n = 11) |
|||||||||
| Low | Mid | High | Low | Mid | High | Low | Mid | High | Low | Mid | High | |
| (A) Blood | ||||||||||||
| JWH 200 | 2.6 | 2.4 | 1.4 | 4.5 | 5.8 | 3.3 | 10.9 | 3.1 | −1.1 | 3.2 | −2.8 | −5.2 |
| RCS4 | 1.6 | 2.2 | 2.4 | 2.8 | 3.1 | 3.0 | 11.7 | 2.9 | 1.7 | 10.4 | 1.4 | 1.7 |
| PB-22 | 0.5 | 1.3 | 2.0 | 6.9 | 3.5 | 3.4 | 9.3 | 4.4 | 0.6 | 12.2 | 2.7 | −2 |
| JWH 250 | 2.8 | 3.2 | 1.6 | 3.4 | 7.0 | 4.4 | 4.8 | −2.4 | −3.8 | 2.1 | −7.4 | −5.3 |
| STS-135 | 2.8 | 2.9 | 2.2 | 11.3 | 5.3 | 2.2 | −10.7 | 4.6 | 1.8 | −3.9 | 4.1 | 2.8 |
| AM2201 | 3.3 | 2.8 | 1.6 | 9.1 | 3.5 | 2.7 | −6.9 | 4.5 | 1.2 | −2.0 | 4.1 | −0.9 |
| MAM2201 | 2.9 | 3.2 | 1.1 | 4.4 | 2.6 | 3.7 | 7.2 | 2.6 | −4.5 | 11.2 | 5.6 | −1.7 |
| JWH 073 | 4.7 | 1.3 | 2.9 | 4.5 | 2.1 | 5.6 | 0.3 | −0.5 | 1.1 | 5.4 | 0.4 | −2.1 |
| XLR11 | 1.9 | 3.0 | 2.5 | 3.3 | 5.9 | 3.4 | −5.9 | −0.7 | −9.0 | −4.2 | −1.8 | −7.4 |
| BB-22 | 2.5 | 7.0 | 5.7 | 5.1 | 5.6 | 5.8 | 14.7 | −1.1 | −1.8 | 8.2 | 1.3 | −2.3 |
| JWH 018 | 1.5 | 2.2 | 1.6 | 3.3 | 6.6 | 3.6 | 3.5 | −0.3 | −9.0 | −0.5 | −4.5 | −9.2 |
| JWH 081 | 2.9 | 2.8 | 3.1 | 7.8 | 3.7 | 4.4 | 17.9 | −0.4 | −1.0 | 9.4 | −0.3 | −2.3 |
| JWH 122 | 6.7 | 4.5 | 4.1 | 10.4 | 16.0a | 16.3a | −8.8 | −16.8 | −9.5 | −0.7 | −6.1 | −5.5 |
| 2NE1 | 8.0 | 6.8 | 5.6 | 17.1a | 19.1a | 17.8a | −8.5 | −16.1 | −12.1 | −4.9 | −4.7 | 1.3 |
| UR144 | 9.9 | 4.2 | 5.7 | 9.1 | 9.5 | 8.4 | −13.1 | −0.5 | −6.5 | −3.1 | −3.3 | −2.8 |
| JWH 210 | 5.2 | 1.2 | 3.2 | 4.0 | 4.8 | 4.0 | 11.7 | −1.3 | −7.0 | 5.4 | −1.0 | −4.6 |
| AKB48 | 4.7 | 5.5 | 5.3 | 7.8 | 7.3 | 7.1 | 1.3 | −6.8 | −5.3 | −12.8 | −7.5 | −8.1 |
| Precision |
Accuracy |
|||||||||||
| Within run (n = 3) |
Between day (n = 11) |
Within run (n = 3) |
Between day (n = 11) |
|||||||||
| Low | Mid | High | Low | Mid | High | Low | Mid | High | Low | Mid | High | |
| (B) Urine | ||||||||||||
| RCS 4 N-COOH | 4.5 | 1.8 | 5.1 | 7.4 | 7.5 | 5.3 | 2.7 | −6.0 | −9.7 | −3.8 | −3.3 | −6.0 |
| JWH 250 N-COOH | N/A | 3.4 | 2.3 | N/A | 5.50 | 4.40 | N/A | −9.7 | −12.9 | N/A | −7.9 | −12.4 |
| JWH 250 N-OH | 5.8 | 7.3 | 1.3 | 9.4 | 11.7 | 7.9 | 5.3 | 1.3 | −10.4 | −3.9 | 1.6 | −8.8 |
| JWH 073 N-COOH | 0.0 | 0.6 | 5.7 | 10.4 | 6.3 | 5.7 | 4.0 | −0.3 | −0.3 | −0.4 | −5.4 | −4.8 |
| JWH 073 N-OH | 9.6 | 4.1 | 8.4 | 7.1 | 6.3 | 6.8 | 5.3 | −1.3 | −6.9 | 0.5 | −4.8 | −8.5 |
| JWH 018 N-COOH | 7.5 | 1.0 | 2.3 | 8.6 | 6.5 | 6.6 | 10.7 | 4.0 | −3.7 | 6.1 | 2.8 | −3.7 |
| JWH 018 N-OH | 8.3 | 6.6 | 7.3 | 6.0 | 8.2 | 6.0 | −4.0 | −11.3 | −7.8 | −7.6 | −12.4 | −10.7 |
| JWH 081 N-OH | 4.7 | 3.0 | 4.6 | 7.0 | 9.3 | 7.6 | −2.7 | −1.0 | −8.4 | −6.8 | −3.8 | −9.2 |
| JWH 210 N-COOH | 2.4 | 4.3 | 7.2 | 7.1 | 6.7 | 6.1 | −2.7 | 14.7 | 6.0 | −2.8 | 8.8 | 2.3 |
| JWH 210 N-OH | 4.9 | 3.3 | 2.9 | 8.3 | 8.4 | 7.2 | −6.7 | 10.0 | −2.5 | −11.5 | 5.3 | −3.9 |
| AM2201 N-OH | 4.7 | 3.4 | 5.0 | 6.8 | 8.4 | 6.1 | −1.3 | −5.3 | −4.8 | −5.6 | −9.4 | −10.4 |
| XLR11 N-OH | 5.9 | 3.0 | 4.3 | 6.8 | 6.8 | 5.2 | 2.7 | −1.0 | −6.0 | −2.3 | −5.2 | −8.4 |
| MAM2201 N-COOH | 2.5 | 1.7 | 1.4 | 4.6 | 4.1 | 5.7 | −6.7 | −9.7 | 1.7 | −9.3 | −6.0 | −3.9 |
| MAM2201 N-OH | 0.0 | 3.3 | 0.8 | 5.7 | 3.8 | 5.1 | −4.0 | −8.7 | −0.5 | −8.4 | −4.7 | −5.0 |
| JWH 122 N-OH | 4.2 | 5.0 | 3.7 | 6.0 | 11.2 | 6.8 | −4.0 | −9.0 | −10.4 | −7.6 | −9.9 | −12.3 |
| UR144 N-COOH | 2.2 | 3.4 | 1.9 | 11.0 | 6.8 | 6.9 | 2.7 | −5.3 | −4.5 | −2.3 | −4.3 | −4.8 |
| UR144 N-OH | 6.9 | 6.5 | 3.7 | 10.6 | 7.7 | 5.2 | 0.0 | −0.7 | −9.6 | 2.9 | −1.5 | −8.5 |
aOutside the acceptable CV range.
The evaluation of matrix effect and analyte recovery was carried out at low and high concentrations within the linear range. Blood method recoveries ranged from 70.6 to 174.4%; while matrix effect ranged from 27.4–146.8%. The matrix effect increases as the analytical run progresses, this was particularly true for late eluting compounds. The increase in matrix effect during the later portion of the chromatographic run may be due to phospholipids, cholesterols and/or column components that elute at the same time as the parent compounds. Matrix effect was minimized in the blood method by using deuterated ISTDs, when available, to compensate for any change in signal throughout the analytical run. Initially, AM2201-d5 was used as the ISTD for AM2201 and MAM2201. However, during method development it was discovered that the standard received was predominately AM2201-d4, not the expected d5 species, therefore the use of AM2201-d5 was discontinued. Due to similarities in retention time and structure, AM2201 and MAM2201 were validated using JWH 073-d7 as their ISTD. For the urine method, recovery and matrix effects were 81.2–107.1% and 13.3–89.0%, respectively. Typically, if the matrix effect exceeds 50% of the signal, the method should be modified to enhance sensitivity. However, the signal at the LOQ was sufficient so no further action was necessary. Results for matrix effect and recovery can be found in Table V.
Table V.
Matrix Effects and Recoveries for Blood (A) and Urine (B) Analytes and Their Designated ISTDs
| Matrix effect |
Recovery |
Matrix Effect |
Recovery |
||||||
|---|---|---|---|---|---|---|---|---|---|
| 0.25 ng/mL | 5.0 ng/mL | 0.25 ng/mL | 5.0 ng/mL | 0.10 ng/mL | 5.0 ng/mL | 0.10 ng/mL | 5.0 ng/mL | ||
| (A) Blood | (B) Urine | ||||||||
| JWH 200-d5 | 127.9 | 111.1 | 77.8 | 73.7 | JWH 073 N-COOH-d5 | 51.2 | 50.0 | 88.5 | 92.4 |
| JWH 200 | 146.8 | 118.7 | 73.7 | 70.6 | RCS 4 N-COOH | N/A | 57.7 | N/A | 86.1 |
| RCS4-d9 | 72.7 | 74.1 | 106.2 | 102.9 | JWH 250 N-COOH | N/A | 29.3 | N/A | 87.5 |
| RCS4 | 75.2 | 74.1 | 103.2 | 99.6 | JWH 250 N-OH | N/A | 50.4 | N/A | 85.9 |
| PB-22-d9 | 72.7 | 72.6 | 105.7 | 100.6 | JWH 073 N-COOH | 30.6 | 29.3 | 95.7 | 98.0 |
| PB-22 | 68.3 | 72.2 | 100.4 | 96.2 | JWH 073 N-OH | 16.1 | 21.6 | 87.4 | 96.5 |
| JWH 250-d5 | 71.7 | 72.0 | 107.6 | 105.6 | JWH 018 N-COOH | 26.8 | 36.7 | 96.9 | 98.8 |
| JWH 250 | 70.6 | 73.3 | 104.3 | 97.5 | JWH 018 N-OH | 13.3 | 16.6 | 94.1 | 95.4 |
| STS-135 | 76.9 | 74.7 | 103.8 | 96.4 | JWH 081 N-OH | 105.5 | 100.7 | 85.3 | 85.3 |
| JWH 073-d7 | 66.6 | 62.7 | 117.7 | 115.4 | JWH 210 N-COOH | 83.7 | 99.2 | 94.5 | 83.4 |
| AM2201 | 71.5 | 69.6 | 94.9 | 93.7 | JWH 210 N-OH | 126.0 | 116.6 | 90.8 | 88.5 |
| MAM2201 | 68.8 | 69.4 | 108.6 | 98.4 | AM2201 N-OH-d5 | 48.3 | 48.4 | 86.4 | 81.6 |
| JWH 073 | 63.8 | 65.3 | 117.0 | 108.7 | AM2201 N-OH | 18.3 | 21.6 | 101.2 | 95.4 |
| XLR11-d5 | 67.2 | 67.2 | 113.3 | 112.1 | XLR11 N-OH | 24.4 | 29.8 | 103.2 | 104.1 |
| XLR11 | 62.3 | 65.8 | 113.4 | 104.6 | MAM2201 N-COOH | 41.5 | 56.2 | 107.1 | 97.0 |
| JWH 018-d9 | 43.0 | 48.9 | 161.4 | 141.6 | MAM2201 N-OH | 36.8 | 50.2 | 104.5 | 97.4 |
| BB-22 | 57.0 | 61.6 | 123.1 | 106.4 | JWH 122 N-OH | 20.3 | 25.8 | 101.5 | 92.3 |
| JWH 018 | 42.4 | 50.6 | 159.8 | 129.7 | UR144 N-COOH | 36.2 | 39.7 | 104.8 | 93.4 |
| JWH 081-d9 | 38.6 | 42.8 | 169.6 | 148.0 | UR144 N-OH | 34.6 | 44.1 | 103.7 | 95.1 |
| JWH 081 | 39.5 | 47.0 | 168.6 | 135.3 | |||||
| JWH 122-d9 | 42.0 | 42.8 | 161.2 | 153.6 | |||||
| JWH 122 | 30.1 | 36.5 | 136.0 | 125.7 | |||||
| 2NE1 | 35.2 | 38.7 | 115.4 | 110.0 | |||||
| UR144-d5 | 37.6 | 27.5 | 148.0 | 146.0 | |||||
| UR144 | 37.7 | 27.4 | 133.7 | 120.0 | |||||
| JWH 210-d9 | 36.8 | 32.5 | 143.1 | 145.1 | |||||
| JWH 210 | 39.3 | 36.7 | 144.4 | 134.1 | |||||
| AKB48 | 31.3 | 31.9 | 174.4 | 168.4 | |||||
No carryover was detected in the retention time windows corresponding with the transitions of nearly all the synthetic cannabinoid parent compounds or their metabolites. The exceptions were JWH 210 N-COOH, JWH 210 N-OH and JWH 081 N-OH which did exhibit carryover above 500 ng/mL.
Specimen analysis
Table VI lists the analytical results for both the blood and urine of the submitted specimens with cases listed in the order in which they were received. Table VI displays only drugs that were detected. The cases were required to have the presence of parent drugs in the blood in addition to metabolites in the urine. The majority of cases (41) were investigative in nature though three postmortem cases are included at the bottom of the table. The concentration range, mean and median for each parent compound detected in the blood are displayed in Table VII.
Table VI.
Blood and Urine Results and Correlation
| Investigative | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Sample | Year | Drugs present in blood in ng/mL |
|||||||||
| AM2201 | JWH 018 | JWH 073 | JWH 081 | JWH 122 | JWH 210 | JWH 250 | RCS4 | UR144 | XLR11 | ||
| 1 | 2010 | 0.28 | |||||||||
| 2 | 2010 | 1.10 | 1.00 | ||||||||
| 3 | 2010 | 0.18 | |||||||||
| 4 | 2010 | 0.28 | |||||||||
| 5 | 2010 | 6.21 | <LOQ | ||||||||
| 6 | 2010 | 3.25 | 0.33 | <LOQ | |||||||
| 7 | 2011 | 0.68 | 0.12 | 2.78 | |||||||
| 8 | 2011 | 1.38 | 0.13 | 0.52 | 0.11 | ||||||
| 9 | 2011 | <LOQ | 0.66 | 0.40 | <LOQ | 0.77 | 0.30 | 0.29 | <LOQ | ||
| 10 | 2011 | 1.06 | 0.50 | <LOQ | 5.00 | 3.20 | 0.20 | ||||
| 11 | 2011 | 0.24 | 0.10 | 1.05 | |||||||
| 12 | 2011 | 2.69 | 0.66 | ||||||||
| 13 | 2011 | 2.61 | |||||||||
| 14 | 2011 | <LOQ | <LOQ | 27.46 | 0.16 | ||||||
| 15 | 2011 | 0.27 | 1.45 | 1.18 | |||||||
| 16 | 2011 | <LOQ | 0.24 | 1.34 | 0.27 | 0.17 | |||||
| 17 | 2011 | 0.18 | 0.95 | 0.38 | <LOQ | ||||||
| 18 | 2011 | 0.15 | 1.30 | 1.14 | 0.29 | ||||||
| 19 | 2011 | 0.62 | <LOQ | 1.17 | |||||||
| 20 | 2011 | <LOQ | 1.05 | 0.39 | |||||||
| 21 | 2011 | 1.90 | <LOQ | 2.60 | 1.43 | ||||||
| 22 | 2012 | 0.18 | 0.24 | 0.29 | |||||||
| 23 | 2012 | <LOQ | 0.18 | ||||||||
| 24 | 2012 | 0.95 | <LOQ | 0.47 | |||||||
| 25 | 2012 | <LOQ | |||||||||
| 26 | 2012 | <LOQ | 0.53 | 0.35 | |||||||
| 27 | 2013 | <LOQ | <LOQ | ||||||||
| 28 | 2013 | <LOQ | |||||||||
| 29 | 2013 | 2.16 | <LOQ | ||||||||
| 30 | 2013 | 0.24 | |||||||||
| 31 | 2013 | 0.12 | |||||||||
| 32 | 2013 | 0.18 | |||||||||
| 33 | 2013 | 0.21 | |||||||||
| 34 | 2013 | <LOQ | |||||||||
| 35 | 2013 | <LOQ | 0.70 | ||||||||
| 36 | 2013 | 1.23 | |||||||||
| 37 | 2013 | 0.10 | |||||||||
| 38 | 2013 | 0.54 | |||||||||
| 39 | 2013 | 0.18 | |||||||||
| 40 | 2013 | 0.99 | <LOQ | 0.11 | 1.89 | ||||||
| 41 | 2013 | 1.09 | |||||||||
| Postmortem | |||||||||||
| 1 | 2010 | 3.16 | |||||||||
| 2 | 2011 | 11.04 | 0.32 | 0.18 | 0.76 | ||||||
| 3 | 2012 | 0.12 | 0.18 | ||||||||
| Metabolites present in urine | |
|---|---|
| Metabolites Detected | Present <LOQ |
| JWH 018 N-COOH, JWH 018 N-OHa, JWH 073 N-COOH | |
| JWH 018 N-COOH, JWH 018 N-OHa, JWH 073 N-COOH, JWH 073 N-OH | |
| JWH 018 N-COOH, JWH 018 N-OHa, JWH 073 N-COOH | |
| JWH 018 N-COOH, JWH 018 N-OHa, JWH 073 N-COOH | |
| JWH 018 N-COOH, JWH 018 N-OHa, JWH 073 N-COOH, JWH 073 N-OH | |
| JWH 018 N-COOH, JWH 018 N-OHa, JWH 073 N-COOH, JWH 073 N-OH | |
| JWH 018 N-COOH, JWH 018 N-OH, JWH 073 N-OH, JWH 081 N-OH, JWH 122 N-OH, JWH 210 N-OHa | RCS 4 N-COOH |
| JWH 018 N-COOH, JWH 018 N-OHa, JWH 073 N-COOH, JWH 073 N-OH, JWH 122 N-OH, JWH 250 N-COOH, JWH 250 N-OH | |
| AM 2201 N-OH, JWH 018 N-COOH, JWH 018 N-OHa, JWH 073 N-COOH, JWH 073 N-OH, JWH 122 N-OH, JWH 210 N-OH, JWH 250 N-COOH, JWH 250 N-OH | MAM 2201 N-COOH |
| AM 2201 N-OH, JWH 018 N-COOHa, JWH 018 N-OH, JWH 073 N-COOH, JWH 073 N-OH, JWH 122 N-OH, JWH 210 N-OH, JWH 250 N-OH, MAM 2201 N-COOH | JWH 250 N-COOH, RCS 4 N-COOH |
| JWH 018 N-COOH, JWH 018 N-OHa | AM 2201 N-OH, JWH 073 N-COOH, JWH 073 N-OH |
| JWH 018 N-COOH, JWH 081 N-OH, JWH 122 N-OHa | MAM 2201 N-COOH |
| JWH 018 N-COOH, JWH 018 N-OHa, JWH 073 N-COOH | |
| AM 2201 N-OH, JWH 018 N-COOH, JWH 018 N-OH, JWH 073 N-COOH, JWH 073 N-OH, JWH 122 N-OHa, MAM 2201 N-COOH | |
| JWH 018 N-COOH, JWH 018 N-OHa, JWH 073 N-COOH, JWH 122 N-OH, JWH 250 N-OH | JWH 073 N-OH, JWH 210 N-OH, JWH 250 N-COOH |
| AM 2201 N-OH, JWH 018 N-COOH, JWH 018 N-OHa, JWH 073 N-COOH, JWH 073 N-OH, JWH 122 N-OH, JWH 250 N-COOH, JWH 250 N-OH | |
| JWH 018 N-COOHa, JWH 018 N-OH, JWH 073 N-COOH,JWH 122 N-OH, JWH 250 N-OH | JWH 250 N-COOH |
| AM 2201 N-OH, JWH 018 N-COOHa, JWH 018 N-OH, JWH 073 N-COOH, JWH 122 N-OH, MAM 2201 N-COOH | JWH 210 N-OH, RCS 4 N-COOH |
| AM 2201 N-OH, JWH 018 N-COOHa, JWH 018 N-OH, JWH 122 N-OH | JWH 073 N-COOH, JWH 073 N-OH |
| AM 2201 N-OH, JWH 018 N-COOH, JWH 018 N-OHa, JWH 073 N-COOH, JWH 122 N-OH | JWH 210 N-OH, MAM 2201 N-COOH |
| AM 2201 N-OH, JWH 018 N-COOH, JWH 018 N-OHa, JWH 073 N-COOH, JWH 073 N-OH, JWH 122 N-OH, JWH 210 N-OH | MAM 2201 COOH <LOQ |
| JWH 018 N-OH, UR 144 N-COOH, UR 144 N-OHa | JWH 018 N-COOH, JWH 122 N-OH |
| AM 2201 N-OH, JWH 018 N-COOHa, JWH 018 N-OH, UR 144 N-COOH, UR 144 N-OH, XLR 11 N-OH | |
| AM 2201 N-OH, JWH 018 N-COOHa, JWH 018 N-OH, JWH 073 N-COOH, JWH 073 N-OH, UR 144 N-COOH, UR 144 N-OH | |
| AM 2201 N-OH, JWH 018 N-COOH, JWH 018 N-OH, JWH 073 N-COOH, UR 144 N-COOHa, UR 144 N-OH | |
| AM 2201 N-OH, JWH 018 N-COOH, JWH 018 N-OHa, JWH 073 N-COOH, JWH 122 N-OH | JWH 210 N-OH |
| UR 144 N-COOHa, UR 144 N-OH, XLR 11 N-OH | |
| UR 144 N-COOHa, UR 144 N-OH, XLR 11 N-OH | |
| AM 2201 N-OH, JWH 018 N-COOH, JWH 018 N-OHa, JWH 073 N-COOH, JWH 073 N-OH | |
| UR 144 N-COOHa, XLR 11 N-OH | |
| UR 144 N-COOHa, UR 144 N-OH, XLR 11 N-OH | |
| UR 144 N-COOHa, UR 144 N-OH, XLR 11 N-OH | |
| UR 144 N-COOHa, UR 144 N-OH, XLR 11 N-OH | |
| UR 144 N-COOHa, UR 144 N-OH, XLR 11 N-OH | |
| UR 144 N-COOHa, UR 144 N-OH, XLR 11 N-OH | |
| UR 144 N-COOHa, UR 144 N-OH, XLR 11 N-OH | |
| UR 144 N-COOHa, UR 144 N-OH, XLR 11 N-OH | |
| UR 144 N-COOHa, UR 144 N-OH, XLR 11 N-OH | |
| UR 144 N-COOHa, UR 144 N-OH, XLR 11 N-OH | |
| AM 2201 N-OH, JWH 018 N-COOH, JWH 018 N-OH, JWH 073 N-COOH, UR 144 N-COOHa, UR 144 N-OH | |
| UR 144 N-COOHa, UR 144 N-OH, XLR 11 N-OH | |
| JWH 018 N-COOH, JWH 018 N-OHa, JWH 073 N-COOH | |
| AM 2201 N-OH, JWH 018 N-COOH, JWH 018 N-OHa, JWH 073 N-COOH, JWH 073 N-OH, JWH 122 N-OH | |
| JWH 018 N-COOHa | |
aThe metabolite present in the highest concentration for each sample.
Table VII.
Range, Mean and Median of Parent Compounds Detected in Blood Specimens
| Number of positives | Range (ng/mL) | Mean (ng/mL) | Median (ng/mL) | |
|---|---|---|---|---|
| AM2201 | 15 | 0.15–11.04 | 2.12 | 0.99 |
| JWH 018 | 23 | 0.10–6.21 | 1.23 | 0.32 |
| JWH 073 | 4 | 0.33–1.00 | 0.58 | 0.40 |
| JWH 081 | 5 | 0.13–2.69 | 1.17 | 0.68 |
| JWH 122 | 16 | 0.12–27.46 | 2.83 | 1.00 |
| JWH 210 | 14 | 0.16–3.20 | 0.97 | 0.58 |
| JWH 250 | 5 | 0.11–0.29 | 0.19 | 0.17 |
| RCS4 | 3 | 0.20–0.29 | 0.19 | 0.25 |
| UR144 | 5 | 0.11–0.24 | 0.18 | 0.18 |
| XLR11 | 17 | 0.10–1.89 | 0.53 | 0.27 |
The results from Table VI support the trend of a rapidly evolving synthetic cannabinoids market. The first set of specimens was submitted to our laboratory in 2010 and contained exclusively JWH 018 and JWH 073. After the initial popularity of JWH 018 and JWH 073, additional JWH compounds emerged on the market in 2011. Presence of JWH 018 persisted in specimens submitted at this time, although JWH 081, JWH 122, JWH 210 and JWH 250 became common additions to the synthetic cannabinoid profile seen in our laboratory. Moreover, of the cases containing these additional JWH compounds, only two failed to confirm for JWH 018. AM2201, which is the fluorinated N-alkyl side chain of JWH 018, also emerged during this time period. Interestingly, there are four cases that show the presence of AM2201 in the absence of JWH 018 and have the JWH 018 metabolites present in urine.
The trend then shifts from JWH-like compounds to the tetramethylcyclopropylindole compounds, represented mainly by UR 144 and XLR11. XLR11 is the fluorinated N-alkyl side chain of UR 144, and this scheme is analogous to the relationship between AM2201 and JWH 018. The majority of the specimens continued to contain multiple parent compounds in the blood. However, if a specimen contained only a single compound, that compound was either JWH 018 or XLR11.
Results listed in Table VI are not from a controlled administration study with known ingredients and standard doses, therefore, only general statements can be made concerning the constituents of the products consumed and the meaning of the analyte concentrations. For example, there were few cases where the JWH compounds or AM2201 had blood concentrations above 5 ng/mL. Analytical results for XLR11 and UR 144 were consistently below 1 ng/mL in the blood. This is consistent with the median blood concentration for all compounds in the study which was at or below 1.0 ng/mL. The postmortem blood concentrations of parent compounds were consistent with parent concentrations found in investigative cases. The exception to this rule was AM2201, whose concentrations were routinely higher. Therefore, it is imperative that the methods used for analyzing synthetic cannabinoids in blood must be sensitive enough to detect subnanogram concentrations.
This study did not monitor all the urinary metabolites of JWH 018 ingestion. The literature reported the carboxypentyl and hydroxypentyl compounds as the most prominent, therefore these metabolites were chosen for urine testing (18, 27, 28). If JWH 018 was detected in the blood, both of the ‘pentyl’ metabolites in addition to the carboxybutyl metabolite of JWH 073 were found in the urine. The JWH 018 N-OH metabolite was found in the highest concentration in the majority of specimens where JWH 018 use was suspected. Ingestion of JWH 073 results in JWH 073 N-COOH as a urinary metabolite. It has been suggested that this same compound might be produced from in vivo demethylation or decarboxylation following JWH 018 use (35). This study does not provide any further insight concerning this discussion as the JWH 073 hydroxybutyl metabolite was also detected in multiple cases. When JWH 073 was detected in the blood, however, both the hydroxybutyl and carboxybutyl metabolites were detected in the urine. Interestingly, there are multiple specimens in which no JWH 073 was found in the blood and yet both ‘butyl’ metabolites were detected in the urine. The probability of a different metabolic pathway for two compounds that differ only by a methyl group on the N-alkyl side chain is low. Chimalakonda et al. reported the primary metabolites of four JWH 073 users as the carboxybutyl and both the 3- and 4-hydroxybutyl compounds (35). Their results are supported by this study.
The proposed metabolic pathway of AM2201 has been presented in other publications (23, 36, 37). When JWH 018 was placed on the emergency schedule list in the USA, AM2201 started to appear in the herbal products, presumably as a pharmacologic replacement for JWH 018. The major legal concern with this compound was that it appeared to share common metabolites with JWH 018. Therefore, it was necessary to distinguish between the use of the two compounds as the use of one was considered illegal while the other was not. Several studies were published showing that the common metabolites between the parent compounds were the hydroxyl- and carboxypentyl produced from JWH 018 (23, 36, 37). This study shows the same metabolic pattern. However, when AM2201 is present in the blood, the hydroxylated N-alkyl side chain with fluorine is detected. This fluorinated metabolite can be used to differentiate between the use of JWH 018 and AM2201. The hydroxylated AM2201 then undergoes defluorination to form the hydroxyl and carboxyl JWH 018 metabolites. Conversely, the JWH 018 metabolites monitored in this study cannot be used to distinguish between AM2201 and JWH 018 ingestion.
The tetramethylcyclopropylindole compounds appear to follow the same metabolic patterns as the naphthylindole compounds. The primary metabolic pathway is via hydroxylation and carboxylation of the N-alkyl side chain. XLR11 is the fluorinated analog of UR 144. The majority of the cases that contain a TMCP compound were positive for XLR11 in the blood. The urine specimen for these cases contained the expected metabolites of XLR11 N-OH, UR 144 N-OH and UR 144 N-COOH. These cases demonstrate the defluorination metabolic route for compounds containing a terminal fluorine on the N-alkyl side chain.
MAM2201, a fluorinated version of JWH 122, was not detected in any of the blood specimens tested. JWH 122 was detected in several specimens along with its corresponding N-hydroxylated metabolites. MAM2201 N-COOH was also detected in those urine specimens. Unfortunately, the presence of these fluorinated/nonfluorinated compounds cannot be used to demonstrate the same metabolic pattern discussed previously in this study.
Conclusion
The presented extraction and LC–MS-MS methods can be used for the identification and quantitation of 15 synthetic cannabinoids in blood and 17 corresponding metabolites in urine. Implementation of both methods in forensic testing can provide significant information for the toxicologist. The analytical results from blood can identify the specific parent synthetic cannabinoid used and may also permit inferences to be made concerning impairment as the pharmacodynamics of this class of compounds matures. In contrast, the urine results can be used to indicate past exposure to synthetic cannabinoids and in some cases provide information concerning the synthetic cannabinoid consumed. There appears to be compelling evidence to suggest synthetic cannabinoids with structural similarities undergo similar metabolic transformations. This concept is illustrated convincingly when comparisons are made between the metabolites generated by structural analogs like JWH 018 and AM2201 as well as XLR11 and UR 144. The study also shows the continuous structural evolution in synthetic cannabinoids in response to the scheduling of drugs.
Disclaimer
The opinions or assertions presented hereafter are the private views of the authors and should not be construed as official or as reflecting the views of the Department of Defense, its branches, the US Army Medical Research and Material Command or the Armed Forces Medical Examiner System.
Funding
This work was funded in part by the ARP Sciences, LLC, Rockville, MD 20850.
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