Journal of Analytical Toxicology, 2016;40:173–186 doi: 10.1093/jat/bkv137 Advance Access Publication Date: 19 January 2016 Article
Article
Analysis of Parent Synthetic Cannabinoids in Blood and Urinary Metabolites by Liquid Chromatography Tandem Mass Spectrometry Jessica L. Knittel*, Justin M. Holler, Jeffrey D. Chmiel, Shawn P. Vorce, Joseph Magluilo Jr, Barry Levine, Gerardo Ramos, and Thomas Z. Bosy Division of Forensic Toxicology, Armed Forces Medical Examiner System, 115 Purple Heart Drive, Dover, DE 19902, USA *Author to whom correspondence should be addressed. Email: [email protected]
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
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Figure 1. Synthetic cannabinoid parent structures and chemical formulas.
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Synthetic Cannabinoids in Blood and Urinary Metabolites 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
Figure 2. Synthetic cannabinoid metabolite structures and chemical formulas.
175 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
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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).
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,
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Table I. Synthetic Cannabinoid Parent Optimal Compound Dependent Parameters Compound
RT (min)
JWH 200-d5
1.02
JWH 200
1.03
RCS4-d 9
4.85
RCS4
4.88
PB-22-d9
4.96
PB-22
4.99
JWH 250-d5
5.09
JWH 250
5.12
STS-135
5.27
JWH 073-d7
5.23
AM2201
4.69
MAM2201
5.10
JWH 073
5.26
XLR11-d5
5.40
XLR11
5.43
JWH 018-d9
5.84
BB-22
5.67
JWH 018
5.88
JWH 081-d9
6.08
JWH 081
6.13
JWH 122-d9
6.43
JWH 122
6.48
2NE1
6.55
UR144-d5
6.83
UR144
6.88
JWH 210-d9
7.09
JWH 210
7.14
AKB48
8.29
MRM transition (m/z)
390.2/155.0 390.2/127.0 385.2/155.0 385.2/127.0 331.2/135.0 331.2/107.0 322.1/135.0 322.1/107.0 368.2/223.2 368.2/144.9 359.2/214.1 359.2/144.9 341.2/121.0 341.2/91.0 336.2/121.0 336.2/91.0 383.3/135.0 383.3/107.0 335.2/155.0 335.2/127.0 360.2/155.0 360.2/127.0 374.2/169.0 374.2/141.0 328.2/154.9 328.2/127.0 335.2/125.0 335.2/236.9 330.2/125.0 330.2/232.1 351.2/155.0 351.2/127.0 385.2/240.1 385.2/143.9 342.2/155.0 342.2/127.0 381.2/185.0 381.2/157.0 372.2/185.0 372.2/157.0 365.2/169.0 365.2/141.0 356.2/169.0 356.2/141.0 365.2/135.1 365.2/107.0 317.2/125.0 317.2/218.8 312.2/125.0 312.2/214.1 379.2/183.0 379.2/223.1 370.2/183.0 370.2/214.1 366.2/135.1 366.2/107.1
Compound dependent parameters CV (V)
CE (eV)
54 54 48 48 52 52 52 52 28 28 30 30 48 48 48 48 62 62 52 52 56 56 60 60 64 64 56 56 56 56 56 56 32 32 52 52 56 56 58 58 56 56 52 52 50 50 56 56 50 50 60 60 54 54 38 38
22 52 22 50 24 44 24 42 12 40 16 40 20 44 20 44 30 44 24 46 26 52 24 46 24 46 22 26 22 24 26 46 14 40 24 44 28 44 26 42 26 42 24 40 28 46 22 24 22 24 28 26 24 24 22 48
RT, retention time; CV, cone voltage; CE, collision energy.
UR-144, XLR11, 2NE1, AM2201-d5, JWH 018-d 9, JWH 073-d7, JWH 081-d 9, JWH 122-d 9, JWH 200-d5, JWH 210-d 9, JWH 250-d5, PB-22-d 9, RCS-4-d 9, 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.
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. Fivehundred 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.
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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).
Table II. Synthetic Cannabinoid Metabolite Optimal Compound Dependent Parameters Compound
JWH 073 N-COOH-d5
RT (min)
MRM Transition (m/z)
Compound dependent parameters DP (V)
EP (V)
CEP (V)
CE (V)
CXP (V)
3.17
363.2/155.2 363.2/127.2 352.1/135.1 352.1/92.1 366.0/121.1 366.0/130.2 352.1/121.0 352.1/130.1 358.2/155.2 358.2/127.1 344.2/155.2 344.2/127.1 372.2/155.2 372.2/127.1 358.2/155.2 358.2/127.2 388.1/185.2 388.1/157.2 400.1/183.2 400.1/155.1 386.1/183.1 386.1/155.2 381.2/155.1 381.2/127.0 376.1/155.1 376.1/127.2 346.2/248.2 346.2/144.0 386.3/169.2 386.3/141.2 390.3/169.2 390.3/141.2 372.2/169.2 372.2/141.2 342.2/125.0 342.2/244.3 328.2/125.0 328.2/244.1
51 51 56 56 56 56 56 56 51 51 61 61 51 51 61 61 66 66 61 61 81 81 66 66 66 66 56 56 51 51 33 63 61 61 51 51 41 41
7.5 7.5 9.5 9.5 4.5 4.5 4.5 4.5 9.5 9.5 8 8 4.5 4.5 10.5 10.5 10.5 10.5 10.5 10.5 10 10 10 10 10.5 10.5 7.5 7.5 11 11 9.5 9.5 10.5 10.5 9 9 10 10
22 22 19 16 19 19 19 19 24 24 22 22 26 26 26 26 20 20 20 20 20 20 24 24 20 20 18 18 22 22 18 18 20 20 36 36 30 30
31 65 31 87 29 51 29 49 33 61 35 65 33 69 33 71 35 57 35 59 33 55 31 69 35 79 29 45 35 59 33 63 33 61 29 35 29 45
4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
RCS4 N-COOH
2.87
JWH 250 N-COOH
2.95
JWH 250 N-OH
3.08
JWH 073 N-COOH
3.19
JWH 073 N-OH
3.28
JWH 018 N-COOH
3.32
JWH 018 N-OH
3.50
JWH 081 N-OH
3.68
JWH 210 N-COOH
3.96
JWH 210 N-OH
4.20
AM2201 N-OH-d5
3.33
AM2201 N-OH
3.35
XLR11 N-OH
3.59
MAM2201 N-COOH
3.62
MAM2201 N-OH
3.66
JWH 122 N-OH
3.83
UR144 N-COOH
3.89
UR144 N-OH
4.11
RT, retention time; DP, declustering potential; EP, entrance potential; CEP, collision cell entrance potential; CE, collision cell; CXP, collision cell exit potential.
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Table III. Linearity and Experimentally Determined LOD, LOQ and ULOL for Blood (A) and Urine (B) Specimens
(A) Blood JWH 200 RCS4 PB-22 JWH 250 STS-135 AM2201 MAM2201 JWH 073 XLR11 BB-22 JWH 018 JWH 081 JWH 122 2NE1 UR144 JWH 210 AKB48 (B) Urine RCS 4 N-COOH JWH 250 N-COOH JWH 250 N-OH JWH 073 N-COOH JWH 073 N-OH JWH 018 N-COOH JWH 018 N-OH JWH 081 N-OH JWH 210 N-COOH JWH 210 N-OH AM2201 N-OH XLR11 N-OH MAM2201 N-COOH MAM2201 N-OH JWH 122 N-OH UR144 N-COOH UR144 N-OH
Linearity (ng/mL)
LOD (ng/mL)
LOQ (ng/mL)
ULOL (ng/mL)
0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10
0.025 0.05 0.025 0.025 0.05 0.05 0.05 0.05 0.05 0.05 0.025 0.025 0.05 0.05 0.05 0.10 0.025
0.05 0.05 0.10 0.025 0.05 0.10 0.10 0.10 0.10 0.05 0.05 0.05 0.05 0.10 0.05 0.10 0.05
50 50 40 50 50 50 40 30 50 50 30 50 10 10 10 50 10
0.25–10 0.50–10 0.25–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10
0.10 0.50 0.25 0.05 0.10 0.05 0.05 0.05 0.025 0.05 0.05 0.05 0.01 0.01 0.05 0.05 0.05
0.25 0.50 0.25 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10
100 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50
LOD, limit of detection; LOQ, limit of quantitation; ULOL, upper limit of linearity.
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. 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.
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 reequilibrate 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). The MS was operated in positive ESI mode. The analysis of the synthetic cannabinoids was operated in MRM acquisition mode.
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Table IV. Precision and Accuracy of the Blood (A) and Urine (B) Assays Precision (% CV)
Accuracy (% difference)
Within run (n = 5)
(A) Blood JWH 200 RCS4 PB-22 JWH 250 STS-135 AM2201 MAM2201 JWH 073 XLR11 BB-22 JWH 018 JWH 081 JWH 122 2NE1 UR144 JWH 210 AKB48
Between day (n = 11)
Low
Mid
High
Low
Mid
High
Low
2.6 1.6 0.5 2.8 2.8 3.3 2.9 4.7 1.9 2.5 1.5 2.9 6.7 8.0 9.9 5.2 4.7 Precision
2.4 2.2 1.3 3.2 2.9 2.8 3.2 1.3 3.0 7.0 2.2 2.8 4.5 6.8 4.2 1.2 5.5
1.4 2.4 2.0 1.6 2.2 1.6 1.1 2.9 2.5 5.7 1.6 3.1 4.1 5.6 5.7 3.2 5.3
4.5 2.8 6.9 3.4 11.3 9.1 4.4 4.5 3.3 5.1 3.3 7.8 10.4 17.1a 9.1 4.0 7.8
5.8 3.1 3.5 7.0 5.3 3.5 2.6 2.1 5.9 5.6 6.6 3.7 16.0a 19.1a 9.5 4.8 7.3
3.3 3.0 3.4 4.4 2.2 2.7 3.7 5.6 3.4 5.8 3.6 4.4 16.3a 17.8a 8.4 4.0 7.1
10.9 3.1 11.7 2.9 9.3 4.4 4.8 −2.4 −10.7 4.6 −6.9 4.5 7.2 2.6 0.3 −0.5 −5.9 −0.7 14.7 −1.1 3.5 −0.3 17.9 −0.4 −8.8 −16.8 −8.5 −16.1 −13.1 −0.5 11.7 −1.3 1.3 −6.8 Accuracy
Within run (n = 3)
(B) Urine RCS 4 N-COOH JWH 250 N-COOH JWH 250 N-OH JWH 073 N-COOH JWH 073 N-OH JWH 018 N-COOH JWH 018 N-OH JWH 081 N-OH JWH 210 N-COOH JWH 210 N-OH AM2201 N-OH XLR11 N-OH MAM2201 N-COOH MAM2201 N-OH JWH 122 N-OH UR144 N-COOH UR144 N-OH
Within run (n = 5)
Between day (n = 11)
Mid
Between day (n = 11) High
Low
−1.1 1.7 0.6 −3.8 1.8 1.2 −4.5 1.1 −9.0 −1.8 −9.0 −1.0 −9.5 −12.1 −6.5 −7.0 −5.3
3.2 10.4 12.2 2.1 −3.9 −2.0 11.2 5.4 −4.2 8.2 −0.5 9.4 −0.7 −4.9 −3.1 5.4 −12.8
Within run (n = 3)
Mid −2.8 1.4 2.7 −7.4 4.1 4.1 5.6 0.4 −1.8 1.3 −4.5 −0.3 −6.1 −4.7 −3.3 −1.0 −7.5
High −5.2 1.7 −2 −5.3 2.8 −0.9 −1.7 −2.1 −7.4 −2.3 −9.2 −2.3 −5.5 1.3 −2.8 −4.6 −8.1
Between day (n = 11)
Low
Mid
High
Low
Mid
High
Low
Mid
High
Low
Mid
High
4.5 N/A 5.8 0.0 9.6 7.5 8.3 4.7 2.4 4.9 4.7 5.9 2.5 0.0 4.2 2.2 6.9
1.8 3.4 7.3 0.6 4.1 1.0 6.6 3.0 4.3 3.3 3.4 3.0 1.7 3.3 5.0 3.4 6.5
5.1 2.3 1.3 5.7 8.4 2.3 7.3 4.6 7.2 2.9 5.0 4.3 1.4 0.8 3.7 1.9 3.7
7.4 N/A 9.4 10.4 7.1 8.6 6.0 7.0 7.1 8.3 6.8 6.8 4.6 5.7 6.0 11.0 10.6
7.5 5.50 11.7 6.3 6.3 6.5 8.2 9.3 6.7 8.4 8.4 6.8 4.1 3.8 11.2 6.8 7.7
5.3 4.40 7.9 5.7 6.8 6.6 6.0 7.6 6.1 7.2 6.1 5.2 5.7 5.1 6.8 6.9 5.2
2.7 N/A 5.3 4.0 5.3 10.7 −4.0 −2.7 −2.7 −6.7 −1.3 2.7 −6.7 −4.0 −4.0 2.7 0.0
−6.0 −9.7 1.3 −0.3 −1.3 4.0 −11.3 −1.0 14.7 10.0 −5.3 −1.0 −9.7 −8.7 −9.0 −5.3 −0.7
−9.7 −12.9 −10.4 −0.3 −6.9 −3.7 −7.8 −8.4 6.0 −2.5 −4.8 −6.0 1.7 −0.5 −10.4 −4.5 −9.6
−3.8 N/A −3.9 −0.4 0.5 6.1 −7.6 −6.8 −2.8 −11.5 −5.6 −2.3 −9.3 −8.4 −7.6 −2.3 2.9
−3.3 −7.9 1.6 −5.4 −4.8 2.8 −12.4 −3.8 8.8 5.3 −9.4 −5.2 −6.0 −4.7 −9.9 −4.3 −1.5
−6.0 −12.4 −8.8 −4.8 −8.5 −3.7 −10.7 −9.2 2.3 −3.9 −10.4 −8.4 −3.9 −5.0 −12.3 −4.8 −8.5
a
Outside the acceptable CV range.
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. 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.
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Table V. Matrix Effects and Recoveries for Blood (A) and Urine (B) Analytes and Their Designated ISTDs Matrix effect
(A) Blood JWH 200-d5 JWH 200 RCS4-d 9 RCS4 PB-22-d9 PB-22 JWH 250-d5 JWH 250 STS-135 JWH 073-d7 AM2201 MAM2201 JWH 073 XLR11-d5 XLR11 JWH 018-d9 BB-22 JWH 018 JWH 081-d9 JWH 081 JWH 122-d9 JWH 122 2NE1 UR144-d5 UR144 JWH 210-d9 JWH 210 AKB48
Recovery
Matrix Effect
0.25 ng/mL
5.0 ng/mL
0.25 ng/mL
5.0 ng/mL
127.9 146.8 72.7 75.2 72.7 68.3 71.7 70.6 76.9 66.6 71.5 68.8 63.8 67.2 62.3 43.0 57.0 42.4 38.6 39.5 42.0 30.1 35.2 37.6 37.7 36.8 39.3 31.3
111.1 118.7 74.1 74.1 72.6 72.2 72.0 73.3 74.7 62.7 69.6 69.4 65.3 67.2 65.8 48.9 61.6 50.6 42.8 47.0 42.8 36.5 38.7 27.5 27.4 32.5 36.7 31.9
77.8 73.7 106.2 103.2 105.7 100.4 107.6 104.3 103.8 117.7 94.9 108.6 117.0 113.3 113.4 161.4 123.1 159.8 169.6 168.6 161.2 136.0 115.4 148.0 133.7 143.1 144.4 174.4
73.7 70.6 102.9 99.6 100.6 96.2 105.6 97.5 96.4 115.4 93.7 98.4 108.7 112.1 104.6 141.6 106.4 129.7 148.0 135.3 153.6 125.7 110.0 146.0 120.0 145.1 134.1 168.4
(B) Urine JWH 073 N-COOH-d5 RCS 4 N-COOH JWH 250 N-COOH JWH 250 N-OH JWH 073 N-COOH JWH 073 N-OH JWH 018 N-COOH JWH 018 N-OH JWH 081 N-OH JWH 210 N-COOH JWH 210 N-OH AM2201 N-OH-d5 AM2201 N-OH XLR11 N-OH MAM2201 N-COOH MAM2201 N-OH JWH 122 N-OH UR144 N-COOH UR144 N-OH
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 (R 2) 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
Recovery
0.10 ng/mL
5.0 ng/mL
0.10 ng/mL
5.0 ng/mL
51.2 N/A N/A N/A 30.6 16.1 26.8 13.3 105.5 83.7 126.0 48.3 18.3 24.4 41.5 36.8 20.3 36.2 34.6
50.0 57.7 29.3 50.4 29.3 21.6 36.7 16.6 100.7 99.2 116.6 48.4 21.6 29.8 56.2 50.2 25.8 39.7 44.1
88.5 N/A N/A N/A 95.7 87.4 96.9 94.1 85.3 94.5 90.8 86.4 101.2 103.2 107.1 104.5 101.5 104.8 103.7
92.4 86.1 87.5 85.9 98.0 96.5 98.8 95.4 85.3 83.4 88.5 81.6 95.4 104.1 97.0 97.4 92.3 93.4 95.1
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
Article
Analysis of Parent Synthetic Cannabinoids in Blood and Urinary Metabolites by Liquid Chromatography Tandem Mass Spectrometry Jessica L. Knittel*, Justin M. Holler, Jeffrey D. Chmiel, Shawn P. Vorce, Joseph Magluilo Jr, Barry Levine, Gerardo Ramos, and Thomas Z. Bosy Division of Forensic Toxicology, Armed Forces Medical Examiner System, 115 Purple Heart Drive, Dover, DE 19902, USA *Author to whom correspondence should be addressed. Email: [email protected]
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
© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
173
174
Figure 1. Synthetic cannabinoid parent structures and chemical formulas.
Knittel et al.
Synthetic Cannabinoids in Blood and Urinary Metabolites 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
Figure 2. Synthetic cannabinoid metabolite structures and chemical formulas.
175 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
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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).
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,
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Table I. Synthetic Cannabinoid Parent Optimal Compound Dependent Parameters Compound
RT (min)
JWH 200-d5
1.02
JWH 200
1.03
RCS4-d 9
4.85
RCS4
4.88
PB-22-d9
4.96
PB-22
4.99
JWH 250-d5
5.09
JWH 250
5.12
STS-135
5.27
JWH 073-d7
5.23
AM2201
4.69
MAM2201
5.10
JWH 073
5.26
XLR11-d5
5.40
XLR11
5.43
JWH 018-d9
5.84
BB-22
5.67
JWH 018
5.88
JWH 081-d9
6.08
JWH 081
6.13
JWH 122-d9
6.43
JWH 122
6.48
2NE1
6.55
UR144-d5
6.83
UR144
6.88
JWH 210-d9
7.09
JWH 210
7.14
AKB48
8.29
MRM transition (m/z)
390.2/155.0 390.2/127.0 385.2/155.0 385.2/127.0 331.2/135.0 331.2/107.0 322.1/135.0 322.1/107.0 368.2/223.2 368.2/144.9 359.2/214.1 359.2/144.9 341.2/121.0 341.2/91.0 336.2/121.0 336.2/91.0 383.3/135.0 383.3/107.0 335.2/155.0 335.2/127.0 360.2/155.0 360.2/127.0 374.2/169.0 374.2/141.0 328.2/154.9 328.2/127.0 335.2/125.0 335.2/236.9 330.2/125.0 330.2/232.1 351.2/155.0 351.2/127.0 385.2/240.1 385.2/143.9 342.2/155.0 342.2/127.0 381.2/185.0 381.2/157.0 372.2/185.0 372.2/157.0 365.2/169.0 365.2/141.0 356.2/169.0 356.2/141.0 365.2/135.1 365.2/107.0 317.2/125.0 317.2/218.8 312.2/125.0 312.2/214.1 379.2/183.0 379.2/223.1 370.2/183.0 370.2/214.1 366.2/135.1 366.2/107.1
Compound dependent parameters CV (V)
CE (eV)
54 54 48 48 52 52 52 52 28 28 30 30 48 48 48 48 62 62 52 52 56 56 60 60 64 64 56 56 56 56 56 56 32 32 52 52 56 56 58 58 56 56 52 52 50 50 56 56 50 50 60 60 54 54 38 38
22 52 22 50 24 44 24 42 12 40 16 40 20 44 20 44 30 44 24 46 26 52 24 46 24 46 22 26 22 24 26 46 14 40 24 44 28 44 26 42 26 42 24 40 28 46 22 24 22 24 28 26 24 24 22 48
RT, retention time; CV, cone voltage; CE, collision energy.
UR-144, XLR11, 2NE1, AM2201-d5, JWH 018-d 9, JWH 073-d7, JWH 081-d 9, JWH 122-d 9, JWH 200-d5, JWH 210-d 9, JWH 250-d5, PB-22-d 9, RCS-4-d 9, 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.
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. Fivehundred 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.
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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).
Table II. Synthetic Cannabinoid Metabolite Optimal Compound Dependent Parameters Compound
JWH 073 N-COOH-d5
RT (min)
MRM Transition (m/z)
Compound dependent parameters DP (V)
EP (V)
CEP (V)
CE (V)
CXP (V)
3.17
363.2/155.2 363.2/127.2 352.1/135.1 352.1/92.1 366.0/121.1 366.0/130.2 352.1/121.0 352.1/130.1 358.2/155.2 358.2/127.1 344.2/155.2 344.2/127.1 372.2/155.2 372.2/127.1 358.2/155.2 358.2/127.2 388.1/185.2 388.1/157.2 400.1/183.2 400.1/155.1 386.1/183.1 386.1/155.2 381.2/155.1 381.2/127.0 376.1/155.1 376.1/127.2 346.2/248.2 346.2/144.0 386.3/169.2 386.3/141.2 390.3/169.2 390.3/141.2 372.2/169.2 372.2/141.2 342.2/125.0 342.2/244.3 328.2/125.0 328.2/244.1
51 51 56 56 56 56 56 56 51 51 61 61 51 51 61 61 66 66 61 61 81 81 66 66 66 66 56 56 51 51 33 63 61 61 51 51 41 41
7.5 7.5 9.5 9.5 4.5 4.5 4.5 4.5 9.5 9.5 8 8 4.5 4.5 10.5 10.5 10.5 10.5 10.5 10.5 10 10 10 10 10.5 10.5 7.5 7.5 11 11 9.5 9.5 10.5 10.5 9 9 10 10
22 22 19 16 19 19 19 19 24 24 22 22 26 26 26 26 20 20 20 20 20 20 24 24 20 20 18 18 22 22 18 18 20 20 36 36 30 30
31 65 31 87 29 51 29 49 33 61 35 65 33 69 33 71 35 57 35 59 33 55 31 69 35 79 29 45 35 59 33 63 33 61 29 35 29 45
4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
RCS4 N-COOH
2.87
JWH 250 N-COOH
2.95
JWH 250 N-OH
3.08
JWH 073 N-COOH
3.19
JWH 073 N-OH
3.28
JWH 018 N-COOH
3.32
JWH 018 N-OH
3.50
JWH 081 N-OH
3.68
JWH 210 N-COOH
3.96
JWH 210 N-OH
4.20
AM2201 N-OH-d5
3.33
AM2201 N-OH
3.35
XLR11 N-OH
3.59
MAM2201 N-COOH
3.62
MAM2201 N-OH
3.66
JWH 122 N-OH
3.83
UR144 N-COOH
3.89
UR144 N-OH
4.11
RT, retention time; DP, declustering potential; EP, entrance potential; CEP, collision cell entrance potential; CE, collision cell; CXP, collision cell exit potential.
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Table III. Linearity and Experimentally Determined LOD, LOQ and ULOL for Blood (A) and Urine (B) Specimens
(A) Blood JWH 200 RCS4 PB-22 JWH 250 STS-135 AM2201 MAM2201 JWH 073 XLR11 BB-22 JWH 018 JWH 081 JWH 122 2NE1 UR144 JWH 210 AKB48 (B) Urine RCS 4 N-COOH JWH 250 N-COOH JWH 250 N-OH JWH 073 N-COOH JWH 073 N-OH JWH 018 N-COOH JWH 018 N-OH JWH 081 N-OH JWH 210 N-COOH JWH 210 N-OH AM2201 N-OH XLR11 N-OH MAM2201 N-COOH MAM2201 N-OH JWH 122 N-OH UR144 N-COOH UR144 N-OH
Linearity (ng/mL)
LOD (ng/mL)
LOQ (ng/mL)
ULOL (ng/mL)
0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10
0.025 0.05 0.025 0.025 0.05 0.05 0.05 0.05 0.05 0.05 0.025 0.025 0.05 0.05 0.05 0.10 0.025
0.05 0.05 0.10 0.025 0.05 0.10 0.10 0.10 0.10 0.05 0.05 0.05 0.05 0.10 0.05 0.10 0.05
50 50 40 50 50 50 40 30 50 50 30 50 10 10 10 50 10
0.25–10 0.50–10 0.25–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10 0.10–10
0.10 0.50 0.25 0.05 0.10 0.05 0.05 0.05 0.025 0.05 0.05 0.05 0.01 0.01 0.05 0.05 0.05
0.25 0.50 0.25 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10
100 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50
LOD, limit of detection; LOQ, limit of quantitation; ULOL, upper limit of linearity.
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. 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.
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 reequilibrate 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). The MS was operated in positive ESI mode. The analysis of the synthetic cannabinoids was operated in MRM acquisition mode.
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Knittel et al.
Table IV. Precision and Accuracy of the Blood (A) and Urine (B) Assays Precision (% CV)
Accuracy (% difference)
Within run (n = 5)
(A) Blood JWH 200 RCS4 PB-22 JWH 250 STS-135 AM2201 MAM2201 JWH 073 XLR11 BB-22 JWH 018 JWH 081 JWH 122 2NE1 UR144 JWH 210 AKB48
Between day (n = 11)
Low
Mid
High
Low
Mid
High
Low
2.6 1.6 0.5 2.8 2.8 3.3 2.9 4.7 1.9 2.5 1.5 2.9 6.7 8.0 9.9 5.2 4.7 Precision
2.4 2.2 1.3 3.2 2.9 2.8 3.2 1.3 3.0 7.0 2.2 2.8 4.5 6.8 4.2 1.2 5.5
1.4 2.4 2.0 1.6 2.2 1.6 1.1 2.9 2.5 5.7 1.6 3.1 4.1 5.6 5.7 3.2 5.3
4.5 2.8 6.9 3.4 11.3 9.1 4.4 4.5 3.3 5.1 3.3 7.8 10.4 17.1a 9.1 4.0 7.8
5.8 3.1 3.5 7.0 5.3 3.5 2.6 2.1 5.9 5.6 6.6 3.7 16.0a 19.1a 9.5 4.8 7.3
3.3 3.0 3.4 4.4 2.2 2.7 3.7 5.6 3.4 5.8 3.6 4.4 16.3a 17.8a 8.4 4.0 7.1
10.9 3.1 11.7 2.9 9.3 4.4 4.8 −2.4 −10.7 4.6 −6.9 4.5 7.2 2.6 0.3 −0.5 −5.9 −0.7 14.7 −1.1 3.5 −0.3 17.9 −0.4 −8.8 −16.8 −8.5 −16.1 −13.1 −0.5 11.7 −1.3 1.3 −6.8 Accuracy
Within run (n = 3)
(B) Urine RCS 4 N-COOH JWH 250 N-COOH JWH 250 N-OH JWH 073 N-COOH JWH 073 N-OH JWH 018 N-COOH JWH 018 N-OH JWH 081 N-OH JWH 210 N-COOH JWH 210 N-OH AM2201 N-OH XLR11 N-OH MAM2201 N-COOH MAM2201 N-OH JWH 122 N-OH UR144 N-COOH UR144 N-OH
Within run (n = 5)
Between day (n = 11)
Mid
Between day (n = 11) High
Low
−1.1 1.7 0.6 −3.8 1.8 1.2 −4.5 1.1 −9.0 −1.8 −9.0 −1.0 −9.5 −12.1 −6.5 −7.0 −5.3
3.2 10.4 12.2 2.1 −3.9 −2.0 11.2 5.4 −4.2 8.2 −0.5 9.4 −0.7 −4.9 −3.1 5.4 −12.8
Within run (n = 3)
Mid −2.8 1.4 2.7 −7.4 4.1 4.1 5.6 0.4 −1.8 1.3 −4.5 −0.3 −6.1 −4.7 −3.3 −1.0 −7.5
High −5.2 1.7 −2 −5.3 2.8 −0.9 −1.7 −2.1 −7.4 −2.3 −9.2 −2.3 −5.5 1.3 −2.8 −4.6 −8.1
Between day (n = 11)
Low
Mid
High
Low
Mid
High
Low
Mid
High
Low
Mid
High
4.5 N/A 5.8 0.0 9.6 7.5 8.3 4.7 2.4 4.9 4.7 5.9 2.5 0.0 4.2 2.2 6.9
1.8 3.4 7.3 0.6 4.1 1.0 6.6 3.0 4.3 3.3 3.4 3.0 1.7 3.3 5.0 3.4 6.5
5.1 2.3 1.3 5.7 8.4 2.3 7.3 4.6 7.2 2.9 5.0 4.3 1.4 0.8 3.7 1.9 3.7
7.4 N/A 9.4 10.4 7.1 8.6 6.0 7.0 7.1 8.3 6.8 6.8 4.6 5.7 6.0 11.0 10.6
7.5 5.50 11.7 6.3 6.3 6.5 8.2 9.3 6.7 8.4 8.4 6.8 4.1 3.8 11.2 6.8 7.7
5.3 4.40 7.9 5.7 6.8 6.6 6.0 7.6 6.1 7.2 6.1 5.2 5.7 5.1 6.8 6.9 5.2
2.7 N/A 5.3 4.0 5.3 10.7 −4.0 −2.7 −2.7 −6.7 −1.3 2.7 −6.7 −4.0 −4.0 2.7 0.0
−6.0 −9.7 1.3 −0.3 −1.3 4.0 −11.3 −1.0 14.7 10.0 −5.3 −1.0 −9.7 −8.7 −9.0 −5.3 −0.7
−9.7 −12.9 −10.4 −0.3 −6.9 −3.7 −7.8 −8.4 6.0 −2.5 −4.8 −6.0 1.7 −0.5 −10.4 −4.5 −9.6
−3.8 N/A −3.9 −0.4 0.5 6.1 −7.6 −6.8 −2.8 −11.5 −5.6 −2.3 −9.3 −8.4 −7.6 −2.3 2.9
−3.3 −7.9 1.6 −5.4 −4.8 2.8 −12.4 −3.8 8.8 5.3 −9.4 −5.2 −6.0 −4.7 −9.9 −4.3 −1.5
−6.0 −12.4 −8.8 −4.8 −8.5 −3.7 −10.7 −9.2 2.3 −3.9 −10.4 −8.4 −3.9 −5.0 −12.3 −4.8 −8.5
a
Outside the acceptable CV range.
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. 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.
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Synthetic Cannabinoids in Blood and Urinary Metabolites
Table V. Matrix Effects and Recoveries for Blood (A) and Urine (B) Analytes and Their Designated ISTDs Matrix effect
(A) Blood JWH 200-d5 JWH 200 RCS4-d 9 RCS4 PB-22-d9 PB-22 JWH 250-d5 JWH 250 STS-135 JWH 073-d7 AM2201 MAM2201 JWH 073 XLR11-d5 XLR11 JWH 018-d9 BB-22 JWH 018 JWH 081-d9 JWH 081 JWH 122-d9 JWH 122 2NE1 UR144-d5 UR144 JWH 210-d9 JWH 210 AKB48
Recovery
Matrix Effect
0.25 ng/mL
5.0 ng/mL
0.25 ng/mL
5.0 ng/mL
127.9 146.8 72.7 75.2 72.7 68.3 71.7 70.6 76.9 66.6 71.5 68.8 63.8 67.2 62.3 43.0 57.0 42.4 38.6 39.5 42.0 30.1 35.2 37.6 37.7 36.8 39.3 31.3
111.1 118.7 74.1 74.1 72.6 72.2 72.0 73.3 74.7 62.7 69.6 69.4 65.3 67.2 65.8 48.9 61.6 50.6 42.8 47.0 42.8 36.5 38.7 27.5 27.4 32.5 36.7 31.9
77.8 73.7 106.2 103.2 105.7 100.4 107.6 104.3 103.8 117.7 94.9 108.6 117.0 113.3 113.4 161.4 123.1 159.8 169.6 168.6 161.2 136.0 115.4 148.0 133.7 143.1 144.4 174.4
73.7 70.6 102.9 99.6 100.6 96.2 105.6 97.5 96.4 115.4 93.7 98.4 108.7 112.1 104.6 141.6 106.4 129.7 148.0 135.3 153.6 125.7 110.0 146.0 120.0 145.1 134.1 168.4
(B) Urine JWH 073 N-COOH-d5 RCS 4 N-COOH JWH 250 N-COOH JWH 250 N-OH JWH 073 N-COOH JWH 073 N-OH JWH 018 N-COOH JWH 018 N-OH JWH 081 N-OH JWH 210 N-COOH JWH 210 N-OH AM2201 N-OH-d5 AM2201 N-OH XLR11 N-OH MAM2201 N-COOH MAM2201 N-OH JWH 122 N-OH UR144 N-COOH UR144 N-OH
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 (R 2) 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
Recovery
0.10 ng/mL
5.0 ng/mL
0.10 ng/mL
5.0 ng/mL
51.2 N/A N/A N/A 30.6 16.1 26.8 13.3 105.5 83.7 126.0 48.3 18.3 24.4 41.5 36.8 20.3 36.2 34.6
50.0 57.7 29.3 50.4 29.3 21.6 36.7 16.6 100.7 99.2 116.6 48.4 21.6 29.8 56.2 50.2 25.8 39.7 44.1
88.5 N/A N/A N/A 95.7 87.4 96.9 94.1 85.3 94.5 90.8 86.4 101.2 103.2 107.1 104.5 101.5 104.8 103.7
92.4 86.1 87.5 85.9 98.0 96.5 98.8 95.4 85.3 83.4 88.5 81.6 95.4 104.1 97.0 97.4 92.3 93.4 95.1
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
