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Structure elucidation and identification of a common metabolite for naphthoylindole-based synthetic cannabinoids using LC-TOF and comparison to a synthetic reference standard
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FSI-7040; No. of Pages 7 Forensic Science International xxx (2013) xxx–xxx
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Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Structure elucidation and identification of a common metabolite for naphthoylindole-based synthetic cannabinoids using LC-TOF and comparison to a synthetic reference standard Dennis P. Lovett a,*, Enrique G. Yanes a, Travis W. Herbelin a, Timm A. Knoerzer b, Joseph A. Levisky b a b
HQ Air Force Drug Testing Laboratory, Lackland Air Force Base, TX 78236-5310, United States HQ United States Air Force Academy/DFC 2355 Fairchild Drive, Suite 2N225 USAF Academy, CO 80840-6230, United States
A R T I C L E I N F O
A B S T R A C T
Article history: Received 16 October 2012 Received in revised form 7 December 2012 Accepted 13 December 2012 Available online xxx
The identification of a predominate metabolite found in urine specimens which test positive for naphthoylindole-based synthetic cannabinoids is reported. The presence of this new metabolite was detected at the Air Force Drug Testing Lab Investigations Division during screening analysis for metabolites of JWH-018 and JWH-073, because it shares the same MRM transitions as the JWH-073 N(3-hydroxybutyl) metabolite. However, the detected peak is chromatographically distinguished from other metabolites due to differences in retention time. This metabolite appears to be a common metabolite for select naphthoylindole-based synthetic cannabinoids that could potentially be used as a common biomarker for their qualitative and quantitative analyses. The new metabolite has been successfully identified as 3-(3-(1-naphthoyl)-1H-indol-1-yl) propanoic acid (1, JWH 072 N-propanoic acid metabolite, Fig. 1) by using various mass spectrometric and liquid chromatographic techniques as well as chemical derivatization. The metabolite identity was confirmed through the comparison of authentic positive urine and a chemically synthesized metabolite standard. Both materials shared the same chromatographic retention time on two separate chromatographic systems, mass fragmentation pattern and exact mass. Full characterization of the synthetic reference material and intermediates by 1H NMR, 13C NMR, IR and HRMS was also conducted. ß 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: UPLC Synthetic cannabinoid metabolites NMR LC-TOF Common biomarker JWH-072 metabolite
1. Introduction Since their introduction into the market place in 2004 as ‘‘legal highs’’, synthetic cannabinoids have rapidly gained popularity in the United States and Europe [1]. Originally sold under the brand name ‘Spice’, the term Spice has become a generic term to include the entire class of ‘‘legal high’’ smoking blends which are sold on the Internet, gas stations and local tobacco shops as incense or potpourri, but are actually inactive plant matter originally adulterated with naphthoylindole containing synthetic cannabinoids such as JWH-018 (2) and JWH-073 (3) and more recently AM-2201 (4) (Fig. 1). In December 2008, THC Pharma (Germany) and AGES PharmMed (Austria) reported independently the presence of JWH-018 in many herbal smoking blends [2]. A direct link was established in 2010 between herbal preparations containing JWH-018 and the psychotropic effects [3]. In July 2012, 14 synthetic cannabinoids were included on the Drug
* Corresponding author. Tel.: +1 210 292 3516. E-mail addresses: [email protected], [email protected] (D.P. Lovett).
Enforcement Agency’s Schedule I list making the possession, manufacture, and consumption of these compounds illegal [4]. It can be expected that black market manufacturers will respond to this scheduling by structural modification of the illicit compounds to create new cannabinoids thus circumventing the new drug law. As a consequence, the work load for forensic and workplace drug testing labs will increase and analyses will become even more challenging. To keep up with the ever-changing synthetic cannabinoid landscape, simpler and faster analytical methods are required. The popularity of Spice has extended to the United States Armed Forces, which resulted in the United States Department of Defense banning all military personnel from possessing or usage of synthetic cannabinoids [5]. A directive was issued to develop both a screening and a confirmation analyses for synthetic cannabinoid metabolites in urine. The resulting process was implemented at the Air Force Drug Testing Lab Investigations Division and analysis of service member samples began in March 2012 [6]. Soon after testing had begun, TIC chromatograms showed the presence of an unidentified peak in all of the specimens that tested positive for one or more of the four metabolites of interest (5–8, Fig. 2). This new peak eluted earlier than the reference metabolites
0379-0738/$ – see front matter ß 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.forsciint.2012.12.012
Please cite this article in press as: D.P. Lovett, et al., Structure elucidation and identification of a common metabolite for naphthoylindole-based synthetic cannabinoids using LC-TOF and comparison to a synthetic reference standard, Forensic Sci. Int. (2013), http://dx.doi.org/10.1016/j.forsciint.2012.12.012
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FSI-7040; No. of Pages 7 D.P. Lovett et al. / Forensic Science International xxx (2013) xxx–xxx
2
O
O N
OH
N
R
O 1
2 (JWH-018) R = CH3 3 (JWH-073) R = H 4 (AM-2201) R = CH2F
Fig. 1. 3-(3-(1-Naphthoyl)-1H-indol-1-yl) propanoic acid, 1; naphthoylindole synthetic cannabinoids 2–4.
and shared multiple reaction monitoring (MRM) transitions with the JWH-073 N-(3-hydroxybutyl) metabolite (7). Based on this evidence, and the fact that the unknown was also extracted along with the other metabolites, it was suspected that 7 and the unknown metabolite shared a similar core structure and consequently belonged to the same synthetic cannabinoid series. A review of current literature reveals that Moran et al. [7,8] had also reported observing an early eluting unknown peak, but did not report its identity nor its potential as a common biomarker for JWH-018, JWH-073 and AM-2201. The present work describes the characterization and identification of a previously unreported metabolite that is common for a select group of naphthoylindole cannabinoids. The metabolite was characterized by using different techniques including LC–MS, LC– MS/MS and LC-TOF which allows the determination of the molecular ion, fragment ions, and accurate mass. The hypothesized metabolite was not commercially available; therefore identity confirmation was achieved by retention time matching to a chemically synthesized metabolite standard that had also been subjected to NMR, IR and LC-TOF analysis. 2. Experimental 2.1. Chemicals, reagents and standards All UPLC solvents were LC–MS grade. Acetonitrile, methanol and water were obtained from Fisher Scientific (Pittsburgh, PA). Formic acid and ammonium acetate (both Optima LC–MS grade) were also obtained from Fisher Scientific. bGlucuronidase (Escherichia coli, Type IX-A, lyophilized powder, 1,000,000– 5,000,000 units/g protein) was purchased from Sigma–Aldrich (St. Louis, MO, USA). Chemicals and reagents used for the synthesis of 3-(3-(1-naphthoyl)-1H-indol-1-yl)
O
OH
propanoic acid (1) and deuterated chloroform (CDCl3) were obtained from Sigma– Aldrich (St. Louis, MO). Reference standards of metabolites: JWH-018 N-(4-hydroxypentyl), JWH-018 Npentanoic acid, JWH-073 N-(3-hydroxybutyl) and JWH-073 N-butanoic acid were obtained from Cerilliant Corporation (Round Rock, TX, USA) in ampoules as 1 mL methanol solutions at 100 mg/mL. The metabolite standard, JWH-018 N-(5hydroxypentyl) was obtained from Cayman Chemicals (Ann Arbor, MI) as a solid in a screw-cap vial. 2.2. Instrumentation 2.2.1. UPLC-TQD; for determination of parent and daughter ions The chromatographic system was a Waters Acquity UPLC (Milford, MA, USA) consisting of sample, solvent and column managers. The solvent manager included a binary pump and the column manager was comprised of a thermostatted column compartment. Chromatographic separation was performed on a Waters Acquity UPLC HSS T3 (2.1 mm 100 mm, 1.8 mm) column which was connected to an Acquity UPLC HSS T3 VanGuard (2.1 mm 5 mm, 1.8 mm) pre-column. The column temperature was maintained at 40 8C. Isocratic conditions (50% A:50% B) were used for the chromatographic separation of the different analytes of interest. The optimal flow rate was set at 0.6 mL/min with a sample injection volume of 10 mL. Mobile phases consisted of LC/MS grade water containing 0.1% formic acid (mobile phase A) and LC/MS grade acetonitrile also containing 0.1% formic acid (mobile phase B). The mass spectrometer was a Waters tandem quadrupole detector (TQD) which was operated in the positive electrospray ionization and multiple reaction monitoring (MRM) mode for selective and sensitive detection. The applied capillary voltage was 3.3 kV. The source temperature was set at 150 8C and desolvation temperature at 450 8C. Nitrogen was used as the nebulizing gas and argon as the collision gas. The desolvation gas flow was set at 600 L/h, cone gas at 50 L/h and collision gas at 0.2 mL/min. The collision cell pressure was approximately 5.28 103 mbar. All data was processed using MassLynx1 software from Waters Corporation. 2.2.2. LC/MSD TOF; for identification and accurate mass determination An Agilent 1100 series HPLC (Agilent Technologies, Palo Alto, CA, USA) was fitted with a reverse-phase Higgins Analytical Phalanx C18 (2.1 mm 150 mm, 5 mm) column. The column temperature was maintained at 25 8C. Mobile phases consisted of mobile phase A (5 mM ammonium formate) and mobile phase B (acetonitrile). A gradient program was used for compounds 11 and 1 consisting of an initial solvent ratio of 50% A and 50% B. The mobile phase gradient was ramped to 95% B over 7 min and held at this proportion for 5 min. The initial 50% A and 50% B mobile phase was re-established in 1 min and held at this value for 3 min between sample runs. For compound 13 an isocratic program was used consisting of 40% mobile phase A and 60% mobile phase B. The flow rate for both programs was 0.3 mL/min with an injection volume of 2 mL. The HPLC system was interfaced to a time-of-flight mass spectrometer (MSD-TOF, Agilent Technologies) operated in the positive electrospray ionization mode and using the following parameters: capillary 3500 V, nebulizer 30 psi g, drying gas 12 L/min, gas temperature 325 8C, fragmentator 125 V, skimmer 65 V, OCT 1RF 250 V. The instrument was calibrated
O
N
N
OH O
5 JWH-018 N-(4-hydroxypentyl)
O
6 JWH-018 N-pentanoic acid
O N
O N
OH
OH 7 JWH-073 N-(3-hydroxybutyl)
8 JWH-073 N-butanoic acid
Fig. 2. Metabolites of interest for JWH-018 and JWH-073.
Please cite this article in press as: D.P. Lovett, et al., Structure elucidation and identification of a common metabolite for naphthoylindole-based synthetic cannabinoids using LC-TOF and comparison to a synthetic reference standard, Forensic Sci. Int. (2013), http://dx.doi.org/10.1016/j.forsciint.2012.12.012
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FSI-7040; No. of Pages 7 D.P. Lovett et al. / Forensic Science International xxx (2013) xxx–xxx prior to each analysis using the solution and procedure recommended by the manufacturer. Spectra were acquired over the m/z 50–1000 range at a scan rate of 1.02 s/spectrum. 2.2.3. GCMS; for reaction monitoring of intermediates during metabolite synthesis Monitoring of the different synthetic steps was conducted on an Agilent 7890A gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) interfaced to an Agilent 5975 C inert XL EI/CI MSD mass spectrometer. The separation was achieved on an Agilent HP-5 MS (30 m 0.250 mm; 0.25 mm) applying two different temperature programs. Temperature program 1 used for compound 11 consisted of 90 8C (2 min), heating 35 8C/min to 325 8C (5.29 min). The mass spectrometer was operated in full scan mode with a mass scan from m/z 40.0 to 300.0. Temperature program 2 used for compounds 1 and 13 consisted of 90 8C (2 min), heating 20 8C/ min to 250 8C (2 min), 30 8C/min to 300 8C (4 min). The mass spectrometer was operated in full scan mode with a mass scan from m/z 40.0 to 500.0. Two microliter were injected at 250 8C in split mode (100:1) with the carrier gas (high purity helium) flow set to 1.0 mL/min. Transfer line temperature was 280 8C and the ion source was held at 230 8C. 2.2.4. NMR spectroscopy The NMR characterization of the synthetic intermediates and final product was carried out on a Varian Mercury Plus 400 MHz NMR spectrometer (VarianNMR Inc., Palo Alto, CA, USA) with an Oxford AS 400 superconducting magnet and a Varian 400 ATB PFG Quad Probe at room temperature. Chemical shifts were referenced to the residual undeuterated solvent peak at 7.26 ppm for 1H and the 13 CDCl3 center peak at 77.00 ppm for 13C. All data were processed using SpinWorks 2.5 software. 2.2.5. IR spectroscopy The IR characterization of the synthetic intermediates and the final product was carried out on a Thermo-Nicolet iS180 spectrometer (Waltham, MA, USA) fitted with a Smart iTR attenuated total reflectance sampling device. 2.3. Reagents and standards preparation The salting out reagent (10 M ammonium acetate solution) was prepared by dissolving 38.55 g of ammonium acetate in a 50 mL volumetric flask with LC/MS grade water (15 mL) to give exactly 50 mL. Solution was sonicated for 2 h to assist with dissolution. The b-glucuronidase solution was prepared as 12.3 units/mL in 0.1 M ammonium acetate by mixing 615,000 modified Fishman units of b-glucuronidase Type IXA (370 mg of 1,661,000 units/g) and 500 mL of 10 M ammonium acetate solution into a 50 mL volumetric flask. The mixture was diluted to the mark with LC/MS grade water, shaken and sonicated for a few seconds to complete the mixing. The solution was transferred into 1.7 mL microcentrifuge tubes and stored at 30 8C until use. Metabolite reference standards were stored at 30 8C according to the manufacture’s recommendations until used. Working solutions were further prepared in methanol and kept in the refrigerator until needed. 2.4. Sample treatment for extraction and concentration of unknown metabolite Thirty urine aliquots were subjected to enzymatic hydrolysis and salting-out assisted liquid–liquid extraction protocol validated in this lab [6]. All microcentrifuge tubes containing 100 mL of authentic positive urine each were treated with 25 mL of b-D-glucuronidase solution and then mixed by vortexing gently for 5 s. The incubation of all tubes was carried out at room temperature over 10 min. After sample hydrolysis, 200 mL of LC/MS grade acetonitrile was added and the tubes were mixed by vortexing for 5 s. Fifty microliter of 10 M ammonium acetate solution was then added and mixed by vortexing for 5 s. The microcentrifuge tubes were centrifuged at 10,000 rpm for 3 min. The upper organic phases of the tubes were removed from each microcentrifuge tube, combined and placed into a TurboVap evaporator. The solvent was reduced to a volume of approximately 0.2 mL using a stream of nitrogen to assist solvent evaporation. The resulting concentrate was prepared for LC–MS analysis by removing 100 mL of the solution and transferring to a clean HPLC vial. The sample was diluted with 100 mL of LC/MS grade water and mixed well by vortexing for 5 s. 2.5. Derivatization of JWH-018 and JWH-073 carboxylate metabolites and authentic urine sample to methyl esters Solutions of JWH-018 N-pentanoic acid (10 mL, 1 mg/mL in DMSO), JWH-073 Nbutanoic acid (10 mL, 1 mg/mL in DMSO) or concentrated extract of authentic positive urine was added to separate micro centrifuge tubes, treated with a solution of 8:1 DMSO/ammonium hydroxide (30 mL) and then mixed by vortexing. Methyl iodide (10 mL) was added to each tube and mixed by vortexing to afford a cloudy suspension. After five min, all tubes were treated with dilute hydrochloric acid (0.1 M, 200 mL) followed by isooctane (200 mL). The mixtures were mixed by
3
vortexing for one min then the tubes were centrifuged for 1 min at 10,000 rpm. The upper layer of each tube was transferred to a separate glass tube and the solvent was removed by under a stream of nitrogen in a 50 8C water bath. The residues were dissolved in acetonitrile (0.5 mL) then mixed by vortexing. LC/MS grade water (0.5 mL) was added to the tubes then the solutions were transferred to HPLC vials and analyzed by LC–MS/MS. Table 1 lists the MRM transitions monitored as well as the cone voltages and collision energies. 2.6. Synthesis of 3-(3-(1-naphthoyl)-1H-indol-1-yl) propanoic acid, (JWH-072 Npropanoic acid, 1) The synthesis of the hypothesized metabolite followed a modification of the published procedure of Martin and Huffman [9] for the connection of the alkyl indole and the naphthalene. The reaction scheme is outlined in Fig. 3. 2.6.1. Synthesis of methyl-3-(1-indol-1-yl) propionate, (11) To an oven dried three-neck 50 mL round bottom flask was charged 10 mL of dry DMSO under N2. Lithium hydride (175 mg, 22 mmol) was added to the stirring DMSO and held for 5 min. Indole (9, 1.17 g, 10 mmol) dissolved in dry DMSO (5 mL) was added over 2 min with observed gas evolution. After stirring for 90 min, methyl 3-bromopropionate (10, 1.20 mL, 11 mmol) was added drop-wise to the reaction mixture. The reaction was poured into stirring 1N HCl (20 mL) and extracted with ethyl acetate (3 20 mL). The pooled organic layers were dried over Na2SO4 prior to removal of solvent to yield 11 as a pale amber oil. The crude material was taken forward without purification. A sample of 11 was purified by silica gel column chromatography using an elution gradient of 9:1–4:1 hexanes:EtOAc (Rf = 0.20, 4:1 hexanes:EtOAc) for the acquisition of analytical spectra. 1H NMR (400 MHz, CDCl3) d 7.61 (1H, d, 7.6 Hz), 7.32 (1H, d, 8.2 Hz), 7.20 (1H, t, 7.4 Hz), 7.11, (2H, m), 6.47 (1H, d, 3.0 Hz), 4.42 (2H, t, 6.9 Hz), 3.63 (3H, s), 2.80 (2H, t, 6.9 Hz); 13C NMR (100 MHz, CDCl3) d 171.71, 135.65, 128.74, 127.98, 121.69, 121.12, 119.56, 109.11, 101.64, 51.96, 41.82, 34.77; IR (ATR) 3735, 3412, 2952, 2359, 2342, 1734, 1512, 1463, 1437, 1398, 1354, 1333, 1333, 1313, 1261, 1202, 1169, 1091, 1062, 1012, 765, 739, 668 cm1; exact mass: m/z = 204.1014 (theoretical: 204.1019). 2.6.2. Synthesis of methyl-3-(3-(1-naphthoyl)-1H-indol-1-yl) propionate, (JWH-072 N-propanoic acid methyl ester, 13) Me2AlCl (1 M in hexanes, 0.72 mL, 0.72 mmol) was added drop-wise to a dry conical vial containing a solution of methyl 3-(1-indol-1-yl) propionate (11, 97 mg, 0.48 mmol) in dry CH2Cl2 (1.4 mL) at 0 8C under N2. The red solution was stirred at 0 8C for 30 min and then a solution of 1-naphthoyl chloride (12, 80 mL, 0.58 mmol) in 0.9 mL of dry CH2Cl2 was added dropwise. The deep red acylation reaction mixture was stirred at 0 8C until the reaction was complete in approximately 30 min as indicated by TLC (Rf = 0.15, 3:1 hexanes:ethyl acetate). The reaction mixture was poured carefully into ice cold 1 M aqueous HCl (10 mL) and extracted with CH2Cl2 (3 5 mL). The combined extracts were washed with three portions of aqueous NaHCO3 (5 mL), dried over Na2SO4 and the solvent was removed in vacuo to give crude methyl ester 13 (121.7 mg, 71%) as a green foam. 1H NMR (400 MHz, CDCl3) d 8.52 (1H, dd, 4.2 Hz, 3.1 Hz), 8.18 (1H, d, 8.2 Hz), 7.96 (1H, d, 8.2 Hz), 7.90 (1H, d, 7.9 Hz), 7.64 (1H, d, 6.9 Hz), 7.46–7.54 (3H, m), 7.41 (1H, s), 7.36–7.39 (3H, m), 4.39 (2H, t, 6.7 Hz), 3.59 (3H, s), 2.81 (2H, d, 6.7 Hz); 13C NMR (100 MHz, CDCl3) d 192.13, 170.92, 138.80, 138.28, 136.62, 133.76, 130.75, 130.14, 128.21, 127.03, 126.80, 126.30, 125.94, 125.89, 124.52, 123.90, 123.09, 123.08, 118.00, 109.63, 52.07, 42.42, 34.18; IR (ATR) 3054, 2951, 1735, 1627, 1610, 1577, 1521, 1484, 1464,1377, 1338, 1261, 1200, 1174, 1137, 1079, 1055, 1014 887, 791, 750 cm1; exact mass: m/z = 358.1449 (theoretical: 358.1443).
Table 1 MS/MS parameters. Compound JWH-073 JWH-073 JWH-018 JWH-018 JWH-073 JWH-073 JWH-018 JWH-018 JWH-018 JWH-018
N-(3-hydroxybutyl) N-(3-hydroxybutyl) N-(4-hydroxypentyl) N-(4-hydroxypentyl) N-butanoic acid N-butanoic acid N-pentanoic acid N-pentanoic acid methyl estera methyl estera
Transition
Cone voltage
Collision energy
344.13 ! 127.02 344.13 ! 154.98 358.10 ! 127.02 358.10 ! 154.98 358.13 ! 127.02 358.13 ! 154.98 372.13 ! 127.02 372.13 ! 154.98 386.13 ! 127.02 386.13 ! 154.98
40 40 36 36 38 38 40 40 40 40
44 24 52 26 46 22 44 24 44 24
Note: The MRM for JWH-073 N-(3-hydroxybutyl) was used to monitor for JWH-072 N-propanoic acid. The MRM for JWH-073 N-butanoic acid was used to monitor for JWH-072 propanoic acid methyl ester. The MRM for JWH-018 N-pentanoic acid was used to monitor for JWH-073 butanoic acid methyl ester. a The cone voltages and collision energies for the JWH-018 methyl ester were not optimized. They were assigned the same values as JWH-018 N-pentanoic acid.
Please cite this article in press as: D.P. Lovett, et al., Structure elucidation and identification of a common metabolite for naphthoylindole-based synthetic cannabinoids using LC-TOF and comparison to a synthetic reference standard, Forensic Sci. Int. (2013), http://dx.doi.org/10.1016/j.forsciint.2012.12.012
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FSI-7040; No. of Pages 7 D.P. Lovett et al. / Forensic Science International xxx (2013) xxx–xxx
4
O
O
+ 9
12
OMe
Br
N H
N
a
Cl
b 11 O
10
OMe
O
O N
OMe
c
N
O 13
1
OH O
a: LiH, DMSO, b: Me2AlCl, toluene, c: NaOH, MeOH Fig. 3. Synthetic scheme for synthesis of 1. 2.6.3. Synthesis of 3-(3-(1-naphthoyl)-1H-indol-1-yl) propanoic acid, (JWH-072 Npropanoic acid, 1) To a stirred solution of methyl 3-(3-(1-naphthoyl)-1H-indol-1-yl) propionate (13, 18.7 mg, 0.052 mmol) in methanol (1 mL) at room temperature was added drop-wise aqueous sodium hydroxide (10%, 0.055 mL, 0. 055 mmol). The reaction was monitored for completeness by GC/MS, and upon completion the solvent was evaporated to dryness. The residue was dissolved in 3 mL of water and extracted with dichloromethane (3 2.5 mL). The aqueous layer was acidified with a small amount of 1N HCl and checked with pH paper. The cloudy solution was extracted with DCM (3 2.5 mL), dried overNa2SO4 and the solvent was removed in vacuo to give JWH-072 N-propionic acid 1 (12 mg, 67%) as a yellow film. The resulting product was purified by silica gel column chromatography with an elution gradient of 3:1–1:1 hexanes:EtOAc. 1H NMR (400 MHz, CDCl3) d 8.97 (1H, br. s), 8.46 (1H, s), 8.14 (1H, d, 8.4 Hz), 7.90 (1H, d, 8.2 Hz), 7.85 (1H, d, 7.8 Hz), 7.58 (1H, d, 6.9 Hz), 7.39–7.47 (4H, m), 7.34 (3H, app. s), 4.32 (2H, t, 6.5 Hz), 2.79 (2H, t, 6.5 Hz); 13C NMR (100 MHz, CDCl3) d 192.45, 175.23, 138.68, 138.50, 136.56, 133.70, 130.68, 130.23, 128.20, 127.01, 126.84, 126.32, 126.09, 125.80, 124.48, 123.99, 123.20, 123.09, 117.88, 109.64, 42.10, 33.86; IR (ATR): 3054, 2961, 1713, 1608, 1574, 1520, 1486, 1464, 1393, 1379, 1261, 1230, 1172, 1079, 907, 888, 790, 730 cm1; exact mass: m/ z = 334.1287 (theoretical: 334.1287).
ions of the m/z 344.2 parent ion (Fig. 4c). The mass spectrum of the daughter scan shows the most intense daughter ions of m/z 127.1 (naphthyl cation) and 155.0 (naphthoyl cation), which are the ions typically seen for synthetic cannabinoids containing an unsubstituted naphthoyl moiety. Other minor product ions observed are m/z 144.2 and 216.2. These results corroborate what had been predicted; the unknown metabolite is closely related to known naphthoylindole metabolites (Fig. 4d). Even though the unknown metabolite and the metabolite standards contain a similar core structure and consequently share the same fragment ions, the information provided by the LC–MS and LC–MS/MS analyses were limited with respect to the unknown metabolite functional group (carboxylated versus hydroxylated metabolite) and its positioning. The earlier elution was indicative that the unknown was a more polar compound than the metabolite standards (5–8) therefore, it seemed reasonable to assume that the metabolite was a carboxylic acid.
3. Results and discussion
3.2. Derivatization of carboxylates 6, 8 and authentic urine sample to methyl esters
3.1. LC–MS/MS analysis of concentrated sample extract Due to the low nanogram concentration levels of the metabolite of interest, it was necessary to increase the extracted metabolite concentrations to enhance the mass spectrometry signals for accurate structure elucidation. The concentration of the metabolite was achieved by repeatedly processing and extracting the analyte of interest from an authentic positive urine sample. The extraction protocol utilized, which was recently validated and published [6], is summarized in the experimental section. This salting out liquid– liquid extraction protocol has demonstrated that high extraction efficiencies are achieved for both hydroxylated and carboxylated metabolites and could be applied for the extraction of a variety of other synthetic cannabinoid metabolites present in urine matrices. The concentrated extract solution was initially analyzed by LC– MS with electrospray ionization in positive mode for the molecular ion characterization and subsequent molecular weight determination of the unknown metabolite. Fig. 4a and b shows the extracted ion chromatogram of the unknown metabolite which elutes at 1.77 min and the corresponding mass spectrum showing the molecular ion at m/z 344.2. These results confirmed the initial observations during the routine urine screening that the unknown metabolite and JWH-073 N-(3-hydroxybutyl) metabolite share a common molecular ion (Table 1). To further elucidate the metabolite structural information, the extract was subjected to LC–MS/MS analysis to obtain molecular fragments. This analysis was achieved by scanning for daughter
To verify that the unknown contained a carboxylic acid, the extract was subjected to a standard methyl ester derivatization protocol that would not form methyl ethers from alcohols. Following the derivatization of the two carboxylic acid standards and the extracted unknown, as described in the experimental section, LC–MS/MS analysis was performed. Conversion of both JWH-073 and JWH-018 carboxylated metabolite standards to their respective methyl esters was indicated by the absence of the standards peaks (at retention times of 1.92 min and 2.32 min, respectively) and the detection of the methyl esters peaks at retention times of 4.93 min and 6.27 min, respectively (Fig. 5a and b). Both methyl esters were detected using the MRM transitions described on Table 1. Fig. 5c and d shows the LC–MS/MS results of the concentrated extract before and after derivatization. Comparing both TIC chromatograms shows the disappearance of the unknown metabolite peak, which elutes at 1.77 min (Fig. 5c), and the appearance of a new peak eluting at 4.05 min (Fig. 5d). This new peak was observed in the MRM channels corresponding to a mass increase of 14 amu, thus indicating the formation of a methyl ester. Also of note is the peak corresponding to the JWH-018 N-(5hydroxypentyl) metabolite (Iso-5) at 2.64 min (Fig. 5c and d) was unchanged by the derivatization conditions and consequently is present at the same retention time. This outcome was a clear indicator that the unknown metabolite was indeed a carboxylic acid and not an alcohol.
Please cite this article in press as: D.P. Lovett, et al., Structure elucidation and identification of a common metabolite for naphthoylindole-based synthetic cannabinoids using LC-TOF and comparison to a synthetic reference standard, Forensic Sci. Int. (2013), http://dx.doi.org/10.1016/j.forsciint.2012.12.012
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5
Fig. 4. (a) Extracted ion chromatogram of unknown metabolite present in positive urine, (b) mass spectrum of unknown metabolite, (c) daughter scan TIC and (d) daughters of m/z 344.2.
3.3. Determination of exact mass For the exact mass determination of the unknown metabolite, the extracted sample solution was subjected to LC/MSD TOF analysis. The accurate mass measurements for the unknown
metabolite resulted in a molecular ion at m/z 344.1295 corresponding to a molecular formula of C22H17NO3. The mass error between the measured and the theoretical mass was 1.7 ppm. Due to limited resources and urine sample availability, the unknown metabolite was not isolated from the concentrated solution
Fig. 5. (a, b) Methyl esters of JWH-018 and JWH-073 carboxylate metabolites, (c) underivatized extract and (d) derivatized extract.
Please cite this article in press as: D.P. Lovett, et al., Structure elucidation and identification of a common metabolite for naphthoylindole-based synthetic cannabinoids using LC-TOF and comparison to a synthetic reference standard, Forensic Sci. Int. (2013), http://dx.doi.org/10.1016/j.forsciint.2012.12.012
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6 Table 2 Quantitative analysis of authentic urine. Sample
1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
127.9 173.3 21.6 132.2 23.2 316.2 11.5 20.8 40.9 35.3 6.1 421.9 207.1 245.6 232.6 5.3 155.6 104.7 9.8 355.9 106.0 23.6 42.2 193.0 549.8
5
Iso-5a
6
8
Sample
0 0 0 0 0 0 8.6 0 0 0 0 298.9 207.4 3.3 215.8 0 168.3 0 0 53.4 43.8 16.2 0 29.0 525.7
31.2 88.0 1.3 51.3 7.0 61.6 7.3 7.0 16.7 7.0 0.7 120.7 73.8 55.2 147.8 1.6 61.5 23.5 4.2 73.7 42.2 6.3 13.7 28.6 227.3
35.5 93.7 5.7 26.4 6.1 86.6 3.4 15.1 34.0 6.7 0.8 2.6 1.9 7.3 3.8 1.0 55.3 90.6 3.9 120.1 9.0 8.0 16.2 305.0 12.7
26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49
Concentration (ng/mL)
a
84.8 147.9 11.6 69.2 12.5 111.4 30.9 25.3 55.4 24.3 1.9 0 0 0.1 0 3.5 351 46.1 15.4 152.3 0 24.9 16.7 56 0
1
5
Iso-5a
6
8
44.9 71.1 0 7.7 0 52.7 191.9 9.5 50.3 3.4 7.9 5.5 7.6 0.4 13.0 6.0 126.6 706.2 5.0 0.6 4.7 8.9 17.7 8.2
18.3 24.1 4.4 7.3 4.5 52.5 51.4 9.2 31.4 7.5 6.3 5.5 5.9 9.8 8.7 4.3 81.8 457.9 7.6 1.2 7.6 11.4 16.9 3.4
20.4 1.7 2.8 10.0 2.5 2.9 2.1 4.8 4.1 6.7 13.3 0.3 0.3 0.4 0.3 0.0 3.3 10.3 0.3 0.0 0.6 0.5 0.6 0.0
Concentration (ng/mL) 62.5 206.3 32.5 21.9 16.8 242.2 105.9 11.1 111.2 23.4 40.4 18.5 52.6 18.8 39.0 17.1 219.1 943.8 17.1 4.7 19.7 26.8 62.3 14.8
22 0.1 31.3 26.2 10.8 0 0 35.5 0 9.9 9.7 0 0 0 0 0 0 0 0 0 0.1 0 0 0
Iso-5 is JWH-018 N-(5-hydroxypentyl) metabolite.
therefore no NMR analysis was performed. From the results described above, the identity of the unknown metabolite was proposed to be 1. 3.4. Synthesis of compound 1 To further confirm the proposed identity of the unknown metabolite, 3-(3-(1-naphthoyl)-1H-indol-1-yl) propanoic acid was synthesized and characterized. Its chromatographic and mass spectrometric data was then compared to the results obtained for the concentrated metabolite extract from the authentic positive urine sample. The carbon and proton NMR spectra are included in the Supplemental section. The chemically synthesized metabolite was analyzed by LC/ MSD TOF for exact mass determination, which resulted in a molecular ion at m/z 344.1287. This corresponds to a molecular formula of C22H17NO3. This exact mass is consistent with the values obtained for unknown metabolite in the extracted authentic positive urine. Another important piece of information that further substantiates the unknown’s identity is the retention time of the metabolite present in the authentic positive urine matches the retention time of the synthetic standard. 3.5. Quantitative examination of positive samples The quantitative examination of 49 samples positive for naphthoylindole synthetic cannabinoids was undertaken using a similar protocol described by Yanes and Lovett [6] in which synthetic 1 was also included in the calibrator standard solution. As presented in Table 2, the results revealed that not only was JWH-072 N-propanoic acid a common metabolite present in all samples, but was also the major carboxylate metabolite in 48 of the 49 specimens. Comparison versus the JWH-018 N-(4-hydroxypentyl) metabolite (5) and its N-(5-hydroxypentyl) isomer indicate that JWH-072 N-propanoic acid is the predominate metabolite in 38 of the 49 samples and is the second most abundant metabolite in the remaining 11 samples. From these preliminary studies, it has
also been observed that a majority of 1 is excreted as the free carboxylic acid unconjugated to b-glucuronic acid. 4. Conclusions A new metabolite with a potential status of being a common biomarker for monitoring naphthoylindole based synthetic cannabinoids has been identified and completely characterized. 3-(3(1-Naphthoyl)-1H-indol-1-yl) propionic acid (1) was observed as a metabolic product in authentic positive urine and its identity was confirmed by the identical chromatographic retention time, fragment ions, and exact mass compared to a chemically synthesized metabolite standard. At this time, the JWH-072 Npropanoic acid metabolite has been determined to be a major metabolic product for JWH-018, JWH-073, and AM-2201. The monitoring of a common biomarker for the unsubstituted naphthoylindole series of synthetic cannabinoids could facilitate the rapid development of qualitative and/or quantitative analyses by requiring only a minimum number of MRM transitions. Furthermore, the preparation of calibration solutions, typically containing a complex mixture of alcohol and carboxylic acid metabolite standards, would be simplified to containing only a few. As the synthetic cannabinoid market continues to evolve, this new paradigm would eliminate the need to monitor the presence of a vast array of metabolites and consequently there would no longer be the necessity to wait for new metabolite reference standards to become commercially available. Acknowledgments The authors gratefully acknowledge LtCol M. Ewy for her assistance and support, the United States Air Force Academy for the use of their chemistry laboratory for the synthetic work, and Mrs. Cynthia Corley for her help with LC-TOF analysis. The authors would like to thank the United States Air Force and ADC Management Services Inc. for funding this project.
Please cite this article in press as: D.P. Lovett, et al., Structure elucidation and identification of a common metabolite for naphthoylindole-based synthetic cannabinoids using LC-TOF and comparison to a synthetic reference standard, Forensic Sci. Int. (2013), http://dx.doi.org/10.1016/j.forsciint.2012.12.012
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.forsciint.2012.12.012. References [1] C. Mustata, M. Torrens, R. Pardo, C. Perez, The Psychonaut web mapping group, M. Farre, spice drugs: cannabinoids as a new designer drugs, Adicciones 21 (2009) 181. [2] European Monitoring Centre for Drugs and Drug Addiction (EMCDDA), Thematic Paper-understanding the ‘Spice’ Phenomenon, Luxembourg, 2009,, p. 3. [3] B.K. Atwood, J.W. Huffman, A. Straiker, K. Mackie, JWH018, a common constituent of ‘Spice’ herbal blends, is a potent and efficacious cannabinoid CB1 agonist, Br. J. Pharmacol. 1 (2010).
7
[4] Food and Drug Administration Safety and Innovation Act, http://www.gpo.gov/ fdsys/pkg/BILLS-112s3187enr/pdf/BILLS-112s3187enr.pdf (accessed 07.09.12). [5] L.N. Sacco, K.M. Finklea, Synthetic drugs: overview and issues for congress, in: Congressional Research Service (CRS) Report for Congress, 2011. [6] E.G. Yanes, D.P. Lovett, High-throughput bioanalytical method for analysis of synthetic cannabinoid metabolites in urine using salting-out sample preparation and LC–MS/MS, J. Chromatogr. B 909 (2012) 42–50. [7] C.L. Moran, V.-H. Huyen Le, K.C. Chimalakonda, A.L. Smedley, F.D. Lackey, S.N. Owen, P.D. Kennedy, G.W. Endres, F.L. Ciske, J.B. Kramer, A.M. Kornilov, L.D. Bratton, P.J. Dobrowolski, W.D. Wessinger, W.E. Fantegrossi, P.L. Prather, L.P. James, A. Radominska-Pandya, J.H. Moran, Quantitative measurement of JWH-018 and JWH-073 metabolites excreted in human urine, Anal. Chem. 83 (2011) 4228–4236. [8] K.C. Chimalakonda, C.L. Moran, P.D. Kennedy, G.W. Endres, A. Uzieblo, P.J. Dobrowolski, E.K. Fifer, J. Lapoint, L.S. Nelson, R.S. Hoffman, L.P. James, A. RadominskaPandya, J.H. Moran, Solid-phase extraction and quantitative measurement of omega and omega-1 metabolites of JWH-018 and JWH-073 in human urine, Anal. Chem. 83 (2011) 6381–6388. [9] B.R. Martin, J.W. Huffman, US Patent Application 2005/0009903 A1.
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FSI-7040; No. of Pages 7 Forensic Science International xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Structure elucidation and identification of a common metabolite for naphthoylindole-based synthetic cannabinoids using LC-TOF and comparison to a synthetic reference standard Dennis P. Lovett a,*, Enrique G. Yanes a, Travis W. Herbelin a, Timm A. Knoerzer b, Joseph A. Levisky b a b
HQ Air Force Drug Testing Laboratory, Lackland Air Force Base, TX 78236-5310, United States HQ United States Air Force Academy/DFC 2355 Fairchild Drive, Suite 2N225 USAF Academy, CO 80840-6230, United States
A R T I C L E I N F O
A B S T R A C T
Article history: Received 16 October 2012 Received in revised form 7 December 2012 Accepted 13 December 2012 Available online xxx
The identification of a predominate metabolite found in urine specimens which test positive for naphthoylindole-based synthetic cannabinoids is reported. The presence of this new metabolite was detected at the Air Force Drug Testing Lab Investigations Division during screening analysis for metabolites of JWH-018 and JWH-073, because it shares the same MRM transitions as the JWH-073 N(3-hydroxybutyl) metabolite. However, the detected peak is chromatographically distinguished from other metabolites due to differences in retention time. This metabolite appears to be a common metabolite for select naphthoylindole-based synthetic cannabinoids that could potentially be used as a common biomarker for their qualitative and quantitative analyses. The new metabolite has been successfully identified as 3-(3-(1-naphthoyl)-1H-indol-1-yl) propanoic acid (1, JWH 072 N-propanoic acid metabolite, Fig. 1) by using various mass spectrometric and liquid chromatographic techniques as well as chemical derivatization. The metabolite identity was confirmed through the comparison of authentic positive urine and a chemically synthesized metabolite standard. Both materials shared the same chromatographic retention time on two separate chromatographic systems, mass fragmentation pattern and exact mass. Full characterization of the synthetic reference material and intermediates by 1H NMR, 13C NMR, IR and HRMS was also conducted. ß 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: UPLC Synthetic cannabinoid metabolites NMR LC-TOF Common biomarker JWH-072 metabolite
1. Introduction Since their introduction into the market place in 2004 as ‘‘legal highs’’, synthetic cannabinoids have rapidly gained popularity in the United States and Europe [1]. Originally sold under the brand name ‘Spice’, the term Spice has become a generic term to include the entire class of ‘‘legal high’’ smoking blends which are sold on the Internet, gas stations and local tobacco shops as incense or potpourri, but are actually inactive plant matter originally adulterated with naphthoylindole containing synthetic cannabinoids such as JWH-018 (2) and JWH-073 (3) and more recently AM-2201 (4) (Fig. 1). In December 2008, THC Pharma (Germany) and AGES PharmMed (Austria) reported independently the presence of JWH-018 in many herbal smoking blends [2]. A direct link was established in 2010 between herbal preparations containing JWH-018 and the psychotropic effects [3]. In July 2012, 14 synthetic cannabinoids were included on the Drug
* Corresponding author. Tel.: +1 210 292 3516. E-mail addresses: [email protected], [email protected] (D.P. Lovett).
Enforcement Agency’s Schedule I list making the possession, manufacture, and consumption of these compounds illegal [4]. It can be expected that black market manufacturers will respond to this scheduling by structural modification of the illicit compounds to create new cannabinoids thus circumventing the new drug law. As a consequence, the work load for forensic and workplace drug testing labs will increase and analyses will become even more challenging. To keep up with the ever-changing synthetic cannabinoid landscape, simpler and faster analytical methods are required. The popularity of Spice has extended to the United States Armed Forces, which resulted in the United States Department of Defense banning all military personnel from possessing or usage of synthetic cannabinoids [5]. A directive was issued to develop both a screening and a confirmation analyses for synthetic cannabinoid metabolites in urine. The resulting process was implemented at the Air Force Drug Testing Lab Investigations Division and analysis of service member samples began in March 2012 [6]. Soon after testing had begun, TIC chromatograms showed the presence of an unidentified peak in all of the specimens that tested positive for one or more of the four metabolites of interest (5–8, Fig. 2). This new peak eluted earlier than the reference metabolites
0379-0738/$ – see front matter ß 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.forsciint.2012.12.012
Please cite this article in press as: D.P. Lovett, et al., Structure elucidation and identification of a common metabolite for naphthoylindole-based synthetic cannabinoids using LC-TOF and comparison to a synthetic reference standard, Forensic Sci. Int. (2013), http://dx.doi.org/10.1016/j.forsciint.2012.12.012
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FSI-7040; No. of Pages 7 D.P. Lovett et al. / Forensic Science International xxx (2013) xxx–xxx
2
O
O N
OH
N
R
O 1
2 (JWH-018) R = CH3 3 (JWH-073) R = H 4 (AM-2201) R = CH2F
Fig. 1. 3-(3-(1-Naphthoyl)-1H-indol-1-yl) propanoic acid, 1; naphthoylindole synthetic cannabinoids 2–4.
and shared multiple reaction monitoring (MRM) transitions with the JWH-073 N-(3-hydroxybutyl) metabolite (7). Based on this evidence, and the fact that the unknown was also extracted along with the other metabolites, it was suspected that 7 and the unknown metabolite shared a similar core structure and consequently belonged to the same synthetic cannabinoid series. A review of current literature reveals that Moran et al. [7,8] had also reported observing an early eluting unknown peak, but did not report its identity nor its potential as a common biomarker for JWH-018, JWH-073 and AM-2201. The present work describes the characterization and identification of a previously unreported metabolite that is common for a select group of naphthoylindole cannabinoids. The metabolite was characterized by using different techniques including LC–MS, LC– MS/MS and LC-TOF which allows the determination of the molecular ion, fragment ions, and accurate mass. The hypothesized metabolite was not commercially available; therefore identity confirmation was achieved by retention time matching to a chemically synthesized metabolite standard that had also been subjected to NMR, IR and LC-TOF analysis. 2. Experimental 2.1. Chemicals, reagents and standards All UPLC solvents were LC–MS grade. Acetonitrile, methanol and water were obtained from Fisher Scientific (Pittsburgh, PA). Formic acid and ammonium acetate (both Optima LC–MS grade) were also obtained from Fisher Scientific. bGlucuronidase (Escherichia coli, Type IX-A, lyophilized powder, 1,000,000– 5,000,000 units/g protein) was purchased from Sigma–Aldrich (St. Louis, MO, USA). Chemicals and reagents used for the synthesis of 3-(3-(1-naphthoyl)-1H-indol-1-yl)
O
OH
propanoic acid (1) and deuterated chloroform (CDCl3) were obtained from Sigma– Aldrich (St. Louis, MO). Reference standards of metabolites: JWH-018 N-(4-hydroxypentyl), JWH-018 Npentanoic acid, JWH-073 N-(3-hydroxybutyl) and JWH-073 N-butanoic acid were obtained from Cerilliant Corporation (Round Rock, TX, USA) in ampoules as 1 mL methanol solutions at 100 mg/mL. The metabolite standard, JWH-018 N-(5hydroxypentyl) was obtained from Cayman Chemicals (Ann Arbor, MI) as a solid in a screw-cap vial. 2.2. Instrumentation 2.2.1. UPLC-TQD; for determination of parent and daughter ions The chromatographic system was a Waters Acquity UPLC (Milford, MA, USA) consisting of sample, solvent and column managers. The solvent manager included a binary pump and the column manager was comprised of a thermostatted column compartment. Chromatographic separation was performed on a Waters Acquity UPLC HSS T3 (2.1 mm 100 mm, 1.8 mm) column which was connected to an Acquity UPLC HSS T3 VanGuard (2.1 mm 5 mm, 1.8 mm) pre-column. The column temperature was maintained at 40 8C. Isocratic conditions (50% A:50% B) were used for the chromatographic separation of the different analytes of interest. The optimal flow rate was set at 0.6 mL/min with a sample injection volume of 10 mL. Mobile phases consisted of LC/MS grade water containing 0.1% formic acid (mobile phase A) and LC/MS grade acetonitrile also containing 0.1% formic acid (mobile phase B). The mass spectrometer was a Waters tandem quadrupole detector (TQD) which was operated in the positive electrospray ionization and multiple reaction monitoring (MRM) mode for selective and sensitive detection. The applied capillary voltage was 3.3 kV. The source temperature was set at 150 8C and desolvation temperature at 450 8C. Nitrogen was used as the nebulizing gas and argon as the collision gas. The desolvation gas flow was set at 600 L/h, cone gas at 50 L/h and collision gas at 0.2 mL/min. The collision cell pressure was approximately 5.28 103 mbar. All data was processed using MassLynx1 software from Waters Corporation. 2.2.2. LC/MSD TOF; for identification and accurate mass determination An Agilent 1100 series HPLC (Agilent Technologies, Palo Alto, CA, USA) was fitted with a reverse-phase Higgins Analytical Phalanx C18 (2.1 mm 150 mm, 5 mm) column. The column temperature was maintained at 25 8C. Mobile phases consisted of mobile phase A (5 mM ammonium formate) and mobile phase B (acetonitrile). A gradient program was used for compounds 11 and 1 consisting of an initial solvent ratio of 50% A and 50% B. The mobile phase gradient was ramped to 95% B over 7 min and held at this proportion for 5 min. The initial 50% A and 50% B mobile phase was re-established in 1 min and held at this value for 3 min between sample runs. For compound 13 an isocratic program was used consisting of 40% mobile phase A and 60% mobile phase B. The flow rate for both programs was 0.3 mL/min with an injection volume of 2 mL. The HPLC system was interfaced to a time-of-flight mass spectrometer (MSD-TOF, Agilent Technologies) operated in the positive electrospray ionization mode and using the following parameters: capillary 3500 V, nebulizer 30 psi g, drying gas 12 L/min, gas temperature 325 8C, fragmentator 125 V, skimmer 65 V, OCT 1RF 250 V. The instrument was calibrated
O
N
N
OH O
5 JWH-018 N-(4-hydroxypentyl)
O
6 JWH-018 N-pentanoic acid
O N
O N
OH
OH 7 JWH-073 N-(3-hydroxybutyl)
8 JWH-073 N-butanoic acid
Fig. 2. Metabolites of interest for JWH-018 and JWH-073.
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FSI-7040; No. of Pages 7 D.P. Lovett et al. / Forensic Science International xxx (2013) xxx–xxx prior to each analysis using the solution and procedure recommended by the manufacturer. Spectra were acquired over the m/z 50–1000 range at a scan rate of 1.02 s/spectrum. 2.2.3. GCMS; for reaction monitoring of intermediates during metabolite synthesis Monitoring of the different synthetic steps was conducted on an Agilent 7890A gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) interfaced to an Agilent 5975 C inert XL EI/CI MSD mass spectrometer. The separation was achieved on an Agilent HP-5 MS (30 m 0.250 mm; 0.25 mm) applying two different temperature programs. Temperature program 1 used for compound 11 consisted of 90 8C (2 min), heating 35 8C/min to 325 8C (5.29 min). The mass spectrometer was operated in full scan mode with a mass scan from m/z 40.0 to 300.0. Temperature program 2 used for compounds 1 and 13 consisted of 90 8C (2 min), heating 20 8C/ min to 250 8C (2 min), 30 8C/min to 300 8C (4 min). The mass spectrometer was operated in full scan mode with a mass scan from m/z 40.0 to 500.0. Two microliter were injected at 250 8C in split mode (100:1) with the carrier gas (high purity helium) flow set to 1.0 mL/min. Transfer line temperature was 280 8C and the ion source was held at 230 8C. 2.2.4. NMR spectroscopy The NMR characterization of the synthetic intermediates and final product was carried out on a Varian Mercury Plus 400 MHz NMR spectrometer (VarianNMR Inc., Palo Alto, CA, USA) with an Oxford AS 400 superconducting magnet and a Varian 400 ATB PFG Quad Probe at room temperature. Chemical shifts were referenced to the residual undeuterated solvent peak at 7.26 ppm for 1H and the 13 CDCl3 center peak at 77.00 ppm for 13C. All data were processed using SpinWorks 2.5 software. 2.2.5. IR spectroscopy The IR characterization of the synthetic intermediates and the final product was carried out on a Thermo-Nicolet iS180 spectrometer (Waltham, MA, USA) fitted with a Smart iTR attenuated total reflectance sampling device. 2.3. Reagents and standards preparation The salting out reagent (10 M ammonium acetate solution) was prepared by dissolving 38.55 g of ammonium acetate in a 50 mL volumetric flask with LC/MS grade water (15 mL) to give exactly 50 mL. Solution was sonicated for 2 h to assist with dissolution. The b-glucuronidase solution was prepared as 12.3 units/mL in 0.1 M ammonium acetate by mixing 615,000 modified Fishman units of b-glucuronidase Type IXA (370 mg of 1,661,000 units/g) and 500 mL of 10 M ammonium acetate solution into a 50 mL volumetric flask. The mixture was diluted to the mark with LC/MS grade water, shaken and sonicated for a few seconds to complete the mixing. The solution was transferred into 1.7 mL microcentrifuge tubes and stored at 30 8C until use. Metabolite reference standards were stored at 30 8C according to the manufacture’s recommendations until used. Working solutions were further prepared in methanol and kept in the refrigerator until needed. 2.4. Sample treatment for extraction and concentration of unknown metabolite Thirty urine aliquots were subjected to enzymatic hydrolysis and salting-out assisted liquid–liquid extraction protocol validated in this lab [6]. All microcentrifuge tubes containing 100 mL of authentic positive urine each were treated with 25 mL of b-D-glucuronidase solution and then mixed by vortexing gently for 5 s. The incubation of all tubes was carried out at room temperature over 10 min. After sample hydrolysis, 200 mL of LC/MS grade acetonitrile was added and the tubes were mixed by vortexing for 5 s. Fifty microliter of 10 M ammonium acetate solution was then added and mixed by vortexing for 5 s. The microcentrifuge tubes were centrifuged at 10,000 rpm for 3 min. The upper organic phases of the tubes were removed from each microcentrifuge tube, combined and placed into a TurboVap evaporator. The solvent was reduced to a volume of approximately 0.2 mL using a stream of nitrogen to assist solvent evaporation. The resulting concentrate was prepared for LC–MS analysis by removing 100 mL of the solution and transferring to a clean HPLC vial. The sample was diluted with 100 mL of LC/MS grade water and mixed well by vortexing for 5 s. 2.5. Derivatization of JWH-018 and JWH-073 carboxylate metabolites and authentic urine sample to methyl esters Solutions of JWH-018 N-pentanoic acid (10 mL, 1 mg/mL in DMSO), JWH-073 Nbutanoic acid (10 mL, 1 mg/mL in DMSO) or concentrated extract of authentic positive urine was added to separate micro centrifuge tubes, treated with a solution of 8:1 DMSO/ammonium hydroxide (30 mL) and then mixed by vortexing. Methyl iodide (10 mL) was added to each tube and mixed by vortexing to afford a cloudy suspension. After five min, all tubes were treated with dilute hydrochloric acid (0.1 M, 200 mL) followed by isooctane (200 mL). The mixtures were mixed by
3
vortexing for one min then the tubes were centrifuged for 1 min at 10,000 rpm. The upper layer of each tube was transferred to a separate glass tube and the solvent was removed by under a stream of nitrogen in a 50 8C water bath. The residues were dissolved in acetonitrile (0.5 mL) then mixed by vortexing. LC/MS grade water (0.5 mL) was added to the tubes then the solutions were transferred to HPLC vials and analyzed by LC–MS/MS. Table 1 lists the MRM transitions monitored as well as the cone voltages and collision energies. 2.6. Synthesis of 3-(3-(1-naphthoyl)-1H-indol-1-yl) propanoic acid, (JWH-072 Npropanoic acid, 1) The synthesis of the hypothesized metabolite followed a modification of the published procedure of Martin and Huffman [9] for the connection of the alkyl indole and the naphthalene. The reaction scheme is outlined in Fig. 3. 2.6.1. Synthesis of methyl-3-(1-indol-1-yl) propionate, (11) To an oven dried three-neck 50 mL round bottom flask was charged 10 mL of dry DMSO under N2. Lithium hydride (175 mg, 22 mmol) was added to the stirring DMSO and held for 5 min. Indole (9, 1.17 g, 10 mmol) dissolved in dry DMSO (5 mL) was added over 2 min with observed gas evolution. After stirring for 90 min, methyl 3-bromopropionate (10, 1.20 mL, 11 mmol) was added drop-wise to the reaction mixture. The reaction was poured into stirring 1N HCl (20 mL) and extracted with ethyl acetate (3 20 mL). The pooled organic layers were dried over Na2SO4 prior to removal of solvent to yield 11 as a pale amber oil. The crude material was taken forward without purification. A sample of 11 was purified by silica gel column chromatography using an elution gradient of 9:1–4:1 hexanes:EtOAc (Rf = 0.20, 4:1 hexanes:EtOAc) for the acquisition of analytical spectra. 1H NMR (400 MHz, CDCl3) d 7.61 (1H, d, 7.6 Hz), 7.32 (1H, d, 8.2 Hz), 7.20 (1H, t, 7.4 Hz), 7.11, (2H, m), 6.47 (1H, d, 3.0 Hz), 4.42 (2H, t, 6.9 Hz), 3.63 (3H, s), 2.80 (2H, t, 6.9 Hz); 13C NMR (100 MHz, CDCl3) d 171.71, 135.65, 128.74, 127.98, 121.69, 121.12, 119.56, 109.11, 101.64, 51.96, 41.82, 34.77; IR (ATR) 3735, 3412, 2952, 2359, 2342, 1734, 1512, 1463, 1437, 1398, 1354, 1333, 1333, 1313, 1261, 1202, 1169, 1091, 1062, 1012, 765, 739, 668 cm1; exact mass: m/z = 204.1014 (theoretical: 204.1019). 2.6.2. Synthesis of methyl-3-(3-(1-naphthoyl)-1H-indol-1-yl) propionate, (JWH-072 N-propanoic acid methyl ester, 13) Me2AlCl (1 M in hexanes, 0.72 mL, 0.72 mmol) was added drop-wise to a dry conical vial containing a solution of methyl 3-(1-indol-1-yl) propionate (11, 97 mg, 0.48 mmol) in dry CH2Cl2 (1.4 mL) at 0 8C under N2. The red solution was stirred at 0 8C for 30 min and then a solution of 1-naphthoyl chloride (12, 80 mL, 0.58 mmol) in 0.9 mL of dry CH2Cl2 was added dropwise. The deep red acylation reaction mixture was stirred at 0 8C until the reaction was complete in approximately 30 min as indicated by TLC (Rf = 0.15, 3:1 hexanes:ethyl acetate). The reaction mixture was poured carefully into ice cold 1 M aqueous HCl (10 mL) and extracted with CH2Cl2 (3 5 mL). The combined extracts were washed with three portions of aqueous NaHCO3 (5 mL), dried over Na2SO4 and the solvent was removed in vacuo to give crude methyl ester 13 (121.7 mg, 71%) as a green foam. 1H NMR (400 MHz, CDCl3) d 8.52 (1H, dd, 4.2 Hz, 3.1 Hz), 8.18 (1H, d, 8.2 Hz), 7.96 (1H, d, 8.2 Hz), 7.90 (1H, d, 7.9 Hz), 7.64 (1H, d, 6.9 Hz), 7.46–7.54 (3H, m), 7.41 (1H, s), 7.36–7.39 (3H, m), 4.39 (2H, t, 6.7 Hz), 3.59 (3H, s), 2.81 (2H, d, 6.7 Hz); 13C NMR (100 MHz, CDCl3) d 192.13, 170.92, 138.80, 138.28, 136.62, 133.76, 130.75, 130.14, 128.21, 127.03, 126.80, 126.30, 125.94, 125.89, 124.52, 123.90, 123.09, 123.08, 118.00, 109.63, 52.07, 42.42, 34.18; IR (ATR) 3054, 2951, 1735, 1627, 1610, 1577, 1521, 1484, 1464,1377, 1338, 1261, 1200, 1174, 1137, 1079, 1055, 1014 887, 791, 750 cm1; exact mass: m/z = 358.1449 (theoretical: 358.1443).
Table 1 MS/MS parameters. Compound JWH-073 JWH-073 JWH-018 JWH-018 JWH-073 JWH-073 JWH-018 JWH-018 JWH-018 JWH-018
N-(3-hydroxybutyl) N-(3-hydroxybutyl) N-(4-hydroxypentyl) N-(4-hydroxypentyl) N-butanoic acid N-butanoic acid N-pentanoic acid N-pentanoic acid methyl estera methyl estera
Transition
Cone voltage
Collision energy
344.13 ! 127.02 344.13 ! 154.98 358.10 ! 127.02 358.10 ! 154.98 358.13 ! 127.02 358.13 ! 154.98 372.13 ! 127.02 372.13 ! 154.98 386.13 ! 127.02 386.13 ! 154.98
40 40 36 36 38 38 40 40 40 40
44 24 52 26 46 22 44 24 44 24
Note: The MRM for JWH-073 N-(3-hydroxybutyl) was used to monitor for JWH-072 N-propanoic acid. The MRM for JWH-073 N-butanoic acid was used to monitor for JWH-072 propanoic acid methyl ester. The MRM for JWH-018 N-pentanoic acid was used to monitor for JWH-073 butanoic acid methyl ester. a The cone voltages and collision energies for the JWH-018 methyl ester were not optimized. They were assigned the same values as JWH-018 N-pentanoic acid.
Please cite this article in press as: D.P. Lovett, et al., Structure elucidation and identification of a common metabolite for naphthoylindole-based synthetic cannabinoids using LC-TOF and comparison to a synthetic reference standard, Forensic Sci. Int. (2013), http://dx.doi.org/10.1016/j.forsciint.2012.12.012
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4
O
O
+ 9
12
OMe
Br
N H
N
a
Cl
b 11 O
10
OMe
O
O N
OMe
c
N
O 13
1
OH O
a: LiH, DMSO, b: Me2AlCl, toluene, c: NaOH, MeOH Fig. 3. Synthetic scheme for synthesis of 1. 2.6.3. Synthesis of 3-(3-(1-naphthoyl)-1H-indol-1-yl) propanoic acid, (JWH-072 Npropanoic acid, 1) To a stirred solution of methyl 3-(3-(1-naphthoyl)-1H-indol-1-yl) propionate (13, 18.7 mg, 0.052 mmol) in methanol (1 mL) at room temperature was added drop-wise aqueous sodium hydroxide (10%, 0.055 mL, 0. 055 mmol). The reaction was monitored for completeness by GC/MS, and upon completion the solvent was evaporated to dryness. The residue was dissolved in 3 mL of water and extracted with dichloromethane (3 2.5 mL). The aqueous layer was acidified with a small amount of 1N HCl and checked with pH paper. The cloudy solution was extracted with DCM (3 2.5 mL), dried overNa2SO4 and the solvent was removed in vacuo to give JWH-072 N-propionic acid 1 (12 mg, 67%) as a yellow film. The resulting product was purified by silica gel column chromatography with an elution gradient of 3:1–1:1 hexanes:EtOAc. 1H NMR (400 MHz, CDCl3) d 8.97 (1H, br. s), 8.46 (1H, s), 8.14 (1H, d, 8.4 Hz), 7.90 (1H, d, 8.2 Hz), 7.85 (1H, d, 7.8 Hz), 7.58 (1H, d, 6.9 Hz), 7.39–7.47 (4H, m), 7.34 (3H, app. s), 4.32 (2H, t, 6.5 Hz), 2.79 (2H, t, 6.5 Hz); 13C NMR (100 MHz, CDCl3) d 192.45, 175.23, 138.68, 138.50, 136.56, 133.70, 130.68, 130.23, 128.20, 127.01, 126.84, 126.32, 126.09, 125.80, 124.48, 123.99, 123.20, 123.09, 117.88, 109.64, 42.10, 33.86; IR (ATR): 3054, 2961, 1713, 1608, 1574, 1520, 1486, 1464, 1393, 1379, 1261, 1230, 1172, 1079, 907, 888, 790, 730 cm1; exact mass: m/ z = 334.1287 (theoretical: 334.1287).
ions of the m/z 344.2 parent ion (Fig. 4c). The mass spectrum of the daughter scan shows the most intense daughter ions of m/z 127.1 (naphthyl cation) and 155.0 (naphthoyl cation), which are the ions typically seen for synthetic cannabinoids containing an unsubstituted naphthoyl moiety. Other minor product ions observed are m/z 144.2 and 216.2. These results corroborate what had been predicted; the unknown metabolite is closely related to known naphthoylindole metabolites (Fig. 4d). Even though the unknown metabolite and the metabolite standards contain a similar core structure and consequently share the same fragment ions, the information provided by the LC–MS and LC–MS/MS analyses were limited with respect to the unknown metabolite functional group (carboxylated versus hydroxylated metabolite) and its positioning. The earlier elution was indicative that the unknown was a more polar compound than the metabolite standards (5–8) therefore, it seemed reasonable to assume that the metabolite was a carboxylic acid.
3. Results and discussion
3.2. Derivatization of carboxylates 6, 8 and authentic urine sample to methyl esters
3.1. LC–MS/MS analysis of concentrated sample extract Due to the low nanogram concentration levels of the metabolite of interest, it was necessary to increase the extracted metabolite concentrations to enhance the mass spectrometry signals for accurate structure elucidation. The concentration of the metabolite was achieved by repeatedly processing and extracting the analyte of interest from an authentic positive urine sample. The extraction protocol utilized, which was recently validated and published [6], is summarized in the experimental section. This salting out liquid– liquid extraction protocol has demonstrated that high extraction efficiencies are achieved for both hydroxylated and carboxylated metabolites and could be applied for the extraction of a variety of other synthetic cannabinoid metabolites present in urine matrices. The concentrated extract solution was initially analyzed by LC– MS with electrospray ionization in positive mode for the molecular ion characterization and subsequent molecular weight determination of the unknown metabolite. Fig. 4a and b shows the extracted ion chromatogram of the unknown metabolite which elutes at 1.77 min and the corresponding mass spectrum showing the molecular ion at m/z 344.2. These results confirmed the initial observations during the routine urine screening that the unknown metabolite and JWH-073 N-(3-hydroxybutyl) metabolite share a common molecular ion (Table 1). To further elucidate the metabolite structural information, the extract was subjected to LC–MS/MS analysis to obtain molecular fragments. This analysis was achieved by scanning for daughter
To verify that the unknown contained a carboxylic acid, the extract was subjected to a standard methyl ester derivatization protocol that would not form methyl ethers from alcohols. Following the derivatization of the two carboxylic acid standards and the extracted unknown, as described in the experimental section, LC–MS/MS analysis was performed. Conversion of both JWH-073 and JWH-018 carboxylated metabolite standards to their respective methyl esters was indicated by the absence of the standards peaks (at retention times of 1.92 min and 2.32 min, respectively) and the detection of the methyl esters peaks at retention times of 4.93 min and 6.27 min, respectively (Fig. 5a and b). Both methyl esters were detected using the MRM transitions described on Table 1. Fig. 5c and d shows the LC–MS/MS results of the concentrated extract before and after derivatization. Comparing both TIC chromatograms shows the disappearance of the unknown metabolite peak, which elutes at 1.77 min (Fig. 5c), and the appearance of a new peak eluting at 4.05 min (Fig. 5d). This new peak was observed in the MRM channels corresponding to a mass increase of 14 amu, thus indicating the formation of a methyl ester. Also of note is the peak corresponding to the JWH-018 N-(5hydroxypentyl) metabolite (Iso-5) at 2.64 min (Fig. 5c and d) was unchanged by the derivatization conditions and consequently is present at the same retention time. This outcome was a clear indicator that the unknown metabolite was indeed a carboxylic acid and not an alcohol.
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Fig. 4. (a) Extracted ion chromatogram of unknown metabolite present in positive urine, (b) mass spectrum of unknown metabolite, (c) daughter scan TIC and (d) daughters of m/z 344.2.
3.3. Determination of exact mass For the exact mass determination of the unknown metabolite, the extracted sample solution was subjected to LC/MSD TOF analysis. The accurate mass measurements for the unknown
metabolite resulted in a molecular ion at m/z 344.1295 corresponding to a molecular formula of C22H17NO3. The mass error between the measured and the theoretical mass was 1.7 ppm. Due to limited resources and urine sample availability, the unknown metabolite was not isolated from the concentrated solution
Fig. 5. (a, b) Methyl esters of JWH-018 and JWH-073 carboxylate metabolites, (c) underivatized extract and (d) derivatized extract.
Please cite this article in press as: D.P. Lovett, et al., Structure elucidation and identification of a common metabolite for naphthoylindole-based synthetic cannabinoids using LC-TOF and comparison to a synthetic reference standard, Forensic Sci. Int. (2013), http://dx.doi.org/10.1016/j.forsciint.2012.12.012
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6 Table 2 Quantitative analysis of authentic urine. Sample
1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
127.9 173.3 21.6 132.2 23.2 316.2 11.5 20.8 40.9 35.3 6.1 421.9 207.1 245.6 232.6 5.3 155.6 104.7 9.8 355.9 106.0 23.6 42.2 193.0 549.8
5
Iso-5a
6
8
Sample
0 0 0 0 0 0 8.6 0 0 0 0 298.9 207.4 3.3 215.8 0 168.3 0 0 53.4 43.8 16.2 0 29.0 525.7
31.2 88.0 1.3 51.3 7.0 61.6 7.3 7.0 16.7 7.0 0.7 120.7 73.8 55.2 147.8 1.6 61.5 23.5 4.2 73.7 42.2 6.3 13.7 28.6 227.3
35.5 93.7 5.7 26.4 6.1 86.6 3.4 15.1 34.0 6.7 0.8 2.6 1.9 7.3 3.8 1.0 55.3 90.6 3.9 120.1 9.0 8.0 16.2 305.0 12.7
26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49
Concentration (ng/mL)
a
84.8 147.9 11.6 69.2 12.5 111.4 30.9 25.3 55.4 24.3 1.9 0 0 0.1 0 3.5 351 46.1 15.4 152.3 0 24.9 16.7 56 0
1
5
Iso-5a
6
8
44.9 71.1 0 7.7 0 52.7 191.9 9.5 50.3 3.4 7.9 5.5 7.6 0.4 13.0 6.0 126.6 706.2 5.0 0.6 4.7 8.9 17.7 8.2
18.3 24.1 4.4 7.3 4.5 52.5 51.4 9.2 31.4 7.5 6.3 5.5 5.9 9.8 8.7 4.3 81.8 457.9 7.6 1.2 7.6 11.4 16.9 3.4
20.4 1.7 2.8 10.0 2.5 2.9 2.1 4.8 4.1 6.7 13.3 0.3 0.3 0.4 0.3 0.0 3.3 10.3 0.3 0.0 0.6 0.5 0.6 0.0
Concentration (ng/mL) 62.5 206.3 32.5 21.9 16.8 242.2 105.9 11.1 111.2 23.4 40.4 18.5 52.6 18.8 39.0 17.1 219.1 943.8 17.1 4.7 19.7 26.8 62.3 14.8
22 0.1 31.3 26.2 10.8 0 0 35.5 0 9.9 9.7 0 0 0 0 0 0 0 0 0 0.1 0 0 0
Iso-5 is JWH-018 N-(5-hydroxypentyl) metabolite.
therefore no NMR analysis was performed. From the results described above, the identity of the unknown metabolite was proposed to be 1. 3.4. Synthesis of compound 1 To further confirm the proposed identity of the unknown metabolite, 3-(3-(1-naphthoyl)-1H-indol-1-yl) propanoic acid was synthesized and characterized. Its chromatographic and mass spectrometric data was then compared to the results obtained for the concentrated metabolite extract from the authentic positive urine sample. The carbon and proton NMR spectra are included in the Supplemental section. The chemically synthesized metabolite was analyzed by LC/ MSD TOF for exact mass determination, which resulted in a molecular ion at m/z 344.1287. This corresponds to a molecular formula of C22H17NO3. This exact mass is consistent with the values obtained for unknown metabolite in the extracted authentic positive urine. Another important piece of information that further substantiates the unknown’s identity is the retention time of the metabolite present in the authentic positive urine matches the retention time of the synthetic standard. 3.5. Quantitative examination of positive samples The quantitative examination of 49 samples positive for naphthoylindole synthetic cannabinoids was undertaken using a similar protocol described by Yanes and Lovett [6] in which synthetic 1 was also included in the calibrator standard solution. As presented in Table 2, the results revealed that not only was JWH-072 N-propanoic acid a common metabolite present in all samples, but was also the major carboxylate metabolite in 48 of the 49 specimens. Comparison versus the JWH-018 N-(4-hydroxypentyl) metabolite (5) and its N-(5-hydroxypentyl) isomer indicate that JWH-072 N-propanoic acid is the predominate metabolite in 38 of the 49 samples and is the second most abundant metabolite in the remaining 11 samples. From these preliminary studies, it has
also been observed that a majority of 1 is excreted as the free carboxylic acid unconjugated to b-glucuronic acid. 4. Conclusions A new metabolite with a potential status of being a common biomarker for monitoring naphthoylindole based synthetic cannabinoids has been identified and completely characterized. 3-(3(1-Naphthoyl)-1H-indol-1-yl) propionic acid (1) was observed as a metabolic product in authentic positive urine and its identity was confirmed by the identical chromatographic retention time, fragment ions, and exact mass compared to a chemically synthesized metabolite standard. At this time, the JWH-072 Npropanoic acid metabolite has been determined to be a major metabolic product for JWH-018, JWH-073, and AM-2201. The monitoring of a common biomarker for the unsubstituted naphthoylindole series of synthetic cannabinoids could facilitate the rapid development of qualitative and/or quantitative analyses by requiring only a minimum number of MRM transitions. Furthermore, the preparation of calibration solutions, typically containing a complex mixture of alcohol and carboxylic acid metabolite standards, would be simplified to containing only a few. As the synthetic cannabinoid market continues to evolve, this new paradigm would eliminate the need to monitor the presence of a vast array of metabolites and consequently there would no longer be the necessity to wait for new metabolite reference standards to become commercially available. Acknowledgments The authors gratefully acknowledge LtCol M. Ewy for her assistance and support, the United States Air Force Academy for the use of their chemistry laboratory for the synthetic work, and Mrs. Cynthia Corley for her help with LC-TOF analysis. The authors would like to thank the United States Air Force and ADC Management Services Inc. for funding this project.
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.forsciint.2012.12.012. References [1] C. Mustata, M. Torrens, R. Pardo, C. Perez, The Psychonaut web mapping group, M. Farre, spice drugs: cannabinoids as a new designer drugs, Adicciones 21 (2009) 181. [2] European Monitoring Centre for Drugs and Drug Addiction (EMCDDA), Thematic Paper-understanding the ‘Spice’ Phenomenon, Luxembourg, 2009,, p. 3. [3] B.K. Atwood, J.W. Huffman, A. Straiker, K. Mackie, JWH018, a common constituent of ‘Spice’ herbal blends, is a potent and efficacious cannabinoid CB1 agonist, Br. J. Pharmacol. 1 (2010).
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[4] Food and Drug Administration Safety and Innovation Act, http://www.gpo.gov/ fdsys/pkg/BILLS-112s3187enr/pdf/BILLS-112s3187enr.pdf (accessed 07.09.12). [5] L.N. Sacco, K.M. Finklea, Synthetic drugs: overview and issues for congress, in: Congressional Research Service (CRS) Report for Congress, 2011. [6] E.G. Yanes, D.P. Lovett, High-throughput bioanalytical method for analysis of synthetic cannabinoid metabolites in urine using salting-out sample preparation and LC–MS/MS, J. Chromatogr. B 909 (2012) 42–50. [7] C.L. Moran, V.-H. Huyen Le, K.C. Chimalakonda, A.L. Smedley, F.D. Lackey, S.N. Owen, P.D. Kennedy, G.W. Endres, F.L. Ciske, J.B. Kramer, A.M. Kornilov, L.D. Bratton, P.J. Dobrowolski, W.D. Wessinger, W.E. Fantegrossi, P.L. Prather, L.P. James, A. Radominska-Pandya, J.H. Moran, Quantitative measurement of JWH-018 and JWH-073 metabolites excreted in human urine, Anal. Chem. 83 (2011) 4228–4236. [8] K.C. Chimalakonda, C.L. Moran, P.D. Kennedy, G.W. Endres, A. Uzieblo, P.J. Dobrowolski, E.K. Fifer, J. Lapoint, L.S. Nelson, R.S. Hoffman, L.P. James, A. RadominskaPandya, J.H. Moran, Solid-phase extraction and quantitative measurement of omega and omega-1 metabolites of JWH-018 and JWH-073 in human urine, Anal. Chem. 83 (2011) 6381–6388. [9] B.R. Martin, J.W. Huffman, US Patent Application 2005/0009903 A1.
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