Accepted Manuscript Title: ANALYSIS OF UR-144 AND ITS PYROLYSIS PRODUCT IN BLOOD AND THEIR METABOLITES IN URINE Author: Piotr Adamowicz Dariusz Zuba Karolina Sekuła PII: DOI: Reference:

S0379-0738(13)00450-7 http://dx.doi.org/doi:10.1016/j.forsciint.2013.10.005 FSI 7374

To appear in:

FSI

Received date: Revised date: Accepted date:

14-6-2013 17-9-2013 5-10-2013

Please cite this article as: P. Adamowicz, D. Zuba, K. Sekula, ANALYSIS OF UR-144 AND ITS PYROLYSIS PRODUCT IN BLOOD AND THEIR METABOLITES IN URINE, Forensic Science International (2013), http://dx.doi.org/10.1016/j.forsciint.2013.10.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ANALYSIS OF UR-144 AND ITS PYROLYSIS PRODUCT IN BLOOD AND THEIR METABOLITES IN URINE Abstract UR-144 [(1-pentyl-1H-indol-3-yl)(2,2,3,3-tetramethylcyclopropyl)methanone] is a synthetic cannabinoid, which has been detected in many herbal blends, resinous samples and powders

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seized from the Polish drug market since the beginning of 2012. This paper presents the case of intoxication by this substance. A complete picture of the symptoms observed by a witness,

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paramedics and medical doctors are given. In the analysis of powder residues from the plastic bag seized from the intoxicated person by gas chromatography-mass spectrometry (GC-MS),

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UR-144 and its major pyrolysis product [1-(1-pentyl-1H-indol-3-yl)-3-methyl-2-(propan-2yl)but-3-en-1-one] were detected. Both substances were also identified in a blood sample

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collected on admission of the patient to hospital using liquid chromatography – triple quadrupole tandem mass spectrometry (LC-QqQ-MS). Blood concentration of UR-144 was 6.1 ng/mL. A urine sample collected at the same time was analyzed by liquid chromatography

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– quadruple time-of-flight tandem mass spectrometry (LC-QTOF-MS). The parent substance and its pyrolysis products were not detected in urine, while their five metabolites were found.

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The experiments allowed the location of derivative groups to be established, and thus elucidate rough structures of the metabolites; a dihydroxylated metabolite of UR-144 and

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mono-, dihydroxylated and carboxylated metabolites of its pyrolysis product were identified.

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Key words: UR-144, synthetic cannabinoids, intoxication, analysis, LC-MS/MS

1 Page 1 of 25

Introduction UR-144 [IUPAC name: (1-pentyl-1H-indol-3-yl)-(2,2,3,3-tetramethylcyclopropyl)methanone] is a synthetic cannabinoid, which was invented in 2006 by Abbott Laboratories [1]. Alternative abbreviations for this substance include TMCP-018, KM-X1, MN-001 and YX-17 [2]. Its chemical structure is presented in Figure 1 and it was compared to JWH-018,

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the first synthetic cannabinoid detected in ‘herbal high’ preparations which is still very popular among drug users [3,4]. It is clearly seen that the structures of both substances are

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similar; they contain common pentyl side chain substituted to nitrogen atom of the indole moiety, which is connected by the bridging carbonyl group with the second ring. JWH-018

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contains a naphthyl ring at this position, while UR-144 has a tetramethylcyclopropyl group. Appearance of UR-144 on the drug market may seem surprising, because the results of

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structure – activity relationship (SAR) study showed that substitution of the naphthyl ring in naphthoylindoles by the tetramethylcyclopropyl moiety increases selectivity of a substance to the CB2 cannabinoid receptor [5]. This feature is desirable when the substance is to be used as

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a medicine, because the CB2 receptors are involved in pain perception. Most synthetic cannabinoids, which have appeared on the drug market in the last four years, have high

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affinity to the CB1 receptors, which are located mainly in the brain and are responsible for the psychoactive effects [6,7]. When compared to delta-9-tetrahydrocannabinol (∆9-THC), which is a partial agonist at both receptors and has binding affinity (Ki) values of 40.7 nmol at CB1

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and 36.4 nmol at CB2, JWH-018 has a Ki of 9 nmol at CB1 and 2.94 nmol at CB2, while these values for UR-144 are 150 and 1.8 nmol at CB1 and CB2, respectively [1]. Despite relatively

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low affinity of UR-144 at CB1 receptor, reports given by drug users on Internet forums clearly show that this substance is very popular and positively assessed for its psychoactive action. UR-144 has been sold mostly as a ‘research chemical’ (‘RC’) in quantities varying

from 0.25 g to 100 g. Although most packages are labeled ‘not for human use’, everyone is aware that they are intended for administration. UR-144 is mostly smoked in ‘joints’ (mixed, e.g., with tobacco, St John's wort or other herbs), and sometimes using a ‘bong’. It can be also taken orally, or vaporized and inhaled. Starting doses of UR-144 reported by the users ranges 0.5 – 2 mg, and typical are between 2.5 and 20 mg (the content in blends is usually from 0.05 to 0.4%). Some users ‘re-dosed’ to prolong the experience leading to several dozen mg smoked in a session [8]. The effects starts in 0.5 – 2 minutes after smoking, the strongest are observed after 3 – 5 minutes, and ends after approximately 1 – 2 hours (after high doses even 2 Page 2 of 25

4 hours). The users compare the effects to those caused by JWH-122 and AM-2201 or marijuana [8,9]. The effects include euphoria, mood elevation, excitation, relaxation, drowsiness, and hallucinations. Users claim that it causes merriment and increased appetite. The most commonly-reported negative effects are anxiety, paranoia, attention disturbance, depression and hallucinations which may persist for several days [8-11]. The effects after high doses are described as psychedelic and especially the first minutes are very intense. Extremely

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high doses can cause overdose with symptoms like tachycardia, nausea, disorientation, dysphoria, blurry vision, inability to communicate, extreme hallucinations and losing

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consciousness [8,12]. Tolerance to UR-144 may develop in users and, as a consequence, users tend to consume larger doses [8].

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UR-144 was first reported in herbal incenses seized in June 2012 in Korea [13], and it has spread very quickly all over the world. This substance has been detected in Europe

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(including Croatia, Sweden, Germany, Finland, Norway and Hungary), Japan and the USA [14-16]. It was the most popular synthetic cannabinoid in 2012 among the samples analyzed in a Russian laboratory [17]. A similar phenomenon was observed in the Institute of Forensic

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Research (IFR, Krakow, Poland). UR-144 was first detected in samples seized in March 2012, and it quickly gained popularity to become the most popular synthetic cannabinoid last year.

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It was found in herbal blends, sometimes sold as ‘herbal highs’ under different brand names, including ‘Sztywny Misza’ (Stiff Misha), ‘Mocarz’ (Athlete) and ‘El Mexicano’, several resinous samples with macroscopic appearance similar to that of hashish, as well as powders.

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This year, prevalence of UR-144 has not diminished. What is more, two analogs of UR-144 were identified in the IFR; XLR-11 is a derivative containing the fluoride atom substituted at

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position 5’ of the side chain, whereas A-834,735 has a (tetrahydropyran-4-yl)methyl group instead of the pentyl chain [18]. Marketing of UR-144 and its analogs seems to be a response of the black market manufacturers to scheduling synthetic cannabinoids by structural modifications, thus circumventing the new drug law. The aforementioned substances are also not controlled in most countries with individual drug listing systems. UR-144 was banned only in a number of countries, including New Zealand and Russia (2012), and United Kingdom and United States (2013). Because administration of substances with unknown action and side-effects to human is ethically questionable, little is known on the metabolism of synthetic cannabinoids, making adequate detection in biological specimens difficult. Notwithstanding, it was found that synthetic cannabinoids are rapidly metabolized, and parent substances are either rarely 3 Page 3 of 25

identified in blood, oral fluid, hair and urine, or their concentrations are at low levels. The urinary metabolites and methods of detection thereof in humans have been reported mainly for naphthoyindoles (JWH-018 [19-21], JWH-073 [22,23], JWH-081, JWH-122 and JWH210 [23]), benzoylindoles (RCS-4 [24] and AM-694 [25]) and phenylacetylindole (JWH-250 [23,26]). In recent studies, metabolism of UR-144 was investigated by Sobolevsky et al [27] and Grigoryev et al [17]. All these publications demonstrate that synthetic cannabinoids are

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subject to extensive metabolism, mainly through hydroxylation (mono-, di- and tri-) and further conjugation with glucuronic or sulphuric acid. Other phase I metabolic pathways

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include dehydrogenation, dehydrogenation and hydroxylation, dihydrodiol formation, dihydrodiol formation and hydroxylation, dealkylation and carboxylation.

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This paper describes the case in which both non-biological (plastic bag with traces of a substance) and biological (blood and urine) materials, collected from a man hospitalized on

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suspicion of poisoning with cannabinoids, were analyzed in the IFR. A complete picture of the symptoms observed by the witness, paramedics and medical doctors is reported. The performed study showed that the patient was intoxicated by UR-144. To our knowledge, no

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formal papers have been published to date on pharmacodynamics, human toxicity, short nor

Case history

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long-term effects of UR-144.

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It is known from the testimony of the witness (brother of the victim) that a 22-year old man was smoking a “joint”, and after a few puffs he lost consciousness with the tightening of

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limbs. Convulsions of the whole body occurred in several minutes. The man was drooling, without contact; his eyes were open, facing straight ahead. At the time, he did not urinate. After some time spasms re-occurred and were lasting about 20 minutes. It was the victim’s first seizure.

On arrival of an ambulance, the man was still unconscious, without contact.

Paramedics reported in the records: generalized spasms of the whole body, dilated and symmetrical pupils, and slow reaction of pupils to light. State of consciousness was rated as moderate (10 in Glasgow Scale). The patient had incomprehensible speech (aphasia), heart rate was 140 bpm, respiratory rate 18/min, systolic blood pressure >89 mmHg, and glucose level 206 mg/dL. The man regained consciousness after about 15 minutes. He was hallucinating and claimed that he saw meerkats. 4 Page 4 of 25

The patient was taken to hospital and admitted to the emergency department at 7:30 p.m. On admission, he was conscious, calm, sleepy, auto- and allopsychic oriented, maintained logical verbal contact. His pupils were still dilated and slowly reactive to light. Eye movements were normal and symmetrical. There were no further seizures. The medical doctor also noted: proper sense, bilateral plantar reflex, heart rate 125/min, pressure 140/56, the body temperature of 37.5 oC. Potassium level in blood was 3.08 mmol/L and glucose –

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198 mg/dL. Toxicological screening tests performed by immunochromatographic and the Roche Kinetic Interaction of Microparticles in Solution (KIMS) assays in the hospital

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laboratory showed the presence of ‘THC’ in urine at concentration of 125 ng/mL. The presence of ethyl alcohol, amphetamine, methamphetamine, MDMA, PCP, benzodiazepines,

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cocaine, opiates, methadone, barbiturates and tricyclic antidepressants was excluded. Urinary catheterization, fluid and electrolyte supplementation were applied, and diazepam (‘Valium’)

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was administered intravenously. Later, the medical doctor reported: proper breathing, heart rate 76/min, pressure 105/50, normal urine output, good appetite and physical fitness, selfmoving, proper senses, normal vision and hearing, good mood, complete orientation but

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impaired memory. The next day the patient was in good condition, however complained of

Reagents and materials

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Experimental

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drowsiness and headache. The following day the man was discharged from hospital.

UR-144 and JWH-018-d9 were purchased from LGC Standards (Dziekanow Lesny,

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Poland). Acetonitrile (MeCN) and methanol (both HPLC-grade), formic acid (98-100%) and β-glucuronidase/arylsulfatase (from Helix pomatia) were bought from Merck (Warsaw, Poland).

Blank blood samples used for the development and validation of the method and for

preparing controls were obtained from a regional blood donation centre. Urine drug-free samples were taken from persons with no history of drug abuse. Biological materials were stored at +4oC before the analysis. Blank blood and urine were screened for common drugs of abuse and the analytes (including UR-144, its open-ring isomer and their metabolites) and the tests were negative.

Standard, calibrators and controls preparation 5 Page 5 of 25

A stock solution of UR-144 (1 mg/mL in methanol) was stored at -22oC. Calibrators were prepared in 0.2 mL of drug-free blood samples by spiking them with UR-144 to the concentrations of 0.5, 1, 2, 5, 10, 20, 50 and 100 ng/mL. Control samples of UR-144 at 10 ng/mL and negative blood controls were also prepared. JWH-018-d9 spiking solution was prepared for use as the internal standard (IS) at the concentration of 10 ng/mL.

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Investigated material Blood and urine samples were collected from the man on admission to the hospital (at 8:00 PM) and sent to the IFR to verify the use of cannabinoids (natural and/or synthetic) by

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the man. A package with the description ‘HARDCORE by Waikiki Ben 0.5g’ containing a

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plastic bag with traces of the powder was also delivered for testing as the reference material. Sample preparation

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Plastic bag with traces of a powder was washed with 0.5 mL of methanol. Then, the obtained solution was centrifuged at 13,000 rpm for 3 min, the supernatant was transferred into the insert for autosampler vials and analyzed by gas chromatography – mass spectrometry

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(GC-MS).

Blood samples (0.2 mL) were placed in Eppendorf vials and 20 μL of 100 ng/mL

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methanolic solution of JWH-018-d9 (IS) was added to obtain a final concentration of 10 ng/mL. The analytes were isolated from the matrix by precipitation with MeCN; six

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hundred microliters of MeCN was added in 50 μL portions, and, after each addition, the samples were vortexed for 10 s. Then, the samples were mixed for 5 min and centrifuged at

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13,000 rpm (15,682 × g) for 3 min, followed by the transfer of the organic layer into 2 mL glass vials. MeCN was evaporated to dryness under nitrogen at 37oC. The dry residues were dissolved in 100 μL of 0.1% formic acid in water (v/v) and transferred to inserts for autosampler vials. The solutions were analyzed by liquid chromatography – triple quadruple tandem mass spectrometry (LC-QqQ-MS). Urine specimens were analyzed without hydrolysis and after enzymatic hydrolysis.

The latter process was performed by addition of 0.1M acetate buffer (pH 5.5) and the enzyme β-glucuronidase/arylsulfatase (50 µL) into 0.5 mL of urine, followed by incubated of the solutions for 60 min at 55oC. Then, the samples were precipitated as described above for blood samples, or diluted with distillated water. The same protocol was used for unhydrolyzed samples. All urine specimens were analyzed by liquid chromatography – quadruple time-offlight tandem mass spectrometry (LC-QTOF-MS). 6 Page 6 of 25

Instrumental analyses GC-MS The analysis of the plastic bag’s content was performed using a gas chromatograph (HP 6890 GC System) coupled to a mass spectrometer (Agilent 5973 Network Mass Selective

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Detector), equipped with a quadruple mass analyzer (Agilent Technologies, USA). The injector was maintained at 280°C. Sample (3 μL) was injected in splitless mode.

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Chromatographic separation was conducted on a HP-5MS capillary column (Agilent Technologies, 30 m length, 0.25 nm inner diameter, 0.25 μm film thickness). Helium was

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used as a carrier gas at the flow rate of 1.0 mL/min. Temperature program consisted of three segments: the initial column temperature (75°C) was maintained for 1 min, then increased

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linearly by 25°C/min up to 280°C, and maintained for 20.80 min. The mass detector was set to positive electron impact mode (EI) and the electron beam energy was 70 eV. The mass

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detector was operating in a full scan mode in range of 29-600 amu. LC-QqQ-MS

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Analyses of blood samples were performed on an Agilent Technologies 1200 series liquid chromatograph connected to a 6460 Triple Quad mass spectrometer. Separation was performed on a Kinetex C18 2.6u 100Å (100 × 4.6 mm) column (Phenomenex), thermostated

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at 25oC. The mobile phase was composed of a mixture of 0.1% formic acid in MeCN (v/v) and 0.1 % formic acid in water (v/v). The flow rate was 0.5 mL/min. The injection volume

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was 10 μL. Screening (qualitative) analyses were conducted in gradient mode (shown in relation to MeCN content): 0 min – 10%, 1 min – 10%, 14 min – 90%, 17 min – 90%, 17.5 min – 10%, 23 min – 10% (total analysis time – 23 min; retention time of UR-144 – 18.7 min, relative retention time – 1.06). Targeted (quantitative) analyses for UR-144 were carried out in isocratic mode: 90% MeCN (total analysis time – 7 min; retention time of UR-144 – 4.35 min, relative retention time – 1.24). Multiple reaction monitoring (MRM) with positive ion detection was used. The following precursor ions and the fragment ions for each compound (quantifiers shown in bold) were monitored: 312.2→125.0, 312.2→55.1, 312.2→214.1 and 312.2→57.10 for UR-144; 312.2→214.1, 312.2→144.0 and 312.2→83.0 for its open-ring isomer, as well as 351.2→127.0 and 351.2→155.0 for JWH-018-d9. The mass detector parameters in targeted method were as follows; capillary voltage: 3500 V, gas 7 Page 7 of 25

flow (nitrogen): 10 L/min and gas temperature: 325°C, sheath gas flow: 11 L/min, sheath gas temperature: 325°C, nebulizer pressure: 40 psi, dwell times: 200 ms. Fragmentor voltage for UR-144 and its isomer was 112 V and 80 V, respectively, and for JWH-018-d9 – 118 V. Collision energies [V] for UR-144, its isomer and JWH-018-d9 transitions were 20, 40, 44 and 24, 52, respectively. Screening method parameters were as above, except for the fragmentor voltage, collision energies and dwell times, which were set for all analysed compounds to

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values: 135 V, 30 V and 50 ms, respectively.

Targeted LC-MS method for quantitative analysis of UR-144 in blood was validated.

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An eight-point blood calibration curve was prepared (number of replicates for each level, n = 3) in the range of 0.5 – 100 ng/mL. The limit of detection (LOD) was estimated as signal to

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noise, S/N = 3 for the transition with the lowest intensity of the three most intense. Limit of quantitation (LOQ) was assumed to be the lowest point from the calibration curve. The assay

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specificity was assessed by analyzing UR-144-free blood collected from ten persons. Extraction recovery for blood (at the concentration of 10 ng/mL, n = 5) was calculated by comparison of the responses (analyte area/IS area) for UR-144 extracted from blood to the

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blank blood spiked with UR-144 after extraction. LC-MS/MS matrix effect (ME) was calculated by comparing the responses of a known amounts (10 ng/mL) of unextracted UR-

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144 (set A, n = 5) with those measured in blank blood spiked after extraction with the same analyte amount (set B, n = 5). The following formula was used: ME [%] = ((B/A) – 1) x 100.

LC-QTOF-MS

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The repeatability (precision error) and bias (accuracy error) were determined at 10 ng/mL.

liquid

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Analyses of urine sample were carried out using an Agilent Technologies 1200 series chromatograph

coupled

with

a

6520

Accurate-Mass

QTOF-MS

detector.

Chromatographic separation was performed on an Ascentis Express C18 (7.5 cm × 2.1 mm × 2.7 µm) column (Supelco), thermostated at 35°C. The mobile phase was composed of a mixture of 0.1% formic acid in MeCN (v/v) and 0.1 % formic acid in water (v/v). The flow rate was 0.3 mL/min. Analyses were conducted in gradient mode (shown in relation to MeCN content): 0 min – 5%, 11 min – 33%, 15 min – 37%, 15.2 min – 5%, 21 min – 5%. The QTOF-MS instrument was operating in electrospray positive ionization mode (+ESI). Nitrogen was used as a drying gas (temp. 300°C; flow 10 L/min) and as a nebulizing gas (pressure 45 psi). Capillary voltage was set at 3000 V and skimmer voltage at 65 V. The fragmentor voltage was 100 V or 300 V. The quadrupole was used as an ion guide in MS 8 Page 8 of 25

mode, and for selection of precursor ions with Δm/z = 1.3 in MS/MS mode. Nitrogen was applied as a collision gas (collision energy was 30 eV). Mass spectra were collected in the range: 80–1000 m/z in MS mode and 60–1000 m/z in MS/MS mode. Spectra were internally mass-corrected in real time using an automatically-introduced reference mass solution containing

two

compounds:

purine

([M+H]+

=

121.050873)

and

HP-921

–

+

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hexakis(1H,1H,3H-tetrafluoropropoxy)phosphazine ([M+H] = 922.009798).

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Results and Discussion

The solution obtained by washing a plastic bag, which was placed inside the package seized

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from the intoxicated person, was subjected to analysis by GC-MS. In the total ion chromatogram, two main peaks were detected at 10.8 and 11.0 min. The EI mass spectra of

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both peaks were similar (Figure 2a,b), although different ion ratios were clearly seen. UR-144 was identified as the first peak by matching its retention time and spectrum against a reference library (SWGDRUG MS Library, ver. 1.7). Formation of the second peak was explained by

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thermal degradation of the cyclopropyl ring in an injection port producing open ring form; therefore, this ion should be treated in GC-MS method as an artefact. The process results from

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the fact that a cyclopropane ring is thermally unstable and it undergoes a variety of ringopening reactions at high temperatures [28]. Similar phenomenon was observed in the analysis of UR-144 analogs containing the cyclopropane ring [18].

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Taking into account that synthetic cannabinoids are usually administered by smoking, thermal degradation of UR-144 during combustion would have to be considered. Therefore,

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blood samples were analysed not only for the parent substance, UR-144, but also its main pyrolysis product – [1-(1-pentyl-1H-indol-3-yl)-3-methyl-2-(propan-2-yl)but-3-en-1-one [15]. The MRM chromatograms of the blood sample obtained by screening method are presented in Figure 3. Peaks corresponding to two substances are seen (at 18.0 min and 18.7 min). UR-144 was identified as the second peak by matching its relative retention time vs IS and spectrum against a standard. The structure of the first peak was confirmed by analysis of the transitions. The ions at m/z 144 and 214 are common in synthetic cannabinoids and correspond to the indole moiety and this ring with untouched N-pentyl side chain. However, the most important is the lack of the ion at m/z 125, which indicates if the intact 2,2,3,3-tetramethylcyclopropyl group is present in the structure of the analyzed substance or not.

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The concentration of UR-144 in blood was determined using the worked-out LC-MS method characterised by the following validation parameters. The method was linear in the range of 0.5 – 100 ng/mL, with R2 = 0.996. The LOD and LOQ were 0.15 ng/mL and 0.5 ng/mL, respectively. The assay was specific, no interferences were observed in the study. Extraction recovery was 62.3%, and matrix effect was –35.3% on average and showed signal suppression. The repeatability was 11.8%, while bias was 9.4%. The performed analyses

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allowed to establish that the concentration of UR-144 in the blood of the patient on admission to the hospital was 6.1 ng/mL.

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Because both UR-144 and its open-ring isomer were identified in the blood of the intoxicated person, the possible existence of two groups of metabolites in urine was assumed.

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Taking into account earlier studies on metabolites of synthetic cannabinoids [17,27,29], several groups of compounds were considered to be present in the QTOFMS spectra of urine

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sample, i.e., despentyl-UR-144 (M+H+ = 242.1545), despentylhydroxy-UR-144 (M+H+ = 258.1494), dehydrated hydroxy-UR-144 (M+H+ = 326.2115), hydroxy-UR-144 (M+H+ = 328.2271), UR-144 N-pentanoic acid (M+H+ = 342.2064), dihydroxy-UR-144 (M+H+ =

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344.2220), and UR-144 N-(5-hydroxypentyl) β-D-glucuronide (M+H+ = 504.2592). Metabolites of the open-ring isomer were expected to be registered at identical m/z values.

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The presence of the parent substance and its main pyrolysis product in urine was also verified (M+H+ = 312.2322).

In the MS experiment performed at a low fragmentor voltage (FV = 100 V), three of

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the expected protonated masses were observed, that is the ions with m/z 328.2279, 342.2070, and 344.2217. Their masses corresponded to monohydroxyl, dihydroxyl and carboxyl

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metabolites of UR-144 or its isomer. The accuracy of mass determination for obtained ions has been calculated and is presented in Table 1. Neither the parent ion nor its main pyrolysis product were detected in urine.

To confirm the presence of the aforementioned metabolites in urine and to elucidate

their structures, the experiments were performed in the targeted MS/MS mode with the fragmentor voltage = 100 V and collision energy = 30 V. At this stage, the aforementioned pseudomolecular ions were selected for further fragmentation. The obtained MS/MS spectra are presented in Figures 4a–e. Two hydroxyl metabolites were identified. The hydroxyl group in the first monohydroxyl metabolite was located on the N-pentyl chain (Fig.4a) as indicated by the presence of the ions at m/z 144.0442 (unchanged indole ring combined with carbonyl group, 10 Page 10 of 25

Δm = -1.4 ppm) and 230.1171 (monohydroxylated N-pentyl chain combined with the aforementioned moiety, Δm = -2.2 pmm). Confirmation of the exact position of the hydroxylation on the alkyl side chain, however, was not possible for the metabolite. The peak around m/z 125.0961 was absent in the spectrum, therefore the presence of 2,4-dimethylpent1-ene moiety in its structure instead of the intact 2,2,3,3-tetramethylcyclopropyl moiety was assumed for this metabolite. The spectrum of the alternative monohydroxyl metabolite is

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presented in Fig.4b. The ion with m/z 214.1228 indicated the lack of hydroxyl group in the side chain and the indole ring, because it corresponded to the unchanged N-pentylindole

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moiety combined with carbonyl group, Δm = 0.9 ppm). These findings were confirmed by the appearance of peaks at m/z 188.1449 (Δm = 8.0 ppm) and m/z 230.1541 (Δm = 0.9 ppm). The

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presence of fragment with m/z 99.0802 suggested that hydroxylation occurred in this case in the 2,4-dimethylpent-1-ene moiety.

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Because the peaks around m/z 144.0444 and 230.1176 were obtained for the both dihydroxyl metabolites (Fig.4c,d), one hydroxyl group was ascribed to the N-pentyl side chain and not to the indole moiety. The peak observed at m/z 99.0799 for the first of these

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metabolites suggested that the other hydroxyl group was bonded to the 2,4-dimethylpent-1ene moiety. For the second one, this group was assumed to be connected to one of the methyl

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groups of the 2,2,3,3-tetramethylcyclopropyl moiety.

The ion observed at m/z 244.0978 in the MS/MS spectrum of the carboxyl metabolite (Fig.4e) corresponded to the mass of the N-pentylindole moiety with one carbonyl and one

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carboxyl group [M(C14H14NO3)+ = 244.0968; Δm = 4.1 ppm]. Because the indole moiety was not modified (the appropriate peak was registered at m/z 144.0441, Δm = -2.1 ppm), this may

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indicate that the carbonyl group was formed on the side chain. This assumption was supported by the presence of the peak at m/z 101.0602, because this mass corresponded to the mass of 4carboxybutylium ion (M+ = 101.0597; Δm = 4.9 ppm). The ion at m/z 125.0961 was also absent in the spectrum of this metabolite, suggesting open structure of the remaining part of the compound. The obtained spectra for five aforementioned substances were consistent with those obtained by other authors [17]. The blood and urine specimens were also analyzed for a wide range of drugs in accordance with routine procedures used of IFR. The blood analyses did not reveal any other substances that could cause the symptoms observed in the patient, while in urine the presence of THC-COOH (11-nor-9-carboxy-∆9-tetrahydrocannabinol) at a concentration of 24.7 ng/mL was shown (by GC-MS method). The positive result for this compound was consistent with 11 Page 11 of 25

those obtained in the hospital, however the determined concentration in the IFR was significantly lower. It may be caused by the use of the non-specific method for determination of THC-COOH in the hospital. It should be also noted that our additional experiments showed that popular immunochemical tests for ‘classical’ cannabinoids do not give the cross-reactions with UR-144. The performed experiments clearly indicated that the patient was intoxicated with UR-

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144. Despite high prevalence of this substance on the drug market, nothing is known about its pharmacodynamic and toxicological properties. To our knowledge, no formal papers have

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been published regarding blood concentrations of UR-144 after administration of ‘legal high’ products containing this compound. Hence, user reports on the Internet about dosage and

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effects must be interpreted with caution. Moreover, even if doses and effects are known, these data cannot be directly associated with UR-144 concentrations in blood.

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According to user reports, UR-144 acted similarly to marijuana as well as other synthetic cannabinoids, e.g. AM-2201. Standard doses of UR-144 taken by the users are 0.5 – 20 mg, which are in consistence with those for Δ9-THC (smoking doses 5 – 20 mg; used for

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medical purposes 2.5 – 10 mg) and JWH-018 (2 – 20 mg). Hence the hydrophobicity of a compound (as measured by its distribution coefficient, logD) affects strongly its

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pharmacokinetics and pharmacodynamics, appropriate calculations were done using ACD/Labs software. The logD values (both at pH 5.5 and 7.4) were 6.84, 6.86 and 6.13 for Δ9-THC, JWH-018 and UR-144, respectively. It clearly indicates that these substances are

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highly lipophilic, and their absorption, distribution, metabolism and excretion are similar. The poor water solubility of cannabinoids causes their absorption across the lipid bilayer

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membranes and fast elimination from blood circulation. One can expect that blood (and urine) levels after administration of similar doses will be comparable. The blood concentration of UR-144 determined in our case, that is 6.1 ng/mL, is similar to concentration of Δ9-THC observed after 1 – 2 hours of its use. The JWH-018 concentration in serum samples analyzed by Dresen et al. [30] varied from 0.3 to 8.17 ng/mL. The broader range of its concentration – 0.1 to 199 ng/mL – was obtained by Shanks et al [31], but the blood level was over 6.0 ng/mL in only 3 of 18 cases. The single blood specimen from a controlled study performed by Kacinko et al. [32] had a JWH-018 concentration of 4.8 ng/mL at 19 min and a concentration of 0.2 ng/mL at 199 min post-dosing. Similar rapid decline was observed by Teske et al. [33]. We estimated that the blood sample was collected from the patient approximately 2 hours after smoking of a ‘legal high’ product containing UR-144 (6:00 PM – 8.00 PM), which 12 Page 12 of 25

confirmed our hypothesis on similarity in blood concentration profiles for different cannabinoids. It also means that the developed and validated LC-QqQ-MS method of determination of UR-144 in blood covers the range of concentrations which may be expected in blood after administration of a typical dose of this drug. It can be used for diagnosis of acute intoxications. The parent substance and its main pyrolysis product were not detected in urine of the

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patient. It is in accordance with the findings in other studies on synthetic cannabinoids, although UR-144 was detected by Sobolevsky et al. [27] at the low level in one urine sample

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out of five. Wolhfarth at al [34] identified AM-2201 in a single specimen out of ten. Our metabolized and excreted in the form of metabolites.

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study confirmed that UR-144, similarly to other synthetic cannabinoids, is almost completely One metabolite of UR-144 and four metabolites of its main pyrolysis product were

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identified in the study. It was shown that monohydroxyl, dihydroxyl and carboxyl derivatives were formed. Our results are consistent with the findings of other authors indicating that hydroxylations and carboxylation are the preferred pathways of metabolism of synthetic

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cannabinoids [17,27,29,34]. According to Sobolevsky et al [27], mono- and dihydroxyl metabolites of UR-144 should be detectable at least 1 week after administration. None

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metabolite with hydroxyl group substituted at the indole ring was found in our case, but hydroxyindoles were rarely noted at all and only when high signals for the other main metabolites were seen [34]. The number of metabolites we detected was significantly lower

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than reported by Grigoryev et al [17], who identified sixteen metabolites of UR-144 and 21 metabolites of the major UR-144 pyrolysis product. These authors pre-concentrated twenty

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urine samples using another LC instrument for the semi-preparative separations. Low level of THC-COOH was also determined in urine of the patient. It indicates

distant usage of cannabis and not recent. According to the statements of the witness and our findings, one can assume that the man did not mix the ‘legal high’ product containing UR-144 with any other drug or alcohol. Therefore, the symptoms described in the ‘Case report’ section may be unambiguously associated with overdose of UR-144. The major symptoms included loss of contact and consciousness, seizures, aphasia, tachycardia, hallucinations followed by drowsiness and impaired memory. Similar effects associated with overdose UR144 were also described by the user on an Internet forum. In people who consumed approximately 100 mg of UR-144, seizures and hallucinations occurred just after

13 Page 13 of 25

administration, while disorientation, insomnia, memory loss and lapses, jerky movements and problems with speech were present even after several days [35]. Conclusions The performed study clearly indicated that the patient was intoxicated with UR-144.

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This substance was found in a blood sample at the concentration of 6.1 ng/mL. Its major pyrolysis product was also identified in this specimen. To our knowledge, this is the first

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report of detection and determination of UR-144 and its isomer in blood after administration of ‘legal high’ product. Both substances were not detected in urine of the patient. It total, five

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metabolites of the aforementioned substances were identified in this specimen. UR-144 was also found in traces of powder from the seized plastic bag. The testimony of the witness and

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medical records showed that the effects caused by this compound were similar to those observed after administration of cannabis products, but more severe. The described case

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shows that the use of UR-144 can be harmful to health and even life.

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Kouhen, B.B. Yao, G.C. Hsieh, M. Pai, C.Z. Zhu, P. Chandran, M.D. Meyer, Indol-3ylcycloalkyl ketones: effects of N1 substituted indole side chain variations on CB(2)

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cannabinoid receptor activity, J. Med. Chem. 53 (2011) 295–315. [2] http://en.wikipedia.org/wiki/UR-144 [7 June 2013]. [3] D. Zuba, B. Byrska, M. Maciów, Comparison of "herbal highs" composition, (2011) Anal. Bioanal. Chem. 400 (2011) 119–126. [4] D. Zuba, B. Byrska, Analysis of the prevalence and coexistence of synthetic cannabinoids in "herbal high" products in Poland, Forensic Toxicol. 31 (2013) 21–30. [5] J.M. Frost, M.J. Dart, K.R. Tietje, T.R. Garrison, G.K. Grayson, A.V. Daza, O.F. ElKouhen, L.N. Miller, L. Li, B.B. Yao, G.C. Hsieh, M. Pai, C.Z. Zhu, P. Chandran, M.D. Meyer, Indol-3-yl-tetramethylcyclopropyl ketones: effects of indole ring substitution on CB2 cannabinoid receptor activity, J. Med. Chem. 51 (2008) 1904–1912.

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[6] J.W. Huffman, D. Dai, Design, synthesis and pharmacology of cannabimimetic indoles, Bioorg. Med. Chem. Lett. 4 (1994) 563–566. [7] J.W. Huffman, L.W. Padgett, Recent developments in the medicinal chemistry of cannabimimetic indoles, pyrroles and indenes, Curr. Med. Chem. 12 (2005) 1395–1411. [8] http://www.drugs-forum.com [7 June 2013]. [9] http://the-tripreport.com/site/tripreport/ur-144-trip-report [7 June 2013].

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[13] H. Choi, S. Heo, E. Kim, B.Y. Hwang, C. Lee, J. Lee, Identification of (1-pentylindol-3-

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yl)-(2,2,3,3-tetramethylcyclopropyl) methanone and its 5-pentyl fluorinated analog in herbal incense seized for drug trafficking, Forensic Toxicol. 31 (2013) 86–92.

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[14] N. Uchiyama, M. Kawamura, R. Kikura-Hanajiri, Y. Goda, URB-754: A new class of designer drug and 12 synthetic cannabinoids detected in illegal products, Forensic Sci Int. 227 (2013) 21–32.

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[15] P. Kavanagh, A. Grigoryev, S. Savchuk, I. Mikhura, A. Formanovsky, UR-144 in products sold via the Internet: Identification of related compounds and characterization of

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pyrolysis products Drug Test. Anal. (2013) doi: 10.1002/dta.1456 [16] https://ednd.emcdda.europa.eu [7 June 2013]. [17] A. Grigoryev, P. Kavanagh, A. Melnik, S. Savchuk, A. Simonov, Gas and liquid

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chromatography-mass spectrometry detection of the urinary metabolites of UR-144 and its major pyrolysis product, J. Anal. Toxicol. 37 (2013) 265–276.

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[18] D. Zuba, B. Geppert, K. Sekuła, C. Żaba, [1-(Tetrahydropyran-4-ylmethyl)-1H-indol-3yl]-(2,2,3,3-tetramethylcyclopropyl)methanone: a new synthetic cannabinoid identified on the drug market, Forensic Toxicol. (2013) doi:10.1007/s11419-013-0191-8. [19] T. Sobolevsky, I. Prasolov, G. Rodchenkov, Detection of JWH-018 metabolites in smoking mixture post-administration urine. Forensic Sci. Int. 200 (2010) 141–147. [20] I. Möller, A. Wintermeyer, K. Bender, M. Jübner, A. Thomas, O. Krug, W. Schänzer, M. Thevis, Screening for the synthetic cannabinoid JWH-018 and its major metabolites in human doping controls, Drug Test. Anal. 3 (2011) 609–620. [21] S. Beuck, I. Möller, A. Thomas, A. Klose, N. Schlörer, W. Schänzer, M. Thevis, Structure characterisation of urinary metabolites of the cannabimimetic JWH-018 using

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chemically synthesised reference material for the support of LC-MS/MS-based drug testing, Anal. Bioanal. Chem. 401 (2011) 493–505. [22] A. Grigoryev, S. Savchuk, A. Melnik, N. Moskaleva, J. Dzhurko, M. Ershov, A. Nosyrev, A. Vedenin, B. Izotov, I. Zabirova, V. Rozhanets, Chromatography-mass spectrometry studies on the metabolism of synthetic cannabinoids JWH-018 and JWH-073, psychoactive components of smoking mixtures. J. Chromatogr. B 879 (2011) 1126–1136.

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[23] M. Hutter, S. Broecker, S. Kneisel, V. Auwärter, Identification of the major urinary

metabolites in man of seven synthetic cannabinoids of the aminoalkylindole type present as

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adulterants in 'herbal mixtures' using LC-MS/MS techniques, J. Mass Spectrom. 47 (2012) 54–65.

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[24] P. Kavanagh, A. Grigoryev, A. Melnik, A. Simonov, The identification of the urinary metabolites of 3-(4-methoxybenzoyl)-1-pentylindole (RCS-4), a novel cannabimimetic, by

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gas chromatography-mass spectrometry, J. Anal. Toxicol. 36 (2012) 303–311. [25] A. Grigoryev, P. Kavanagh, A. Melnik, The detection of the urinary metabolites of 1-[(5fluoropentyl)-1H-indol-3-yl]-(2-iodophenyl)methanone (AM-694), a high affinity

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cannabimimetic, by gas chromatography - mass spectrometry, Drug Test. Anal. 5 (2013) 110– 115.

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[26] A. Grigoryev, A. Melnik, S. Savchuk, A. Simonov, V. Rozhanets, Gas and liquid chromatography-mass spectrometry studies on the metabolism of the synthetic phenylacetylindole cannabimimetic JWH-250, the psychoactive component of smoking

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mixtures, J. Chromatogr. B 879 (2011) 2519–2526. [27] T. Sobolevsky, I. Prasolov, G. Rodchenkov, Detection of urinary metabolites of AM-

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2201 and UR-144, two novel synthetic cannabinoids, Drug Test. Anal. 4 (2012) 745–753. [28] H.N.C. Wong, M.Y. Hon, C.W. Tse, Y.C. Yip, J. Tanko, T. Hudlicky, Use of cyclopropanes and their derivatives in organic synthesis, Chem. Rev. 89 (1989) 165–198. [29] N. de Brabanter, S. Esposito, L. Geldof, L. Lootens, P. Meuleman, G. Leroux-Roels, K. Deventer, P. van Eenoo, In vitro and in vivo metabolisms of 1-pentyl-3-(4-methyl-1naphthoyl)indole (JWH-122), Forensic Toxicol. (2013) doi: 10.1007/s11419-013-0179-4. [30] S. Dresen, N. Ferreirós, H. Gnann, R. Zimmermann, W. Weinmann, Detection and identification of 700 drugs by multi-target screening with a 3200 Q TRAP® LC-MS/MS system and library searching, Anal. Bioanal. Chem. 396 (2010) 2425-2434. [31] K.G. Shanks, T. Dahn, A.R. Terrell, Detection of JWH-018 and JWH-073 by UPLC-MSMS in postmortem whole blood casework, J. Anal. Toxicol. 36 (2012) 145–152. 16 Page 16 of 25

[32] S.L. Kacinko, A. Xu, J.W. Homan, M.M. McMullin, D.M. Warrington, B.K. Logan, Development and validation of a liquid chromatography-tandem mass spectrometry method for the identification and quantification of JWH-018, JWH-073, JWH-019, and JWH-250 in human whole blood, J. Anal. Toxicol. 35 (2011) 386–393. [33] J. Teske, J.P. Weller, A. Fieguth, T. Rothämel, Y. Schulz, H.D. Tröger, Sensitive and rapid quantification of the cannabinoid receptor agonist naphthalen-1-yl-(1-pentylindol-3-

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yl)methanone (JWH-018) in human serum by liquid chromatography-tandem mass spectrometry, J. Chromatogr. B 878 (2010) 2659–2663.

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[34] A. Wohlfarth, K.B. Scheidweiler, X. Chen, H.F. Liu, M.A. Huestis, Qualitative

confirmation of 9 synthetic cannabinoids and 20 metabolites in human urine using LC-

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MS/MS and library search, Anal. Chem. 85 (2013) 3730–3738.

[35] http://www.bluelight.ru/vb/threads/654828-UR-144-massive-overdose-WARNING

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[7 June 2013].

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Figures descriptions Figure 1. Chemical structures of UR-144 (a) and JWH-018 (b). Figure 2. EI spectra (GC-MS) of UR-144 (a) and its major pyrolysis product (b) detected in

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Figure 3. MRM chromatograms (LC-QqQ-MS) of the blood sample.

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the traces from the plastic bag.

Figure 4. The MS/MS spectra (LC-QTOFMS) of the metabolites of UR-144 and its major

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pt

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pyrolysis products detected in urine.

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Table 1. The metabolites detected in urine with accuracy of their mass determination Measured Calculated ∆m [ppm] m/z value m/z value

Metabolite

RT [min]

hydroxy-UR-144

7.8

C21H30NO2 328.2279

328.2271

2.4

UR-144 N-pentanoic acid

8.2

C21H28NO3 342.2070

342.2064

1.8

dihydroxy-UR-144

6.7

C21H30NO3 344.2217

344.2220

-0.9

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pt

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Formula

19 Page 19 of 25

ANALYSIS OF UR-144 AND ITS PYROLYSIS PRODUCT IN BLOOD AND THEIR METABOLITES IN URINE Piotr Adamowicz, Dariusz Zuba, Karolina Sekuła

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Institute of Forensic Research, Westerplatte 9, 31-033 Krakow, Poland

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Corresponding author:

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Piotr Adamowicz Institute of Forensic Research,

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Westerplatte 9, 31-033 Krakow, Poland E-mail: [email protected]

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Tel. +48 12 6185743

20 Page 20 of 25

Figure 1

b

O

N

Ac ce p

te

d

M

an

us

cr

N

O

ip t

a

Page 21 of 25

Figure 2

Fig. 2.

214

100

a

90

ip t

80

60 50

cr

Relative intensity

70

40

us

30

144

20 10

229 238

0 0

20

40

60

80

100

120

140

160

100

180

200

220

311

252

240

260

280

300

320

340

240

260

280

300

320

340

214

229

M

b

90

an

44

296

80

ed

60 50

ce pt

40 30 20 10 0

Ac

Relative intensity

70

0

20

40

60

80

100

120

140

160

180

200

220

m/z

Page 22 of 25

Ac

ce pt

ed

M

an

us

cr

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Figure 3

Page 23 of 25

Figure 4

Fig. 4.

a O

O +

80

N OH

m/z 328.2271

+

HC

N

N H

CH3

230.1171

60

N

H3C

40

H3C

69.0702

C

+

cr

CH3

CH3

CH3

83.0848

186.1254

100

150

b CH3

OH

H3C

m/z 328.2271

CH3

CH3

+

C

40 20

Ac ce p +

C

CH3

N OH

m/z 344.2220

OH

CH3 H

O

200

250

CH3

300

+

O

CH2

+

C

N +

CH2

99.0799

N

N H

144.0445

CH3

186.1268

130.0650

69.0703

60

+

m/z 150

80

Relative intensity

CH3

+

C

N

100

OH

100

H3C

CH3

O

H3C

+

N

214.1228 230.1541

N

50

H O

300

CH3

188.1449

CH

OH

60

0

c

250

d

N

200

+

te

Relative intensity

80

M

+

99.0802

100

m/z

an

50

H O

CH3

+

0

H3C

OH

CH2

20

us

+

Relative intensity

H3C H O

C

ip t

100

+

144.0442

C

230.1175

OH N

40

81.0698

20

172.0753

+

C O

CH3

m/z

0 50

100

150

200

250

300

Page 24 of 25

d

CH3 OH N OH

m/z 344.2220

O +

CH3

80

Relative intensity

+

HC

60

C

O +

C

CH3

N

69.0705 N H

40 20

CH3

cr

H O

CH3

ip t

H3C +

230.1168

144.0448

100

0 100

150

200

us

50

100

H3C H O

CH3

+

+

H2C

244.0978 O +

C

40

M

O

101.0602 20

+

C

N

N H OH

d

OH

300

O

OH

60

83.0489

0

100

O

m/z 150

200

250

300

Ac ce p

50

te

Relative intensity

m/z 342.2064 O

CH3

m/z

250

144.0441

80

N

an

e

OH

Page 25 of 25