Accepted Manuscript Title: A liquid chromatography–mass spectrometry method based on class characteristic fragmentation pathways to detect the class of indole-derivative synthetic cannabinoids in biological samples Author: Monica Mazzarino Xavier de la Torre Francesco Botr`e PII: DOI: Reference:

S0003-2670(14)00715-6 http://dx.doi.org/doi:10.1016/j.aca.2014.06.003 ACA 233303

To appear in:

Analytica Chimica Acta

Received date: Revised date: Accepted date:

19-3-2014 12-5-2014 3-6-2014

Please cite this article as: Monica Mazzarino, Xavier de la Torre, Francesco Botr`e, A liquid chromatographyndashmass spectrometry method based on class characteristic fragmentation pathways to detect the class of indolederivative synthetic cannabinoids in biological samples, Analytica Chimica Acta http://dx.doi.org/10.1016/j.aca.2014.06.003 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.

A liquid chromatography – mass spectrometry method based on class characteristic fragmentation pathways to detect the class of indole-derivative synthetic cannabinoids in biological samples

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Monica Mazzarino1, Xavi er de la Torre1, Francesco Botrè1,2*

1: Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Largo Giulio Onesti, 1, 00197

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Rome, Italy

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2: Dipartimento di Medicina Sperimentale, “Sapienza” Università di Roma, Viale Regina Elena

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324, 00161 Rome, Italy

Prof. Francesco Botrè

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Federazione Medico Sportiva Italiana

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Scientific Director, Laboratorio Antidoping,

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Corresponding Author:

Largo Giulio Onesti, 1 00197 Rome, Italy

phone: +39-06-36859600 fax: +39-06-8078971

email: [email protected]

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Graphical Abstract The capability of different LC-MS/MS acquisition modes for the detection of known and unknown synthetic cannabinoids in different biological fluids was investigated.

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The results demonstrated that the complementary use of different precursor ion scan methods represents the best option for the detection of the largest amount of analytes (including also unknown and/or not commercially available metabolites). The overall performance of the method

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suggests that it could be successfully applied not only for routine use in anti-doping laboratories,

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but also for various applications in the field of analytical, clinical and forensic toxicology.

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Highlights

► Known and unknown synthetic cannabinoids are detected by LC-MS/MS

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► Common mass spectral fragments are recognized for each sub-class of compounds ► Efficacy tested on 15 representative aminoalkylindole-cannabinoids

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► Metabolites (obtained in vitro by human liver microsomes) are also detectable ► The method is suitable also for the detection of still unknown target analytes

Abstract

This article describes a liquid chromatographic/tandem mass spectrometric method, based on the use of precursor ion scan as the acquisition mode, specifically developed to detect indole-derived cannabinoids (phenylacetylindoles, naphthoylindoles and benzoylindoles) in biological fluids (saliva, urine and blood). The method is designed to recognize one or more common “structural markers”, corresponding to mass spectral fragments originating from the specific portion of the molecular structure that is common to the aminoalkylindole analogues and that is fundamental for their pharmacological classification. As such, the method is also suitable for detecting unknown substances, provided they contain the targeted portion of the molecular structure.

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The pre-treatment procedure consists in a liquid/liquid extraction step carried out at neutral pH: this is the only pretreatment in the case of analyses carried out in saliva, while it follows an enzymatic hydrolysis procedure, in the case of urine samples, or a protein precipitation step, in the case of blood samples. The chromatographic separation is achieved using an octadecyl reversephase 5 µm fused-core particle column; while the mass spectrometric detection is carried out by a

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triple-quadrupole instrument in positive electrospray ionization and precursor ion scan as

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acquisition mode, selecting, as mass spectral fragments, the indole (m/z 144), the carbonylnaphthalenyl (m/z 155) and the naphthalenyl (m/z 127) moieties.

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Once developed and optimized, the analytical procedure was validated in term of sensitivity (lower limits of detection in the range of 0.1-0.5 ng mL-1), specificity (no interference were detected

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at the retention times of the analytes under investigation), recovery (higher than 65% with a

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satisfactory repeatability: CV % lower than 10), matrix effect (lower than 30% for all the biological specimens tested), repeatability of the retention times (CV% lower than 0.1 ), robustness, and carry

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blank samples).

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over (the positive reference samples at a concentration 20 times the LLOD value did not affect the

The suitability of the proposed procedure, both as a targeted and an untargeted approach, was verified by analyzing samples containing synthetic cannabinoids and/or their metabolites and samples obtained from the incubation of synthetic cannabinoids with human liver microsomes.

Keywords: Anti-Doping Analysis; Drug analysis; Liquid Chromatography – Mass Spectrometry; Precursor ion scan; Structural marker; Synthetic Cannabinoids.

1. Introduction

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Marijuana, hashish and other pharmacologically active extracts of Cannabis sativa are among the most extensively produced and consumed illicit substances in the world. The psychoactive effects of marijuana are mainly due to the presence of delta-9-tetrahydrocannabinol, which exhibits partial agonistic activity at CB1 cannabinoid receptors (responsible of the physiological responses such as analgesia and hypothermia), located primarily in the brain and neuronal cells, and at the

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peripheral receptor CB2 (responsible for immunomodulatory effects), which is primarily expressed

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in the immune system [1-3].

Since the discovery of delta-9-tetrahydrocannabinol, cannabinoid designer drugs were

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synthesized for biomedical research purposes in order to evaluate the potential of novel active principles, capable of selectively activating the cannabinoid receptors and to separate therapeutic

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action from the unwanted psychotropic effects. At the same time, specific mechanisms of action

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were also deeply investigated, also considering the potential pharmaco-toxicological effects of marijuana and its analogues [3-12]. Due to difficulties in isolating anti-inflammatory and analgesic

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properties, only a few of these compounds are in medical use today (i.e. nabilone).

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At present, synthetic cannabinoids have found great popularity into the world of “recreational” drug use, being among the most widely diffused of the new psychoactive substances. In fact, starting around 2004, smokable herbal products, containing cannabinoid designer drugs as “legal” alternatives to marijuana, were synthesized in order to circumvent existing drug law [13-19]. These products are typically sold as “herbal blend” or “incense” and can be easily bought also via the Internet, with little or no restriction. A large variety of synthetic cannabinoids with enormous structural diversity are available in the market, and it can be forecasted that more compounds with the same pharmacological properties will appear in the near future. At present, synthetic cannabinoids are mainly categorized into 2 groups: the classical structures related to delta-9tetrahydrocannabinol, and the non-classical structures such as aminoalkylindole, 1,5-diarylpyrazole, quinolines, arylsulfonamides, and eicosanoids [14, 17].

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The constant addition of new entries to the family of synthetic cannabinoids, together with the broad physico-chemical variety of the single compounds belonging to this class, makes their detection in herbal products and in biological fluids a demanding challenge for forensic toxicology laboratories. For indeed, the conventional cannabinoid screening tests, mostly based on immunoassays, are ineffective in detecting with sufficient specificity this class of compounds.

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Furthermore, the newly developed cannabinoid designer drugs cannot be covered by the targeted

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analytical procedures based on the use of gas or liquid chromatography (LC) coupled to mass spectrometry (MS), due to the lack of reference materials. In addition, the urine analyses are further

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complicated by the fact that these substances are rapidly biotransformed into a large number of metabolites, mostly of them unknown or not fully characterized yet. The development of new

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analytical procedures becomes therefore necessary in order to drastically reduce the time necessary

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to identify newly synthesized variants of cannabinoid designer drugs. So far, numerous analytical procedures have been developed to detect synthetic cannabinoids in herbal products [20-24] and

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biological specimens such as hair [25-27], oral fluid [28-29], blood [30-33] and urine [34-39]. In

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this context, analytical strategies capable of detecting the widest range of compounds are focused on the use of high-resolution mass spectrometry techniques [24, 40-41]. The samples are first screened in full-scan acquisition mode in order to obtain the elemental compositions of all the compounds detected; a subsequent analysis by multistage tandem LC-MS measuring products ions with accurate mass detection is carried out for each suspected signal in order to obtain information to elucidate the chemical structure.

We are here proposing an alternative approach, still based on LC-MS/MS, but focused on the recognition of specific molecular portions, recurrent within the same pharmacological class of compounds [42-46]. More specifically, we have developed an analytical procedure capable of detecting known and unknown aminoalkylindole analogues and/or their metabolites in biological specimens (saliva, plasma/serum hair and urine), primarily, but not exclusively, for doping control

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purpose. The method is based on the use of triple quadrupole-mass spectrometer with electrospray ionization (ESI) and precursor ion scanning as acquisition mode. The overall performance of the newly developed method was evaluated taking into account the guidelines of the World Anti-Doping Agency (WADA), including the minimum required performance limit (MRPL) for the class of cannabimimetics [48]. The efficacy of the proposed

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strategy was tested analyzing samples obtained incubating the synthetic cannabinoids with human

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liver microsomes and comparing the results with the data reported in the literature [34, 40-41, 49-

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50].

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2.1 Chemicals and Reagents

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2. Experimental

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The aminoalkylindole analogues: JWH007 (1-pentyl-2-methyl-3-(1-naphthoyl)indole), JWH015 (1propyl-2-methyl-3-(1 naphthoyl)indole), JWH018 (1-pentyl-3-(1- naphthoyl)indole), JWH073 (1butyl-3-(1-naphthoyl)indole), JWH098 (1-pentyl-2-methyl-3-(4-methoxy-1- naphthoyl)indole), JWH122

(1-pentyl-3-(4-methyl-1-naphthoyl)indole),

JWH182

(1-pentyl-3-(4-ethyl-1-

naphthoyl)indole), JWH200 (1-[2-(4-morpholinyl)ethyl]-3-(1-naphthoyl)indole), JWH210 (1pentyl-3-(4-ethylnaphthoyl)indole), JWH249 (1-pentyl-3-(2-bromophenylacetyl)indole), JWH250 (1-pentyl-3-(2-methoxyphenylacetyl)indole), JWH251 (1-pentyl-3-(2-methylphenylacetyl)indole), JWH302

(1-pentyl-3-(3-methoxyphenylacetyl)indole),

naphthoyl)indole), WIN 55,212-2

JWH424

(1-pentyl-3-(8-bromo-1-

(2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo-1,4-

benzooxazin6-yl)-1-naphthalenylmethanone), JWH018 4-hydroxy indole metabolite, JWH018 N(5-hydroxypentyl) metabolite, JWH018 N-pentanoic acid metabolite, JWH073 4-hydroxy indole metabolite and JWH073 N-butanoic acid metabolite were purchased from Cayman Chemicals (Ann

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Arbor, MI, USA). The urine samples containing the metabolites of JWH018 and JWH073 were part of an educational study conducted by the World Association of Anti-Doping Scientists (WAADS). The chromatographic columns evaluated were: Gemini® C6-phenyl, Kinetex® C18, Synergi® C18 and kinetex® PFP from Phenomenex (Milano, Italy) and Discovery® C18 and Ascentis® C18 from Supelco (Sigma-Aldrich, Milano, Italy).

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All chemicals (ammonium formate, formic acid, acetonitrile, methanol, dimethylsufoxide,

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sodium phosphate, sodium hydrogen phosphate, tert-butylmethyl ether) were of analytical or HPLC grade and provided by Carlo Erba (Milano, Italy) and Sigma-Aldrich (Milano, Italy). The ultrapure

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water used was of Milli-Q-grade (Millipore Italia, Vimodrone, Milano, Italy).

The reagents (sodium phosphate and tris-HCl buffers and the NADPH regenerating system

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consisting of magnesium chloride hexahydrate, NADP+, glucose-6-phosphate and glucose-6phosphate dehydrogenase) and the enzymatic proteins (human liver microsomes pooled from 50

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(Milano, Italy).

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individual donors, 20 mg mL-1) for the in vitro studies were purchased from BD Biosciences

2.2 In vitro procedure

Stock solutions (0.5, 1, 5 and 10 mM) of each substrate (JWH007, JWH015, JWH018, JWH073, JWH098, JWH122, JWH182, JWH200, JWH210, JWH249, JWH250, JWH251, JWH302, JWH4242, WIN 55,212-2) were prepared separately in methanol. Reaction mixtures were prepared in 100 mM phosphate buffer, pH 7.4, and a NADPH regenerating system consisting of 3.3 mM magnesium chloride, 1.3 mM NADP+, 3.3 mM glucose-6-phosphate and 0.4 U mL-1 glucose-6phosphate dehydrogenase at substrate concentrations of 10 µM (close to the KM). One sample (negative control) containing all reaction components but not the enzymatic protein was also added to the batch to monitor the potential non-enzymatic reactions within the incubation period. The reaction was initiated by adding 6.5 µL of the enzymatic protein (human liver microsomes pooled from 50 individual donors, final concentration 1 mg mL-1) and the phase-I reaction was carried out

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in an incubation volume of 250 µL for 4 h at 37 °C. The overall reaction was terminated by the addition of 250 µL of ice-cold acetonitrile. The reaction mixtures were then centrifuged (12000 rpm for 10 min) before sample purification. All incubations were performed in triplicate.

2.3 Sample preparation

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2.3.1 In vitro samples

To the samples obtained by the in vitro incubation, up to 1 mL of phosphate buffer (1 M, pH 7.4)

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and 50 L of the internal standard (ISTD: deuterated JWH015 final concentration 5 ng mL-1) were

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added and a liquid/liquid extraction was carried out with 5 mL tert-butylmethyl ether for 5 minutes on a mechanical shaker. After centrifugation the organic layer was evaporated to dryness under N2

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stream at a temperature of 75 °C. The residue was reconstituted in 50 µL of mobile phase and an

2.3.2 Urine samples

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aliquot of 10 µL was injected into the liquid chromatography-mass spectrometry system.

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To 2 mL of urine, up to 1 mL phosphate buffer (pH 7.4), 50 L of -glucuronidase from E. coli

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and 50 L of internal standard (ISTD: deuterated JWH015 final concentration 5 ng mL-1) were added and the sample was incubated for 60 minutes at 50 ºC. After hydrolysis a liquid/liquid extraction was carried out with 7 mL of tert-butylmethyl ether for 5 minutes on a mechanical shaker; after centrifugation the organic phase layer was transferred and evaporated to dryness under N2 stream at a temperature of 75 °C. The dried extract was reconstituted in 50 µL of mobile phase and 10 µL were injected into the liquid chromatography-mass spectrometry system.

2.3.3 Blood samples To 1 mL of plasma/serum, up to 1 mL of acetonitrile containing 0.1% of formic acid was added and the mixture was centrifuged at 12000 rpm for 10 min. To the supernatant 1 mL of phosphate buffer (1 M, pH 7.4) and 50 L of the internal standard (ISTD: deuterated JWH015 final concentration 5 ng mL-1) were added and a liquid/liquid extraction was carried out with 5 mL tert-

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butylmethyl ether for 5 minutes on a mechanical shaker. After centrifugation the organic layer was evaporated to dryness under N2 stream at a temperature of 75 °C. The residue was reconstituted in 50 µL of mobile phase and an aliquot of 10 µL was injected into the liquid chromatography-mass spectrometry system. 2.3.3 Saliva samples

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To 1 mL of saliva, up to 1 mL phosphate buffer (pH 7.4) and 50 L of internal standard (ISTD:

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deuterated JWH015 final concentration 5 ng mL-1) were added and a liquid/liquid extraction was carried out with 5 mL tert-butylmethyl ether for 5 minutes on a mechanical shaker. After

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centrifugation the organic layer was evaporated to dryness under N2 stream at a temperature of 75 °C. The residue was reconstituted in 50 µL of mobile phase and an aliquot of 10 µL was injected

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into the liquid chromatography-mass spectrometry system.

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2 . 4 I n s t r u m e nt a l C o n d it io n s 2.4.1 Liquid chromatography conditions

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All LC experiments were performed using an Agilent 1200 Rapid Resolution Series HPLC pump with binary gradient system and automatic injector (Agilent Technologies S.p.A, Cernusco sul Naviglio, Milano, Italy), evaluating the chromatographic performance of the previously mentioned LC columns . The solvents used were: water containing 0.1% formic acid and 5 mM of ammonium formate (eluent A) and acetonitrile containing 0.1% formic acid (eluent B). The gradient program started at 25% B and increased to 90% B in 6 min, and then after 2 min to 100% B in 2 min. The column was flushed for 5 min at 100% B and finally re-equilibrated at 25% B for 3 min. The flow rate was set at 250 L min-1 and the column temperature at 30 °C. 2.4.2 Triple quadrupole system conditions All experiments were performed using an Applied Biosystems (Applera Italia, Monza, Italy) API4000 instrument with positive electrospray ionization using a curtain gas pressure of 25 psi, a ion source temperature of 500 °C, an ion source gas 1 pressure of 35 psi, an ion source gas 2

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pressure of 40 psi, a declustering voltage of 80 V, an entrance potential of 10 V and a needle voltage of 5000 V. The experiments were performed using precursor ion scan (PIS) and selected reaction monitoring (SRM) as acquisition modes (the mass spectral fragments and ion transitions selected are reported in the Table 1 and Figure 2), employing collision-induced dissociation (CID) using nitrogen as collision gas at 5.8 mPa, obtained from a dedicated nitrogen generator system

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Parker-Balston model 75-A74, gas purity 99.5% (CPS analitica Milano, Italy). The collision energies (CE) were optimized by infusion of the standard solutions of the cannabimimetics

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available in our laboratory at a concentration of 10 g mL-1 (see the Table 1). For this purpose, 1

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mL syringe operated by a syringe pump at a flow-rate of 10 µL min-1 was utilized. All aspects of instrument control, method setup parameters, sample injection and sequence operation were

2.5 Validation Parameters

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controlled by the Applied Biosystems Analyst software version 1.5.1.

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Experiments were performed using at least 20 saliva, blood and urine blank samples, to

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determine all parameters (lower limits of detection, specificity, ion suppression/enhancement, retention time repeatability, recovery, robustness and carry over) required for the validation of a qualitative analytical procedure.

For the lower limits of detection (LLOD) determination, 20 saliva, blood and urine blank samples spiked with the standards available in our laboratory at a concentration of 1 ng mL-1 were used. Serial dilutions were made and the LLOD was reported as the lowest concentration at which a compound could be identified in all twenty urines tested with a signal-to-noise (S/N) ratio greater than 3. The specificity was studied by analyzing the 20 samples of saliva, urine and blood to demonstrate that no interferences were detected at the retention times of the analytes studied. The effect of the saliva, urine and blood matrix on the ion suppression and ion enhancement was assessed analyzing 20 different blank samples and water only with continuous co-infusion of the

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cannabimimetics available in our laboratory (10 μg mL-1 at a flow rate of 10 μL min-1) via a Tconnector. The retention time repeatability was evaluated using the 20 stock positive saliva, blood and urine samples spiked with the synthetic cannabinoids at a concentration 5-times higher than the LLOD

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value. The recovery from the biological fluids considered was determined using positive saliva, blood

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and urine samples spiked with the synthetic cannabinoids at a concentration of 5 ng mL-1. Twenty

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blank saliva, blood and urine samples were fortified with the 15 cannabimimetics under investigation before sample preparation, while another set of twenty blank specimens were

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extracted according to the described protocol followed by the same addition of all compounds studied into the organic layer before evaporation. To both sets of samples, the internal standard

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(ISTD) was added into the organic layer before evaporation. Recovery was then calculated by

liquid/liquid extraction.

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comparison of mean peak area ratios of analytes and ISTD of samples fortified prior to and after

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Carryover was evaluated by analyzing negative samples after injection of a negative sample fortified with the compounds under investigation at a concentration 20-times higher than the LLODs.

The robustness of the method was demonstrated by analyzing the twenty positive samples described above once a week for seven weeks, randomly changing the instrument and the operator involved in the instrumental analysis and in the preparation of the urine samples.

3. Results and Discussion

3.1 Mass spectrometric parameters optimization Page 11 of 36

Instrumental parameters in MS/MS were optimized by infusing the reference standards of synthetic cannabinoids available in our laboratory dissolved in the mobile phases at a concentration of 10 µg mL-1. Different collision energies (20, 30, 40, 45, 50 and 60 eV) were evaluated in order to obtain information about the fragmentation patterns of the compounds considered in this study and

molecular structure that is common to the aminoalkylindole analogues.

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to select and optimize the mass spectral fragments formed from the characteristic portions of the

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In Figures 2A-C the product ion spectra (using a collision energy of 45 eV) of the 15 synthetic cannabinoids (11 naphthoylindoles and 4 phenylacetylindoles) available in our laboratory are

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reported. As can be noticed the fragment ion corresponding to the indole portion (m/z 144) is found in all product ion spectra of the indole-derived cannabinoids (phenylacetylindoles, naphthoylindoles

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and benzoylindoles) without any substitution at the indole moiety, whereas the fragment ions

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corresponding to the naphthoyl (m/z 127) and carbonylnaphthalenyl (m/z 155) portions are present in the product ion spectra of the naphthoylindole derivatives without any substitution at the

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naphthyl moiety.

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Based on the above information, three different LC-MS/MS methods (precursor ion scan of m/z 127 and m/z 155 for the naphthoylindoles without any substitution at the naphthyl moiety; of m/z 144 for the different class of aminoalkylindole analogues without any substitution at the indole moiety) were developed.

The suitability of the proposed precursor ion scan methods in detecting compounds belonging to the aminoalkylindole group was first evaluated analyzing a standard mixture of 15 cannabimimetics (11 naphthoylindoles and 4 phenylacetylindoles) at a concentration of 1 ng mL-1. In Figure 3, the results obtained using the precursor ion scan of m/z 155, 127 and 144 were reported. As expected the seven naphthoylindoles (JWH007, JWH015, JWH018, JWH073, JWH182, JWH200 and WIN 55,212-2) without any substitution at the naphthyl moiety were detected selecting the product ions at m/z 127 and 155; whereas the indole-derived cannabinoids without any substituent at the indole moiety (JWH018, JWH073, JWH122, JWH210, JWH249, JWH250, JWH251, JWH302 and

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JWH424) were detected selecting the fragment ion at m/z 144. On the contrary the JWH098 was no detected using the precursor ion methods developed in this study due to the presence of substitutions at both the indole and naphthyl moieties confirming our hypothesis. The precursor ion scan experiments were then applied to blank saliva, serum and plasma samples spiked with the 15 cannabinoids considered at a concentration of 5 ng mL-1 and pre-treated using

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the procedures reported in the experimental part in order to evaluate the matrices effects. The results

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obtained confirm the previous data showing that 14 compounds were clearly detected utilizing the precursor ion scan methods developed in this study (7 using the precursor ion scan at m/z 155 and

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m/z 127 specific of the naphthoylindoles without any substitution at the naphthyl moiety and 9 using the precursor ion scan at m/z 144 specific of the aminoalkylindoles without any substituent at

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times of the aminoalkylindoles (see Figures 4A-C).

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the indole moiety), furthermore no matrices interferences were detected in the range of retention

Concerning the urinary matrix, urine samples containing the metabolites (mono-hydroxylated at

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the indole or N-alkyl side chain, and carboxylated at the N-alkyl side chain) of JWH018 and

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JWH073 were analyzed using the precursor ion scan methods developed in this study. As it can be seen in Figures 5A-C all the metabolites were detected using the precursor ion scan at m/z 155 being all modified at the indole moiety or N-alkyl side chain. Furthermore the metabolites carboxylated or hydroxylated only at the N-alkyl side chain can be, also, detected using the precursor ion scan at m/z 144 but with reduced sensitivity, being the fragments at m/z 155 and 127 the most abundant ions in the spectra of naphthoylindoles at the selected collision energy.

3.2 Chromatographic separation optimization In parallel to the development of the mass spectrometric acquisition methods, the chromatographic separation was optimized in order to distinguish the unknown compounds from other peaks from the background and to obtain a good resolution between the different cannabimimetics with special emphasis to the compounds with same molecular weight and

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fragmentation pattern (i.e. JWH073 and JWH015), excluding in this way incorrect mass assignment. For this purpose stationary phases with different selectivity (phenyl-hexyl, pentafluorophenyl and octadecyl), column sizes (length: 100 and 150 mm; particle size: 4, 5 and 2.6 µm) and solvent gradients were evaluated. The best results in terms of selectivity, peak shape, and overall time of analysis, were obtained using the octadecyl reverse-phase with fused-core particles

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(Figure 6).

3.3 Validation Parameters

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The newly developed methods were validated in term of repeatability of relative retention time, specificity, carry over, ion suppression/enhancement, sensitivity and recovery.

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Good sensitivity was obtained for all the compounds tested with lower limits of detection ranging from 0.1 to 0.5 ng mL-1 (see Table 2) and thus in conformity with the Minimum

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Performance Required Limits (MRPL: 1 ng mL-1) set by the WADA [48].

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Good repeatability of the relative retention times (CV% lower than 0.1) was registered in all the

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matrices tested. The analyses performed on the 20 negative samples of each matrix confirmed that the methods did not show any significant interference at the retention times of the analytes of interest, and therefore it has adequate selectivity (see Figures 4A-C and 5). Carry over was tested by analyzing blank saliva, urine and blood samples after blank samples spiked with the cannabimimetics available in our laboratory at a concentration 20 times the LLOD value. The procedure was carried out twice and showed that the positive reference samples did not affect the blank samples.

The test for ion suppression/enhancement effects by post column split-infusion of analytes yielded no significant matrix effect (lower than 30%) at the retention times of the cannabimimetics considered while 20 different saliva, urine and blood samples were injected (see Table 1). Finally, the recovery, in all the matrices evaluated, was higher than 65% for all the compounds tested (see Table 1), with a satisfactory repeatability (CV % lower than 10).

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3.4 Analysis of samples obtained following incubation of synthetic cannabinoids with human liver microsomes The suitability of the developed precursor ion scan methods in detecting unknown compounds was evaluated analyzing samples obtained after incubation of JWH015 and JWH210 with human liver microsomes. In Figures 7-8 and in Tables 3-4 were reported the results obtained analyzing the

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in vitro phase I metabolism of the JWH015 (Table 3 and Figure 7) and JWH210 (Table 4 and

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Figure 8) using the precursor ion methods developed in this study. For JWH015, 18 metabolites were formed: 10 metabolites were detected using the precursor ion scan of m/z 155 and 127, and

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eight metabolite utilizing the precursor ion scan of m/z 144 (see Table 3 and Figure 7). On the contrary for the JWH210, all the 18 metabolites formed were detected using the precursor ion scan

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of m/z 144 (Table 4 and Figure 8). In this case in fact, due to the presence of an ethyl group at the

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naphthyl moiety, no metabolites were found using precursor ion scan of m/z 155 and 127. For both compounds (JWH015 and JWH210) the biotransformation pathways detected consist in mono-

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hydroxylation, di-hydroxylation, tri-hydroxylation, carboxylation, N-dealkylation, dehydratation

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and combination of them, confirming the data reported in literature [34, 40-41, 49-50].

4. Conclusions

The application of very specific and sensitive “targeted” analytical approaches is limited to the analyses of preselected compounds of known molecular structure; as such, this approach cannot allow the detection and identification of any new, previously unknown, chemical structure. Considering that one of the most used doping strategy is based on the use of designer substances, synthesized specifically to elude legislative controls, and, as such, either still totally unknown, or at least not fully characterized yet, alternative approaches have to be proposed in order to drastically

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reduce the time necessary to identify the newly synthesized variants of those designer drugs. Supporting information can be preliminarily gathered by a combination of computational approaches and in vitro toxicology methods [51]. In this paper we have investigated the capabilities of different LC-MS/MS acquisition modes for the detection of known and unknown synthetic cannabinoids in different biological fluids. The

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complementary use of different precursor ion scan methods seems to be the best option for the

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detection of the largest amount of analytes (including also unknown and/or not commercially available) belonging to the group of the aminoalkylindoles. The compounds considered in this study

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and their metabolites can be easily detected selecting the fragment ions corresponding to the indole (m/z 144), carbonylnaphthalenyl (m/z 155) and naphthalenyl (m/z 127) moieties; however the

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robustness of this approach can be increased by introducing other common residues (i.e. those at

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m/z 91 and 121, corresponding to the phenyl and metoxyphenyl moieties of the phenylacetylindoles, or those corresponding to the N-alkyl chain) and/or by combining the precursor ion scan acquisition

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mode with product ion scan acquisition mode using the information-dependent acquisition (ID) that

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allows to obtained both molecular weight information and structural details. . The overall performance of the method suggests that it could be successfully applied not only for routine use in anti-doping laboratories but also for various applications in the field of clinical and forensic toxicology.

The added value with respect to SRM based methods lies in its capability of being used for the early detection of any new drugs/metabolites which shares the same portion(s) of the molecular structure selected for the analysis in precursor ion scanning. Once a suspicious signal is observed, additional studies utilizing various mass spectrometric analyses including, among others, selective derivatization, different ionization techniques, different acquisition modes, additional spectrometric investigations (i.e. by 1H-NMR), as well as synthesis and characterization of the suspected compound, are necessary to prove the structure of the potentially prohibited substance.

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5. Acknowledgements

This work has been supported by a Research Grant of the Italian Department of Health (“Ministero della Salute, Commissione per la vigilanza sul doping e sulla tutela sanitaria delle

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attività sportive”).

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6.

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FIGURE captions

Figure 1. General molecular structures of the classes of synthetic cannabinoids considered in the present study. A: naphthoylindoles; B: benzoylindoles; C: phenylacetylindoles. Common MS fragments

Page 24 of 36

are indicated by the circles (m/z 144 for all the three classes, and m/z 155 and m/z 127, for the “A” class only). Figure 2. Product ion spectra of 15 synthetic cannabinoids (A: JWH007, JWH015, JWH018, JWH073, and JWH098; B: JWH122, JWH182, JWH200, JWH210, and JWH249; C: JWH250, JWH251,

cr

study and reported in the experimental part and a collision energy of 45 eV.

ip t

JWH302, JWH424, and WIN 55,212-2) utilizing the mass spectrometric conditions set up in this

us

Figure 3.

Chromatograms of a standard mixture of the 15 synthetic cannabinoids under investigation at a

an

concentration of 5 ng mL-1. Acquisition mode precursor ion scan of m/z 155 (A), 127 (B) and m/z 144 (C). Peaks identity: 1. JWH200; 2. WIN 55,212-2; 3. JWH250; 4. JWH015, 5. JWH302, 6.

Ac ce pt e

Figure 4.

d

JWH122, 14. JWH210, 15.JWH424.

M

JWH073 7. JWH251; 8. JWH182; 9. JWH249; 10. JWH018; 11. JWH007; 12. JWH098, 13.

Chromatograms of negative saliva (A), serum (B) and plasma (C) samples (blue line) overlaid to the chromatograms of the same blank samples spiked with 15 synthetic cannabinoids at a concentration of 5 ng mL-1 (black line) and with a standard mixture of the same 15 synthetic cannabinoids at a concentration of 5 ng mL-1 (grey line). Acquisition mode precursor ion scan of m/z 155, 127 and 144. Peaks identity: 1. JWH200; 2. WIN 55,212-2; 3. JWH250; 4. JWH015, 5. JWH302, 6. JWH073 7. JWH251; 8. JWH182; 9. JWH249; 10. JWH018; 11. JWH007; 12. JWH098, 13. JWH122, 14. JWH210, 15. JWH424.

Figure 5. Chromatograms of a negative urine sample spiked with the hydroxylated and carboxylated metabolites of JWH018 and JWH073. Acquisition mode: precursor ion scan of m/z 155, 127 and Page 25 of 36

144. Peaks identity: M1. JWH018 N-(5-hydroxypentyl) metabolite, M2. JWH018 N-pentanoic acid metabolite, M3. JWH073 N-butanoic acid metabolite, M4. JWH018 4-hydroxy indole metabolite, M5. JWH073 4-hydroxy indole metabolite.

Figure 6

ip t

Extracted chromatograms of a standard mixture of 15 synthetic cannabinoids at a concentration of 5

cr

ng mL-1 using different fixed phases and column sizes. Acquisition mode SRM utilizing the ion

us

transitions reported in Table 1.

Figure 7.

an

Chromatograms of samples obtained after incubation of JWH015 with human liver microsomes.

M

Acquisition mode precursor ion scan of m/z 155, 127 and 144. Peaks identity: M1: dihydrodiol formation and N-dealkylation; M2: dihydrodiol formation and N-dealkylation; M3: tri-

d

hydroxylation; M4: tri-hydroxylation, M5: dihydrodiol formation and N-dealkylation; M6: triand N-dealkylation, M8: di-hydroxylation; M9:

Ac ce pt e

hydroxylation; M7: monohydroxylation

dihydrodiol formation; M10: monohydroxylation

and N-dealkylation; M11: Di-hydroxylation;

M12: dihydrodiol formation; M13: monohydroxylation; M14: monohydroxylation; M15: Ndealkylation; M16: dehydratation and monohydroxylation; M17: monohydroxylation; M18: dehydratation.

Figure 8.

Chromatograms of samples obtained after incubation of JWH210 with human liver microsomes. Acquisition mode precursor ion scan of m/z 144. Peaks identity: M1: dihydrodiol formation and Ndealkylation; M2: tri-hydroxylation; M3: tri-hydroxylation; M4: tri-hydroxylation, M5: trihydroxylation;

M6:

carboxylation

and

monohydroxylation;

M7:

N-dealkylation

anmonohydroxylation, M8: carboxylation and monohydroxylation; M9: dihydroxylation; M10:

Page 26 of 36

dihydroxylation; M11: dehydration and dihydroxylation; M12: dehydration and dihydroxylation; 13: carboxylation; M14: dihydrodiol formation; M15: dihydroxylation; M16: monohydroxylation;

ip t

M17: dehydratation and monohydroxylation; M18: dehydratation.

cr

Table 1 Molecular weights, structural information, retention times, and mass spectrometric parameters of all synthetic cannabinoids considered in this study (see Figure 1 for the general molecular

us

structures).

Precursor ion scan

R3

Rt (min)

Structural markers (m/z)

CH3 CH3

C5H11 C3H7

11.7 10.6

127, 155 127, 155

H

C5H11

11.4

127, 144, 155

R1

R2

11 JWH007 4 JWH015

355 327

Naphthoylindoles Naphthoylindoles

H H

10

341

Naphthoylindoles

H

JWH018 JWH073

327

Naphthoylindoles

H

12 13 8

JWH098 JWH122 JWH182

385 355 383

Naphthoylindoles Naphthoylindoles Naphthoylindoles

OCH3 CH3 C3H7

1

JWH200

384

Naphthoylindoles

14 15

JWH210 JWH424

2 9 3 7 5

CE (eV)

Ion Transitions (m/z)

CE (eV)

50, 45 50, 45 50, 50, 45 50, 45, 40

356/127; 356/155 328/127; 328/155

50, 45 50, 45

342/127; 342/155

50, 45

328127; 328/144 328/155

50, 45, 40

H

C4H9

10.9

127, 144, 155

CH3 H H

C5H11 C5H11 C5H11 2morpholin4 -yl-ethyl C5H11 C5H11 2morpholin 4‐yl‐methyl C5H11 C5H11 C5H11 C5H11

11.9 12.0 11.2

185

35

385/185

35

144 144

45 50

370/144; 370/169 384/144

45, 35 50

127, 144, 155

50, 50, 35

385/127; 385/155

50, 40

144, 155 144

45, 45 50

370/144; 370/155 422/144

45, 45 50

8.3

127, 155

50, 45

427/127;427 /155

50, 45

11.3 10.5 11.1 10.7

144 144

50 50

385/144, 336/121; 336/144

50, 45 50, 45

144

45

320/105; 320/144

50, 45

144

50

336/121; 336/144

50, 45

d

6

M

Class

an

Compoun d

MW (Da)

ID

Selected Reaction Monitoring

5.6

369 421

Naphthoylindoles Naphthoylindoles

C2H5 Br

H H

12.5 13.5

WIN 55,212-2

426

Naphthoylindoles

H

H

JWH249 JWH250 JWH251 JWH302

384 335 319 335

Phenylacetylindoles Phenylacetylindoles Phenylacetylindoles Phenylacetylindoles

Br OCH3 CH3 OCH3

H H H H

Ac ce pt e

H

H

Page 27 of 36

Table 2 Lower limits of detection, matrix effect and recovery of the 14 synthetic cannabinoids and the 5 metabolites under investigation detectable with the precursor ion methods developed in this study Lower Limit of Detection -1 (ng mL )

metabolite

---

---

---

---

---

---

Ac ce pt e

metabolite M3 JWH073 N-butanoic acid metabolite M4 JWH018 4-hydroxy indole metabolite M5 JWH073 4-hydroxy indole metabolite

---

Urin e ----------------------------30

Saliv a 84 85 75 82 85 75 75 75 72 85 80 75 65 87 ---

---

Plasma/Se Urine rum 79 --80 --77 --75 --83 --67 --83 --77 --68 --76 --80 --77 --66 --88 ----75

cr

ip t

Plasma/S erum 30 20 20 20 20 30 20 25 30 30 30 30 30 20 ---

0.4

---

---

30

---

---

70

0.4

---

---

30

---

---

70

0.3

---

---

35

---

---

75

0.3

---

---

35

---

---

75

d

M2 JWH018 N-pentanoic acid

Recovery (%)

us

Plasma/S Saliv Urine erum a 0.2 --30 0.2 --20 0.2 --30 0.2 --20 0.5 --20 0.5 --30 0.5 --30 0.4 --20 0.5 --20 0.5 --30 0.1 --20 0.4 --30 0.5 --30 0.1 --20 --0.3 ---

an

Saliv a 11 JWH007 0.2 4 JWH015 0.3 10 JWH018 0.3 6 JWH073 0.4 13 JWH122 0.5 8 JWH182 0.5 1 JWH200 0.5 14 JWH210 0.5 9 JWH249 0.5 3 JWH250 0.1 7 0.4 JWH251 5 JWH302 0.5 15 JWH424 0.5 2 WIN 55,212-2 0.2 M1 JWH018 N-(5-OH-pentyl) ---

Matrix Effect (%)

M

I. Compound D.

Page 28 of 36

Table 3 Metabolites of JWH015 identified after incubation with human liver microsomes MW (Da)

----

----

X

320

----

----

X

378 378

-------

-------

X X

320

----

----

X

378

----

----

302

X

X

360 362

X ----

X ----

302

X

360 362 344 344 286

X ---X X X

X ---X X X

---X ----------

X

X

----

X X X

X X X

----------

342

X

------X

cr X

us

M

344 326 328

ip t

378

----

d

Tri-hydroxylation (M1) Dihydrodiol formation and Ndealkylation (M2) Tri-hydroxylation (M3) Tri-hydroxylation (M4) Dihydrodiol formation and Ndealkylation (M5) Tri-hydroxylation (M6) Monohydroxylation and Ndealkylation (M7) Di-hydroxylation (M8) Dihydrodiol formation (M9) Monohydroxylation and Ndealkylation (M10) Di-hydroxylation (M11) Dihydrodiol formation (M12) Monohydroxylation (M13) Monohydroxylation (M14) N-dealkylation (M15) Dehydratation and monohydroxylation (M16) Monohydroxylation (M17) Dehydratation (M18) JWH015

Triple quadrupole (precursor ion scan) m/z 155 m/z 127 m/z 144

an

Compound

Ac ce pt e

Table 4 Metabolites of JWH210 identified after incubation with human liver microsomes Compound

Dihydrodiol formation and Ndealkylation (M1) Tri-hydroxylation (M2) Tri-hydroxylation (M3) Tri-hydroxylation (M4) Tri-hydroxylation (M5) Carboxylation and monohydroxylation (M6) N-dealkylation and monohydroxylation (M7) Carboxylation and monohydroxylation (M8) Dihydroxylation (M9) Dihydroxylation (M10) Dehydration and dihydroxylation (M11) Dehydration and dihydroxylation (M12) Carboxylation (M13) Dihydrodiol formation (M14) Dihydroxylation (M15) Monohydroxylation (M16)

MW (Da)

Triple quadrupole (precursor ion scan) m/z 155 m/z 127 m/z 144

332

----

----

X

418 418 418 418

-------------

-------------

X X X X

416

----

----

X

316

----

----

X

416

----

----

X

402 402

-------

-------

X X

400

----

----

X

400

----

----

X

400 404 402 386

-------------

-------------

X X X X

Page 29 of 36

384

----

----

X

368 370

-------

-------

X X

an

us

cr

ip t

Dehydratation and monohydroxylation (M17) Dehydration (M18) JWH210

Ac ce pt e

d

M

Figure 1

Figure 2ab Page 30 of 36

ip t cr us an M d Ac ce pt e

Figure 2c

Page 31 of 36

ip t cr us an Ac ce pt e

d

M

Figure 3

Page 32 of 36

ip t cr us an M d Ac ce pt e

Figure 4

Page 33 of 36

ip t cr us an M d Ac ce pt e

Figure 5

Figure 6

Page 34 of 36

ip t cr us an M d Ac ce pt e

Figure 7

Page 35 of 36

ip t cr us an M Ac ce pt e

d

Figure 8

Page 36 of 36