Street-Like Synthesis of Krokodil Results in the Formation of an

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THE STREET-LIKE SYNTHESIS OF KROKODIL RESULTS IN THE FORMATION OF AN ENLARGED CLUSTER OF KNOWN AND NEW MORPHINANS WITH THERAPEUTIC POTENTIAL José Soares, Emanuele Alves, André Silva, Natália Figueiredo, João F. Neves, Sara Cravo, Maria Rangel, Annibal Netto, Félix Carvalho, Ricardo Jorge Dinis-Oliveira, and Carlos Afonso Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.7b00126 • Publication Date (Web): 14 Jul 2017 Downloaded from http://pubs.acs.org on July 29, 2017

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Chemical Research in Toxicology

The Street-Like Synthesis of Krokodil Results in the Formation of an Enlarged Cluster of Known And New Morphinans with Therapeutic Potential

Running Head: “krokodil” synthesis and analysis

José Xavier Soares1*†, Emanuele Amorim Alves2,3,4,5*†, André M. N. Silva6, Natália Guimarães de Figueiredo7, João F. Neves8, Sara Manuela Cravo8, Maria Rangel9, Annibal Duarte Pereira Netto10, Félix Carvalho1, Ricardo Jorge Dinis-Oliveira*1,2,4Ψ, and Carlos Manuel Afonso*8,11Ψ †

The first two authors contributed equally to this work.

Ψ

Co-principal investigators of this study.

1

LAQV, REQUIMTE, Department of Chemical Sciences, Laboratory of Applied Chemistry, Faculty of Pharmacy, University of Porto, José Viterbo Ferreira Street nº 228, 4050-313 Porto, Portugal 2 UCIBIO, REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Porto, José Viterbo Ferreira Street nº 228 4050-313 Porto, Portugal 3 Department of Public Health and Forensic Sciences, and Medical Education, Faculty of Medicine, University of Porto, Porto, Portugal, Prof. Hernâni Monteiro Alameda, 4200319 Porto, Portugal 4 EPSJV – Polytechnic School of Health Joaquim Venâncio, Oswaldo Cruz Foundation, Brasil 4.365 Avenue, Manguinhos, 21.040-900, Rio de Janeiro, Brazil 5 IINFACTS - Institute of Research and Advanced Training in Health Sciences and Technologies, Department of Sciences, University Institute of Health Sciences (IUCS), CESPU, CRL, Gandra, Portugal, Central de Gandra Street, 1317, 4585-116 Gandra, PRD 6 LAQV, REQUIMTE, Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, Campo Alegre Street, 4169-007 Porto, Portugal 7 Laboratory of Tobacco and Derivatives, Analytical Chemistry Division, National Institute of Technology, Venezuela Avenue, 82, Praça Mauá, 20081-312 Rio de Janeiro, Brazil 8 Department of Chemical Sciences, Laboratory of Organic and Pharmaceutical Chemistry, Faculty of Pharmacy, University of Porto, José Viterbo Ferreira Stree nº 228, 4050-313 Porto, Portugal 9 LAQV, REQUIMTE, Institute of Science Abel Salazar, University of Porto, José Viterbo Ferreira Street nº 228, 4050-313 Porto, Portugal 10 Department of Analytical Chemistry, Chemistry Institute, Fluminense Federal University, Niterói, Brazil, Outeiro de São João Batista, Valonguinho Campus, Centro, Niterói, 24020-150, Rio de Janeiro, Brasil 1

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11

Interdisciplinary Center of Marine and Environmental Investigation (CIIMAR/CIMAR), General Norton de Matos Avenue 4450-208 Matosinhos, Portugal

*Corresponding authors: José Xavier Soares, [email protected] Emanuele Alves, [email protected] Ricardo Jorge Dinis-Oliveira, [email protected] Carlos Afonso, [email protected] Mailing address: Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Porto, Portugal, Rua José Viterbo Ferreira nº 228, 4050-313 Porto, Portugal Fax: 00351 226093390 Phone: +351 220428597

TABLE OF CONTENTS (TOC) GRAPHIC

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ABSTRACT

“Krokodil” is the street name for a homemade injectable drug that has been used as a cheap substitute for heroin. Codeine is the opioid starting material for krokodil synthesis and desomorphine is the opioid claimed to be the main component of krokodil and the main responsible for its addictive and psychoactive characteristics. However, due to its peculiar manufacture, using cheap raw materials, krokodil is composed by a large and complex mixture of different substances. In order to shed some light upon the chemical complexity of krokodil, its profiling was conducted by reverse phase high performance liquid chromatography coupled to photodiode array detector (RP-HPLCDAD) and by liquid chromatography coupled to high resolution tandem mass spectrometry (LC-ESI-IT-Orbitrap-MS). Besides desomorphine, codeine and morphine, profiting from the high resolution mass spectrometry (HRMS) data, an endeavor to study the morphinans content in krokodil was set for the first time. Considering codeine as the only morphinan precursor and the possible chemical transformations that can occur during krokodil synthesis, the morphinan chemical space was designed and 95 compounds were defined. By making use of the morphinan chemical space in krokodil, the exact masses featured by HRMS, and the morphinan mass fragmentations patterns, a targeted identification approach was designed and implemented. The proposed 95 morphinans were searched using the full scan chromatogram of krokodil and findings were validated by mass fragmentation of the correspondent precursor ions (MS2 spectra). Following this effort, a total of 54 morphinans were detected, highlighting the fact that these additional morphinans may contribute to the psychotropic effects of krokodil.

Keywords: krokodil; opioids; morphinans; LC-ESI-IT-Orbitrap-MS; analgesia.

INTRODUCTION

“Krokodil” is a complex psychotropic drug mixture with iodine odor resulting from a homemade synthesis process using easy access starting materials and used as a cheap substitute for heroin.1-9 Krokodil firstly appeared around 2002/3 in Siberia and its use 3



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spread throughout other Russian areas and former Soviet Republic countries and more recently across Europe, namely Germany, Czech Republic, France, Belgium, Sweden, Norway, Poland, Spain, Portugal and also USA.9-11 Desomorphine is the semi-synthetic opioid claimed to be the main component of krokodil and considered to be responsible for its addictive and psychoactive effects. However, due to its nature, the synthesis produces not only desomorphine but also other side-products as previously described in krokodil samples and biological fluids of people who inject krokodil (PWIK).2,12 Several toxic effects have been reported and claimed to be related to impurities, namely jaw osteonecrosis presenting as alveolar process exposure in the oral cavity, infections by HIV and hepatitis A, B, and C, skin and venous damage, including ulcers, abscesses, scaly and rough phlebitis, like a crocodile skin around the injection sites, gangrene and limb amputations.1-9 Recently, we observed that repeated subcutaneous administrations of krokodil causes skin necrosis and toxicity to internal organs in Wistar rats, leading to cardiac and renal dysfunction. The impurities, particularly the high phosphorous concentrations of krokodil batches may explain many of the signs and symptoms present by PWIK.13 Besides pleasure, clinical cases related to PWIK and also animals exposed to krokodil suggests high pain tolerance to the corrosive and necrotizing effects.13,14 Therefore, we hypothesized that, besides desomorphine, krokodil may uncover new morphinan derivatives capable of relieving moderate and severe pain and contribute to the addictive effects. Aiming to identify additional morphinans (as a representative skeleton of a large class of opioids), a suitable analytical methodology, capable of identifying complex mixtures is required. A great variety of separation techniques coupled to mass spectrometry (MS) have been used for the identification of different compounds in clinical and forensic toxicology due to its capacity to generate information both on the molecular mass and the structure of molecules.15-22 With the advent of high resolution mass spectrometry (HRMS), exact masses could be determined and new perspectives were opened for liquid chromatography (LC)-HRMS allowing the study of complex mixtures. Since its first description and its commercial introduction in 2005, the Orbitrap mass analyzer has demonstrated great sensitivity and high resolving power.23-25 This high-resolution mass analyzer is particularly attractive due to its great sensitivity and scan rate, which is ideal for online coupling with liquid chromatography separation. Indeed, coupling LC to Orbitrap mass spectrometers 4


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equipped with an electrospray ionization source (ESI) has been providing an analytical instrument of great utility in different areas of analytical and forensic chemistry.15,26 Therefore, the use of LC-HRMS data analysis with the aid of informatics tools, such as Mass Frontier® and Compound Discoverer®, may be considered of great value for the identification of substances in krokodil.27 Therefore, the goals of this work were to contribute a better knowledge of krokodil providing new insights into the chemical profile and detecting the different morphinans present in krokodil. Morphinans were identified by comparison with reference standards (desomorphine, codeine and morphine) and by making use of exact masses and mass fragmentations provided by LC-ESI-IT-Orbitrap-MS.

MATERIAL AND METHODS

Reagents and standards

For krokodil synthesis, gasoline, alkali solutions for cleaning pipes and matchboxes were purchased from local retail stores in Porto, Portugal. Hydrochloric acid 37% was purchased from VWR Prolabo®. Codeine-containing capsules, iodine tinctures, hydrogen peroxide and commercial ethanol 96% were purchased from local pharmacies in Porto, Portugal. For high performance liquid chromatography (HPLC) analysis, Chromasolv acetonitrile and LC-MS grade formic acid were obtained from SigmaAldrich®. Ultrapure (MilliQ) water was used throughout the study.

Krokodil samples

All the krokodil samples were produced by street-like synthesis as previous described.28 The synthesis procedure was repeated ten times, and a representative pool of the obtained products were used for further analysis. 5



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Reversed phase high performance liquid chromatography with photodiode array detection (RP-HPLC-DAD) conditions

For photodiode array detection (DAD) analysis, a Dionex Ultimate 3000 HPLC Basic system (Thermo Scientific), equipped with an auto-sampler and a DAD detector was used. For chromatographic separation, a C18 Hypersil GOLD with 1.9 µm particle size, 50 mm L and 2.1 mm ID (Thermo Scientific) equipped with a C18 BEH VanGuard (5 mm L × 2.1 mmID) (Waters) guard column was used. Column temperature was set to 40ºC. Eluent A was aqueous formic acid (1%) and eluent B was formic acid (1%) in acetonitrile. Samples (10 µL) were injected directly into the column and washed over for 5 minutes with an isocratic flux of 98% eluent A and 2% eluent B at a flow rate of 300 µl/min. Subsequently to the initial 5 min wash, a 30 min linear gradient from 2% to 40% buffer B was applied. The eluent composition was then raised to 100% buffer B over 15 min and thus maintained for 10 additional min in order to allow column cleaning. The column was re-equilibrated in 98% buffer A/2% buffer B before any further analysis. DAD spectra were acquired for the full length of the chromatographic run between 200 and 600 nm. Chromeleon 7.1 SR2 software Thermo Fisher Scientific managed chromatographic data. Prior to use, mobile phase solvents were degassed in an ultrasonic bath for 15 min. The identification of desomorphine, codeine and morphine was established based on the comparison with standard retention time under the same chromatographic conditions and UV/Vis spectrum and by co-elution.

Reversed phase high performance liquid chromatography with electrospray ionization coupled to high resolution tandem mass spectrometry detection conditions

Krokodil samples were analyzed by LC-ESI-IT-Orbitrap-MS. Before analysis, the full krokodil reaction mixture was diluted ten times in the initial eluent composition. An Accela 600 HPLC system (Thermo Scientific, Bremen, Germany), equipped with an auto-sampler, was coupled on-line to a LTQ-Orbitrap XL mass spectrometer (Thermo Scientific, Bremen, Germany) operated with an electrospray ionization (ESI) source. 6


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For chromatographic separation, a C18 Hypersil GOLD with 1.9 µm particle size, 50 mm L and 2.1 mm ID (Thermo Scientific) equipped with a C18 BEH VanGuard (5 mm L × 2.1 mmID) (Waters) guard column was used. Column temperature was set to 40 ºC. Eluent A was aqueous formic acid (1%) and eluent B was formic acid (1%) in acetonitrile. Samples (10 µl) were injected directly into the column and washed over for 5 minutes with an isocratic flux of 98% eluent A and 2% eluent B at a flow rate of 300 µl/min. Subsequently to the initial 5 min wash, a 30 min linear gradient from 2% to 40% buffer B was applied. The eluent composition was then raised to 100% buffer B over 15 min and maintained for 10 additional min in order to allow column cleaning. The column was re-equilibrated in 98% buffer A/2% buffer B before any further analysis. The mass spectrometer was operated in the positive ion mode, with a spray voltage of 3.1 kV and a transfer capillary temperature of 275ºC. Sheath and Auxiliary gas (nitrogen) flows were set to 40 and 10 (arbitrary units), respectively. Tube lens voltage was set to 120 V. MS survey scans were acquired at an Orbitrap resolution of 60000 for an m/z range from 100 to 1000. Tandem MS (MS/MS) data were acquired either in the linear ion trap or in the Orbitrap at a resolution a 7500. A data dependent method with dynamic exclusion was employed for MS2 acquisition: the top 3 most intense ions were selected for collision induced dissociation (CID). CID settings were 50% normalized collision energy, 2 Da isolation window, 30 ms activation time and an activation Q of 0.250. A window of 45 s was used for dynamic exclusion. Automatic gain control was enabled and target values were 1.00e6 for the Orbitrap and 1.00e4 for LTQ MSn analysis. The identification of desomorphine, codeine and morphine was established based on the comparison with standard retention time under the same chromatographic conditions and mass fragmentation spectra (MS2).

Data acquisition and processing

HPLC-DAD data were recorded with Chromeleon 7.1 SR2 software Thermo Fisher Scientific managed chromatographic data (LC-ESI-IT-Orbitrap-MS with Xcalibur software version 2.1). The acquired data set from the injection of four similar samples was processed using Compound Discoverer 2.0.0.303 with the following nodes: select 7



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spectra (default values); filter centroids (S/N threshold = 3; minimum peak intensity = 1000); align retention times (default values); a generate expected compound node for each parent compound (apply dealkylation = false; apply dearylation = false; phase I = none; phase II = none; ions = [M+H]+1, [M+Na]+1, [M+NH4]+1; remaining were set to default values); find expected compounds (mass tolerance = 5 ppm; minimum peak intensity = 100000; minimum number of isotopes = 2; remaining were set to default values); group expected compounds (default values); merge features (retention time tolerance: 0.5 minutes; remaining were set to default values); FISH scoring (default values). Spectra were searched against an internal database of ninety five morphinans attained from the krokodil chemical space (Table S1). Further analysis of fragment spectra was performed with Mass Frontier version 7.0.5.9. Retention times were selected considering the lower ∆Mass (ppm). Morphinan relative abundance (percentage) was calculated dividing the respective peak area by the peak areas sum of all detected morphinans. Manual inspections of chromatograms and mass spectra were conducted with XcaliburTM version 2.1. Chemical structures were drawn with ChemDraw Professional version 16.0.0.82.

RESULTS AND DISCUSSION

Krokodil chemical profiling

Due to the raw materials, homemade character, and the harsh reaction conditions used in its manufacturing, krokodil is a very complex mixture, composed by a great variety of substances with different chemical and physicochemical characteristics.2,28 Initial studies in krokodil were previously performed using normal phase HPLC-DAD and GC-MS methodologies.12,28 Organic extracts of krokodil and GC derivatization reagents were used in those analyses. In order to deeply explore the chemical composition of krokodil, several studies were performed in samples of crude krokodil, starting with a RP-HPLC-DAD analysis. Since no extraction steps or derivatization reactions were used, the loss of substances during these procedures was eliminated, and a more complete profile was possible. The DAD 3D chromatogram is showed in Figure

8


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1A (260-400 nm). The chromatogram reveals the expected complexity of krokodil, with several substances eluting along a wide range of polarities. Desomorphine has been considered the chemical marker for the identity of krokodil.2 However, other morphinans (i.e., 3,6-dideoxy-dihydromorphine and 4,5epoxymorphinan-3-ol) have been found in krokodil samples, which may play an important role in the additive and psychotropic effects.12,28 Consequently, in order to better understand the krokodil morphinan composition a LC-ESI-IT-Orbitrap-MS analysis was made, using the optimized RP-HPLC-DAD conditions. Morphinans are alkaloid derivatives with basic nitrogen making them suitable for ESI analysis in positive mode.29 LC-ESI-IT-Orbitrap-MS is able to couple the power of the chromatographic separation to the power of MS structure elucidation and resolution with accurate masses.30 A total ion current (TIC) chromatogram of crude krokodil in positive mode is showed in Figure 1B. This provided another perspective over the complex krokodil profile, revealing the compounds that can be ESI positive ionizable compounds, such as morphinans. Desomorphine, codeine, and for the first time morphine were identified in the crude krokodil by comparison with retention times, exact masses of the precursor ions and mass fragmentation patterns of standards analyzed under the same chromatographic conditions (Figure 1C; Table 1). Additionally, the identification of desomorphine, codeine and morphine was established by co-injection with the reference standards. As far as we known, morphine is reported for the first time in krokodil. Furthermore, the understanding of the fragmentation behavior of morphinans in the analytical conditions used in this work is important for establishing morphinan fragmentation patterns. Therefore, the mass fragmentation spectra of desomorphine, codeine and morphine were interpreted (Figure 2) using Mass Frontier® and manual interpretation. Mass Frontier® uses a set of general fragmentation rules and a fragmentation library to predict the structure of the different fragments.31 Manual interpretation of spectra was conducted accordingly with mass fragmentation rules and with morphine and codeine fragmentation pathways described in literature.28,31-33 The three morphinans showed a similar fragmentation behavior. All three show the piperidine ring loss, leading to the base peak (m/z 215) in desomorphine and to the intense peaks in codeine (m/z 243) and morphine (m/z 229). Other typical fragmentation behaviors were observed. When a α,β-unsaturated hydroxyl moiety is present, like in 9



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morphine and codeine, base peaks are produced by losing piperidine ring and CO, leading respectively to fragments m/z 201 and m/z 215.32 In addition, when an alcohol hydroxyl group is present in ring C, a loss of water was observed, leading to intense peaks.32 When a phenolic hydroxyl group is present in ring A and alcohol hydroxyl group is not present in ring C, the loss of water was verified leading to a medium intensity peak, like in desomorphine (m/z 197). Moreover, the loss of H2 from different fragments was also a very common feature, like in desomorphine m/z 213.32

Targeted detection of morphinans in krokodil

The identification of other morphinans could represent a pivotal importance to fully understand the psychotropic effects of krokodil but it is even more interesting to explain the apparent profound analgesia to tolerate the injection of a corrosive and caustic formulation (pH

Street-Like Synthesis of Krokodil Results in the Formation of an

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