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Street-Like Synthesis of Krokodil Results in the Formation of an Enlarged Cluster of Known and New Morphinans José Xavier Soares,*,†,‡‡ Emanuele Amorim Alves,*,‡,§,∥,⊥,‡‡ André M. N. Silva,# Natália Guimaraẽ s de Figueiredo,∇ Joaõ F. Neves,○ Sara Manuela Cravo,○ Maria Rangel,◆ Annibal Duarte Pereira Netto,¶ Félix Carvalho,*,‡ Ricardo Jorge Dinis-Oliveira,*,‡,§,⊥,‡‡ and Carlos Manuel Afonso*,○,††,‡‡ †

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 ‡ UCIBIO, REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, José Viterbo Ferreira Street No. 228 4050-313 Porto, Portugal § Department of Public Health and Forensic Sciences, and Medical Education, Faculty of Medicine, University of Porto, Prof. Hernâni Monteiro Alameda, 4200-319 Porto, Portugal ∥ EPSJV−Polytechnic School of Health Joaquim Venâncio, Oswaldo Cruz Foundation, Brazil 4.365 Avenue, Manguinhos, 21.040-900 Rio de Janeiro, Brazil ⊥ IINFACTS-Institute of Research and Advanced Training in Health Sciences and Technologies, Department of Sciences, University Institute of Health Sciences (IUCS), CESPU, CRL, Central de Gandra Street, 1317, 4585-116 Gandra, Portugal # LAQV, REQUIMTE, Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, Campo Alegre Street, 4169-007 Porto, Portugal ∇ Laboratory of Tobacco and Derivatives, Analytical Chemistry Division, National Institute of Technology, Venezuela Avenue, 82, Praça Mauá, 20081-312 Rio de Janeiro, Brazil ○ 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 ◆ LAQV, REQUIMTE, Institute of Science Abel Salazar, University of Porto, José Viterbo Ferreira Street No. 228, 4050-313 Porto, Portugal ¶ Department of Analytical Chemistry, Chemistry Institute, Fluminense Federal University, Outeiro de São João Batista, Valonguinho Campus, Centro, Niterói, 24020-150, Rio de Janeiro, Brazil †† Interdisciplinary Center of Marine and Environmental Investigation (CIIMAR/CIMAR), General Norton de Matos Avenue, 4450-208 Matosinhos, Portugal S Supporting Information *

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 claimed to be the main opioid of krokodil and the main component responsible for its addictive and psychoactive characteristics. However, due to its peculiar manufacture, using cheap raw materials, krokodil is composed of 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 a photodiode array detector (RP-HPLC-DAD) 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. continued... Received: May 11, 2017 Published: July 14, 2017 © 2017 American Chemical Society

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DOI: 10.1021/acs.chemrestox.7b00126 Chem. Res. Toxicol. 2017, 30, 1609−1621

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Chemical Research in Toxicology

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.

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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.

INTRODUCTION “Krokodil” is a complex psychotropic drug mixture with iodine odor resulting from a homemade synthesis process using easy access starting materials and is used as a cheap substitute for heroin.1−9 Krokodil first appeared around 2002/3 in Siberia, and its use spread throughout other Russian areas and the former Soviet Republic countries and more recently across Europe, namely, Germany, Czech Republic, France, Belgium, Sweden, Norway, Poland, Spain, and Portugal, and also the USA.9−11 Desomorphine is the semisynthetic 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 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 phosphorus concentrations of krokodil batches, may explain many of the signs and symptoms present in PWIK.13 Besides pleasure, clinical cases related to PWIK and also animals exposed to krokodil suggest 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 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 informatic 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

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MATERIALS 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 96% ethanol 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 Sigma-Aldrich. Ultrapure (Milli-Q) water was used throughout the study. Krokodil Samples. All of the krokodil samples were produced by street-like synthesis as previous described.28 The synthesis procedure was repeated 10 times, and a representative pool of the obtained products was used for further analysis. 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 autosampler 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 40 °C. 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 for 5 min with an isocratic flux of 98% eluent A and 2% eluent B at a flow rate of 300 μL/min. Subsequent 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 an 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 by Thermo Fisher Scientific was used to manage 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 the UV/vis spectrum and by coelution. 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 10 times in the initial eluent composition. An Accela 600 HPLC system (Thermo Scientific, Bremen, Germany), equipped with an autosampler, was coupled online to a LTQ-Orbitrap XL mass spectrometer (Thermo Scientific, Bremen, Germany) operated with an ESI source. 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 °C. 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 for 5 min with an isocratic flux of 98% eluent A and 2% eluent B at a flow rate of 300 μL/min. Subsequent 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 minutes in order to allow column cleaning. The column 1610


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times were selected considering the lower ΔMass (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 Xcalibur, version 2.1. Chemical structures were drawn with ChemDraw Professional, version 16.0.0.82.

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 275 °C. 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, and Thermo Fisher Scientific managed the 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 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 min; remaining were set to default values); and FISH scoring (default values). Spectra were searched against an internal database of 95 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

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RESULTS AND DISCUSSION Krokodil Chemical Profiling. Because of the raw materials, homemade character, and the harsh reaction conditions used in its manufacturing, krokodil is a very complex mixture, composed of 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 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 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 4,5-epoxymorphinan-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 conducted, using the optimized RP-HPLC-DAD conditions. Morphinans are alkaloid derivatives with basic nitrogen making

Figure 1. Krokodil chromatograms obtained by different detection methodologies. (A) RP-HPLC-DAD in 3D wavelength detection; (B) full MS scan chromatograms (TIC) of crude krokodil (black line); (C) chromatogram of morphine (RT = 1.96), codeine (RT = 8.21), and desomorphine (RT = 10.41) standards. 1611


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Chemical Research in Toxicology them suitable for ESI analysis in positive mode.29 LC-ESI-ITOrbitrap-MS is able to couple the power of 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, 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 the 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 morphine and codeine, base peaks are produced by losing the 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 an 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 < 1).2,33 It is clear that all morphinans present in krokodil have to be originated from codeine, the only possible precursor of this class of compounds present in the starting materials. From the codeine precursor, three types of conditions can be outlined in eight different chemical transformations (Figure 3a−i): (a) The codeine to desomorphine synthetic pathway; three different reactions are involved (a, b, and c), dehydroxylation at position number 6, reduction of double bond between positions 7 and 8, and O-demethylation of the methoxyl group at position 3; (b) the previously reported desomorphine byproducts, dehydroxylation at position number 3 and N-demethylation (d,e);12,28 (c) codeine byproducts; considering the highly reductive environment of krokodil manufacture,28 it is reasonable to assume that a 4,5-epoxy cleavage can also occur as is the case with the already described methoxyl cleavage at position 3. The epoxy cleavage will lead to two possible types of products, a phenol bearing an hydroxyl group at position 4 or an alcohol at position 5 (f,g). The phenol obtained from epoxy cleavage products can be further dehydroxylated (h) as is the case with the already described dehydroxilation at position 3 (d). The alcohol obtained from epoxy cleavage products can be further dehydroxylated (i) as is the case with the already described dehydroxilation at position 6 (a). Considering these logical and reasoned chemical transformations, it is reasonable to hypothesize that they can occur in similar ways with all morphinan based compounds present in krokodil, as long as the necessary chemical conditions are present. Figure 4 depicts one way to achieve the prototypic morphinan structure molecule from codeine. Bearing this in mind and combining the considered chemical transformations of codeine and derivatives, a total of 95 different compounds can be

Table 1. Unequivocal Identification of Morphine, Codeine, and Desomorphine

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Figure 2. MS2 fragmentation spectra and fragmentation pathway of desomorphine, codeine, and morphine. Morphinan rings A to E are provided in red.

performed by the software (Table S1). A hit was defined when the exact masses of the precursor ion were compatible with the exact mass of a molecular formula proposed in the library. In this matching analysis, beyond finding the same exact masses, for hit validation two additionally acceptance criteria were also considered: compatibility of the isotopic peak mass ([M + H]+1) and its relative abundance, and repeatability, which was evaluated by analyzing four replicates, considering only hits those that matched in at least three. The second level of identification was focused in analyzing the mass fragmentation of each hit precursor ion (MS2 spectra). Taking into account the similarity of the already discussed fragmentation pathways of desomorphine, codeine, and morphine (Figure 5, bold lines), it is reasonable to assume that other morphinans with similar structural features will present similar fragmentation behaviors. Dashed lines depict some representative examples of such morphinans and their respective fragmentation pathways, showing the characteristic neutral losses of the piperidine ring (−CH2CHNHCH3, Δm/z = 57.06), water (-H2O, Δm/z = 18.01) or hydrogen (-H2, Δm/z = 2.01), as happens with desomorphine, codeine, and morphine. Accordingly, other morphinans will yield fragments with m/z values that are dependent on parent ion chemical structure.

rationally produced and represent the morphinans chemical space in krokodil (Table S1). Because of the homemade uncontrolled production, the probability of the formation of the hypothesized morphinans and their relative amounts can be variable. Nevertheless, chemical transformation occurring on labile positions is certainly favored. It is also noteworthy that several of the proposed morphinans are constitutional isomers (same molecular formula, different atoms’ connectivity). As a consequence of the presence of isomers, from 95 admitted compounds 30 molecular formulas are possible to establish. The above-mentioned chemical space can be subdivided into three categories: (i) morphinans that have already been identified in krokodil;2,12,22 (ii) morphinans that have not been identified in krokodil, but structures have been reported in other contexts; and (iii) morphinans that, as far as we know, have never been reported at all. In order to put in evidence the morphinans that are a part of krokodil, a two-level identification strategy was implemented. The first level of identification consisted in finding in the MS TIC chromatogram of krokodil the 95 proposed morphinans. For this, Compound Discover was used. First, a library with the exact masses of the proposed morphinans was built, and a screening on krokodil MS full scan chromatogram for these exact masses was 1613


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Figure 3. Krokodil chemical space. The inner circle summarizes the proposed chemical transformations involved in krokodil manufacture. Outer circles provide the rationale that justifies each chemical transformation. (a) dihydroxylation at C6; (b) reduction of C7-C8 double bond; (c) O-demethylation at C3; (d) dihydroxylation at C3; (e) N-demethylation; (f) ether cleavage at C4; (g) ether cleavage at C5; h) dihydroxylation at C5; i) dehydroxylation at C4.

concerning the performance of analytical methods and the interpretation of results.30 Desomorphine, codeine, and morphine which were already identified by coelution with standards as previously described were also put in evidence using this approach. Other 51 morphinans were detected, making a total of 54 morphinans revealed (Table 2), in the total of 95 possible morphinans that could be formed in krokodil. For each morphinan, the molecular formula and the compatible chemical structure are provided (Table 3). For six of the detected morphinans, only one compatible structure can be written, according to our reasoning and the consequently proposed chemical space (Table S1). For the remaining 40 three

Consequently, at this second level of identification, hits were admitted as morphinans if they showed a morphinan compatible fragmentation behavior. Finally, only validated hits that fulfill both identification level requirements were definitely considered as morphinans. Morphinans detected in krokodil samples are summarized in Table 2. Morphinans were identified with high accuracy (Δm/z = −1.89−2.81 ppm) and similar to what is reported for other Orbitrap-based methods.33−35 In addition, for each validated hit, the mass of the three most abundant fragments obtained from MS2 fragmentation are reported. These fragments were considered in order to fulfill the requirements of EU Decision (implementing, 2002) 1614


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Figure 4. One representative synthetic pathway from codeine to morphinan. (a) dehydroxylation; (b) reduction; (c) O-demethylation; (d) dehydroxylation; (e) N-demethylation; (f) ether cleavage; (h) dehydroxylation. Gray arrows represent the other chemical transformations that could occur for each product.

Figure 5. Morphinan fragmentation pattern. Fragments obtained from codeine, morphine, and desomorphine are represented with solid lines. Dashed lines are used to indicate the fragmentation of other morphinans.

being identified for the first time in krokodil. Assuming that codeine is the only possible morphinan precursor and that possible chemical transformations followed the rationale behind our reaction mechanism proposal, a total of 95 morphinans were defined.28 This chemical space is the cornerstone of a targeted identification approach based on the exact masses feature and mass fragmentations patterns provided by LC-ESI-IT-Orbitrap-MS. In this approach, the 95 proposed morphinans were searched using the MS TIC chromatogram of krokodil, and findings were validated by mass fragmentation of the correspondent precursor ions (MS2 spectra). Following this strategy, 54 morphinans were detected. Summarizing the work done so far, it is plausible to hypothesize that the high psychoactive and analgesic effects of krokodil cannot be explained only by the presence of desomorphine, the so far claimed chemical marker of krokodil. Indeed, even performed under controlled conditions (e.g., same laboratory and operator), composition varies between batches. While desomorphine is the main opioid in some batches, in others it is present in lower amounts. This variability is certainly reflected in real samples of krokodil. The presence of other morphinans might additively or synergistically maximize the pharmacokinetics and/or pharmacodynamics of opioids, but that needs to be further clarified. PWIK and simultaneous producers of krokodil may have offered, even in a totally accidental way, an analgesic new therapeutic arsenal that is truly incomparable. In our opinion, it might be the source of new therapeutic molecules, although the proof of

morphinans, with different retention times, some share the same molecular formula (isomers). Codeine, the starting material of krokodil, and morphine were found in trace amounts, corresponding each one to 1% of the total morphinans. Desomorphine is the most abundant morphinan present in krokodil (Table 2, RT = 10.03), corresponding to near 45%. The second most abundant morphinan (Table 2, RT = 17.10, C17H21NO), close to 14%, corresponds to one of the six proposed compounds. It is reasonable to admit that this morphinan could correspond to 3,6-dideoxy-dihydromorphine since it was already found at a similar concentration in krokodil.12,28 The majority of morphinans correspond to a cluster, forming a complex mixture.

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CONCLUSIONS Built on top of our previous reports,28,33 the data presented here, resulting from RP-HPLC-DAD analysis and MS TIC analysis by LC-ESI-IT-Orbitrap-MS, provided new insights about the chemical profile of crude krokodil. Because of its chemical complexity, krokodil analytical characterization is challenging. In this work, a representative sample that resulted from 10 streetmimetic syntheses was used. However, according to our experience, the krokodil composition being always very complex can greatly vary depending on the manufacture/synthesis conditions. Desomorphine, codeine, and morphine were detected and confirmed by co-injection with reference standards, morphine 1615


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Chemical Research in Toxicology Table 2. Exact Mass and Mass Fragmentation of the Morphinans Identified in Krokodil RT (min)

fwhm (min)

% area of morphinans

formula

isotopic mass

precursor ion m/z

error (ppm)

number of isotopes

Frag. 1 m/z

Frag. 2 m/z

Frag. 3 m/z

22.61 25.72 22.29 17.35 14.54 13.45 22.12 15.01 17.10 20.29 19.15 15.28 21.17 12.84 12.81 9.87 11.54 14.55 11.60 12.69 17.27 13.64 18.96 5.89 21.18 3.62 5.06 14.39 6.90 15.66 11.80 19.25 21.53 10.03 12.95 12.34 2.36 17.26 15.28 11.34 15.15 1.87 17.01 13.42 20.89 5.08 4.37 1.52 2.92 4.49 3.48 8.09 5.38 10.93

0.13 0.14 0.13 0.15 0.10 0.07 0.15 0.10 0.18 0.13 0.10 0.11 0.10 0.09 0.11 0.07 0.10 0.16 0.10 0.08 0.11 0.08 0.09 0.12 0.10 0.10 0.14 0.11 0.28 0.12 0.10 0.10 0.10 0.36 0.12 0.12 0.06 0.11 0.10 0.09 0.11 0.10 0.13 0.08 0.10 0.18 0.13 0.07 0.13 0.14 0.12 0.18 0.14 0.09

0.59 0.79 1.43 2.19 0.19 0.07 0.54 0.13 14.02 0.05 0.12 0.52 0.12 0.37 0.23 1.51 0.07 2.55 0.09 0.03 0.01 0.03 0.03 3.11 0.37 0.03 0.07 0.40 0.14 2.68 1.87 0.75 0.06 45.46 5.13 6.15 0.04 0.16 0.53 0.20 0.95 1.11 0.11 0.22 0.12 0.08 0.01 0.15 2.30 0.24 0.23 1.43 0.13 0.04

C16H19N C16H21N C17H21N C16H19NO C16H19NO C16H19NO C17H23N C16H21NO C17H21NO C17H21NO C17H21NO C17H21NO C17H21NO C17H21NO C16H19NO2 C16H19NO2 C16H19NO2 C17H23NO C17H23NO C17H23NO C16H21NO2 C16H21NO2 C16H21NO2 C17H19NO2 C18H23NO C17H21NO2 C17H21NO2 C17H21NO2 C17H21NO2 C17H21NO2 C17H21NO2 C17H21NO2 C17H21NO2 C17H21NO2 C17H21NO2 C17H21NO2 C16H19NO3 C17H23NO2 C17H23NO2 C17H23NO2 C18H21NO2 C17H19NO3 C18H23NO2 C18H23NO2 C18H23NO2 C17H21NO3 C17H21NO3 C17H21NO3 C17H21NO3 C17H23NO3 C17H23NO3 C18H21NO3 C18H23NO3 C18H23NO3

225.15175 227.16740 239.16740 241.14666 241.14666 241.14666 241.18305 243.16231 255.16231 255.16231 255.16231 255.16231 255.16231 255.16231 257.14158 257.14158 257.14158 257.17796 257.17796 257.17796 259.15723 259.15723 259.15723 269.14158 269.17796 271.15723 271.15723 271.15723 271.15723 271.15723 271.15723 271.15723 271.15723 271.15723 271.15723 271.15723 273.13649 273.17288 273.17288 273.17288 283.15723 286.14389 285.17288 285.17288 285.17288 287.15214 287.15214 287.15214 287.15214 289.16779 289.16779 300.15952 301.16779 301.16779

226.15935 228.17484 240.17496 242.15424 242.15399 242.15405 242.19064 244.16962 256.16928 256.16995 256.16983 256.16974 256.16983 256.17007 258.14896 258.14905 258.14908 258.18515 258.18527 258.18527 260.16437 260.16467 260.16476 270.14902 270.18530 272.16449 272.16452 272.16458 272.16461 272.16476 272.16479 272.16470 272.16467 272.16473 272.16473 272.16486 274.14377 274.18024 274.18008 274.18033 284.16443 285.13649 286.17935 286.18018 286.18015 288.15939 288.15936 288.15948 288.15952 290.17505 290.17514 299.15214 302.17508 302.17505

−1.42 −0.70 −1.17 −1.22 −0.21 −0.46 −1.31 −0.11 1.21 −1.41 −0.93 −0.58 −0.93 −1.89 −0.39 −0.75 −0.86 0.35 −0.12 −0.12 0.53 −0.64 −1.00 −0.60 −0.23 0.06 −0.05 −0.28 −0.39 −0.95 −1.06 −0.73 −0.62 −0.84 −0.84 −1.29 0.01 −0.30 0.26 −0.63 0.27 −0.42 2.81 −0.07 0.04 0.09 0.20 −0.22 −0.33 0.07 −0.24 −0.32 −0.03 0.07

4 4 3 4 3 3 3 3 4 2 3 4 3 3 4 3 3 4 3 2 2 2 2 3 3 3 3 4 3 4 3 5 2 3 3 3 3 3 3 3 3 3 2 3 3 2 2 3 2 3 3 4 3 3

211.09 184.00 225.06 197.02 199.01 199.04 185.04 201.03 197.01 211.03 199.02 198.99 211.04 199.00 215.00 215.03 227.05 199.02 227.05 215.00 229.08 217.03 203.04 194.99 213.03 215.04 215.03 215.01 227.11 241.05 215.04 241.00 182.97 215.00 215.01 215.01 198.97 148.91 217.02 199.00 194.98 201.02 229.06 229.06 229.02 213.02 212.99 194.99 231.01 215.03 215.02 215.04 194.97 194.98

198.05 185.02 199.06 199.01 183.98 197.02 128.92 199.02 198.98 213.04 225.01 196.95 213.05 197.02 229.04 212.98 215.04 201.03 215.04 229.04 203.03 214.95 182.98 213.04 211.07 227.07 197.02 229.04 216.00 215.04 148.89 182.97 223.01 197.00 148.92 229.03 231.03 217.01 148.93 196.98 227.02 229.02 211.05 211.07 197.04 270.11 231.01 270.09 199.01 213.02 212.99 243.04 227.04 226.99

166.94 186.05 212.10 225.04 225.07 156.96 156.98 227.03 181.00 225.06 197.04 225.02 199.02 156.95 241.01 197.03 148.90 227.07 148.90 241.01 185.05 243.09 229.07 192.95 195.03 216.00 216.01 196.99 214.98 213.04 172.96 223.05 215.98 212.99 194.99 216.01 213.04 197.00 196.99 225.04 192.96 211.02 227.01 209.03 243.04 194.99 194.97 213.02 213.00 241.05 241.04 282.10 192.91 192.99

concept for the activity and potential clinical use of the identified and especially of the unknown compounds needs to be further addressed. Previous structure−activity relationship studies demonstrated that the amine nitrogen (mostly protonated at physiological pH) is the most important functional group for

opioid receptor affinity since it permits receptor anchoring through an essential electrostatic bond with the Asp residue of the μ-receptor conserved in all G protein-coupled receptors.36−38 The basic nitrogen is most often tertiary for optimal activity.39 Since the krokodil synthesis occurs in a highly reductive 1616


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Chemical Research in Toxicology Table 3. Formula, Retention Time, and Compatibles Chemical Structures for the Morphinans Identified in Krokodil

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Chemical Research in Toxicology Table 3. continued

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Chemical Research in Toxicology Table 3. continued

a

Only one chemical structure is compatible with the detected molecular formula. bIdentified by co-injection with reference standards.

care in the context of terminal neoplasm, polytraumatization, burns, adjuvant or postsurgery, acute pulmonary edema, and multiple medical specialties can now benefit from research on these new alternative morphinans that we first described in krokodil.

environment, chemical transformations mainly lead to the loss of oxygen-containing functional groups, and therefore, more lipophilic compounds are produced. The phenolic hydroxyl in C3 has been claimed to be important for biological activity since the moiety C3−O is part of the pharmacophore. Indeed, the substitution of phenolic hydroxyl in C3 for -H, -OCH3, or − OCOCH3 reduces opioid activity.39 This can be easily understood by the fact of that the analgesic activity of codeine can be attributed to the O-demethylation to morphine. The C6 hydroxyl is believed to engage in a week hydrogen bond with an Asn residue present in receptors.40 Despite this reported drug−receptor interaction, the loss of the C6 hydroxyl group results in an approximate 10-fold increase of activity due to a significant increase in lipophilicity.40 Finally, if the 7,8 double bond is reduced, the C ring becomes significantly more flexible, and opioid activity increases. If the C7−C8 double bond is reduced, the C ring adopts a more stable chair conformation which promotes the increase of opioid activity.40 Finally, morphinan based analogues, without the 4,5-epoxy bridge, C7−C8 double bond, and 6-hydroxyl group, showed stereoselective opioid activity.41 In this context, we will further study whether we can design the “good part of krokodil” through the elimination of necrotic impurities that greatly cause morbidity and may even cause death. Refractory therapies with morphine and other opioids, namely, for severe pain of multiple nature/ causes such as acute myocardial infarction, patients in palliative

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.7b00126.

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Library with the proposed 95 morphinans (PDF)

AUTHOR INFORMATION

Corresponding Authors

*(J.X.S.) E-mail: jfxsoares@ff.up.pt. *(E.A.) E-mail: [email protected] *(F.C.) E-mail: felixdc@ff.up.pt. *(R.J.D.-O.) E-mail: [email protected] *(C.A.) E-mail: cafonso@ff.up.pt ORCID

Ricardo Jorge Dinis-Oliveira: 0000-0001-7430-6297 Author Contributions ‡‡

J.X.S. and E.A.A. contributed equally to this work. F.C., R.J.D.-O. and C.M.A are co-principal investigators of this study.

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Chemical Research in Toxicology Funding

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E.A. and A.D.P.N. acknowledge Brazilian agency Conselho ́ e Tecnológico (CNPq) Nacional de Desenvolvimento Cientifico (process 245844/2012-0) for research grants and scholarship. J.S. thanks National Funds from FCT (Fundaçaõ para a Ciência e a Tecnologia), FEDER under Program PT2020 (project 007265 -UID/QUI/50006/2013), and through the FCT PhD Programmes and by Programa Operacional Potencial Humano (POCH), specifically by the BiotechHealth Programme (Doctoral Programme on Cellular and Molecular Biotechnology Applied to Health Sciences), reference PD/00016/2012 for for financial support. J.S. also thanks FCT and POPH (Programa Operacional Potencial Humano) for his Ph.D. grant (SFRH/BD/98105/2013). F.C. received financial support from the European Union (FEDER funds POCI/01/0145/FEDER/007728) and National Funds (FCT/MEC, Fundaçaõ para a Ciência e Tecnologia and Ministério da Educaçaõ e Ciência) under the Partnership Agreement PT2020 UID/MULTI/04378/2013, as well as the project NORTE-010145-FEDER-000024, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement (DESignBIOtecHealth-New Technologies for three Health Challenges of Modern Societies: Diabetes, Drug Abuse and Kidney Diseases), through the European Regional Development Fund (ERDF). R.D.-O. acknowledges FCT for his Investigator Grant (IF/01147/2013). S.C. and C.A. acknowledge FCT through the strategic project CEQUIMED-UP (Pest-OE/SAU/UI4040/2014). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS CID, collision induced dissociation; DAD, photodiode array detector; ESI, electrospray ionization; HPLC, high performance liquid chromatography; HRMS, high resolution mass spectrometry; IT, ion trap; LC, liquid chromatography; LC-ESI-ITorbitrap-MS, iquid chromatography with electrospray ionization high resolution tandem mass spectrometry; LTQ, linear trap quadrupole; MS, mass spectrometry; MS/MS, tandem mass spectrometry; PWIK, people who inject krokodil; RP-HPLCDAD, reversed phase high performance liquid chromatography with photodiode array detection; RT, retention time; TIC, total ion current

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