Linköping University | Department of Physics, Chemistry and Biology Type of thesis, 16 hp | Educational Program: Physics, Chemistry and Biology Spring or Autumn term 2020 | LITH-IFM-G-EX—20/3846--SE
Characterization of hepatocyte derived metabolites of various New
Psychoactive Substances using LC-QTOF-MS
Sarah Ingvarsson
Examinator: Johan Dahlén
Date 20-06-18
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Division, Department
Department of Physics, Chemistry and Biology Linköping University
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ISRN: LITH-IFM-G-EX--20/3846--SE
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Språk Language Svenska/Swedish Engelska/English ________________ Rapporttyp Report category Licentiatavhandling Examensarbete C-uppsats D-uppsats Övrig rapport _____________ Title
Characterization of hepatocyte derived metabolites of various New Psychoactive Substances using LC-QTOF-MS.
Author
Sarah Ingvarsson
Abstract
New psychoactive substances are becoming increasingly common in many parts of the world, and some of them are marketed as “legal highs” and are produced to circumvent the drug legislation, and they come in many unregulated forms. The aim of this research was to characterize the metabolites of a new psychoactive substance and hence provide the fundamental data needed for further research of toxicity and future drug testing. The synthetic
cannabinoid 4-fluoro-CUMYL-5-fluoro-PICA was incubated in cryopreserved hepatocytes for 1, 3 and 5 hour and then the formed metabolites was analyzed with an LC-QTOF-MS method, data analysis was performed by using the software MassHunter Qualitative Analysis.
For 4-fluoro-CUMYL-5-fluoro-PICA a total of ten metabolites were identified, with three hydroxylations, two oxidative defluorinations to carboxylation, three oxidative defluorination and two fluoropentyl dealkylation. The metabolite with the highest intensity was oxidative defluorination.
Abstract
New psychoactive substances are becoming increasingly common in many parts of the world, and some of them are marketed as “legal highs” and are produced to circumvent the drug legislation, and they come in many unregulated forms. The aim of this research was to characterize the metabolites of a new psychoactive substance and hence provide the fundamental data needed for further research of toxicity and future drug testing. The synthetic
cannabinoid 4-fluoro-CUMYL-5-fluoro-PICA was incubated in cryopreserved hepatocytes for 1, 3 and 5 hour and then the formed metabolites was analyzed with an LC-QTOF-MS method, data analysis was performed by using the software MassHunter Qualitative Analysis.
For 4-fluoro-CUMYL-5-fluoro-PICA a total of ten metabolites were identified, with three hydroxylations, two oxidative defluorinations to carboxylation, three oxidative defluorination and two fluoropentyl dealkylation. The metabolite with the highest intensity was oxidative defluorination.
Abbreviations
NPS: New Psychoactive Substances
LC-QTOF-MS: Liquid Chromatography Quadrupole Time of Flight Mass Spectrometry Rt: Retention time
Ppm: Parts per million LC: Liquid Chromatography
TOF-MS: Time of Flight Mass Spectrometry EIC: Extracted Ion Chromatogram
EMCDDA: European Monitoring Centre for Drugs and Drug Addiction m/z: mass to charge ratio
Table of contents Abstract ... 1 Abbreviations ... 2 1. Introduction ... 4 1.1 Project purpose ... 4 1.2 Expected results ... 4 2. Theory ... 5 2.1 Synthetic Cannabinoids ... 5 2.2 Drug metabolism ... 5
2.3 Cryopreserved human hepatocytes ... 6
2.4 Liquid Chromatography ... 6
2.5 Mass Spectrometry ... 6
2.5.1 The ion source ... 7
2.5.2 The mass analyzer ... 7
3. Material and Methods ... 9
3.1 Chemicals ... 9
3.2 Incubation with hepatocytes and sample preparation ... 9
3.3 LC-QTOF-MS analysis ... 9
3.4 Data analysis ... 10
4. Results ... 11
4.1 Experimental results ... 11
4.1.1 Metabolic patterns of 4-fluoro-Cumyl-5-fluoro-PICA ... 11
4.1.2 Summary of results ... 17
5. Discussion ... 18
5.1 Hydroxylated and dihydroxylated metabolites... 18
5.2 Metabolites generated by N-dealkylation ... 18
5.3 Metabolites generated by oxidative defluorination ... 18
5.4 Metabolites generated by oxidative defluorination to carboxylation ... 19
5.5 Impact ... 19
5.6 Ethical implications ... 19
6. Conclusion ... 20
Acknowledgement ... 20
1. Introduction
The consumption of new psychoactive substances is rising worldwide at an alarming rate and the consumption is a growing problem in many countries. On the European illicit drug market, it has been shown that the number of NPS that have appeared on the drug market has increased and that NPS designed as legal substitutes to the illicit drugs are relatively easily available to the public through retail shops and the online market. New psychoactive substances are commonly called designer drugs and are designed to mimic effects of established illicit drugs such as cannabis and cocaine and are constantly changing chemically so the detection in biological matrices is challenging. The introduction of NPS on the illicit drug market is a potential health and social risk and has led to challenges for the analytical laboratories, keeping screening methods up to date with all relevant drugs can be hard to achieve [6-8].
Synthetic cannabinoids have been on the market since 2008 and have been linked to toxic effects, kidney injury, seizures, myocardial infraction, and death, while the mechanisms for these effects are still unknown. Synthetic cannabinoids are ongoing public health challenges, even with continuing efforts to identify these compounds, to characterize their pharmacological and toxicological properties, and to limit their manufacture, distribution, and use [12]. At the end of 2017 European Union Early Warning System reported a total of 251 compounds with cannabinoid receptor activity [11]. Some of the synthetic cannabinoid agonists were banned in some countries but since then uncontrolled structural variants of synthetic cannabinoid agonists soon replaced them [9].
Synthetic cannabinoids have also been a problem in Sweden, as evident by the death investigations conducted by the National Board of Forensic Medicine. The synthetic cannabinoids have the same effect as conventional drugs, but some of them are not covered by the drug legislation and are free to distribute, sell and use [22]. Some of the synthetic cannabinoids have a rapid and extensive metabolism and are mainly extracted in human urine as metabolites while the parent drugs are rarely found in urine. Therefore, identification of parent in blood or metabolites in urine is required to show an intake [28].
The demand for forensic analysis of NPSs is high, and through incubation of drugs with human hepatocytes and liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) analysis, it is possible to identify the biotransformations and potential metabolites of NPS. This will reveal many important aspects of the effects of these substances, and lay the important groundwork needed for future attempts to understand the physiological effects of NPS [14].
1.1 Project purpose
The aim of this research was to characterize the metabolites of a new psychoactive substance and hence provide the fundamental data needed for further research of toxicity and future drug testing [13]. The aims were to find the main metabolites of the parent 4-fluoro-CUMYL-5-fluoro-PICA, determine structures of possible metabolites and to identify the metabolic pathway for the compound.
1.2 Expected results
The result of the study will be the identification of biotransformation and to find potential metabolites of the
synthetic cannabinoid 4-fluoro-CUMYL-5-fluoro-PICA [14]. The biotransformation could tentatively be hydroxylation, oxidative defluorination to carboxylation, oxidative defluorination and fluoropentyl dealkylation. The metabolic pathway of 4-fluro-CUMYL-5-fluoro-PICA provide a very important insight in the pharmacokinetics and
biotransformation of these substances and to this day, primary human hepatocyte cell cultures are still considered the golden standard of in vitro liver modeling [14].
2. Theory
Synthetic cannabinoids are distributed on the illegal market in Europe and Sweden. Incubating these drugs with human hepatocytes and analyzing the incubates with an LC-QTOF-MS method allow the identification of potential metabolites. This section describes the theoretical background of the methods and important aspects for this study.
2.1 Synthetic Cannabinoids
Synthetic cannabinoids (Figure 1) include various psychoactive substances designed to mimic the effect of cannabis [11]. Synthetic cannabinoids were developed as pharmacological tools to probe the endocannabinoid system and as novel pharmacotherapies [28]. Synthetic cannabinoids comprise the largest group of new psychoactive substances that are monitored by the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA). These new psychoactive substances are not covered by the drug legislation and they are accessible widely and sold as legally herbal highs. With their cannabis-like intoxicating effects, these compounds have been a challenge for the current drug policy, because of new variants that are emerging and their tendency for acute harm [11].
Synthetic cannabinoids produce different health challenges compared to cannabis because synthetic cannabinoids are full cannabinoid receptor agonists and their metabolites tend to retain higher cannabinoid receptor affinity than delta-9-tetrahydrocannabinol (THC). This makes them more potent and increase the risk of toxicity, abuse, psychosis, acute kidney injury and fatality. The structures of synthetic cannabinoids vary, and some of them also have actions at non-cannabinoid receptor for example µ- and δ-opioid, and 5-HT-receptors. Synthetic cannabinoids are usually more common among young adults, men, homeless and who use cannabis and other drugs [11].
The endogenous cannabinoid system includes endogenous cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2) [16]. The CB1 receptor are distributed in the brain regions, especially the limbic system, the frontal cortex, and including the amygdala and hippocampus, pons and medulla, and sensory and motor areas. The CB2 gene is located in various regions of the brain but less so than CB1. Immune cells express high levels of CB2 and there is a hierarchy of CB2 expression within the immune system. Synthetic cannabinoids are functionally like THC, and that is the active part of cannabis. Synthetic cannabinoids bind to the same cannabinoid receptors in the brain and other organs as THC and the endogenous ligands, 2-arachidonylglycerol and anandamide, which interact with both CB1 and CB2 receptor [30]. Synthetic cannabinoids often have rapid and extensive metabolism, and the metabolites can be found in urine, but the parent of synthetic cannabinoids are rarely found in urine [28].
2.2 Drug metabolism
Drug metabolism occurs in the lungs, heart, kidney, and blood, but the most important organ for drug metabolism is the liver. The liver is containing many hepatic enzymes, and especially the cytochrome P450 family; a group of enzymes containing heme. Many drugs have low solubility in aqueous systems, such as urine, and need to be more hydrophilic to be easily eliminated. Drug metabolism occurs in phase I and phase II. Cytochrome P450 are a major part of the oxidative metabolism, of the part of drug metabolism called phase I. Phase I of biotransformation is the oxidative pathway, where the compound becomes more polar, which is performed by Cytochrome P450-dependent monooxygenases and flavin monooxygenases. This process is followed by Phase II reactions, the metabolites are conjugated by hepatocytes with endogenous molecules by acetylate ion, sulfate ion, glucuronidation, methylate ion and mercapture formation, this result in derivatives that are more soluble, and ease their elimination. [23].
Figure 1. The structure of the synthetic cannabinoid 4-fluoro-CUMYL-5-fluoro-PICA.
2.3 Cryopreserved human hepatocytes
Primary cultures of hepatocytes are used to reproduce drug metabolism in vitro in biomedical research, and for the purpose of drug development. The recent developments in culturing, isolation and cryopreserved human
hepatocytes has been successful. Human hepatocytes are used in drug development and are important for evaluation of human specific drug properties such as drug toxicity and metabolic fate [27].
2.4 Liquid Chromatography
Liquid chromatography (LC) is one of the most important and widely used technique for analysis of chemical mixtures [19]. Several separation modes can be used to separate compounds, depending on the choice of mobile and stationary phase. Two major separations modes are reversed phase chromatography and normal phase
chromatography. In reversed phase, its stationary phase is nonpolar or weakly polar, and the mobile phase solvent is polar. In normal phase, a polar stationary phase is used, and the solvent is less polar. A liquid sample is injected and loaded in a column packed with small porous particle and that is the stationary phase. The sample is then
transported along the column with the mobile phase. The molecules in the sample are separated based on their different affinity for the stationary and mobile phase. The components then elute from the column and are detected [20].
High performance liquid chromatography (HPLC) is an advance type of LC and instead of the solvent is travelling by gravity, the solvent in HPLC is travelling under high pressure [19-20]. The columns containing fine particles that give high resolution separations. The HPLC system consists of a high pressure chromatograph column, a solvent delivery system (a pump), an autosampler, a sample injection valve, a mass spectrometer, which work as a detector, solvent reservoirs, a photodiode array absorbance detector, and a computer to display the results. The column is in the oven, and the door to the oven is normally closed to keep the column at a constant temperature [26].
2.5 Mass Spectrometry
Mass Spectrometry (MS) is almost a hundred years old and in modern analytical chemistry MS has become a dominant force. With a spectacular range of capabilities and application, it also provides unparalleled levels of sensitivity and selectivity for trace analysis. MS is both a separation method and a spectrometric method. The connection of mass spectrometry with a separation technique such as gas chromatography, liquid chromatography, capillary electrophoresis and a second stage of mass spectrometry has established it as the standard for trace analysis [17]. A mass spectrometer has three essential components: the ion source, the mass analyzer, and the detector [18].
2.5.1 The ion source
The ion source is the part of the mass spectrometer that ionizes the analyte under analysis. The resulting ions that are coming from the ion source will be separated by the mass analyzer according to their mass to charge ratio (m/z). Electrospray ionization (ESI) and Atmospheric pressure chemical ionization (APCI) are some of the techniques used in mass spectrometry [18].
2.5.1.1 Electrospray ionization
Liquid from the chromatography column and a coaxial flow of N2 gas are entering the steel nebulizer capillary. For positive ion mass spectrometry, the spray chamber is held at -3500 V and the nebulizer 0 V. For the negative ion mass spectrometry, all the voltages are reversed. Fine aerosol of charged particles is created of the strong electric field ate the nebulizer outlet, combined with the coaxial flow of N2 gas. In the mass spectrometer, the glass capillary attracts the positive ions from the aerosol and leading them to the mass spectrometer by an even more negative potential of -4500 V. In the spray chamber, gas is flowing from atmospheric pressure and transports ions through the capillary to the exit, and the pressure is reduced by a vacuum pump. Charged liquid is exiting the capillary and forms a cone and then breaks into fine droplets, and then they evaporate, leaving the ions in the gas phase [24].
2.5.1.2 Atmospheric pressure chemical ionization
In APCI, a coaxial flow of N2 gas and heat convert eluate into a fine mist, and then the analyte and solvent evaporate. In the ion source of a mass spectrometer, APCI creates new ions from gas-phase reactions between molecules and ions. High voltage is applied to a metal needle in the path of the aerosol. Around the metal needle, an electric corona is formed, which is a plasma containing charged particles, and eject electrons into the aerosol and creating ions [25].
2.5.2 The mass analyzer
A mass analyzer is the component of the mass spectrometer that takes ionized masses and separates them based on m/z and bring them to the detector [18]. There are several types off mass analyzers that can be used for separation of ions, for example: Quadrupole mass analyzer, Time of Flight mass analyzer and Quadrupole Ion Trap mass analyzers. In this study Time of Flight and Quadrupole mass analyzers were used.
2.5.2.1 Time of Flight Mass Spectrometer (TOF-MS)
Inside the source region (Figure 2) ions can be produced by electron ionization in the gas phase or by laser irradiation of a solid sample on the surface of the backplate. To get the ions into the drift region about 20 000 V is applied to the backplate to accelerate the ions and remove them from the ion source. In the drift region there is no magnetic or electric field and there is no longer any acceleration. If the ions have the same kinetic energy, but different masses, the lighter ions will travel faster than the heavier ions. The TOF-MS is an evacuated, about one-meter long tube with the detector at one end and the source at the other end. Ions expelled from the source drift to the detector in order of increasing mass, because the lighter ones travel faster [5].
The TOF-MS (Figure 2) is designed for improved resolving power and can tell the exact molecular formula of a particular ion. All the ions do not arise from the source with the same kinetic energy and that is a limitation on resolving power. Ions that are formed close to the backplate is accelerated through a higher voltage difference than ions that are formed close to the grid and the ions near the backplate gains more kinetic energy. Among the ions there is some dealing of kinetic energies even when there is no acceleration voltage present [5].
Lighter ions with less than average kinetic energy and heavier ions with more than average kinetic energy will reach the detector at the same time [5].
The reflectron, an electrostatic mirror is turning around the ions to improve the resolving power. The reflectron is several hollow rings held at increasingly positive potential and have an end grid whose potential that is more positive than the accelerating potential on the backplate of the source. When the ions are entering the reflectron they are slowed down, stopped, and then reflected. If the ion that enters the reflectron has high kinetic energy it penetrates further before it is turned around. In front of the detector the reflected ions reach a new space focus plane at the grid. Regardless of ions initial kinetic energies all ions of the same mass reach this grid at the same time [5].
A quadrupole time of flight (QTOF) is high-resolution mass spectrometer and have the ability to measure accurate m/z with up to four decimals, that provides identification of a substance. The quadrupole selects a specific precursor ion, and in a collision cell the ion is fragmented, and the fragments are then detected. The detected fragments can then be structurally identified [29].
Figure 2. Schematic picture of TOF-MS inspired by Harris DC. Quantitative Chemical Analysis. The positive ions are accelerated out of the source by voltage +V periodically applied to the backplate. The heavier ions travel not as fast as the lighter one and therefore lighter ions reach the detector sooner than heavier ions.
2.5.2.2 Quadrupole
Quadrupole mass analyzers can be used in mass spectrometry to filter out ions within a limited m/z range only. The analyzer uses a combination of radio frequency (RF) and direct-current (DC) potentials. A quadrupole contains four parallel cylindric metal rods and are arranged in a symmetrically, square configuration. The opposite rods are connected electrically. At any given time, the two pairs will have the same potentials of the same magnitude, but they have opposite signs. Ions that are emerging from the source enter the analyzer region between the rods and travel parallel to the rods. Only ions within a certain narrow m/z range will have a stable course through the
quadrupole, at given values of the RF frequency, RF and DC potential. The m/z range for the ions that are allowed to pass through, are depending on the ratio between the RF and DC potentials, and the ions that do not have a stable course will collide with the rods and never reach the detector [18, 21]
A single quadrupole system contains one mass filtering quadrupole and a triple quadrupole system consist of three quadrupole Q1, Q2 and Q3. Q2 is called the collision cell while Q1 and Q2 are working as mass filters. Quadrupoles can be used in scanning or filtering mode. The triple quadrupole system have a higher selectivity, which results in less interference of co-eluting compounds and matrix, better signal to noise allowing quantitation with lower limits of quantitation, and better accuracy and reproducibility at low concentrations [29].
3. Material and Methods
3.1 Chemicals
From Thermo Fischer Scientific (Gothenburg, Sweden) L-glutamine, Hepes buffer, Acetonitrile and Williams E medium were obtained. Cryopreserved human hepatocytes (Liverpool, 20-donor pool) and in vitro Gro HT thawing medium were obtained from Bioreclamation IVT (Brussels, Belgium). 4-fluoro-CUMYL-5-fluoro-PICA was obtained from Cayman Chemical (Ann Arbor, MI, USA). Methanol was purchased from Merck (Darmstadt, Germany).
3.2 Incubation with hepatocytes and sample preparation
0.4% Trypan blue solution (Sigma-Aldrich, Stockholm, Sweden) was available in the laboratory.
Cryopreserved hepatocytes were thawed by using a beaker of water (400-600ml) and then poured into 48 ml of InVitro Gro HT media, heated to 37 °C for 30 minutes, and then centrifuged at 100 g-force for 5 minutes at room temperature. The supernatant was removed by using a pipette, and the remaining pellet was re-suspended in 50 ml of supplemented Williams E medium fortified with 1.5 ml L-glutamine (2mM final concentration) and 3.0 ml HEPES buffer (20 mM). A second washing step with fortified Williams E medium was performed. The remaining pellet was re-suspended in 2 ml Williams E medium. Cell counting was performed with 0.4 % Trypan blue solution and with a mix counting solution (10 x dilution) containing 140 µl Williams E buffer, 40 µl Trypan blue solution (0,4%) and 20 µl diluted cells. By using a microscope, a minimum of 5 squares were counted and the hepatocyte volume was adjusted to get 2 million cells/ml.
4-fluoro-CUMYL-5-fluoro-PICA was diluted to 10 µM in 2 ml of Williams E medium from 1 mg/ml stock solutions and 50 µl was added with a pipette into 96-well-plates. Then 50 µl of the diluted hepatocyte solution was applied into every well, making the final drug concentration 5 µM. The plates were placed in the incubator at 37 °C. The incubation was then terminated after 1, 3 and 5 hours by addition of 100 μl ice-cold acetonitrile to each well, then the plates were covered with foil, and mixed with vortex on plate mixer 600 rpm for 2 minutes. The plates were then placed in the freezer for at least 10 minutes, followed by centrifugation for 15 minutes at 1100 g-force at 4 ° C, then from each well, 100 µl supernatant was pipetted into the injection plate used for LC-QTOF analysis.
Time zero sample were precipitated with acetonitrile before addition of the drug. Degradation (50 μl Williams E buffer instead of cells, incubation without the hepatocytes) and negative (50 μl Williams E buffer instead of drug solution, incubation without substrate) controls were also prepared.
3.3 LC-QTOF-MS analysis
Agilent 1290 infinity Ultra-High-Performance Liquid Chromatography (UHPLC)-system (Agilent Technologies, Kista Sverige) with an Acquity HSS T3 column (150 mm x 2.1 mm, 1.8 µm; Waters, Sollentuna, Sweden) fitted with an Acquity Van Guard pre column (Waters, Sollentuna, Sweden) was used to analyze the sample, and
chromatographically separate the sample. The solvent composition was 99% mobile phase A, 0.1% formic acid in water, and 1 % mobile phase B, 0.1 % formic acid in Acetonitrile. The gradient was initiated at 1 % B and held for 0.6 minutes, ramped to 20 % at 0.7 minutes, increased to 85% at 13 minutes and 95 % at 15 minutes, held until 18 minutes before decreasing to 1 % B within 0.1 minutes and re- equilibration for 0.9 minutes. The column
temperature was 60 °C and the flow rate 0.500 mL/min. An Agilent 6550 iFunnel QTOF mass spectrometer (Agilent Technologies, Kista, Sweden) with a dual Agilent Jet Stream electrospray ionization source was coupled to the Agilent 1290 UHPLC-system. Gas temp 150 °C, Gas flow 18 L/min, Nebulizer 50 psig, sheath gas temperature 375 °C and sheat gas flow 11 L/min, the source parameters were, Vcap 3500, Fragmentor 380, Skimmer 165 and Octopole RF Peak 750. The MS min (m/z) was 100, and max (m/z) 950. MS scan rate was 6 spectra/second, and MS/MS scan rate was 10 spectra/second.
3.4 Data analysis
Data analysis was performed by using the software Agilent MassHunter Qualitative analysis. A library for potential metabolites was constructed and the formulas for the potential metabolites were manually assembled. The metabolite library was analyzed in MassHunter and potential metabolites were evaluated according to several parameters. The retention time (Rt) for the metabolite should be between 3-15 minutes, peak area > 20 000 counts and should have a Gaussian peak shape. The difference between the observed mass and the mass of the target compound (in parts per million) should be within ± 5 ppm. The score is based on how well the compound matches the, mass isotope pattern and retention time of the target compound and should have about 80/100.The following parameters were used to further evaluation of each metabolite.
All the metabolites MS/MS spectra are compared with the MS/MS spectra for the parent, the similarities and differences can be used to identify where the metabolites are modified. From the MS/MS spectrum, the parent and metabolite structures could be identified through the fragment ions. The accurate masses and the monoisotopic masses from the fragment ions where compared.
4. Results
The results presented in this part, including MS/MS spectra for the parent (Figure 5) and metabolites (Figure 6, 7, 8 and 9), extracted ion chromatograms (EIC), proposed biotransformations and map of metabolic pathways.
4.1 Experimental results
The results from the experimental part were obtained by incubating the drug with human hepatocytes and formed metabolites were analyzed with an LC-QTOF-MS method. Data analysis was performed by using the software MassHunter Qualitative Analysis. A metabolic pathway was then executed.
4.1.1 Metabolic patterns of 4-fluoro-Cumyl-5-fluoro-PICA
A total of ten metabolites were detected in the hepatocyte samples (Figure 3.) and labeled M1-M10, and the parent is labeled M. The parent compound, 4-fluoro-CUMYL-5-fluoro-PICA was highly abundant. Appendix 1 summarizes retention times (Rt), formulas, m/z, biotransformations, mass errors and peak area of the parent and metabolites. The combined extracted ion chromatogram (Figure 3) of 4-fluoro-CUMYL-5-fluoro-PICA metabolites after 5-hour incubation with human hepatocytes, obtained with LC-QTOF-MS. All ten metabolites were occurring in the 3 h and 5 h samples.
Figure 3. Combined extracted ion chromatogram of 4-fluoro-CUMYL-5-fluoro-PICA metabolites after 5h incubation with human hepatocytes, obtained with LC-QTOF-MS. Ten metabolites labelled M1-M10 and the parent (M).
M1 M2 M3 M4 M5 M6 M7 M9 M8 M10 4-fluoro-CUMYL-5-fluoro-PICA M
Metabolic pathway of 4-fluoro-CUMYL-5-fluoro-PICA are shown in Figure 4. The proposed metabolic structures are labelled M1-M10 and the transformation, m/z and Rt are presented in table 1.
Table 1. Metabolites, and their labelled Id, transformation, m/z, and Rt.
Metabolite Transformation m/z Rt
4F-CUMYL-5F-PICA Parent compound 385.2086 10.70
M1 Oxidative defluorination + O + Gluc 575.2400 5.50
M2 Fluoropentyl dealkylation + O 313.1347 6.23
M3 Monohydroxylation + Gluc 577.2356 6.79
M4 Oxidative defluorination to Carboxylation + O 413.1871 6.80
M5 Oxidative defluorination + O 399.2078 6.92
M6 Dihydroxylation + Gluc 593.2305 6.94
M7 Fluoropentyl dealkylation 297.1398 7.96
M8 Oxidative defluorination to Carboxylation 397.1922 8.40
M9 Oxidative defluorination 383.2130 8.65
M10 Monohydroxylation 401.2035 8.74
The MS/MS spectra for 4-fluoro-CUMYL-5-fluoro-PICA, and seven fragments are presented in Figure 5. The fragments are labelled A, B, C, D, E, F and G.
Figure 5. Parent MS/MS spectra of 4-fluoro-CUMYL-5-fluoro-PICA. Dashed arrows denote formation of possible fragments.
A A C C B 109.0447 E E D D F F 144.0443 G G B
The hydroxylated metabolites M10 (monohydroxylation), M3 (monohydroxylation + glucuronidation) and M6 (dihydroxylation + glucuronidation) are shown in Figure 6. For metabolite M10 six fragments are presented with m/z 109.0448, m/z 134,0600, m/z 137.0761, m/z 160.0393, m/z 222.1289 and m/z 248.1081. For metabolite M3 six fragments are presented with m/z 134,0600, m/z 137.0761, m/z 160.0393, m/z 222.1289, m/z 248.1081 and m/z 265.1347, and for metabolite M6 seven fragments are presented with m/z 109.0448, m/z 137.0761, m/z 176.0342, m/z 220.1132, m/z 238.1238, m/z 264.1030 and m/z 281.1296. M10 M3 M6
11
134.0589 265.1338 134.0594 160.0415 264.1010 + Glucuronide A B+O C D+O E+O F+O B+O C D+O E+O F+O G+O C D+2O -H2O E+2O A G+2O F+2O m/z 401.2035 m/z 593.2305 m/z 577.2356 + GlucuronideThe oxidative defluorinated metabolites M9 (oxidative defluorination), M5 (oxidative defluorination + O) and M1 (oxidative defluorination + O + glucuronidation) are presented in Figure 7. For metabolite M9 seven fragments are presented with m/z 109.0448, m/z 118.0651, m/z 137.0761, m/z 144.0444, m/z 186.1277, m/z 204.1383 and m/z 230.1176. For metabolite M5 seven fragments are presented with m/z 109.0448, m/z 134.0600, m/z 137.0761, m/z 160.0393, m/z 202.1226, m/z 220.1332 and m/z 246.1125. For metabolite M1 seven fragments are presented with m/z 109.0448, m/z 134.0600, m/z 137.0761, m/z 160.0393, m/z 202.1226, m/z 220.1332 and m/z 246.1125.
Figure 7. MS/MS spectra (Intensity on the Y-axis and m/z on the X-axis) of metabolites M9 (oxidative defluorination), M5 (oxidative defluorination + O) and M1 (oxidative defluorination + O + glucuronidation) on the left side along with their proposed structure and assignment of fragments on the right side.
M1
11
M5 M9 134.0589 134.0580 118.0639 144.0431 Glucuronide A B C D -H2O E-F+O F-F+O A B+O C -H2O D+O E-F+2O F-F+2O A B+O C -H2O D+O E-F+2O F-F+2O + m/z 575.2400 m/z 399.2078 m/z 383.2130The oxidative defluorination to carboxylation metabolites M8 (oxidative defluorination to carboxylation) and M4 (oxidative defluorination to carboxylation + O) are shown in Figure 8. For metabolite M8 seven fragments are presented with m/z 109.0448, m/z 118.0651, m/z 137.0761, m/z 172.1121, m/z 200.1070, m/z 218.1176 and m/z 244.0968. For metabolite M4 six fragments are presented with m/z 134.0600, m/z 137.0761, m/z 188.0706, m/z 216.1019, m/z 234.1125 and m/z 260.0917.
Figure 8. MS/MS spectra (Intensity on the Y-axis and m/z on the X-axis) of metabolite M8 (oxidative defluorination to carboxylation) and M4 (oxidative defluorination to carboxylation + O) on the left side along with their proposed structure and assignment of fragments on the right side. M8
11
M4 E-F+2O F-F+2O -H2O, -CO2 -CO C B A 134.0581 118.0649 B+O C D+C2H4O -H2O, D+C4H8O E-HF+3O F-F+3O m/z 413.1871 m/z 397.1922The fluoropentyl dealkylation metabolites M7 (fluoropentyl dealkylation) and M2 (fluoropentyl dealkylation + O) are shown in Figure 9. For metabolite M7 five fragments are presented with m/z 109.0448, m/z 118.0651, m/z 137.0761, m/z 144.0444 and m/z 161.0709. For metabolite M2 five fragments are presented with m/z 109.0448, m/z 134.0600, m/z 137.0761, m/z 160.0393 and m/z 177.0659.
Figure 9. MS/MS spectra (Intensity on the Y-axis and m/z on the X-axis) of metabolite M7 (fluoropentyl dealkylation)and M2 (fluoropentyl dealkylation + O) on the left side along with their proposed structure and assignment of fragments on the right side.
4.1.2 Summary of results
For 4-fluoro-CUMYL-5-fluoro-PICA a total of ten metabolites were identified, with three hydroxylations, two oxidative defluorinations to carboxylation, three oxidative defluorination and two fluoropentyl dealkylation. The metabolite with the highest intensity was oxidative defluorination.
M2 M7 137.0761 A D A C B 137.0742 G-C5H10F B+O C 160.0395 G-C5H10F+O F-C5H10F+O m/z 313.1347 m/z 297.1398
5. Discussion
This study was preformed to examine the characterization of the metabolites of a new psychoactive substance the synthetic cannabinoid 4-fluoro-CUMYL-5-fluoro-PICA. The identification of biotransformation and potential metabolites of new psychoactive substance, and mapping of metabolic pathways are included in the study. Ten metabolites were identified for compound 4-fluoro-CUMYL-5-fluoro-PICA, they are presented in Figure 3. The metabolites included two monohydroxylations, one dihydroxylations, two oxidative defluorinations to carboxylation, three oxidative defluorination and two fluoropentyl dealkylation (table 1, Appendix). The metabolic pathway and the characteristic fragments for 4-fluoro-CUMYL-5-fluoro-PICA seem to be very similar comparing to other synthetic cannabinoids, like CUMYL-PICA and 4-fluoro-CUMYL- BINACA from previous studies (Åstrand and Vikingsson with others 2018, Kevin and Lefever with others 2017), this indicates that the ten metabolites that was identified for 4-fluoro-CUMYL-5-fluoro-PICA are real metabolites.
The fragmentation of the parent compound was observed and identified in the MS/MS spectra, and seven fragment ions labelled A to G in Figure 5 was found to be characteristic for the parent. The fragment ions: m/z 109.0447 (A) corresponding to the fluorophenyl ion, m/z 118.0651 (B) to the indole group, m/z 137.0761 (C) to the cumyl group, m/z 144.0444 (D) to the indole acylium ion, m/z 206.1339 (E) to the 5-fluoropentyl indole, m/z 232.1132 (F) to the indole acylium ion with a fluoropentyl chain, and m/z 249.1398 (G) to the indole amide ion with a fluoropentyl chain. When these fragments are present in the metabolites MS/MS spectra, it helps to sort out modifications to respective metabolite structure.
5.1 Hydroxylated and dihydroxylated metabolites
The MS/MS spectrum for metabolite M10 (Figure 6) displayed the fragment ions m/z 109.0448, m/z 137.0761, which indicate an intact fluorophenyl and an intact cumyl group. M10 is hydroxylated at the indole, the presence of
fragment ions m/z 134.0600 and m/z 160.0393 strongly suggest that the hydroxy group is located at the indole. The metabolite M3 is showing the same fragments as M10 but with higher m/z and is proposed to be a glucuronide of M10. The MS/MS spectrum for metabolite M6 (Figure 6) showed fragment which excludes modifications at the cumyl group (m/z 137.0761). M6 is dihydroxylated at the indole, the presence of fragment ions m/z 150.0550 and m/z 176.0342 strongly suggest that the hydroxy groups is located at the indole. The fragment ion m/z 238.1238 indicate a 5-fluoropentyl dihydroxyindole ion, and water loss generated m/z 220.1132.
5.2 Metabolites generated by N-dealkylation
The MS/MS spectrum for metabolite M7 (Figure 9) displayed the fragment ions m/z 109.0448, m/z 118.0651, m/z 137.0761, and m/z 144.0444, which indicate an intact fluorophenyl group, an intact indole group and an intact cumyl group, respectively. The fragment ion at m/z 160.0393 was unique for M7 and correspond to the indole amide ion. The MS/MS spectrum of M2 (Figure 9) showed fragment which excludes modifications at the fluorophenyl group and at the cumyl group (m/z 109.0448, and m/z 137.0761). M2 is hydroxylated at the indole, the presence of fragment ions m/z 134.0600 and m/z 160.0393 strongly suggest that the hydroxy group is located at the indole. The unique fragment ion for metabolite M2 was m/z 177.0659, which correspond to the hydroxyindole amide ion.
5.3 Metabolites generated by oxidative defluorination
The MS/MS spectrum for metabolite M9 (Figure 7) displayed the fragment ions m/z 109.0448, m/z 118.0651, m/z 137.0761, and m/z 144.0444 which indicate an intact fluorophenyl group, an intact indole group and an intact cumyl group, respectively. The fragment ion m/z 204.1383 indicate an indole with a pentanol chain, and water loss
generated m/z 186.1277. The MS/MS spectrum for metabolite M5 (Figure 7) showed fragment which excludes modifications at the fluorophenyl group and at the cumyl group (m/z 109.0448, and m/z 137.0761). M5 is
hydroxylated at the indole, the presence of fragment ions m/z 134.0600 and m/z 160.0393 strongly suggest that the hydroxy group is located at the indole. The fragment ion m/z 220.1332 indicate a hydroxyindole with a pentanol chain, and water loss occurred during fragmentation (m/z 202.1332). The metabolite M1 (Figure 7.) is showing the same fragments as M5 but with higher m/z and is proposed to be a glucuronide of M5.
5.4 Metabolites generated by oxidative defluorination to carboxylation
The MS/MS spectrum for metabolite M8 (Figure 8) displayed fragment ions m/z 118.0651 and m/z 137.0761, which indicate an intact indole and an intact cumyl group. The fragment ion m/z 218.1175 indicate an indole with a
pentanoic acid chain, and water loss occurred during fragmentation (m/z 200.1070). The fragment ion m/z 244.0968 indicates an indole acylium ion with a pentanoic acid chain, and m/z 200.1070 was generated by CO2 loss. The fragment ion m/z 200.1070 can be generated through water loss or by CO2 loss. The ion m/z 172.1121 was generated by loss of CO. The MS/MS spectra for metabolite M4 (Figure 8) showed fragments which excludes modifications at the intact cumyl group (m/z 137.0761). M4 is hydroxylated at the indole, and the presence of fragment ion m/z 134.0600 strongly suggest that the hydroxy group is located at the indole. The fragment ion m/z 234.1125 indicates a hydroxyindole with a pentanoic acid chain, and water loss generated m/z 216.1019, this ion was also identified as an hydroxyindole acylium ion with a butyl chain, which shows that the fragmentation can occur in different ways for the same m/z. Fragmentation ion m/z 260.0917 indicates a indole acylium with a pentanoic acid chain, while ion m/z 188.0706 indicates a indole acylium with a ethanoic chain.
The difficulty of this study was to analyze all of data for the potential metabolites, there is always a little chance that a real metabolite was overlooked. To identify the metabolites, it is important to analyze all the parameters that are described in 3.4 Data analysis, but also to predict and understand the metabolism. There is also a possibility that metabolites not qualified by the parameters but could be real metabolites anyway. The metabolic pathway gives information of the metabolites structure, and information if metabolites possibly are missing, for example a monohydroxylated with glucuronidation metabolite should not exist without the corresponding monohydroxylated metabolite. The metabolic pathway is a good way of verifying observed metabolites and identify potential missing metabolites. In this study the metabolic pathway in Figure 4 shows that a dihyroxylated metabolite is possible missing, because it is very rare that a dihyroxylated + glucuronide metabolite is generated and the dihydroxylated metabolite are not. This shows that maybe the dehydroxylated metabolite is overlooked or it is a possibility for the dihydroxylated + glucuronidation metabolite to be generated without the dihydroxylated metabolite.
5.5 Impact
Through incubation of 4-fluoro-CUMYL-5-fluoro-PICA with cryopreserved human hepatocytes and LC-QTOF-MS analysis, we got a much better understanding of the metabolism of this drug. Such an understanding is important to develop new effective screening methods which in turn are important to the legal system. When a new psychoactive substance appears on the drug market toxicological screening is key for scheduling as a narcotic, to support the healthcare system, and for evidence in criminal cases, where a correct results could be the difference between a guilty or not guilty verdict.
5.6 Ethical implications
To understand the physiological effects on new psychoactive substances, further research is needed on the substances. And this could be considered important for the public as some compounds can be dangerous to the public health.
6. Conclusion
The number and diversity of synthetic cannabinoid increase in many parts of the world, and there is a concern for public health. The comparison of the metabolic pathway for 4-Fluoro-CUMYL-5-Fluoro-PICA and the synthetic cannabinoids CUMYL-PICA and 4-fluoro-CUMYL-BINACA shows that there is similarity to the metabolic pathways, and that it might be possible to predict the metabolism of future synthetic cannabinoids. Predicting metabolic pathways and metabolite structures is important for future cases.
The preformed metabolism study in human hepatocytes resulted in identification to a total of 10 metabolites. The major metabolites were produced by oxidative defluorination, oxidative defluorination to carboxylation,
fluoropentyl dealkylation, oxidative defluorination + O and monohydroxylation, and the minor metabolites were produced by dihydroxylation, fluoropentyl dealkylation + O, oxidative defluorination to carboxylation + O, and oxidative defluorination + O + glucuronide .Through the interpreting of the MS/MS spectra all the metabolite structures could be determined.
Acknowledgement
I am very grateful to have had the opportunity to do my thesis work at the National Board of Forensic Medicine. I would like to thank my tutors Henrik Green, Svante Vikingsson and Shimpei Watanabe for all your help and support in this project, and for making it possible for me to do a theoretical thesis work. I would also thank the great Friday meetings with the whole NPS-group.
I would like to thank Johan Dahlén, he has been a big part of my entire education and I am very grateful that Johan agreed to be my examiner.
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Appendix 1
Table 1. 4-fluoro-CUMYL-5-fluoro-PICA metabolites with assigned Id, biotransformation, formula, Rt, m/z value, peak area, and Ppm (min,
