Title: In vitro metabolic fate of nine LSD-based new psychoactive substances and their analytical detectability in different urinary screening procedures

Authors: Lea Wagmann, Lilian H. J. Richter, Tobias Kehl, Franziska Wack, Madeleine Pettersson Bergstrand, Simon D. Brandt, Alexander Stratford, Hans H. Maurer & Markus R. Meyer

This is a post-peer-review, pre-copyedit version of an article published in Analytical and Bioanalytical Chemistry (Volume 411, Pages 4751–4763(2019), DOI: 10.1007/s00216-018-1558-9 The final authenticated version is available online at: https://doi.org/10.1007/s00216-018-1558-9

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1 2 3 In vitro metabolic fate of nine LSD-based new psychoactive substances and 4 5 their analytical detectability in different urinary screening procedures 6 7 8 9 10 11 Lea Wagmann1, Lilian H. J. Richter1, Tobias Kehl1, Franziska Wack1, 12 13 Madeleine Pettersson Bergstrand1,2,3, Simon D. Brandt4, Alexander Stratford5, 14 15 Hans H. Maurer1, and Markus R. Meyer1 16 17 18 19 20 1 Department of Experimental and Clinical Toxicology, Institute of Experimental and 21 For Peer Review 22 Clinical Pharmacology and Toxicology, Center for Molecular Signaling (PZMS), 23 24 Saarland University, Homburg, Germany 25 26 2 27 Department of Laboratory Medicine, Division of Clinical Pharmacology, Karolinska 28 Institutet, Stockholm, Sweden 29 30 3 31 Department of Laboratory Medicine, Division of Clinical Chemistry, Karolinska 32 33 Institutet, Stockholm, Sweden 34 35 4 School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, 36 37 Liverpool, UK 38 39 5 Synex Synthetics BV, Maastricht, The Netherlands 40 41 42 43 Markus R. Meyer 44 [email protected] 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 7 of 49 Analytical & Bioanalytical Chemistry

1 2 3 Abstract 4 5 The market of new psychoactive substances (NPS) is characterized by a high turnover and 6 7 thus provides several challenges for analytical toxicology. The analysis of urine samples often 8 9 requires detailed knowledge about metabolism given that parent compounds may either be 10 11 present only in small amounts or may not even be excreted. Hence, knowledge of the 12 metabolism of NPS is a prerequisite for the development of reliable analytical methods. The 13 14 main aim of this work was to elucidate for the first time the pooled human liver S9 fraction 15 16 metabolism of the nine d-lysergic acid diethylamide (LSD) derivatives 1-acetyl-LSD (ALD- 17 52), 1-propionyl-LSD (1P-LSD), 1-butyryl-LSD (1B-LSD), N6-ethyl-nor-LSD (ETH-LAD), 18 19 1-propionyl-N6-ethyl-nor-LSD (1P-ETH-LAD), N6-allyl-nor-LSD (AL-LAD), N-ethyl-N- 20 21 cyclopropyl lysergamideFor (ECPLA), Peer (2’S,4’S)-lysergic Review acid 2,4-dimethylazetidide (LSZ), and 22 23 lysergic acid morpholide (LSM-775) by means of liquid chromatography coupled to high 24 resolution tandem mass spectrometry. Identification of the monooxygenase enzymes involved 25 26 in the initial metabolic steps was performed using recombinant human enzymes and their 27 28 contribution confirmed by inhibition experiments. Overall, N-dealkylation, hydroxylation, as 29 30 well as combinations of these steps predominantly catalyzed by CYP1A2 and CYP3A4 were 31 found. For ALD-52, 1P-LSD, and 1B-LSD deacylation to LSD was observed. The obtained 32 33 mass spectral data of all metabolites is essential for reliable analytical detection particularly in 34 35 urinalysis and for differentiation of the LSD-like compounds as biotransformations also led to 36 structurally identical metabolites. However, in urine of rats after the administration of 37 38 expected recreational doses and using standard urine screening approaches, parent drugs or 39 40 metabolites could not be detected. 41 42 43 44 45 KEYWORDS 46 47 48 Lysergamides, NPS, metabolism, urinalysis, LC-HRMS/MS 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 Introduction 4 5 6 The discovery of the potent psychoactive effects of d-lysergic acid diethylamide (LSD, see 7 Fig. 1) in 1943 triggered a new era in psychopharmacological research and subsequently 8 9 became a household name in popular culture [1]. For the purpose of extending the knowledge 10 11 of lysergamide-based structure-activity relationships, several LSD-based substances have 12 13 been investigated [2-9]. In recent years, a renewed interest in the use of LSD within the 14 clinical context emerged as documented by increasing numbers of studies involving human 15 16 volunteers [10-12]. At the same time, a number of LSD-based substances appeared as 17 18 ‘research chemicals’ on the new psychoactive substances (NPS) market, predominantly in the 19 20 form of blotters and powders. These substances included 1-acetyl-LSD (ALD-52), 1- 21 propionyl-LSD (1P-LSD)For [13], 1-butyryl-LSDPeer Review (1B-LSD), N6-ethyl-nor-LSD (ETH-LAD) 22 23 [14], 1-propionyl-ETH-LAD (1P-ETH-LAD) [14], N6-allyl-nor-LSD (AL-LAD) [15], N- 24 25 ethyl-N-cyclopropyl lysergamide (ECPLA) [16], (2’S,4’S)-lysergic acid 2,4-dimethylazetidide 26 (LSZ) [15], and lysergic acid morpholide (LSM-775) [17] (Fig. 1). 27 28 NPS in general were reported to the EU Early Warning System at a rate of one per week 29 30 in 2016 being a big issue in forensic or clinical toxicology and the health care system in 31 32 general [18]. The ability to detect newly emerging drugs of abuse in samples obtained from 33 biological matrices requires the development of reliable screening methods. Particularly the 34 35 analysis of urine samples benefits from knowing the drug’s metabolic transformation patterns, 36 37 especially in cases where the parent compound may only be present in small amounts or not 38 39 even be excreted. In case of LSD, its metabolite 2-oxo-3-hydroxy LSD was reported to be 40 present at higher concentrations and detectable for a longer time than LSD itself in urine 41 42 samples of LSD users [19]. The detection of 2-oxo-3-hydroxy LSD in urine has been 43 44 subsequently described as part of some clinical investigations [20, 21]. In vivo studies in 45 rodents or in vitro studies using human hepatocytes or human liver cell fractions such as 46 47 pooled human liver microsomes (pHLM) or pooled human liver S9 (pS9) are established tools 48 49 for metabolite formation and subsequent identification [22-25]. Several studies showed 50 51 comparable metabolic profiles to human urine samples [26-28]. To date, no information on 52 the phase I and II metabolism of the nine LSD derivatives investigated in this study is 53 54 available. Therefore, the aim of the present study was first to identify the main in vitro phase I 55 56 and II metabolites of ALD-52, 1P-LSD, 1B-LSD, ETH-LAD, 1P-ETH-LAD, AL-LAD, 57 58 ECPLA, LSZ, and LSM-775 for toxicological screening purposes using pS9 incubations and 59 subsequent analysis by liquid chromatography coupled to high resolution tandem mass 60 spectrometry (LC-HRMS/MS). LSD was also investigated and results compared to literature 3 Page 9 of 49 Analytical & Bioanalytical Chemistry

1 2 3 to aid development of the experimental conditions. In addition, the monooxygenase enzymes 4 5 involved in the initial metabolic steps were investigated to assess the potential impact of 6 7 interindividual variations that may occur during metabolism or as a consequence of drug-drug 8 and drug-food interactions. As no information about the monooxygenases involved in the 9 10 metabolism of LSD is available so far, it was also included in this study. Finally, detectability 11 12 studies using rat urine collected after the administration of expected recreational doses of the 13 14 LSD-based NPS and standard urine screening approaches (SUSAs) were conducted. 15 16 17 18 19 Experimental 20 21 Chemicals and enzymesFor Peer Review 22 23 24 ALD-52, 1P-LSD, 1B-LSD, ETH-LAD, 1P-ETH-LAD, AL-LAD, ECPLA, LSZ, and LSM- 25 26 775 were available from previous studies and provided by Synex Synthetics (Maastricht, The 27 Netherlands). LSD and LSD-d3 were supplied by LGC Standards (Wesel, Germany). 28 29 Isocitrate, isocitrate dehydrogenase, superoxide dismutase, 3’-phosphoadenosine-5’- 30 31 phosphosulfate (PAPS), S-(5’-adenosyl)-L-methionine (SAM), dithiothreitol (DTT), reduced 32 glutathione (GSH), carnitine acetyltransferase, acetylcarnitine, acetyl coenzyme A (AcCoA), 33 34 magnesium chloride (MgCl2), potassium dihydrogenphosphate (KH2PO4), dipotassium 35 36 hydrogenphosphate (K2HPO4), Tris hydrochloride, ketoconazole, and alpha-naphthoflavone 37 + 38 from Sigma-Aldrich (Taufkirchen, Germany), and NADP were from Biomol (Hamburg, 39 Germany). Acetonitrile (LC-MS grade), ammonium formate (analytical grade), formic acid 40 41 (LC-MS grade), methanol (LC-MS grade), and all other chemicals and reagents (analytical 42 43 grade) were from VWR (Darmstadt, Germany). The baculovirus-infected insect cell 44 microsomes (Supersomes) containing 1 nmol/mL of human cDNA-expressed CYP1A2, 45 46 CYP2A6, CYP2B6, CYP2C8, CYP2C9 (2 nmol/mL), CYP2C19, CYP2D6, CYP2E1 47 48 (2 nmol/mL), CYP3A4, CYP3A5 (2 nmol/mL), or FMO3 (5 mg protein/mL), and pHLM 49 50 (20 mg microsomal protein/mL, 330 pmol total CYP/mg protein), pS9 (20 mg microsomal 51 protein/mL), UGT reaction mixture solution A (25 mM UDP-glucuronic acid), and UGT 52 53 reaction mixture solution B (250 mM Tris HCl, 40 mM MgCl2, and 125 µg/mL alamethicin) 54 55 were obtained from Corning (Amsterdam, The Netherlands). After delivery, the enzymes and 56 57 liver cell preparations were thawed at 37 °C, aliquoted, snap-frozen in liquid nitrogen, and 58 stored at -80 °C until use. LSD and all other lysergamide test drugs were dissolved in 59 60 methanol at a concentration of 1 mg/mL, each, and stored at -20 °C.

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1 2 3 4 5 6 In vitro incubations for metabolism studies 7 8 As previously described by Richter et al. [29], the final incubation volume was 150 µL. 9 10 Incubations were performed using pS9 (2 mg microsomal protein/mL) after preincubation for 11 12 10 min at 37 °C with 25 µg/mL alamethicin (UGT reaction mixture solution B), 90 mM 13 2+ + 14 phosphate buffer (pH 7.4), 2.5 mM Mg , 2.5 mM isocitrate, 0.6 mM NADP , 0.8 U/mL 15 isocitrate dehydrogenase, 100 U/mL superoxide dismutase, 0.1 mM AcCoA, 2.3 mM 16 17 acetylcarnitine, and 8 U/mL carnitine acetyltransferase. Thereafter, 2.5 mM UDP-glucuronic 18 19 acid (UGT reaction mixture solution A), 40 µM PAPS, 1.2 mM SAM, 1 mM DTT, 10 mM 20 GSH, and 25 µM substrate (LSD or analog) were added. All given concentrations are the final 21 For Peer Review 22 concentrations in one reaction tube. The organic solvent content in the final incubation 23 24 mixtures was always below 1% [30]. The reaction was initiated by addition of substrate and 25 26 the reaction mixture was incubated for a maximum of 480 min. After 60 min, an aliquot of 27 60 µL of the incubation mixture was transferred to a reaction tube containing 20 µL ice-cold 28 29 acetonitrile for termination of the reactions. The remaining mixture was incubated for 30 31 additional 420 min and thereafter stopped by addition of 30 µL ice-cold acetonitrile. 32 33 Afterwards, mixtures were cooled for 30 min at -18 °C, centrifuged for 2 min at 10.000 x g, 34 and a volume of 60 µL of the supernatants transferred into autosampler vials, followed by 35 36 injection of 1 µL onto the LC-HRMS/MS system. Blank samples without substrate and 37 38 control samples without pS9 were prepared to confirm the absence of interfering compounds 39 and to identify compounds not formed by metabolism, respectively. All incubations were 40 41 performed in duplicates (n = 2). 42 43 44 45 46 Monooxygenases activity screening and inhibition studies using selective CYP inhibitors 47 48 49 According to previously published procedures [31], microsomal incubations (n = 2 each) were 50 51 performed at 37 °C for 30 min using a substrate concentration of 25 µM (LSD or analog) and 52 CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, 53 54 CYP3A5 (75 pmol/mL each), or FMO3 (0.25 mg protein/mL). Reference incubations with 55 56 pHLM (1 mg microsomal protein/mL) were used as positive control. Control samples without 57 58 enzymes were prepared to assess formation of compounds that did not originate from 59 metabolism. Besides enzymes and substrates, the incubation mixtures (final volume, 50 µL) 60 contained 90 mM phosphate buffer (pH 7.4), 5 mM Mg2+, 5 mM isocitrate, 1.2 mM NADP+, 5 Page 11 of 49 Analytical & Bioanalytical Chemistry

1 2 3 0.5 U/mL isocitrate dehydrogenase, and 200 U/mL superoxide dismutase. For incubations 4 5 with CYP2A6 or CYP2C9, phosphate buffer was replaced with 90 mM Tris buffer, 6 7 respectively, according to the manufacturer’s recommendation. Reactions were initiated by 8 addition of the enzyme preparation and terminated by addition of 50 µL of ice-cold 9 10 acetonitrile, containing 5 µM LSD-d3 as internal standard. The mixture was centrifuged for 11 12 2 min at 18,407 x g (rcf). Seventy µL of the supernatant were transferred into an autosampler 13 14 vial and 1 µL was injected onto the LC-HRMS/MS system. 15 According to a previous study [32], the influence of ketoconazole (CYP3A4 inhibitor), alpha- 16 17 naphthoflavone (CYP1A2 inhibitor), or a mixture of them (1 µM, respectively) on the 18 19 formation of monooxygenase-dependent metabolites was assessed using pHLM (1 mg 20 microsomal protein/mL), 25 µM substrate (LSD or one of the analogs), and LSD-d as 21 For Peer Review 3 22 internal standard. All incubations were performed at 37 °C for 20 min with six replicates in 23 24 each test set. All other incubation settings were the same as described for the monooxygenase 25 26 activity screening. As measure of the metabolite formation in reference incubations without 27 inhibitor, peak areas of all metabolites were summed up and divided by the peak area of the 28 29 internal standard. This was also done for control incubations without pHLM and peak area 30 31 ratios were subtracted from that in reference incubations. The result was referred to as 100% 32 33 and compared to peak area ratios in test incubations with inhibitor. Significance of inhibition 34 was tested using a one-way ANOVA followed by Dunnett's multiple comparison test 35 36 (significance level, P < 0.001, 99.9% confidence intervals) by GraphPad Prism 5.00 software 37 38 (GraphPad Software, San Diego, USA). 39 40 41 42 43 LC‐HRMS/MS instrumentation 44 45 A Thermo Fisher Scientific (TF, Dreieich, Germany) Dionex UltiMate 3000 Rapid Separation 46 47 (RS) UHPLC system with a quaternary UltiMate 3000 RS pump and an UltiMate 3000 RS 48 49 autosampler was used, controlled by the TF Chromeleon software version 6.80, and coupled 50 51 to a TF Q-Exactive Plus equipped with a heated electrospray ionization II source (HESI-II). 52 Mass calibration was performed prior to analysis according to the manufacturer’s 53 54 recommendations using external mass calibration. Gradient elution was performed on a TF 55 56 Accucore PhenylHexyl column (100 mm x 2.1 mm inner diameter, 2.6 µm particle size). The 57 mobile phases consisted of 2 mM aqueous ammonium formate containing formic acid (0.1%, 58 59 v/v) and acetonitrile (1%, v/v, pH 3, eluent A) and 2 mM ammonium formate in 60 acetonitrile/methanol (50:50, v/v) containing formic acid (0.1%, v/v) and water (1%, v/v, 6 Analytical & Bioanalytical Chemistry Page 12 of 49

1 2 3 eluent B). The gradient and flow rate were programmed as follows: 0-10 min 10% B to 50% 4 5 B, 10-12 min hold 98% B, and 12-14 min hold 10% B, constantly at a flow rate of 6 7 0.5 mL/min. HESI-II conditions have been employed as described previously by Wagmann et 8 al. [33]: heater temperature, 438 °C; ion transfer capillary temperature, 269 °C; sheath gas, 53 9 10 arbitrary units (AU); auxiliary gas, 14 AU; sweep gas, 3 AU; spray voltage, 3.50 kV, and S- 11 12 lens RF level, 60.0. Mass spectrometric analysis was performed in positive full scan mode 13 2 14 and targeted MS mode using an inclusion list. Pick others mode was activated to ensure the 15 recording of MS2 spectra of precursor ions not in the inclusion list. The settings for full scan 16 17 data acquisition were as follows: resolution, 35 000; automatic gain control (AGC) target, 18 19 1e6; maximum injection time (IT), 120 ms; scan range, m/z 100-700. The settings for the 20 targeted MS2 mode using an inclusion list were as follows: resolution, 17 500; AGC target, 21 For Peer Review 22 2e5; maximum IT, 250 ms; isolation window, m/z 1.0; high collision dissociation cell with 23 24 stepped normalized collision energy, 17.5, 35.0, 52.5. TF Xcalibur Qual Browser software 25 26 version 2.2 SP1.48 was used for data evaluation. 27 28 29 30 31 Collection of rat urine samples for detectability studies 32 33 In accordance to previous publications [34], the in vivo studies were performed using rat 34 35 urine samples from male Wistar rats (Charles River, Sulzfeld, Germany) for toxicological 36 37 diagnostic reasons according to corresponding German law (Bundesministerium der Justitz 38 und für Verbraucherschutz, http://www.gesetze-im-internet.de/tierschg/index.html). Before 39 40 drug administration, blank urine was collected over a period of 24 h. Animals had water ad 41 42 libitum during collection of urine over a 24 h period and were housed in a metabolism cage. 43 44 Urine was collected separated from feces. For the detectability studies, doses of 0.01 mg/kg 45 body weight (BW) were administered. The urine samples were analyzed directly and 46 47 remaining was aliquoted and stored at -20 °C. 48 49 50 51 52 Rat urine analysis by LC-HRMS/MS, LC-linear ion trap MS, and gas chromatography- 53 54 MS 55 56 57 To check the detectability and analytical differentiation of the LSD-based NPS after estimated 58 recreational user´s dose administrations, three SUSAs were performed. The settings were 59 60 used as described in the given references for the LC-HRMS/MS [35], LC linear ion trap MS

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1 2 3 (LC-MSn) [36, 37], and gas chromatography (GC)-MS [38, 39] SUSAs. Briefly, the following 4 5 sample preparations were used: a simple urine precipitation with acetonitrile for the LC-based 6 7 SUSAs and a liquid-liquid extraction after acidic hydrolysis followed by and acetylation for 8 the GC-MS SUSA. Detection limits for LSD and its derivatives by the aforementioned 9 10 methods was studied using methanolic pure substance solutions at various concentrations 11 12 (0.1, 1, 10, and 100 ng/mL). The lowest concentration resulting in an MS2 spectrum was 13 14 defined as limit of identification (LOI). 15 16 17 18 Results and discussion 19 20 Identification of metabolites 21 For Peer Review 22 23 Human metabolites of LSD were already described [19, 40-43]. To confirm suitable 24 25 incubation conditions and detection methods concerning the in vitro assay, LSD was also 26 incubated with pS9 and analyzed by LC-HRMS/MS. In general, MS1 data was screened for 27 28 potential exact precursor masses (PM) of expected metabolites. Afterwards, the fragmentation 29 30 pattern in the MS2 spectrum was interpreted and compared to that of the parent compound for 31 32 confirmation and possible assignment of the position. This procedure was the same for LSD 33 and its analogs. 34 35 The lysergamide test drugs and all identified phase I and II metabolites are listed in 36 37 Table 1, which contains their ID, the calculated exact mass of the protonated precursor 38 (M+H+), elemental composition, determined retention time (RT) and three most abundant 39 40 fragment ions (FI) in MS2. In total, the pS9 incubations resulted in detection of several 41 42 tentative metabolites. LSD: six metabolites; ALD-52: seven metabolites; 1P-LSD: eight 43 44 metabolites; 1B-LSD: seven metabolites; ETH-LAD: five metabolites; 1P-ETH-LAD: seven 45 metabolites; AL-LAD: eleven metabolites; ECPLA: eight metabolites; LSZ: nine metabolites, 46 47 and LSM-775: four metabolites. 48 49 The LSD metabolites were comparable to those described before: N-deethyl LSD (ID 2, 50 6 51 also referred to as lysergic acid monoethylamide, LAE) and N -demethyl LSD (ID 3, also 52 referred to as nor-LSD) could be unambiguously identified. Two hydroxy LSD isomers were 53 54 detected, but it has to be stated that the final positions of the hydroxy groups could not be 55 56 determined based on fragmentation patterns. However, hydroxy LSD isomer 2 (ID 5) and the 57 corresponding glucuronide (ID 7) are expected to be the same as described by Steuer et al. in 58 59 plasma samples [42] based on their fragmentation. Dihydroxy LSD (ID 6) is expected to be 60 the same compound referred to as 2-oxo-3-hydroxy LSD elsewhere, which is widely known 8 Analytical & Bioanalytical Chemistry Page 14 of 49

1 2 3 as main metabolite of LSD [19, 20, 44-46]. Only the hydroxy LSD isomer with the hydroxy 4 5 group located at the diethylamide part of the molecule (also referred to as lysergic acid ethyl- 6 7 hydroxy ethylamide, LEO) and trihydroxy LSD were described to be present as minor signals 8 in human plasma or urine but not detected in the in vitro pS9 incubations. However, it should 9 10 be kept in mind that quantitative determinations of the metabolites' concentrations were 11 12 beyond the scope of this study. Under the investigated conditions, these two metabolites 13 14 might have been formed in concentrations below detectability or the chosen incubation time 15 was not sufficient for their formation, as multistep reactions were already described to need a 16 17 longer incubation time [47]. Furthermore, LSD hydroxy isomer 1 (ID 4) was expected to be 18 19 hydroxylated in position 4 or 5 due to the fragmentation pattern, which was not described 20 before (discussed in detail below for 1-depropionyl hydroxy 1P-LSD isomer 1, ID 4). Iso- 21 For Peer Review 22 LSD, a diastereomer of LSD, which is formed during the production of LSD under basic 23 24 conditions, was previously used as additional marker for LSD consumption and shown to be 25 26 metabolized in the human body. The spectra of LSD and iso-LSD differed only in the 27 intensity of particular FI [42]. For several LSD-based NPS, a smaller signal with the same 28 29 mass could be detected eluting after the parent compound. The MS2 only showed differences 30 31 in the intensity of some FI and therefore, these signals were expected to be generated by the 32 33 iso-forms. However, due to the lack of reference material, their identity could not be 34 confirmed. 35 36 Due to the large number of LSD-based drugs used during this study, some 37 38 representative examples were chosen to illustrate the general identification procedure. 39 Furthermore, all compounds and their metabolites showed comparable fragmentation patterns. 40 41 The procedure used for the tentative identification of the metabolites based on the HRMS/MS 42 43 spectra was employed according to the example described for 1P-LSD (ID 11). Brandt et al. 44 2 45 previously published an MS spectrum of 1P-LSD recorded using electrospray ionization 46 accurate mass quadrupole time of flight tandem mass spectrometry [13]. The observed 47 48 fragmentation pattern corresponded to the MS2 spectrum of 1P-LSD using orbitrap-based 49 2 50 tandem mass spectrometry. The MS spectra of LSD (ID 1) and 1P-LSD (ID 11) can be found 51 in Fig. 2. Briefly, the FI at m/z 337.1910 (C H N O +) formed after loss of N- 52 21 25 2 2 53 + methylmethanimine (C2H5N) and the FI at m/z 279.1491 (C18H19N2O ) formed after loss of 54 + 55 N,N-diethylformamide (C5H11NO) from the protonated molecule (C23H30N3O2 ) were 56 2 57 characteristic for 1P-LSD and allowed for a differentiation of the MS spectrum of LSD since 58 both FI carried the additional 1-propionyl moiety (C H O). These FI correspond to the FI at 59 3 5 + + 2 60 m/z 281.1648 (C18H21N2O ) and at m/z 223.1229 (C15H15N2 ) detected in the MS spectrum of

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1 2 3 LSD (Fig. 2). The last-named FI (m/z 223.1229) represented the N6-methylated 9,10- 4 5 didehydroergoline core and was the FI with the highest abundance in the MS2 spectrum of 1P- 6 1 7 LSD, most probably formed after elimination of the N -propionyl moiety from the FI at m/z 8 279.1491. Also, the FI at m/z 208.0756 (C H NO+) had a high abundance and was also 9 14 10 10 detectable in the MS2 spectrum of LSD. However, it has to be mentioned that a second FI 11 + 12 with the nominal mass of m/z 208 but the exact mass of at m/z 208.0995 (C14H12N2 ) was 13 14 detectable with a comparable abundance to 208.0756 in both spectra, which was most 15 probably formed after loss of methyl (CH3) from FI at m/z 223.1229. In addition, the FI at m/z 16 + + 17 128.1069 (C7H14NO ) and m/z 74.0964 (C4H12N ) representing the diethylamide part were 18 19 detectable in the spectra of 1P-LSD and LSD. The HRMS/MS spectra of all parent 20 compounds and the most abundant, unique metabolites after pS9 incubation allowing the 21 For Peer Review 22 identification of the parent compound (with exception of the ETH-LAD metabolite that could 23 24 also be formed after intake of 1P-ETH-LAD) can be found in the electronic supplementary 25 26 material in Fig. S1. 27 In total, eight metabolites of 1P-LSD were identified in the pS9 incubations (see Table 28 29 1). 1-Depropionylation led to the formation of LSD (ID 1, PM at m/z 324.2070). Therefore, 30 31 1P-LSD can be considered as LSD prodrug, which was consistent with preliminary data 32 33 reported by Brandt et al. who incubated 1P-LSD in human serum [13]. An additional N- 34 deethylation led to formation of 1-depropionyl-N-deethyl 1P-LSD (ID 2, PM at m/z 35 36 296.1757). The loss of an ethylene group (- 28.0313 u, C2H4) resulted in the FI at m/z 37 + 2 38 253.1335 (C16H17N2O ) in comparison to m/z 281.1648 in the MS spectrum of LSD. Other 39 FI, for example at m/z 223.1229 or m/z 208.0756 remained unchanged, whereas the FI at m/z 40 41 74.0964 was not detectable in the MS2 spectrum of 1-depropionyl-N-deethyl 1P-LSD (ID 2). 42 43 N6-demethylation instead of N-deethylation resulted in formation of 1-depropionyl-N6- 44 45 demethyl 1P-LSD (ID 3, PM at m/z 310.1913). The FI at m/z 223.1229 was shifted to m/z 46 + 209.1073 (C14H13N2 ) corresponding to the loss of methylene (- 14.0156 u, CH2). Other FI, 47 48 for example at m/z 281.1648 or m/z 74.0964 were unchanged compared to LSD. Two isomers 49 50 of 1-depropionyl-hydroxy 1P-LSD (ID 4 and 5, PM at m/z 340.2019) were detectable. In the 51 MS2 spectrum of isomer 1 (ID 4, RT 3.4 min, Fig. 2), an initial loss of water (- 18.0105 u, 52 53 H2O) was detectable. Therefore, 1-depropionyl-hydroxy 1P-LSD isomer 1 was expected to 54 55 carry an aliphatic hydroxy group. Based on the fragmentation pattern, a hydroxylation at the 56 2 57 diethylamide part can be excluded because the FI at m/z 74.0964 was present in the MS 58 spectrum. Due to the detection of m/z 297.1597 (C H N O +), a hydroxylation at the N6- 59 18 21 2 2 60 methyl group and position 7 can be excluded, which led to the indication that the hydroxy

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1 2 3 group might have been located at position 4 or 5. However, an unambiguous assignment was 4 5 not possible based on the fragmentation pattern in the MS2 spectrum. In case of 1- 6 7 depropionyl-hydroxy 1P-LSD isomer 2 (ID 5, RT 3.6 min) no water loss was detectable and 8 the hydroxy group should therefore most likely be located at an aromatic position [48]. The FI 9 10 at m/z 74.0964 and m/z 297.1597 were both present. Due to these FI, the hydroxy group is 11 12 most probably located at the indole part of the 9,10-didehydroergoline core. Furthermore, 1- 13 14 depropionyl-dihydroxy 1P-LSD (ID 6, PM at m/z 356.1968) was detected with one aliphatic 15 and one aromatic hydroxy group. Due to the fragmentation pattern, this metabolite could be 16 17 the result of the combination of both monohydroxy isomers (ID 4 and 5). However, the 18 19 metabolite 2-oxo-3-hydroxy LSD was reported to be the main urinary excretion product of 20 LSD [19, 41]. Possible metabolic pathways resulting in its formation were discussed by Klette 21 For Peer Review 22 et al. [40]. Nevertheless, its protonated mass at m/z 356 corresponds to the mass of 1- 23 24 depropionyl-dihydroxy 1P-LSD (ID 6). Also the prominent FI at m/z 237 and m/z 338 25 26 described by Klette et al. were present in the spectrum of 1-depropionyl-dihydroxy 1P-LSD 27 (ID 6). Due to this fact and the proposed fragmentation pattern, both compounds were 28 29 expected to be identical. The aforementioned metabolites of 1P-LSD were also found in 30 31 incubations with LSD and are therefore not suitable as urinary markers to discriminate 32 33 between the intake of LSD and 1P-LSD. In general, structurally identical metabolites have the 34 same ID in Table 1 to highlight these observations. Nevertheless, two 1P-LSD metabolites not 35 36 evolved from LSD were identified. These metabolites were formed from 1P-LSD by N- 37 6 38 deethylation (ID 12, PM at m/z 352.2019) and N -demethylation (ID 13, PM at m/z 366.2176), 39 respectively. In case of N-deethyl 1P-LSD (ID 12, Fig. 2), the FI at m/z 309.1597 40 + 41 (C19H21N2O2 ) corresponded to the FI at m/z 337.1910 in the spectrum of 1P-LSD shifted by - 42 43 28.0313 u, the loss of an ethylene group (C2H4). The FI at m/z 279.1491 was unchanged. In 44 6 45 case of N -demethyl 1P-LSD (1P-nor-LSD) (ID 13), the FI at m/z 279.1491 was shifted by - 46 14.0156 u due to the loss of methylene (CH2) while the FI at m/z 337.1910 was unchanged. 47 48 However, it is possible that N6-demethyl 1P-LSD (ID 13) was also generated during the 49 50 metabolic transformation of 1P-ETH-LAD although it was not detected in the pS9 51 52 incubations. In conclusion, N-deethyl 1P-LSD (ID 12) was the only metabolite that could only 53 be formed from 1P-LSD and from no other LSD-type NPS investigated in this study. LSD 54 55 could also be formed after intake of ALD-52 or 1B-LSD and in both cases, LSD and its 56 57 metabolites were detected in the pS9 incubations. In case of ALD-52, the two specific 58 metabolites N-deethyl ALD-52 (ID 9, PM at m/z 338.1863) and N6-demethyl ALD-52 (nor- 59 60 ALD-52) (ID 10, PM at m/z 352.2019) were additionally identified and in case of 1B-LSD,

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1 2 3 only hydroxy 1B-LSD (ID 15, PM at m/z 410.2438) was detected. These findings clearly 4 5 demonstrated that urinary screening results have to be interpreted with care and that a positive 6 7 urine screening result for LSD or its metabolites must not always be caused by an intake of 8 LSD itself. 9 10 11 12 13 Proposed metabolic pathways 14 15 16 The proposed metabolic pathways of 1P-LSD are given in Figure 3. The proposed metabolic 17 2 18 pathways and MS data of the other compounds can be found in the electronic supplementary 19 20 material (Fig. S1-S10). 21 For Peer Review 22 23 24 25 Monooxygenases activity screening and inhibition studies using selective CYP inhibitors 26 27 In order to investigate the ability of monooxygenases to catalyze the initial metabolic phase I 28 29 steps, an activity screening study using the ten most important CYP isoenzymes and FMO3 30 31 was performed. Incubations with pHLM were used as control. These data are only qualitative 32 and do not reflect a quantitative contribution of the investigated monooxygenases to hepatic 33 34 clearance. Such an assessment requires the collection of enzyme kinetic data or inhibition 35 36 experiments [31, 32, 49]. Results are listed in Table 2. CYP3A4 was found to be involved in 37 6 38 the N -demethylation of LSD, ALD-52, 1P-LSD, 1B-LSD, ECPLA, LSZ, and LSM-775, but 39 also in the N6-deethylation of ETH-LAD and 1P-ETH-LAD and N6-deallylation of AL-LAD. 40 41 Only in case of LSD, CYP2C19 showed additional N6-demethylation activity. CYP3A4 also 42 43 catalyzed the N-deethylation of LSD and all other lysergamide test drugs, which resulted in 44 the formation of N-mono ethylamide analog. Only CYP2C9 also catalyzed the N-deethylation 45 46 of LSD and ECPLA. Aliphatic or aromatic hydroxylations were also catalyzed by CYP3A4 47 48 for all investigated test drugs and additionally by CYP1A2 with the exception of the 1- 49 50 acylated LSD derivatives and ETH-LAD. CYP2D6 was found to be involved in the 51 hydroxylation of ALD-52, 1B-LSD, and ETH-LAD. In pHLM incubations, the 1-deacylation 52 53 of ALD-52, 1P-LSD, 1B-LSD, and 1P-ETH-LAD was additionally observed. However, this 54 55 step was expected to be catalyzed by amidases and was not observed in incubations with the 56 57 recombinant monooxygenases. In pHLM incubations with AL-LAD, ECPLA, and LSZ, also 58 the corresponding lysergic acid metabolite was detected, and in case of ECPLA and LSZ, also 59 60

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1 2 3 lysergic acid amide. However, these metabolites could not be detected in incubations with 4 5 recombinant enzymes and may also be the result of amidase-catalyzed reactions. 6 7 The involvement of CYP1A2 and especially CYP3A4 in the metabolism of the investigated 8 lysergamides is noteworthy. To confirm these results and to assess the importance of their 9 10 contribution to the metabolism of these substances in the human liver, pHLM incubations in 11 12 presence of selective CYP inhibitors were performed [32]. LSD, 1B-LSD, ETH-LAD, AL- 13 14 LAD, ECPLA, LSZ, and LSM-775 were incubated with the CYP1A2 inhibitor alpha- 15 naphthoflavone, the CYP3A4 inhibitor ketoconazole or a mixture of them. For ALD-52, 1P- 16 17 LSD, 1B-LSD, ETH-LAD, and 1P-ETH-LAD, only ketoconazole was used because only 18 19 CYP3A4 was shown to be involved in their initial metabolic steps. Results are summarized in 20 Figure 4. Formation of CYP-dependent metabolites in reference incubations without inhibitor 21 For Peer Review 22 were set to 100% and compared to metabolite formation in incubations with inhibitor. For all 23 24 test drugs, a significant reduction of metabolite formation in incubations with inhibitors was 25 26 observed. In case of LSD, co-incubations with alpha-naphthoflavone decreased the metabolite 27 formation by more than 50% and with ketoconazole and a mixture of both inhibitors by more 28 29 than 80% in comparison to the incubations without inhibitor. Metabolite formation in co- 30 31 incubations of the 1-acylated lysergamides (ALD-52, 1P-LSD, 1B-LSD, and 1P-ETH-LAD) 32 33 with ketoconazole was reduced by more than 80%. In case of ETH-LAD, ketoconazole led to 34 a reduction of the metabolite formation by around 70%. The formation of AL-LAD, ECPLA, 35 36 and LSZ metabolites was significantly increased by both inhibitors, alpha-naphthoflavone and 37 38 ketoconazole, and co-incubations with their mixture resulted in a decrease of more than 70% 39 in case of AL-LAD and around 90% for ECPLA and LSZ. Only in case of LSM-775, co- 40 41 incubations with alpha-naphthoflavone did not result in a statistically significant reduction of 42 43 formed metabolites, in contrast to co-incubations with ketoconazole and their mixture that led 44 45 to decreased metabolite formation of around 70% or 80%, respectively. Residual metabolite 46 formations were most probably caused by incomplete enzyme inhibition and/or the 47 48 involvement of further CYP isoforms in the metabolic transformation of the test drugs. 49 50 Nevertheless, the inhibition experiments confirmed the importance of CYP1A2 and especially 51 52 CYP3A4 in the metabolism of LSD analogs. CYP1A2 is known to be expressed 53 polymorphically in humans, resulting in large interindividual variation of activity [50]. For 54 55 example, polymorphisms resulting in lower CYP1A2 activity may lead to a decreased 56 57 metabolic transformation of LSD-like substances that could result in an increased half-life of 58 the drug. However, the results of the inhibition experiments suggest that CYP3A4 is even 59 60 more important in the metabolism of LSD-related substances. Inhibition of CYP3A4 by co-

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1 2 3 consumed drugs or food ingredients may also lead to decreased metabolic transformation. 4 5 Nevertheless, LSD is not expected to exhibit remarkable acute toxicity [51, 52], but nothing is 6 7 known about the toxicity of the LSD-based NPS. 8 9 10 11 12 Toxicological detectability 13 14 For toxicological detectability studies, a dose of 0.01 mg/kg BW was administered to rats. 15 16 Assuming a human BW of 60 kg, the doses corresponded to an expected recreational dose of 17 n 18 136 µg in accordance to the dose by factor approach [53]. The SUSAs by GC-MS, LC-MS , 19 20 and LC-HRMS/MS were used and the results are briefly described in the following. 21 For Peer Review 22 23 24 25 GC-MS standard urine screening approach 26 27 No metabolites or parent compounds could be identified in the rat urine by the GC-MS 28 29 SUSA. This was most probably caused by the low stability of LSD and its derivatives against 30 31 acidic hydrolysis and heat [54]. Nevertheless, the sample preparation was not changed as this 32 is an established procedure used in toxicological routine analysis [55, 39]. Other reason could 33 34 be minor excretion in rat urine and/or insufficient sensitivity of the used GC-MS apparatus. 35 36 Also at and below 100 ng/mL of methanolic pure substance solutions, the GC-MS procedure 37 38 was not able to detect the analytes. 39 40 41 42 n 43 LC-MS standard urine screening approach 44 45 No metabolites or parent compounds could be identified in the rat urine by the LC-MSn 46 47 SUSA. most probably caused by insufficient sensitivity. The LOIs using methanolic pure 48 49 substance solutions were as follows: 1 ng/mL for 1P-LSD, 10 ng/mL for LSD, ALD-52, 50 ETH-LAD, AL-LAD, ECPLA, LSZ, and LSM-775, and 100 ng/mL for 1B-LSD and 1P- 51 52 ETH-LAD. One reason for the lack of sensitivity might be the extremely broad peaks under 53 54 the used standard conditions. 55 56 57 58 59 LC-HRMS/MS standard urine screening approach 60

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1 2 3 Only a single ALD-52 metabolite, 1-deacetyl-N-deethyl ALD-52 (ID 2), was identified in the 4 5 rat urine samples after a recreational user´s dose. However, this metabolite is not unique and 6 7 can also be formed after intake of LSD, 1P-LSD, or 1B-LSD. No other metabolites or parent 8 compounds could be identified by the LC-HRMS/MS SUSA. The LOIs using methanolic 9 10 pure substance solutions were as follows: 1 ng/mL for LSD, 1P-LSD, ETH-LAD, AL-LAD, 11 12 ECPLA, and LSZ, and 10 ng/mL for ALD-52, 1B-LSD, 1P-ETH-LAD, and LSM-775. 13 14 Therefore, for detection and differentiation of an intake of LSD or its derivatives after 15 expected recreational doses, a more selective sample workup, concentration step, and more 16 17 selective MS settings, may help in detecting the parent drugs and/or unique metabolites 18 19 identified in this study. For example solid-phase extraction by mixed-mode cartridges, 20 optionally after enzymatic conjugate cleavage, followed by LC-HRMS/MS using an adapted 21 For Peer Review 22 separation method and selective product ion scan or multiple-reaction monitoring should meet 23 24 these requests. 25 26 27 28 29 Conclusions 30 31 32 The present study demonstrated that ALD-52, 1P-LSD, 1B-LSD, ETH-LAD, 1P-ETH-LAD, 33 AL-LAD, ECPLA, LSZ, and LSM-775 were biotransformed to several phase I and few phase 34 35 II metabolites in vitro. Many metabolites can originate from different parent compounds 36 37 making their differentiation difficult, but this study also identified unique metabolites 38 overcoming this problem. The mass spectral data obtained during these studies is essential for 39 40 establishing reliable detection methods, especially for urinalysis. However, selective sample 41 42 workup and MS settings are needed for reliable analytical detection and differentiation. 43 44 CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 were found to be involved in the 45 initial metabolic steps. Inhibition experiments confirmed the major influence of CYP1A2 and 46 47 CYP3A4, therefore genetic polymorphisms of CYP1A2 might have an impact on the 48 49 metabolism of these NPS. Furthermore, drug-drug or drug-food interactions might occur in 50 51 case of co-consumption with CYP1A2 or CYP3A4 inhibitors resulting in decreased metabolic 52 transformation of LSD analogs, but further investigations are needed for an assessment of 53 54 possible clinical effects. The authors´ standard urine screening procedures are most likely not 55 56 able to allow the detection of the studied NPS. Alternative analytical strategies need to be 57 used instead. 58 59 60

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1 2 3 Acknowledgement 4 5 6 The authors would like to thank Armin A. Weber and the “Stiftelsen för Klinisk farmakologi 7 & farmakoterapi” for support. 8 9 10 11 12 13 Compliance with ethical standards 14 15 Conflict of interest: The authors declare that there are no conflicts of interest. 16 17 18 19 20 Electronic supplementary material 21 For Peer Review 22 23 Electronic supplementary material may be found online. 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 Table 1: LSD, ALD-52, 1P-LSD, 1B-LSD, ETH-LAD, 1P-ETH-LAD, AL-LAD, ECPLA, LSZ, LSM-775, and 4 their phase I and II metabolites along with their identification numbers (ID), the exact mass of the protonated 5 molecule (M+H+), elemental composition, retention time (RT), and the three most abundant fragment ions (FI A- 6 C) in MS2 sorted by decreasing intensity. The parent compounds are given in bold and the metabolites were 7 sorted by increasing mass and RT. 8 9 10 ID Compound Exact Elemental RT, FI A FI B FI C 11 mass, m/z composition min 12 1 LSD 324.2070 C20H26N3O 5.3 223.1229 208.0995 281.1648

13 2 N-Deethyl LSD 296.1757 C18H22N3O 3.3 223.1229 208.0756 253.1335 6 14 3 N -Demethyl LSD 310.1913 C19H24N3O 5.3 209.1073 74.0964 237.1022 15 4 Hydroxy LSD isomer 1 340.2019 C20H26N3O2 3.4 221.1073 239.1184 322.1913 16 5 Hydroxy LSD isomer 2 340.2019 C H N O 3.6 239.1184 297.1597 224.0706 17 20 26 3 2 6 Dihydroxy LSD 356.1968 C H N O 2.5 237.1022 74.0964 313.1552 18 20 26 3 3 19 7 Hydroxy LSD isomer 2 516.2340 C26H34N3O8 2.0 239.1184 340.2019 224.0706 20 glucuronide 21 8 ALD-52 For366.2176 PeerC22H 28ReviewN3O2 6.2 265.1335 223.1229 208.0756 22 2 1-Deacetyl-N-deethyl 296.1757 C18H22N3O 3.3 223.1229 208.0756 253.1335 23 ALD-52 6 24 3 1-Deacetyl-N -demethyl 310.1913 C19H24N3O 5.3 209.1073 74.0964 237.1022 25 ALD-52 26 1 1-Deacetyl ALD-52 324.2070 C20H26N3O 5.3 223.1229 208.0995 281.1648 27 9 N-Deethyl ALD-52 338.1863 C H N O 4.5 265.1335 223.1229 295.1441 28 20 24 3 2 4 1-Deacetyl-hydroxy 340.2019 C H N O 3.4 221.1073 239.1184 322.1913 29 20 26 3 2 30 ALD-52 isomer 1 31 5 1-Deacetyl-hydroxy 340.2019 C20H26N3O2 3.6 239.1184 297.1597 224.0706 32 ALD-52 isomer 2 6 33 10 N -Demethyl ALD-52 352.2019 C21H26N3O2 6.2 251.1178 209.1073 74.0964

34 11 1P-LSD 380.2332 C23H30N3O2 7.2 223.1229 279.1491 208.0756

35 2 1-Depropionyl-N- 296.1757 C18H22N3O 3.3 223.1229 208.0756 253.1335 36 deethyl-1P-LSD 37 6 3 1-Depropionyl-N - 310.1913 C19H24N3O 5.3 209.1073 74.0964 237.1022 38 demethyl 1P-LSD 39 1 1-Depropionyl 1P-LSD 324.2070 C H N O 5.3 223.1229 208.0995 281.1648 40 20 26 3 41 4 1-Depropionyl-hydroxy 340.2019 C20H26N3O2 3.4 221.1073 239.1184 322.1913 42 1P-LSD isomer 1 43 5 1-Depropionyl-hydroxy 340.2019 C20H26N3O2 3.6 239.1184 297.1597 224.0706 44 1P-LSD isomer 2

45 12 N-Deethyl 1P-LSD 352.2019 C21H26N3O2 5.6 223.1229 279.1491 208.0756

46 6 1-Depropionyl- 356.1968 C20H26N3O3 2.5 237.1022 74.0964 313.1552 47 dihydroxy 1P-LSD 48 6 13 N -Demethyl 1P-LSD 366.2176 C22H28N3O2 7.2 209.1073 265.1335 74.0964 49 14 1B-LSD 394.2489 C H N O 8.0 293.1648 223.1229 208.0756 50 24 32 3 2 2 1-Debutyryl-N-deethyl- 296.1757 C H N O 3.3 223.1229 208.0756 253.1335 51 18 22 3 52 1B-LSD 6 53 3 1-Debutyryl-N - 310.1913 C19H24N3O 5.3 209.1073 74.0964 237.1022 54 demethyl 1B-LSD 55 1 1-Debutyryl 1B-LSD 324.2070 C20H26N3O 5.3 223.1229 208.0995 281.1648

56 4 1-Debutyryl-hydroxy 340.2019 C20H26N3O2 3.4 221.1073 239.1184 322.1913 57 1B-LSD isomer 1 58 5 1-Debutyryl-hydroxy 340.2019 C20H26N3O2 3.6 239.1184 297.1597 224.0706 59 1B- LSD isomer 2 60 6 1-Debutyryl-dihydroxy 356.1968 C20H26N3O3 2.5 237.1022 74.0964 313.1552 17 Page 23 of 49 Analytical & Bioanalytical Chemistry

1 2 3 1B- LSD

4 15 Hydroxy 1B-LSD 410.2438 C24H32N3O3 5.7 309.1597 223.1229 208.0995 5 16 ETH-LAD 338.2226 C21H28N3O 5.6 237.1386 208.0995 309.1835 6 17 N-Deethyl ETH-LAD 310.1913 C H N O 3.6 237.1386 208.0995 281.1522 7 19 24 3 3 N6-Deethyl ETH-LAD 310.1913 C H N O 5.3 209.1073 74.0964 237.1022 8 19 24 3 9 18 Hydroxy ETH-LAD 354.2176 C21H28N3O2 3.8 253.1335 297.1603 196.0762 10 19 Dihydroxy ETH-LAD 370.2125 C21H28N3O3 2.8 251.1178 279.1128 313.1552 11 20 Hydroxy ETH-LAD 530.2496 C27H36N3O8 2.4 253.1335 354.2176 325.1784 12 glucuronide 13 21 1P-ETH-LAD 394.2489 C24H32N3O2 7.6 293.1648 237.1386 208.0756

14 17 1-Depropionyl-N-deethyl 310.1913 C19H24N3O 3.6 237.1386 208.0995 281.1522 15 1P-ETH-LAD 16 6 3 1-Depropionyl-N - 310.1913 C19H24N3O 5.3 209.1073 74.0964 237.1022 17 deethyl 1P-ETH-LAD 18 16 1-Depropionyl 1P-ETH- 338.2226 C H N O 5.6 237.1386 208.0995 309.1835 19 21 28 3 20 LAD 21 18 1-Depropionyl-hydroxyFor 354.2176 PeerC21H 28ReviewN3O2 3.8 253.1335 297.1603 196.0762 22 1P-ETH-LAD 23 22 N-Deethyl 1P-ETH-LAD 366.2176 C22H28N3O2 5.9 293.1648 208.0995 237.1648

24 19 1-Depropionyl- 370.2125 C21H28N3O3 2.8 251.1178 279.1128 313.1552 25 dihydroxy 1P-ETH-LAD 26 20 1-Depropionyl-hydroxy 530.2496 C27H36N3O8 2.4 253.1335 354.2176 325.1784 27 1P-ETH-LAD 28 glucuronide 29 23 AL-LAD 350.2226 C H N O 6.2 208.0995 309.1835 182.0838 30 22 28 3 6 31 24 N -Deallyl-N-deethyl 282.1600 C17H20N3O 3.2 209.1073 237.1027 134.0964 32 AL-LAD 6 33 25 N -Allyl-norlysergic acid 295.1441 C18H19N2O2 2.9 70.0656 254.1049 238.0868 6 34 3 N -Deallyl AL-LAD 310.1913 C19H24N3O 5.3 209.1073 74.0964 237.1022

35 26 N-Deethyl AL-LAD 322.1913 C20H24N3O 4.2 281.1522 208.0995 182.0838

36 27 N-Deethyl-hydroxy AL- 338.1863 C20H24N3O2 2.6 297.1471 225.1022 207.0916 37 LAD 38 28 Hydroxy AL-LAD 366.2176 C22H28N3O2 3.8 224.0944 325.1784 198.0787 39 isomer 1 40 29 Hydroxy AL-LAD 366.2176 C H N O 4.3 223.0871 325.1784 297.1603 41 22 28 3 2 42 isomer 2 43 30 Hydroxy AL-LAD 366.2176 C22H28N3O2 5.0 207.0922 247.1235 265.1340 44 isomer 3 45 31 Dihydroxy AL-LAD 382.2125 C22H28N3O3 3.3 221.0714 263.1178 313.1552 46 isomer 1

47 32 Dihydroxy AL-LAD 382.2125 C22H28N3O3 4.1 128.1069 255.1133 223.0871 48 isomer 2 49 33 Hydroxy AL-LAD 542.2496 C H N O 3.1 325.1784 224.0944 501.2105 50 28 36 3 8 isomer 1 glucuronide 51 34 ECPLA 336.2070 C H N O 6.0 223.1235 208.0756 86.0964 52 21 26 3 53 35 Lysergic acid amide 268.1444 C16H18N3O 1.5 223.1235 208.0762 180.0813 54 36 Lysergic acid 269.1284 C16H17N2O2 2.2 223.1235 254.1049 238.0868 55 37 N-Deethyl ECPLA 308.1757 C19H22N3O 3.3 223.1235 208.0762 265.1340 6 56 38 N -Demethyl ECPLA 322.1913 C20H24N3O 5.9 209.1078 237.1027 86.0964 57 Hydroxy ECPLA 39 352.2019 C H N O 3.0 239.1184 224.0944 86.0964 58 isomer 1 21 26 3 2 59 Hydroxy ECPLA 40 352.2019 C H N O 4.0 239.1184 221.1078 334.1913 60 isomer 2 21 26 3 2

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1 2 3 Hydroxy ECPLA 41 352.2019 C H N O 4.2 239.1184 86.0964 224.0706 4 isomer 3 21 26 3 2 5 42 Dihydroxy ECPLA 368.1968 C21H26N3O3 3.3 237.1027 222.0555 86.0964 6 43 LSZ 336.2070 C H N O 5.3 223.1235 140.1070 208.0995 7 21 26 3 35 Lysergic acid amide 268.1444 C H N O 1.5 223.1235 208.0762 180.0813 8 16 18 3 9 36 Lysergic acid 269.1284 C16H17N2O2 2.2 223.1235 254.1049 238.0868 10 Hydroxy lysergic acid 44 284.1393 C16H18N3O2 1.0 221.1078 266.1287 239.1184 11 amide 6 12 45 N -Demethyl LSZ 322.1913 C20H24N3O 5.2 209.1073 140.1070 237.1027 13 46 Hydroxy LSZ isomer 1 352.2019 C21H26N3O2 3.7 239.1184 224.0711 140.1070

14 47 Hydroxy LSZ isomer 2 352.2019 C21H26N3O2 3.9 221.1078 334.1913 249.1027 15 48 Hydroxy LSZ isomer 3 352.2019 C21H26N3O2 4.3 208.0762 223.1235 334.1913 16 49 Dihydroxy LSZ 368.1968 C21H26N3O3 2.8 237.1027 222.0555 265.0977 17 Hydroxy LSZ isomer 1 18 50 528.2340 C H N O 2.1 239.1184 352.2019 140.1070 glucuronide 27 34 3 8 19 20 51 LSM-775 338.1863 C20H24N3O2 3.5 223.1235 208.0762 295.1446 6 21 52 N -Demethyl LSM-775For324.1706 PeerC19H 22ReviewN3O2 3,4 209.1078 237.1027 183.0922 Hydroxy LSM-775 22 53 354.1812 C H N O 1.5 221.1078 336.1706 249.1027 23 isomer 1 20 24 3 3 24 Hydroxy LSM-775 54 354.1812 C H N O 1.7 221.1078 336.1706 249.1027 25 isomer 2 20 24 3 3 26 Hydroxy LSM-775 55 354.1812 C H N O 2.1 239.1184 224.0706 311.1395 27 isomer 3 20 24 3 3 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 Table 2: General involvement of tested monooxygenases in the initial metabolic steps. n.d. = not detected 4 5 Compound N6-Dealkylation N-Deethylation Hydroxylation 6 CYP2C19 CYP2C9 CYP1A2 7 LSD 8 CYP3A4 CYP3A4 CYP3A4 9 CYP2D6 ALD-52 CYP3A4 CYP3A4 10 CYP3A4 11 12 1P-LSD CYP3A4 CYP3A4 CYP3A4 13 14 CYP2D6 1B-LSD CYP3A4 CYP3A4 15 CYP3A4 16 CYP2D6 17 ETH-LAD CYP3A4 CYP3A4 18 CYP3A4 19 20 1P-ETH-LAD CYP3A4 CYP3A4 CYP3A4 21 For Peer Review CYP1A2 22 AL-LAD CYP3A4 CYP3A4 23 CYP3A4 24 CYP2C19 CYP2C9 CYP1A2 ECPLA 25 CYP3A4 CYP3A4 CYP3A4 26 CYP1A2 27 LSZ CYP3A4 n.d. CYP3A4 28 CYP1A2 29 LSM-775 CYP3A4 n.d. 30 CYP3A4 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 References 4 5 6 1. Stevens J. Storming Heaven: LSD and the American dream. New York: Grove 7 Press; 1987. 8 9 2. Hoffman AJ, Nichols DE. Synthesis and LSD-like discriminative stimulus properties 10 in a series of N(6)-alkyl norlysergic acid N,N-diethylamide derivatives. J Med Chem. 11 1985;28(9):1252-5. 12 13 14 3. Oberlender R, Pfaff RC, Johnson MP, Huang XM, Nichols DE. Stereoselective 15 LSD-like activity in d-lysergic acid amides of (R)- and (S)-2-aminobutane. J Med 16 Chem. 1992;35(2):203-11. 17 18 4. Huang X, Marona-Lewicka D, Pfaff RC, Nichols DE. Drug discrimination and 19 receptor binding studies of N-isopropyl lysergamide derivatives. Pharmacol Biochem 20 Behav. 1994;47(3):667-73. 21 For Peer Review 22 5. Monte AP, Marona-Lewicka D, Kanthasamy A, Sanders-Bush E, Nichols DE. 23 24 Stereoselective LSD-like activity in a series of d-lysergic acid amides of (R)- and (S)- 25 2-aminoalkanes. J Med Chem. 1995;38(6):958-66. 26 27 6. Nichols DE, Frescas S, Marona-Lewicka D, Kurrasch-Orbaugh DM. Lysergamides 28 of isomeric 2,4-dimethylazetidines map the binding orientation of the diethylamide 29 moiety in the potent hallucinogenic agent N,N-diethyllysergamide (LSD). J Med 30 Chem. 2002;45(19):4344-9. 31 32 33 7. Shulgin A, Shulgin A. Tihkal: The Continuation. Transform Press, Berkley (CA). 34 1997. 35 36 8. Pfaff RC, Huang X, Marona-Lewicka D, Oberlender R, Nichols DE. Lysergamides 37 revisited. NIDA Res Monogr. 1994;146:52-73. 38 39 9. Nichols DE. LSD and its lysergamide cousins. Heffter Rev Psychedelic Res. 40 2001;2:80-7. 41 42 10. Nichols DE. Psychedelics. Pharmacol Rev. 2016;68(2):264-355. 43 44 45 11. Nichols DE. Dark Classics in Chemical Neuroscience: Lysergic Acid Diethylamide 46 (LSD). ACS Chem Neurosci. 2018;9(10):2331-43. 47 48 12. Liechti ME. Modern Clinical Research on LSD. Neuropsychopharmacology. 49 2017;42(11):2114-27. 50 51 13. Brandt SD, Kavanagh PV, Westphal F, Stratford A, Elliott SP, Hoang K et al. 52 Return of the lysergamides. Part I: Analytical and behavioural characterization of 1- 53 propionyl-d-lysergic acid diethylamide (1P-LSD). Drug Test Anal. 2016;8(9):891-902. 54 55 56 14. Brandt SD, Kavanagh PV, Westphal F, Elliott SP, Wallach J, Stratford A et al. 57 Return of the lysergamides. Part III: Analytical characterization of N(6) -ethyl-6- 58 norlysergic acid diethylamide (ETH-LAD) and 1-propionyl ETH-LAD (1P-ETH-LAD). 59 Drug Test Anal. 2017;9(10):1641-9. 60

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1 2 3 15. Brandt SD, Kavanagh PV, Westphal F, Elliott SP, Wallach J, Colestock T et al. 4 Return of the lysergamides. Part II: Analytical and behavioural characterization of 5 6 N(6) -allyl-6-norlysergic acid diethylamide (AL-LAD) and (2'S,4'S)-lysergic acid 2,4- 7 dimethylazetidide (LSZ). Drug Test Anal. 2017;9(1):38-50. 8 9 16. Halberstadt AL, Klein LM, Chatha M, Valenzuela LB, Stratford A, Wallach J et al. 10 Pharmacological characterization of the LSD analog N-ethyl-N-cyclopropyl 11 lysergamide (ECPLA). Psychopharmacology (Berl). 2018. 12 13 17. Brandt SD, Kavanagh PV, Twamley B, Westphal F, Elliott SP, Wallach J et al. 14 Return of the lysergamides. Part IV: Analytical and pharmacological characterization 15 16 of lysergic acid morpholide (LSM-775). Drug Test Anal. 2017. 17 18 18. EMCDDA. European Drug Report 2017: Trends and Developments. Publications 19 of of the European Union 2017. 20 21 19. Canezin J, CailleuxFor A, Turcant Peer A, Le BouilReview A, Harry P, Allain P. Determination of 22 LSD and its metabolites in human biological fluids by high-performance liquid 23 chromatography with electrospray tandem mass spectrometry. J Chromatogr B 24 Biomed Sci Appl. 2001;765(1):15-27. 25 26 27 20. Dolder PC, Liechti ME, Rentsch KM. Development and validation of a rapid 28 turboflow LC-MS/MS method for the quantification of LSD and 2-oxo-3-hydroxy LSD 29 in serum and urine samples of emergency toxicological cases. Anal Bioanal Chem. 30 2015;407(6):1577-84. 31 32 21. Dolder PC, Schmid Y, Haschke M, Rentsch KM, Liechti ME. Pharmacokinetics 33 and Concentration-Effect Relationship of Oral LSD in Humans. Int J 34 35 Neuropsychopharmacol. 2015;19(1). 36 37 22. Diao X, Huestis MA. Approaches, Challenges, and Advances in Metabolism of 38 New Synthetic Cannabinoids and Identification of Optimal Urinary Marker 39 Metabolites. Clin Pharmacol Ther. 2017;101(2):239-53. 40 41 23. Welter-Luedeke J, Maurer HH. New Psychoactive Substances: Chemistry, 42 Pharmacology, Metabolism, and Detectability of Amphetamine Derivatives With 43 Modified Ring Systems. Ther Drug Monit. 2016;38(1):4-11. 44 45 46 24. Meyer MR. New psychoactive substances: an overview on recent publications on 47 their toxicodynamics and toxicokinetics. Arch Toxicol. 2016;90(10):2421-44. 48 49 25. Meyer MR. Toxicokinetics of NPS: Update 2017. Handb Exp Pharmacol. 2018. 50 51 26. Mogler L, Wilde M, Huppertz LM, Weinfurtner G, Franz F, Auwarter V. Phase I 52 metabolism of the recently emerged synthetic cannabinoid CUMYL-PEGACLONE 53 and detection in human urine samples. Drug Test Anal. 2018. 54 55 27. Watanabe S, Vikingsson S, Roman M, Green H, Kronstrand R, Wohlfarth A. In 56 57 Vitro and In Vivo Metabolite Identification Studies for the New Synthetic Opioids 58 Acetylfentanyl, Acrylfentanyl, Furanylfentanyl, and 4-Fluoro-Isobutyrylfentanyl. AAPS 59 J. 2017;19(4):1102-22. 60

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1 2 3 28. Welter J, Meyer MR, Wolf EU, Weinmann W, Kavanagh P, Maurer HH. 2- 4 methiopropamine, a thiophene analogue of methamphetamine: studies on its 5 6 metabolism and detectability in the rat and human using GC-MS and LC-(HR)-MS 7 techniques. Anal Bioanal Chem. 2013;405(10):3125-35. 8 9 29. Richter LHJ, Flockerzi V, Maurer HH, Meyer MR. Pooled human liver 10 preparations, HepaRG, or HepG2 cell lines for metabolism studies of new 11 psychoactive substances? A study using MDMA, MDBD, butylone, MDPPP, MDPV, 12 MDPB, 5-MAPB, and 5-API as examples. J Pharm Biomed Anal. 2017;143:32-42. 13 14 30. Chauret N, Gauthier A, Nicoll-Griffith DA. Effect of common organic solvents on 15 16 in vitro cytochrome P450-mediated metabolic activities in human liver microsomes. 17 Drug Metab Dispos. 1998;26(1):1-4. 18 19 31. Wagmann L, Meyer MR, Maurer HH. What is the contribution of human FMO3 in 20 the N-oxygenation of selected therapeutic drugs and drugs of abuse? Toxicol Lett. 21 2016;258:55-70. For Peer Review 22 23 32. Meyer GM, Meyer MR, Wink CS, Zapp J, Maurer HH. Studies on the in vivo 24 contribution of human cytochrome P450s to the hepatic metabolism of glaucine, a 25 26 new drug of abuse. Biochem Pharmacol. 2013;86(10):1497-506. 27 28 33. Wagmann L, Brandt SD, Kavanagh PV, Maurer HH, Meyer MR. In vitro 29 monoamine oxidase inhibition potential of alpha-methyltryptamine analog new 30 psychoactive substances for assessing possible toxic risks. Toxicol Lett. 31 2017;272:84-93. 32 33 34. Welter J, Kavanagh P, Meyer MR, Maurer HH. Benzofuran analogues of 34 35 amphetamine and methamphetamine: studies on the metabolism and toxicological 36 analysis of 5-APB and 5-MAPB in urine and plasma using GC-MS and LC-(HR)- 37 MS(n) techniques. Anal Bioanal Chem. 2015;407(5):1371-88. 38 39 35. Helfer AG, Michely JA, Weber AA, Meyer MR, Maurer HH. Orbitrap technology 40 for comprehensive metabolite-based liquid chromatographic-high resolution-tandem 41 mass spectrometric urine drug screening - exemplified for cardiovascular drugs. Anal 42 Chim Acta. 2015;891:221-33. 43 44 45 36. Caspar AT, Westphal F, Meyer MR, Maurer HH. LC-high resolution-MS/MS for 46 identification of 69 metabolites of the new psychoactive substance 1-(4-ethylphenyl-)- 47 N-[(2-methoxyphenyl)methyl] propane-2-amine (4-EA-NBOMe) in rat urine and 48 human liver S9 incubates and comparison of its screening power with further MS 49 techniques. Anal Bioanal Chem. 2018;410(3):897-912. 50 51 37. Wissenbach DK, Meyer MR, Remane D, Philipp AA, Weber AA, Maurer HH. 52 Drugs of abuse screening in urine as part of a metabolite-based LC-MSn screening 53 54 concept. Anal Bioanal Chem. 2011;400(10):3481-9. 55 56 38. Meyer MR, Lindauer C, Welter J, Maurer HH. Dimethocaine, a synthetic cocaine 57 analogue: studies on its in-vivo metabolism and its detectability in urine by means of 58 a rat model and liquid chromatography-linear ion-trap (high-resolution) mass 59 spectrometry. Anal Bioanal Chem. 2014;406(7):1845-54. 60

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1 2 3 39. Maurer HH, Pfleger K, Weber AA. Mass spectral data of drugs, poisons, 4 pesticides, pollutants and their metabolites. Weinheim: Wiley-VCH; 2016. 5 6 7 40. Klette KL, Anderson CJ, Poch GK, Nimrod AC, ElSohly MA. Metabolism of 8 lysergic acid diethylamide (LSD) to 2-oxo-3-hydroxy LSD (O-H-LSD) in human liver 9 microsomes and cryopreserved human hepatocytes. J Anal Toxicol. 2000;24(7):550- 10 6. 11 12 41. Poch GK, Klette KL, Hallare DA, Manglicmot MG, Czarny RJ, McWhorter LK et 13 al. Detection of metabolites of lysergic acid diethylamide (LSD) in human urine 14 specimens: 2-oxo-3-hydroxy-LSD, a prevalent metabolite of LSD. J Chromatogr B 15 16 Biomed Sci Appl. 1999;724(1):23-33. 17 18 42. Steuer AE, Poetzsch M, Stock L, Eisenbeiss L, Schmid Y, Liechti ME et al. 19 Development and validation of an ultra-fast and sensitive microflow liquid 20 chromatography-tandem mass spectrometry (MFLC-MS/MS) method for 21 quantification of LSD andFor its metabolitesPeer Review in plasma and application to a controlled 22 LSD administration study in humans. Drug Test Anal. 2017;9(5):788-97. 23 24 43. Dolder PC, Liechti ME, Rentsch KM. Development and validation of an LC- 25 26 MS/MS method to quantify lysergic acid diethylamide (LSD), iso-LSD, 2-oxo-3- 27 hydroxy-LSD, and nor-LSD and identify novel metabolites in plasma samples in a 28 controlled clinical trial. J Clin Lab Anal. 2018;32(2). 29 30 44. Favretto D, Frison G, Maietti S, Ferrara SD. LC-ESI-MS/MS on an ion trap for the 31 determination of LSD, iso-LSD, nor-LSD and 2-oxo-3-hydroxy-LSD in blood, urine 32 and vitreous humor. Int J Legal Med. 2007;121(4):259-65. 33 34 35 45. Jang M, Kim J, Han I, Yang W. Simultaneous determination of LSD and 2-oxo-3- 36 hydroxy LSD in hair and urine by LC-MS/MS and its application to forensic cases. J 37 Pharm Biomed Anal. 2015;115:138-43. 38 39 46. Johansen SS, Jensen JL. Liquid chromatography-tandem mass spectrometry 40 determination of LSD, ISO-LSD, and the main metabolite 2-oxo-3-hydroxy-LSD in 41 forensic samples and application in a forensic case. J Chromatogr B Analyt Technol 42 Biomed Life Sci. 2005;825(1):21-8. 43 44 45 47. Richter LHJ, Maurer HH, Meyer MR. New psychoactive substances: Studies on 46 the metabolism of XLR-11, AB-PINACA, FUB-PB-22, 4-methoxy-alpha-PVP, 25-I- 47 NBOMe, and meclonazepam using human liver preparations in comparison to 48 primary human hepatocytes, and human urine. Toxicol Lett. 2017;280:142-50. 49 50 48. Niessen WM, Correa RA. Interpretation of MS-MS Mass Spectra of Drugs and 51 Pesticides. Hoboken, New Jersey: John Wiley & Sons; 2016. 52 53 49. Wink CS, Meyer GM, Meyer MR, Maurer HH. Toxicokinetics of lefetamine and 54 55 derived diphenylethylamine designer drugs-Contribution of human cytochrome P450 56 isozymes to their main phase I metabolic steps. Toxicol Lett. 2015;238(3):39-44. 57 58 50. Koonrungsesomboon N, Khatsri R, Wongchompoo P, Teekachunhatean S. The 59 impact of genetic polymorphisms on CYP1A2 activity in humans: a systematic review 60 and meta-analysis. Pharmacogenomics J. 2017.

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1 2 3 51. Nichols DE, Grob CS. Is LSD toxic? Forensic Sci Int. 2018;284:141-5. 4 5 52. Passie T, Halpern JH, Stichtenoth DO, Emrich HM, Hintzen A. The pharmacology 6 7 of lysergic acid diethylamide: a review. CNS Neurosci Ther. 2008;14(4):295-314. 8 9 53. Sharma V, McNeill JH. To scale or not to scale: the principles of dose 10 extrapolation. Br J Pharmacol. 2009;157(6):907-21. 11 12 54. Crews C. Analysis of Ergot Alkaloids. Toxins (Basel). 2015;7(6):2024-50. 13 14 55. Meyer MR, Schmitt S, Maurer HH. Studies on the metabolism and detectability of 15 the emerging drug of abuse diphenyl-2-pyrrolidinemethanol (D2PM) in rat urine using 16 GC-MS and LC-HR-MS/MS. J Mass Spectrom. 2013;48(2):243-9. 17 18 19 20 21 For Peer Review 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 Legends to the figures 4 5 6 Fig. 1: Chemical structures of LSD and nine LSD-based new psychoactive substances. 7 Structural changes in comparison to LSD are marked in red. 8 9 Fig. 2: HRMS/MS spectra and retention times (RT) of LSD (ID 1) and 1P-LSD (ID 11) as 10 11 well as two of their metabolites (hydroxy LSD isomer 1, ID 4 and N-deethyl 1P-LSD, ID 12) 12 13 to support described mass spectral based metabolite identification. Numbering according to 14 Table 1. 15 16 17 Fig. 3: Postulated metabolic pathways of 1P-LSD studied by in vitro incubations with pooled 18 human liver S9 fraction. Numbering according to Table 1. 19 20 Fig. 4: Percentage formation of CYP-dependent metabolites in pHLM co-incubations of LSD 21 For Peer Review 22 or LSD analogs with the selective CYP1A2 inhibitor alpha-naphthoflavone, the selective 23 CYP3A4 inhibitor ketoconazole, or a mixture of both inhibitors. Formation in reference 24 incubations without inhibitor was referred to as 100%. 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 For Peer Review 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 33 of 49 Analytical & Bioanalytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 For Peer Review 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Analytical & Bioanalytical Chemistry Page 34 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 For Peer Review 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 35 of 49 Analytical & Bioanalytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 For Peer Review 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Analytical & Bioanalytical Chemistry Page 36 of 49

1 2 3 Analytical and Bioanalytical Chemistry 4 5 6 7 Electronic Supplementary Material 8 9 10 11 In vitro metabolic fate of nine LSD-based new psychoactive substances and 12 13 their analytical detectability in different urinary screening procedures 14 15 16 17 18 19 Lea Wagmann, Lilian H. J. Richter, Tobias Kehl, Franziska Wack, Madeleine 20 21 Pettersson Bergstrand,For Simon Peer D. Brandt, Review Alexander Stratford, Hans H. Maurer, 22 23 and Markus R. Meyer 24 25 26

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 For Peer Review 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53

54 Fig. S1: HRMS/MS spectra of LSD-based new psychoactive substances and their most 55 56 abundant, unique metabolites along with their retention times (RT). Numbering according to 57 58 Table 1 in the manuscript. 59 60 Analytical & Bioanalytical Chemistry Page 38 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 For Peer Review 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 Fig. S1 (continued) 55 56 57 58 59 60 Page 39 of 49 Analytical & Bioanalytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 For Peer Review 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 Fig. S1 (continued) 55 56 57 58 59 60 Analytical & Bioanalytical Chemistry Page 40 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 For Peer Review 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 Fig. S1 (continued) 55 56 57 58 59 60 Page 41 of 49 Analytical & Bioanalytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 For Peer Review 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 Fig. S2: Metabolic pathways of LSD studied in incubations with pooled human liver S9 45 46 fraction. Numbering according to Table 1 in the manuscript. 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Analytical & Bioanalytical Chemistry Page 42 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 For Peer Review 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 Fig. S3: Metabolic pathways of ALD-52 studied in incubations with pooled human liver S9 45 fraction. Numbering according to Table 1 in the manuscript. 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 43 of 49 Analytical & Bioanalytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 For Peer Review 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Fig. S4: Metabolic pathways of 1B-LSD studied in incubations with pooled human liver S9 46 fraction. Numbering according to Table 1 in the manuscript. 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Analytical & Bioanalytical Chemistry Page 44 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 For Peer Review 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 Fig. S5: Metabolic pathways of ETH-LAD studied in incubations with pooled human liver S9 44 45 fraction. Numbering according to Table 1 in the manuscript. 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 45 of 49 Analytical & Bioanalytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 For Peer Review 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 Fig. S6: Metabolic pathways of 1P-ETH-LAD studied in incubations with pooled human liver 45 46 S9 fraction. Numbering according to Table 1 in the manuscript. 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Analytical & Bioanalytical Chemistry Page 46 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 For Peer Review 22 23 24 25 26 27 28 29 30 31 32 33 Fig. S7: Metabolic pathways of AL-LAD studied in incubations with pooled human liver S9 34 35 fraction. Numbering according to Table 1 in the manuscript. 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 47 of 49 Analytical & Bioanalytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 For Peer Review 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 Fig. S8: Metabolic pathways of ECPLA studied in incubations with pooled human liver S9 42 43 fraction. Numbering according to Table 1 in the manuscript. 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Analytical & Bioanalytical Chemistry Page 48 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 For Peer Review 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 Fig. S9: Metabolic pathways of LSZ studied in incubations with pooled human liver S9 42 43 fraction. Numbering according to Table 1 in the manuscript. 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 49 of 49 Analytical & Bioanalytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Fig. S10: Metabolic pathways of LSM-775 studied in incubations with pooled human liver S9 17 18 fraction. Numbering according to Table 1 in the manuscript. 19 20 21 For Peer Review 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60