The versatile binding landscape of the TAAR1 pocket for LSD and other antipsychotic drug molecules.

Increasing global concerns about psychoactive substance addiction and psychotic disorders highlight the need for comprehensive research into the structure-function relationship governing ligand recognition between these substances and their receptors in the brain. Recent studies indicate the significant involvement of trace amine-associated receptor 1 (TAAR1) in the signaling regulation of the hallucinogen lysergic acid diethylamide (LSD) and other antipsychotic drugs. This study presents structures of the TAAR1-Gs protein complex recognizing LSD, which exhibits a polypharmacological profile, and the partial agonist RO5263397, which is a drug candidate for schizophrenia and addiction. Moreover, we elucidate the cross-species recognition and partial activation mechanism for TAAR1, which holds promising implications from a drug discovery perspective. Through mutagenesis, functional studies, and molecular dynamics (MD) simulations, we provide a comprehensive understanding of a versatile TAAR1 pocket in recognizing various ligands as well as in the ligand-free state, underpinning the structural basis of its high adaptability. These findings offer valuable insights for the design of antipsychotic drugs.

Modification of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method targeting lysergic acid diethylamide (LSD) and its primary metabolite (OH-LSD) to include nine LSD analogs.

A variety of LSD analogs have emerged in recent years with dual purposes of avoiding prosecution from possession while providing new options for those willing to experiment with hallucinogenic drugs. In this study, a previously published automated sample preparation method for LSD and its primary metabolite (OH-LSD) was utilized to extract LSD, OH-LSD, and nine LSD analogs from urine. The liquid chromatography tandem mass spectrometry (LC-MS/MS) method was modified from the previously published LC conditions to utilize a different analytical column and gradient elution program. Mobile phases of 10 mM ammonium formate with 0.1% formic acid in deionized water (mobile phase A) and 0.1% formic acid in methanol (mobile phase B) were employed. The method was validated to ANSI/ASB Standard 036 with a 0.1 ng/mL limit of detection for all analytes and was utilized for the analysis of 325 urine specimens. Although no LSD analogs were observed in the samples analyzed, this validated method was demonstrated to be suitable for the analysis of these compounds in laboratories seeking to expand their testing scope. Automated sample preparation allows for the efficient analysis of these analytically challenging compounds with minimal manual handling. Additionally, there was no increased analytical time burden when the LC column and gradient were modified to target nine additional analytes. Detection may improve as new reference standards are developed to allow laboratories to focus on the metabolic products of these analogs. For now, this validated procedure can assist with the routine analysis and surveillance of these emerging substances.

An innovative approach for selective and robust screening of NBOHs, NBOMes, and LSD in forensic samples using a 3D-Printed electrochemical double cell.

Lysergic acid diethylamide (LSD) and two phenethylamine classes (NBOHs and NBOMes) are the main illicit drugs found in seized blotter papers. The preliminary identification of these substances is of great interest for forensic analysis. In this context, this work constitutes the inaugural demonstration of an efficient methodology for the selective detection of LSD, NBOHs, and NBOMes, utilizing a fully 3D-printed electrochemical double cell (3D-EDC). This novel 3D-EDC enables the use of two working electrodes and/or two supporting electrolytes (at different pHs) in the same detection system, with the possibility of shared or individual auxiliary and pseudo-reference electrodes. Thus, the selective voltammetric detection of these substances is proposed using two elegant strategies: (i) utilizing the same 3D-EDC platform with two working electrodes (boron-doped diamond (BDD) and 3D-printed graphite), and (ii) employing two pH levels (4.0 and 12.0) with 3D-printed graphite electrode. This comprehensive framework facilitates a fast, robust, and uncomplicated electrochemical analysis. Moreover, this configuration enables a rapid and sensitive detection of LSD, NBOHs, and NBOMes in seized samples, and can also provide quantitative analysis. The proposed method showed good stability of the electrochemical response with RSD <9 % for Ip and <5 % for Ep, evaluating all oxidation processes observed for studied analytes (n = 7) at two pH levels, using the same and different (n = 3) working electrodes. It demonstrates a broad linear range (20-100 and 20-70 µmol L-1) and a low LOD (1.0 µmol L-1) for quantification of a model molecule (LSD) at the two pHs studied. Hence, the 3D-EDC combined with voltammetric techniques using BDD and 3D-printed graphite electrodes on the same platform, or only with this last sensor at two pH values, provide a practical and robust avenue for preliminary identification of NBOHs, NBOMes, and LSD. This method embodies ease, swiftness, cost-efficiency, robustness, and selectivity as an on-site screening tool for forensic analysis.

Structural pharmacology and therapeutic potential of 5-methoxytryptamines.

Psychedelic substances such as lysergic acid diethylamide (LSD) and psilocybin show potential for the treatment of various neuropsychiatric disorders1-3. These compounds are thought to mediate their hallucinogenic and therapeutic effects through the serotonin (5-hydroxytryptamine (5-HT)) receptor 5-HT2A (ref. 4). However, 5-HT1A also plays a part in the behavioural effects of tryptamine hallucinogens5, particularly 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT), a psychedelic found in the toxin of Colorado River toads6. Although 5-HT1A is a validated therapeutic target7,8, little is known about how psychedelics engage 5-HT1A and which effects are mediated by this receptor. Here we map the molecular underpinnings of 5-MeO-DMT pharmacology through five cryogenic electron microscopy (cryo-EM) structures of 5-HT1A, systematic medicinal chemistry, receptor mutagenesis and mouse behaviour. Structure-activity relationship analyses of 5-methoxytryptamines at both 5-HT1A and 5-HT2A enable the characterization of molecular determinants of 5-HT1A signalling potency, efficacy and selectivity. Moreover, we contrast the structural interactions and in vitro pharmacology of 5-MeO-DMT and analogues to the pan-serotonergic agonist LSD and clinically used 5-HT1A agonists. We show that a 5-HT1A-selective 5-MeO-DMT analogue is devoid of hallucinogenic-like effects while retaining anxiolytic-like and antidepressant-like activity in socially defeated animals. Our studies uncover molecular aspects of 5-HT1A-targeted psychedelics and therapeutics, which may facilitate the future development of new medications for neuropsychiatric disorders.

Discovery and Structure-Activity Relationships of 2,5-Dimethoxyphenylpiperidines as Selective Serotonin 5-HT2A Receptor Agonists.

Classical psychedelics such as psilocybin, lysergic acid diethylamide (LSD), and N,N-dimethyltryptamine (DMT) are showing promising results in clinical trials for a range of psychiatric indications, including depression, anxiety, and substance abuse disorder. These compounds are characterized by broad pharmacological activity profiles, and while the acute mind-altering effects can be ascribed to their shared agonist activity at the serotonin 2A receptor (5-HT2AR), their apparent persistent therapeutic effects are yet to be decidedly linked to activity at this receptor. We report herein the discovery of 2,5-dimethoxyphenylpiperidines as a novel class of selective 5-HT2AR agonists and detail the structure-activity investigations leading to the identification of LPH-5 [analogue (S)-11] as a selective 5-HT2AR agonist with desirable drug-like properties.

Analytical and behavioral characterization of N-ethyl-N-isopropyllysergamide (EIPLA), an isomer of N6 -ethylnorlysergic acid N,N-diethylamide (ETH-LAD).

Preclinical investigations have shown that N-ethyl-N-isopropyllysergamide (EIPLA) exhibits lysergic acid diethylamide (LSD)-like properties, which suggests that it might show psychoactive effects in humans. EIPLA is also an isomer of N6 -ethylnorlysergic acid N,N-diethylamide (ETH-LAD), a lysergamide known to produce psychedelic effects in humans that emerged as a research chemical. EIPLA was subjected to analysis by various forms of mass spectrometry, chromatography (GC, LC), nuclear magnetic resonance (NMR) spectroscopy, and GC condensed-phase infrared spectroscopy. The most straightforward differentiation between EIPLA and ETH-LAD included the evaluation of mass spectral features that reflected the structural differences (EIPLA: N6 -methyl and N-ethyl-N-isopropylamide group; ETH-LAD: N6 -ethyl and N,N-diethylamide group). Proton NMR analysis of blotter extracts suggested that EIPLA was detected as the base instead of a salt, and two blotter extracts suspected to contain EIPLA revealed the detection of 96.9 ± 0.5 µg (RSD: 0.6%) and 85.8 ± 2.8 µg base equivalents based on LC-MS analysis. The in vivo activity of EIPLA was evaluated using the mouse head-twitch response (HTR) assay. Similar to LSD and other serotonergic psychedelics, EIPLA induced the HTR (ED50 = 234.6 nmol/kg), which was about half the potency of LSD (ED50 = 132.8 nmol/kg). These findings are consistent with the results of previous studies demonstrating that EIPLA can mimic the effects of known psychedelic drugs in rodent behavioral models. The dissemination of analytical data for EIPLA was deemed justifiable to aid future forensic and clinical investigations.

A simple, quick and non-destructive approach for sampling drugs of abuse in tablets and blotter for qualitative analysis by paper spray mass spectrometry.

This study presents the development of a simple, fast, and inexpensive approach for the direct analysis of new psychoactive substances (NPS) in seized tablets and blotter paper, with improved sample preservation and increased analytical frequency. Paper triangles were gently rubbed against the surface of the samples containing synthetic drugs and then subjected to analysis by paper spray ionization mass spectrometry (PS-MS). Seized samples containing lysergic acid diethylamide (LSD) and several other substances from the classes of amphetamines, N-benzyl-substituted phenethylamines, synthetic cathinones, and synthetic cannabinoids, were analysed. Three types of paper were tested (filter paper, blotter paper, and synthetic paper) and several combinations of spray solvents were studied for the optimization. All samples were weighed and photographed before and after sequences of analysis in order to attest to the sample preservation. The results revealed that the approach is excellent for sample preservation, with less than 5% of mass loss even after 27 consecutive analyses. Moreover, no significant signal decreases were observed in mass spectrometry (MS) even after the experiments. It was possible to unequivocally identify illicit substances from seized samples (pills and blotter paper). By overcoming the solubilization and wet extraction process used for sample preparation, the waste was restricted to a volume of only 10 µL of solvent for the PS-MS analysis. The main advantage of our approach over existing methods is the sample preparation, which is simple and quick since the samples are just rubbed against the PS paper. This brings enormous benefits in terms of analytical frequency, economy of time and low consumption of solvents. Another important point is that the sample can remain intact for further analysis, which is crucial in forensic analysis.

Psychedelics promote plasticity by directly binding to BDNF receptor TrkB.

Psychedelics produce fast and persistent antidepressant effects and induce neuroplasticity resembling the effects of clinically approved antidepressants. We recently reported that pharmacologically diverse antidepressants, including fluoxetine and ketamine, act by binding to TrkB, the receptor for BDNF. Here we show that lysergic acid diethylamide (LSD) and psilocin directly bind to TrkB with affinities 1,000-fold higher than those for other antidepressants, and that psychedelics and antidepressants bind to distinct but partially overlapping sites within the transmembrane domain of TrkB dimers. The effects of psychedelics on neurotrophic signaling, plasticity and antidepressant-like behavior in mice depend on TrkB binding and promotion of endogenous BDNF signaling but are independent of serotonin 2A receptor (5-HT2A) activation, whereas LSD-induced head twitching is dependent on 5-HT2A and independent of TrkB binding. Our data confirm TrkB as a common primary target for antidepressants and suggest that high-affinity TrkB positive allosteric modulators lacking 5-HT2A activity may retain the antidepressant potential of psychedelics without hallucinogenic effects.

Analytical profile of the lysergamide 1cP-AL-LAD and detection of impurities.

The development of novel lysergamides continues to occur, based on both the needs of psychedelic medicine and commercial interest in new recreational substances. The present study continues the authors' research on novel lysergamides and describes the analytical profile of 1-cyclopropanoyl-AL-LAD (IUPAC name: 1-(cyclopropanecarbonyl)-N,N-diethyl-6-(prop-2-en-1-yl)-9,10-didehydroergoline-8ß-carboxamide; 1cP-AL-LAD), using various chromatographic, mass spectrometric, and spectroscopic methods. Analysis of a powdered sample of 1cP-AL-LAD, obtained from an online vendor, by high performance liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry in full scan/AutoMS/MS mode revealed the detection of 17 impurities based on high-resolution tandem mass spectral data; tentative determination of their identity was based on mass spectral grounds alone, though detection of AL-LAD and 1P-AL-LAD was confirmed using available reference standards. Other tentative compound identifications included 1-acetyl-AL-LAD and several other substances potentially reflecting oxidation of the N6 -allyl group as well as other positions on the ergoline ring system. These data may assist those interested in the chemistry of lysergamides. Finally, 1cP-AL-LAD was also detected in samples of "blotters" sold online for recreational use.

Effects of repeated lysergic acid diethylamide (LSD) on the mouse brain endocannabinoidome and gut microbiome.

BACKGROUND AND PURPOSE: Psychedelics elicit prosocial, antidepressant and anxiolytic effects via neuroplasticity, neurotransmission and neuro-immunomodulatory mechanisms. Whether psychedelics affect the brain endocannabinoid system and its extended version, the endocannabinoidome (eCBome) or the gut microbiome, remains unknown. EXPERIMENTAL APPROACH: Adult C57BL/6N male mice were administered lysergic acid diethylamide (LSD) or saline for 7 days. Sociability was assessed in the direct social interaction and three chambers tests. Prefrontal cortex and hippocampal endocannabinoids, endocannabinoid-like mediators and metabolites were quantified via high-pressure liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). Neurotransmitter levels were assessed via HPLC-UV/fluorescence. Gut microbiome changes were investigated by 16S ribosomal DNA sequencing. KEY RESULTS: LSD increased social preference and novelty and decreased hippocampal levels of the N-acylethanolamines N-linoleoylethanolamine (LEA), anandamide (N-arachidonoylethanolamine) and N-docosahexaenoylethanolamine (DHEA); the monoacylglycerol 1/2-docosahexaenoylglycerol (1/2-DHG); the prostaglandins D2 (PGD2 ) and F2α (PGF2α ); thromboxane 2 and kynurenine. Prefrontal eCBome mediator and metabolite levels were less affected by the treatment. LSD decreased Shannon alpha diversity of the gut microbiota, prevented the decrease in the Firmicutes:Bacteroidetes ratio observed in saline-treated mice and altered the relative abundance of the bacterial taxa Bifidobacterium, Ileibacterium, Dubosiella and Rikenellaceae RC9. CONCLUSIONS AND IMPLICATIONS: The prosocial effects elicited by repeated LSD administration are accompanied by alterations of hippocampal eCBome and kynurenine levels, and the composition of the gut microbiota. Modulation of the hippocampal eCBome and kynurenine pathway might represent a mechanism by which psychedelic compounds elicit prosocial effects and affect the gut microbiome.

Signaling snapshots of a serotonin receptor activated by the prototypical psychedelic LSD.

Serotonin (5-hydroxytryptamine [5-HT]) 5-HT2-family receptors represent essential targets for lysergic acid diethylamide (LSD) and all other psychedelic drugs. Although the primary psychedelic drug effects are mediated by the 5-HT2A serotonin receptor (HTR2A), the 5-HT2B serotonin receptor (HTR2B) has been used as a model receptor to study the activation mechanisms of psychedelic drugs due to its high expression and similarity to HTR2A. In this study, we determined the cryo-EM structures of LSD-bound HTR2B in the transducer-free, Gq-protein-coupled, and ß-arrestin-1-coupled states. These structures provide distinct signaling snapshots of LSD's action, ranging from the transducer-free, partially active state to the transducer-coupled, fully active states. Insights from this study will both provide comprehensive molecular insights into the signaling mechanisms of the prototypical psychedelic LSD and accelerate the discovery of novel psychedelic drugs.

Psychedelics without hallucinations?

[Figure: see text].

Structure-based discovery of nonhallucinogenic psychedelic analogs.

Drugs that target the human serotonin 2A receptor (5-HT2AR) are used to treat neuropsychiatric diseases; however, many have hallucinogenic effects, hampering their use. Here, we present structures of 5-HT2AR complexed with the psychedelic drugs psilocin (the active metabolite of psilocybin) and d-lysergic acid diethylamide (LSD), as well as the endogenous neurotransmitter serotonin and the nonhallucinogenic psychedelic analog lisuride. Serotonin and psilocin display a second binding mode in addition to the canonical mode, which enabled the design of the psychedelic IHCH-7113 (a substructure of antipsychotic lumateperone) and several 5-HT2AR ß-arrestin-biased agonists that displayed antidepressant-like activity in mice but without hallucinogenic effects. The 5-HT2AR complex structures presented herein and the resulting insights provide a solid foundation for the structure-based design of safe and effective nonhallucinogenic psychedelic analogs with therapeutic effects.

Preparation of yolk-shell structure NH2-MIL-125 magnetic nanoparticles for the selective extraction of nucleotides.

Yolk-shell structure magnetic metal-organic framework nanoparticles were prepared via post solvothermal method and employed as a magnetic solid-phase extraction adsorbent for selective pre-concentration of 5'-ribonucleotides by π stacking interaction, hydrogen bonding, and the strong interaction between titanium ions (Ti4+) and phosphate group. The properties of the materials were confirmed by scanning electron microscopy, transmission electron microscopy, X-ray diffraction, X-ray photoelectron spectrometry, vibrating sample magnetometer, infrared spectroscopy, thermogravimetric analysis, and Brunauer-Emmett-Teller analysis. The main parameters affecting the adsorption-desorption process, including adsorbent amount, incubation time, incubation temperature, sample pH, shaking speed, elution solution, and elution time, were systematically optimized. Finally, 1.0 mg of adsorbent mixed with 1.0 mL sample solution (10.0 mmol⋅L-1 NaCl, pH 3.0) and shaken at 135 rpm for 5 min at 40 °C, washed with 1.0 mL Na3PO4-NH3∙H2O under vortex for 5 min were selected as optimized adsorption-desorption conditions. The binding performance of adsorbent towards five nucleotides was evaluated by static adsorption experiments. The data are well-fitted to the Langmuir isotherm model and the maximum adsorption capacity is 27.8 mg g-1 for adenosine 5'-monophosphate. The limit of detection of the method is 19.44-38.41 ng mL-1. Under the optimal conditions, the adsorbent was successfully applied to magnetic solid-phase extraction and high performance liquid chromatography determination of five nucleotides in octopus, chicken, fish, and pork samples.

Biosynthesis, total synthesis, and biological profiles of Ergot alkaloids.

While the use of ergot alkaloids in folk medicine has been practiced for millennia, systematic investigations on their therapeutic potential began about 100 years ago. Subsequently, Albert Hofmann's discovery of lysergic acid diethylamide (LSD) and its intense psychedelic properties garnered worldwide attention and prompted further studies of this compound class. As a result, several natural ergot alkaloids were discovered and unnatural analogs were synthesized, and some were used to treat an array of maladies, including Alzheimer's and Parkinson's disease. While LSD was never commercially approved, recent clinical studies have found it can be an innovative and effective treatment option for several psychiatric disorders. Ongoing biosynthetic and total synthetic investigations aim to understand the natural origins of ergot alkaloids, help develop facile means to produce these natural products and enable their continued use as medicinal chemistry lead structures. This review recounts major developments over the past 20 years in biosynthetic, total synthetic, and pharmaceutical studies. Many ergot alkaloid biosynthetic pathways have been elucidated, with some of them subsequently applied toward "green" syntheses. New chemical methodologies have fostered a fast and efficient access to the ergoline scaffold, prompting some groups to investigate biological properties of natural product-like ergot alkaloids. Limited pharmaceutical applications have yet to completely bypass the undesirable side effects of ergotism, suggesting further studies of this drug class are likely needed and will potentially harness major therapeutic significance.

Validation of a New Sensitive Method for the Detection and Quantification of R and S-Epimers of Ergot Alkaloids in Canadian Spring Wheat Utilizing Deuterated Lysergic Acid Diethylamide as an Internal Standard.

Ergot sclerotia effect cereal crops intended for consumption. Ergot alkaloids within ergot sclerotia are assessed to ensure contamination is below safety standards established for human and animal health. Ergot alkaloids exist in two configurations, the R and S-epimers. It is important to quantify both configurations. The objective of this study was to validate a new ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for quantification of six R and six S-epimers of ergot alkaloids in hard red spring wheat utilizing deuterated lysergic acid diethylamide (LSD-D3) as an internal standard. Validation parameters such as linearity, limit of detection (LOD), limit of quantification (LOQ), matrix effects, recovery and precision were investigated. For the 12 epimers analyzed, low LOD and LOQ values were observed, allowing for the sensitive detection of ergot epimers. Matrix effects ranged between 101-113% in a representative wheat matrix. Recovery was 68.3-119.1% with an inter-day precision of <24% relative standard deviation (RSD). The validation parameters conform with previous studies and exhibit differences between the R and S-epimers which has been rarely documented. This new sensitive method allows for the use of a new internal standard and can be incorporated and applied to research or diagnostic laboratories.

Free energy calculations of the functional selectivity of 5-HT2B G protein-coupled receptor.

G Protein-Coupled Receptors (GPCRs) mediate intracellular signaling in response to extracellular ligand binding and are the target of one-third of approved drugs. Ligand binding modulates the GPCR molecular free energy landscape by preferentially stabilizing active or inactive conformations that dictate intracellular protein recruitment and downstream signaling. We perform enhanced sampling molecular dynamics simulations to recover the free energy surfaces of a thermostable mutant of the GPCR serotonin receptor 5-HT2B in the unliganded form and bound to a lysergic acid diethylamide (LSD) agonist and lisuride antagonist. LSD binding imparts a ∼110 kJ/mol driving force for conformational rearrangement into an active state. The lisuride-bound form is structurally similar to the apo form and only ∼24 kJ/mol more stable. This work quantifies ligand-induced conformational specificity and functional selectivity of 5-HT2B and presents a platform for high-throughput virtual screening of ligands and rational engineering of the ligand-bound molecular free energy landscape.

ᶫ-Leucine Loading and Release in MIL-100 Nanoparticles.

Synthesis of the MIL-100 metal-organic framework particles was carried out by hydrothermal (HT) and microwave (MW)-assisted methods. Transmission electron microscopy showed formation of microparticles in the course of hydrothermal synthesis and nanoparticles for microwave-assisted synthesis. Powder X-ray diffraction confirmed formation of larger crystallites for hydrothermal synthesis. Particle aggregation in aqueous solution was observed by dynamic light scattering. However, the stability of both samples could be improved in acetic acid solution. Nitrogen sorption isotherms showed high porosity of the particles. ᶫ-leucine molecule was used as a model molecule for loading in the porous micro- and nanoparticles. Loading was estimated by FTIR spectroscopy and thermogravimetric analysis. UV-VIS spectroscopy quantified ᶫ-leucine release from the particles in aqueous solution. Cytotoxicity studies using the HeLa cell model showed that the original particles were somewhat toxic, but ᶫ-leucine loading ameliorated the toxic effects, likely due to signaling properties of the amino acid.

Structure of a Hallucinogen-Activated Gq-Coupled 5-HT2A Serotonin Receptor.

Hallucinogens like lysergic acid diethylamide (LSD), psilocybin, and substituted N-benzyl phenylalkylamines are widely used recreationally with psilocybin being considered as a therapeutic for many neuropsychiatric disorders including depression, anxiety, and substance abuse. How psychedelics mediate their actions-both therapeutic and hallucinogenic-are not understood, although activation of the 5-HT2A serotonin receptor (HTR2A) is key. To gain molecular insights into psychedelic actions, we determined the active-state structure of HTR2A bound to 25-CN-NBOH-a prototypical hallucinogen-in complex with an engineered Gαq heterotrimer by cryoelectron microscopy (cryo-EM). We also obtained the X-ray crystal structures of HTR2A complexed with the arrestin-biased ligand LSD or the inverse agonist methiothepin. Comparisons of these structures reveal determinants responsible for HTR2A-Gαq protein interactions as well as the conformational rearrangements involved in active-state transitions. Given the potential therapeutic actions of hallucinogens, these findings could accelerate the discovery of more selective drugs for the treatment of a variety of neuropsychiatric disorders.

[Identification and Analysis of LSD Derivatives in Illegal Products as Paper Sheet].

To prevent the abuse of new psychoactive substances (NPS), a total of 2372 substances and two plants are controlled as "Designated Substances" in Japan as of September 2019. Although the distribution of these substances has decreased for the past three years, newly-emerged NPS are still being found. In this study, we detected four lysergic acid diethylamide (LSD) derivatives as designer drugs from four paper sheet products, which were obtained from 2014 to 2017 in Japan. The compounds were identified as 4-Acetyl-N,N-diethyl-7-methyl-4,6,6a,7,8,9-hexahydroindolo[4,3-fg]quinoline-9-carboxamide (ALD-52), N,N,7-triethyl-4,6,6a,7,8,9-hexahydroindolo[4,3-fg]quinoline-9-carboxamide (ETH-LAD), 7-Allyl-N,N-diethyl-4,6,6a,7,8,9-hexahydroindolo[4,3-fg]quinoline-9-carboxamide (AL-LAD), N,N-diethyl-7-methyl-4-propionyl-4,6,6a,7,8,9-hexahydroindolo[4,3-fg]quinoline-9-carboxamide (1P-LSD), by GC-MS, LC-MS, LC-Q-TOF-MS and NMR analyses. Further, we studied the extraction methods of LSD derivatives from paper sheet, and the analytical conditions of GC-MS, LC-MS and LC-FL(fluorescence). Among LSD derivatives, 1P-LSD have been controlled as designated substances (Shitei Yakubutsu) under the Pharmaceutical and Medical Device Act in Japan since April 2016. For the legislation of the other derivatives identified in this study, the evaluation of their pharmacological properties are now in progress.