Identification of 1-Butyl-Lysergic Acid Diethylamide (1B-LSD) in Seized Blotter Paper Using an Integrated Workflow of Analytical Techniques and Chemo-Informatics

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Article Identiﬁcation of 1-Butyl-Lysergic Acid Diethylamide (1B-LSD) in Seized Blotter Paper Using an Integrated Workﬂow of Analytical Techniques and Chemo-Informatics

Emmanouil Tsochatzis 1, Joao Alberto Lopes 1,*, Fabiano Reniero 1, Margaret Holland 1, Jenny Åberg 2 and Claude Guillou 1,* 1 European Commission, Joint Research Centre (JRC), I-20127 Ispra, Italy; [email protected] (E.T.); [email protected] (F.R.); [email protected] (M.H.) 2 Swedish Customs Laboratory, Box 6055, SE-171 06 Solna, Sweden; [email protected] * Correspondence: [email protected] (J.A.L.); [email protected] (C.G.); Tel.: +32-1457-3032 (J.A.L.); +39-0332785678 (C.G.) Received: 24 January 2020; Accepted: 5 February 2020; Published: 7 February 2020

Abstract: The rapid dispersion of new psychoactive substances (NPS) presents challenges to customs services and analytical laboratories, which are involved in their detection and characterization. When the seized material is limited in quantity or of a complex nature, or when the target substance is present in very small amounts, the need to use advanced analytical techniques, efficient workflows and chemo-informatics tools is essential for the complete identification and elucidation of these substances. The current work describes the application of such a workflow in the analysis of a single blotter paper, seized by Swedish customs, that led to the identification of a lysergic acid diethylamide (LSD) derivative, 1-butyl-lysergic acid diethylamide (1B-LSD). Such blotter paper generally contains an amount in the range of 30–100 ug. This substance, which is closely related to 1-propionyl-lysergic acid diethylamide (1P-LSD), seems to have only recently reached the drug street market. Its identification was made possible by comprehensively combining gas chromatography with mass spectrometry detection (GC–MS), liquid chromatography coupled with high-resolution tandem MS (LC–HR-MS/MS), Orbitrap-MS and both 1D and 2D nuclear-magnetic-resonance (NMR) spectroscopy. All the obtained data have been managed, assessed, processed and evaluated using a chemo-informatics platform to produce the effective chemical and structural identification of 1B-LSD in the seized material.

Keywords: blotter paper sample; 1-butyl-lysergic acid diethylamide (1B-LSD); HR–MS/MS; NMR; GC–MS

1. Introduction During the past decade, there has been a significant increase in the circulation of new psychoactive substances (NPS) within the EU. This evolution in the drugs market, and the speed at which new substances are being created, is a cause of great concern for regulatory bodies. In 2016, NPS were reported to the EU Early Warning System at a rate of one per week, and it is estimated that a similar trend continued during the following years [1]. At the EU level, two agencies have a particular interest in this field—the European Monitoring Center for Drugs and Drug Addiction (EMCDDA) and EUROPOL [1–3]. Customs authorities are responsible for controlling the flow of goods into the EU, acting as the first control and contact point for NPS. However, the majority of customs laboratories are not equipped for

Molecules 2020, 25, 712; doi:10.3390/molecules25030712 www.mdpi.com/journal/molecules Molecules 2020, 25, x FOR PEER REVIEW 2 of 10

Customs authorities are responsible for controlling the flow of goods into the EU, acting as the first control and contact point for NPS. However, the majority of customs laboratories are not equipped for the analysis of these substances, as they normally lack the advanced analytical and chemo-informatics tools that enable the complete identification and characterization of any new and/or relatively unknown NPS. These tools and expertise are available at the European Commission’s Joint Research Centre (JRC), and, therefore, a collaborative project with the Customs Laboratories European Network (CLEN) has been established. Whenever an EU customs laboratory cannot completely identify an unknown seized substance/mixture, it is sent to the JRC for additional analysis. Since 2014, dozens of substances have been identified in the frame of this collaboration, including someMolecules which2020, 25 ,were 712 previously unreported [3]. 2 of 10 One of the most well-known and characterized psychoactive substances is d-lysergic acid diethylamidethe (LSD), analysis which of these substances,is most commonly as they normally circulated lack the advanced through analytical small pieces and chemo-informatics of paper called “blotter paper” and toolsis widespread that enable the all complete over identificationthe world and because characterization of its strong of any new hallucinogenic and/or relatively unknowneffect [4]. In recent years, structurallyNPS. These modified tools and expertiseLSD-type are available NPS have at the been European found Commission’s on the market, Joint Research as is Centre reported (JRC), in several and, therefore, a collaborative project with the Customs Laboratories European Network (CLEN) has scientific papersbeen established. [5–9]. WheneverOne of anthem, EU customs 1-propionyl-d-lysergic laboratory cannot completely acid identify diethylamide an unknown seized(1P-LSD), was identified andsubstance characterized/mixture, it isafter sent toextracti the JRCon for from additional a seized analysis. sample Since 2014, by applying dozens of substances 1H and 13 haveC-NMR along with GC–MSbeen and identified UHPLC-qTOF-MS in the frame of this analysis collaboration, [5]. including Another some closely which related were previously LSD derivative, unreported [3]. 1-butanoyl- One of the most well-known and characterized psychoactive substances is d-lysergic acid d-lysergic aciddiethylamide diethylamide (LSD), which (1B-LSD), is most commonly appears circulated to have through already small reached pieces of the paper street called market, “blotter although no seizure ofpaper” this and drug is widespread has yet been all over reported the world (F becauseigure of1). its The strong only hallucinogenic articles published effect [4]. In present the analysis of commercialrecent years, structurally standards, modified using LSD-type some NPSanalytical have been techniques found on the (GC–MS, market, as isNMR, reported LC-MS) in [9], or the screeningseveral procedures scientific papers for the [5–9 ].detection One of them, of 1-propionyl-d-lysergic nine LSD derivatives acid diethylamide in rat urine, (1P-LSD), including was 1B-LSD, identified and characterized after extraction from a seized sample by applying 1H and 13C-NMR using LC–HR-MS/MSalong with GC–MS [10]. and UHPLC-qTOF-MS analysis [5]. Another closely related LSD derivative, The current1-butanoyl-d-lysergic study reports acid the diethylamide integrated (1B-LSD), approach appears used to have for already the extraction reached the street and market, identification of 1B-LSD, fromalthough a blotter no seizure paper of sample this drug has(labeled yet been “1B-LS reportedD (Figure Blotters1). The (25 only MCG)”) articles publishedfound in present a package seized the analysis of commercial standards, using some analytical techniques (GC–MS, NMR, LC-MS) [9], by the Swedishor the customs screening on procedures 05/11/2018 for the at detection Arlanda of nine airport. LSD derivatives The chemical in rat urine, identification including 1B-LSD, was performed using GC–MS,using HR–MS, LC–HR-MS NMR/MS [10 and]. chemo-informatics tools.

Figure 1. ChemicalFigure 1. Chemicalstructures, structures, molecular molecular formulas formulas and molecular molecular masses masses (Da) of (Da) 1-butyl-lysergic of 1-butyl-lysergic acid acid diethylamide (1B-LSD) and 1-propionyl-lysergic acid diethylamide (1P-LSD). diethylamide (1B-LSD) and 1-propionyl-lysergic acid diethylamide (1P-LSD). The current study reports the integrated approach used for the extraction and identification of 2. Results 1B-LSD, from a blotter paper sample (labeled “1B-LSD Blotters (25 MCG)”) found in a package seized by the Swedish customs on 05/11/2018 at Arlanda airport. The chemical identification was performed using GC–MS, HR–MS, NMR and chemo-informatics tools. 2.1. GC-MS Analysis 2. Results From the chromatogram of the GC–MS analysis (Figure S1), it could be concluded that the matrix was quite complex.2.1. GC-MS The Analysis main peak was among the last to be eluted, with a retention time of 23.5 min, and was identifiedFrom as the potentially chromatogram 1B-LSD of the GC–MS (see Figure analysis (Figure2), in agreement S1), it could be with concluded the fragmentation that the matrix patterns was quite complex. The main peak was among the last to be eluted, with a retention time of 23.5 min, of 1P-LSD andand wasthe identified 1B-LSD, as potentiallyas reported 1B-LSD by (seeBrandt Figure 2et), inal. agreement [5,9]. The with complete the fragmentation identification patterns has been verified andof confirmed 1P-LSD and theby 1B-LSD,the results as reported obtained by Brandt from et NMR al. [5,9]. and The HR-MS. complete identification has been verified and confirmed by the results obtained from NMR and HR-MS.

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Figure 2. MS spectrum resulting from the GC–MS peak, identified as 1B-LSD. FigureFigureFigure 2. 2.MSMS 2.spectrum MS spectrum spectrum resulting resulting resulting from from from the the GCGC–MSthe–MS GC–MS peak,peak, peak, identifiedidentified identified as as 1B-LSD. 1B-LSD.as 1B-LSD. Figure 2. MS spectrum resulting from the GC–MS peak, identified as 1B-LSD. The resultingFigure identification 2. MS spectrum of resulting the most from rele thevant GC–MS fragments, peak, identified as received as 1B-LSD. from the chemo- The resultingFigure identification 2. MS spectrum of resulting the most from rele thevant GC–MS fragments, peak, identified as received as 1B-LSD. from the chemo- TheinformaticsThe resultingThe resulting resulting tool identificationidentificationFigure for theidentification 2. GC–MS MS spectrum of of the analysis, the mostof resulting themost relevant are most presentedrele from fragments,vantrele thevant GC fragments,in–MS Tablefragments, as peak, received 1. Nevertheless, identifiedas as fromreceived received theas 1B-LSD. chemo-informatics itfrom shall from bethe thehighlighted chemo- chemo- informaticsThe resulting toolFigure for theidentification 2. GC–MS MS spectrum analysis, of resulting the are most presented from rele thevant GC in–MS Tablefragments, peak, 1. Nevertheless, identified as received as 1B-LSD. it shall from be thehighlighted chemo- informaticstoolinformaticsthat for theallThe tool GC–MS GC–MS resulting for tool theFigure fragmentationfor analysis, GC–MS theidentification 2. GC–MS MS areanalysis, spectrum presented patterns analysis, of areresulting the werepresented are in most Table presented fromconsiste rele1 the. in Nevertheless,vant GCTablent in with–MS Tablefragments, 1. peak,those Nevertheless, 1. Nevertheless, itidentified reported shall as received be as by highlightedit 1B-LSD. shall itBrandt shall from be be ethighlighted thehighlighted thatal. [9].chemo- all informaticsthat allThe GC–MS resulting tool fragmentationfor theidentification GC–MS patternsanalysis, of the were are most presented consiste relevantnt in with Tablefragments, those 1. Nevertheless, reported as received by itBrandt shall from be et highlightedtheal. [9].chemo- thatGC–MS allinformaticsthat GC–MS all fragmentationThe GC–MS resultingfragmentation tool fragmentationfor patternstheidentification GC–MS patterns were patterns analysis, consistentof were the were are consistemost presented with consiste relent those vantwithnt in reportedwith Table fragments,those those 1. reported byNevertheless, reported Brandt as received by et byBrandt al. itBrandt [shall9 ].from et be etal. thehighlightedal. [9]. [9].chemo- thatinformatics allThe GC–MS resulting tool fragmentationfor theidentification GC–MS patterns analysis, of the were are most presented consiste relevantnt in with Tablefragments, those 1. Nevertheless, reported as received by itBrandt shall from beet thehighlightedal. [9].chemo- informaticsthat allThe GC–MS resulting toolTable fragmentationfor 1. theGC–MS-identifiedidentification GC–MS patternsanalysis, of fragmentsthe were are most presented consiste for rele 1B-LvantntSD in with Table(ACD/Spectrusfragments, those 1. Nevertheless, reported as Processor received by itBrandt 2017.2.1).shall from be et highlightedthe al. [9].chemo- informaticsthat all GC–MSTable toolTable 1. fragmentationforGC–MS-identified 1. theGC–MS-identified GC–MS patternsanalysis, fragments fragments were are for presented consiste 1B-LSD for 1B-L (ACDntSD in with Table(ACD/Spectrus/Spectrus those 1. Nevertheless, reported Processor Processor by 2017.2.1). itBrandt 2017.2.1).shall be et highlightedal. [9]. thatinformatics allTable GC–MS toolTable1. GC–MS-identified fragmentationfor 1. theGC–MS-identified GC–MS patterns analysis, fragments fragments were are for presented consiste1B-L for 1B-LSD nt(ACD/SpectrusSD in with Table(ACD/Spectrus those 1. Nevertheless, reported Processor Processor by 2017.2.1). itBrandt 2017.2.1).shall be et highlightedal. [9]. that all GC–MSFragmentTable fragmentation 1. GC–MS-identified New patterns fragments were consiste for 1B-LntSD with (ACD/Spectrus those reported Processor by Brandt 2017.2.1). et al. [9]. thatNo. all GC–MSFragmentTable fragmentation 1. GC–MS-identified New patternsFormula fragments were consiste for 1B-LntSD with (ACD/SpectrusLabel those reported Processorm/z by Exp.1 Brandt 2017.2.1). 1 RI et Exp.al. [9]. (%)2 2 No. FragmentFragmentTable New 1. GC–MS-identified StructureNew fragments Formula for 1B-LSD Label (ACD/Spectrusm /Processorz Exp. 2017.2.1).1 RI Exp. (%) 2 No.Fragment FragmentStructure New New Formula Label m/z Exp. 1 RI Exp. (%) 2 No. TableStructure 1. GC–MS-identified Formula fragments for 1B-LSD (ACD/SpectrusLabel Processorm/z Exp.1 2017.2.1). 1 RI Exp. (%)2 2 No. No. FragmentTableStructure 1. GC–MS-identified New O FormulaFormula fragments for 1B-LSDLabel (ACD/SpectrusLabel m/z Processorm/z Exp. Exp. 2017.2.1). RIRI Exp. Exp. (%) (%) 1 2 No. StructureFragmentOTableStructure 1. GC–MS-identified New O Formula fragments for 1B-LSD (ACD/SpectrusLabel Processorm/z Exp. 2017.2.1). RI Exp. (%) O N 1 2 O N 1 No.1 FragmentStructureO New O N C24H31N3O2(+)C24H31N3O2(Formula + ) MLabelM 393.2m/z393.2 Exp. RI 100.0 Exp.100.0 (%) O + 1 2 FragmentNStructure NNew N No.1 O O C24H31N3O2(+)Formula LabelM m/z393.2 Exp. RI Exp.100.0 (%) O N + 1 2 No.1 FragmentN NNewN C24H31N3O2(+)Formula LabelM m/z393.2 Exp. RI Exp.100.0 (%) N Structure+ O 1 1 O N O N C24H31N3O2(+)C24H31N3O2(+) M M 393.2393.2 1 100.0100.0 2 No. Structure+ + N Formula Label m/z Exp. RI Exp. (%) O N NO N O 1 StructureN N C24H31N3O2(+) M 393.2 100.0 O + O O N N 1 N C24H31N3O2(+) M 393.2 100.0 O O N + O O N N 12 N N C24H31N3O2(+)C18Η15Ν2Ο2(+) M-C6H16NM 393.2 291.1 100.0 79.3 O + 2 O N N N C18H15N2O2(+) M-C6H16N 291.1 79.3 12 N N N C24H31N3O2(+)C18Η15Ν2Ο2(+) M-C6H16NM 393.2 291.1 100.0 79.3 N O + 12 N N + C24H31N3O2(+)C18Η15Ν2Ο2(+) M-C6H16NM 393.2 291.1 100.0 79.3 2 O + N C18Η15Ν2Ο2(+) M-C6H16N 291.1 79.3 2 N N +N C18Η15Ν2Ο2(+) M-C6H16N 291.1 79.3 O + 2 N N+ C18Η15Ν2Ο2(+) M-C6H16N 291.1 79.3 O N + C 2 + O C18Η15Ν2Ο2(+) M-C6H16N 291.1 79.3 N N+ HN C 3 + + C16H15N2O(+) M-C8H16NO 251.1 15.5 2 N CN O C18Η15Ν2Ο2(+) M-C6H16N 291.1 79.3 HN N 23 + + O C18C16H15N2O(+)Η15Ν2Ο2(+) M-C8H16NOM-C6H16N 291.1251.1 79.315.5 HN + NC C 3 3 N + O C16H15N2O(+)C16H15N2O(+) M-C8H16NOM-C8H16NO 251.1 251.1 15.5 15.5 2 + C18Η15Ν2Ο2(+) M-C6H16N 291.1 79.3 3 HN O N NC C16H15N2O(+) M-C8H16NO 251.1 15.5 HN + 3 + O C16H15N2O(+) M-C8H16NO 251.1 15.5 NH N +NC 3 + C16H15N2O(+) M-C8H16NO 251.1 15.5 N CH + O 3 HNN N CH+C C16H15N2O(+) M-C8H16NO 251.1 15.5 4 CH+ + O C15H13N2(+) M-C9H18NO2 221.1 58.665 3 HNN NCHCHC C16H15N2O(+) M-C8H16NO 251.1 15.5 4 N CH+ + O C15H13N2(+) M-C9H18NO2 221.1 58.665 HNN NCH C 34 + O C16H15N2O(+)C15H13N2(+) M-C9H18NO2M-C8H16NO 251.1221.1 58.665 15.5 N CH N CH+ 4 34 HN CH NCHN C16H15N2O(+)C15H13N2(+)C15H13N2(+) M-C9H18NO2M-C9H18NO2M-C8H16NO 221.1 251.1221.1 58.665 58.665 15.5 N CH+ 4 4 N NNCHN C15H13N2(+)C15H13N2(+) M-C9H18NO2M-C9H18NO2 221.1221.1 58.665 58.665 N +CH 4 N + CHNN C15H13N2(+) M-C9H18NO2 221.1 58.665 5 N C N CH+O C12H19N2O(+) M-C12H12NO 207.0 69.3 4 + CHN C15H13N2(+) M-C9H18NO2 221.1 58.665 C+ N CH+O 5 N H2C N CH C12H19N2O(+) M-C12H12NO 207.0 69.3 4 C N CHO C15H13N2(+) M-C9H18NO2 221.1 58.665 5 N N + N C12H19N2O(+) M-C12H12NO 207.0 69.3 4 H2C C N N O C15H13N2(+) M-C9H18NO2 221.1 58.665 5 + H C N C12H19N2O(+) M-C12H12NO 207.0 69.3 C 2 O + NN 5 H C C N O C12H19N2O(+) M-C12H12NO 207.0 69.3 5 5 2 + N C12H19N2O(+)C12H19N2O(+) M-C12H12NOM-C12H12NO 207.0 207.0 69.3 69.3 C +O 5 N C12H19N2O(+) M-C12H12NO 207.0 69.3 6 H2C HN2CH + N NC C12H9N2(+) M-C12H11NO 181.0 45.6 C +O 56 HHN2C + N NC+ N C12H19N2O(+)C12H9N2(+) M-C12H12NOM-C12H11NO 207.0181.0 69.3 45.6 6 C C O C12H9N2(+) M-C12H11NO 181.0 45.6 5 HHN2C +N + N C12H19N2O(+) M-C12H12NO 207.0 69.3 C OC O 56 HHNC N N C12H19N2O(+)C12H9N2(+) M-C12H12NOM-C12H11NO 207.0181.0 69.345.6 2 + + O + NH C N 6 6 HNH C2C N+ O C12H9N2(+)C12H9N2(+) M-C12H11NOM-C12H11NO 181.0181.0 45.6 45.6 7 HN NH + C7H14NO(+) M-C17H20N2O 128.1 20.6 6 + O C CH C12H9N2(+) M-C12H11NO 181.0 45.6 6 HN NHN + N 2 C12H9N2(+) M-C12H11NO 181.0 45.6 7 + C7H14NO(+) M-C17H20N2O 128.1 20.6 67 HN NH OC OCHN 2 C7H14NO(+)C12H9N2(+) M-C17H20N2OM-C12H11NO 181.0 128.1 45.620.6 O +CH 67 HN + C+ N 2 C7H14NO(+)C12H9N2(+) M-C17H20N2OM-C12H11NO 181.0 128.1 45.620.6 + NH N OC +OCH 68 NH + CO 2 C5H10NO(+)C12H9N2(+) M-C19H21N2OM-C12H11NO 181.0 100.0 45.631.4 7 HN NH O + N C7H14NO(+) M-C17H20N2O 128.1 20.6 7 N C+OCH C7H14NO(+) M-C17H20N2O 128.1 20.6 78 CH+N OC N 2 C7H14NO(+)C5H10NO(+) M-C17H20N2OM-C19H21N2O 128.1100.0 20.631.4 8 NH 2 + C5H10NO(+) M-C19H21N2O 100.0 31.4 +NNH C OCH 2 7 78 ONH O + C7H14NO(+)C5H10NO(+)C7H14NO(+ ) M-C17H20N2OM-C17H20N2OM-C19H21N2O 128.1 128.1100.0 20.6 20.631.4 9 +NH +CHOCH C4H10N(+) M-C20H21N2O2 72.0 90.8 7 + NHNNHC 2+ C7H14NO(+) M-C17H20N2O 128.1 20.6 89 N C CH+CH+2 C5H10NO(+)C4H10N(+) M-C20H21N2O2M-C19H21N2O 100.072.0 31.490.8 8 79 NHN CO C5H10NO(+)C7H14NO(+)C4H10N(+) M-C19H21N2OM-C20H21N2O2M-C17H20N2O 100.0 128.172.0 31.4 20.690.8 8 1 +CHCH+2 C5H10NO(+)2 M-C19H21N2O 100.0 31.4 9 m/z Exp.: N C experimentalO mC4H10N(+)/z; RI Exp. (%): RelativeM-C20H21N2O2 Intensity (%) of experimental72.0 m/z. 90.8 8 1 NH CH+ C5H10NO(+)2 M-C19H21N2O 100.0 31.4 1 NHm/z Exp.:N C Oexperimental+ m/z; 2 RI Exp. (%): Relative Intensity (%) of experimental m/z. 8 89 m/z Exp.:+NH CH+experimental C5H10NO(+)mC4H10N(+)C5H10NO(/z; RI Exp.+ (%): ) RelativeM-C19H21N2OM-C20H21N2O2M-C19H21N2O Intensity (%) of100.0 experimental 100.072.0 m/ 31.4z. 31.490.8 9 1 CHN C + C4H10N(+)2 M-C20H21N2O2 72.0 90.8 89 m/z Exp.:NH CHexperimental + C5H10NO(+)mC4H10N(+)/z; RI Exp. (%): RelativeM-C20H21N2O2M-C19H21N2O Intensity (%) of experimental 100.072.0 m/z. 31.490.8 1 2 9 m/z Exp.:NH CHexperimental + mC4H10N(+)/z; RI Exp. (%): RelativeM-C20H21N2O2 Intensity (%) of experimental72.0 m/z. 90.8 9 1 1 NH 2 C4H10N(+)2 M-C20H21N2O2 72.0 90.8 m/z Exp.: m/z experimentalExp.: CHexperimental+ m/z; m RI/z ;Exp. RI Exp.(%): (%):Relative Relative Intensity Intensity (%) (%) of experimental of experimental m/ mz. /z. 9 9 1 m/z Exp.: CHexperimental mC4H10N(+)/zC4H10N(; 2 RI Exp.+ )(%): RelativeM-C20H21N2O2M-C20H21N2O2 Intensity (%) of 72.0experimental72.0 m/z 90.8. 90.8 1 m/z Exp.: experimental m/z; 2 RI Exp. (%): Relative Intensity (%) of experimental m/z. 1 m/zz Exp.: experimentalm zm2/z; 2 RI Exp. (%): Relative Intensity (%) of experimentalm z m/z. / Exp.: experimental / ; RI Exp. (%): Relative Intensity (%) of experimental / .

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Molecules 2020, 25, x FOR PEER REVIEW 4 of 10 2.2. UHPLC–HR-MS Experiment Molecules2.2. UHPLC–HR-MS 2020, 25, x FOR PEERExperiment REVIEW 4 of 10 The full-scan UHPLC–qTOF-MS analysis (positive mode) of the blotter paper methanol extract revealed2.2. UHPLC–HR-MS theThe TICfull-scan of a relativelyUHPLC–qTOF-MS Experiment complex anal mixture,ysis (positive in accordance mode) of withthe blotter what paper had beenmethanol observed extract in the revealed the TIC of a relatively complex mixture, in accordance with what had been observed in the + GC–MS analysisThe full-scan (Figure UHPLC–qTOF-MS S2). The extraction analysis of (positive the ion mode) chromatogram of the blotter (XIC) paper with methanol the [M extract+ H] of GC–MS analysis (Figure S2). The extraction of the ion chromatogram (XIC) with the [M + H]+ of 1B- 1B-LSDrevealed (m/z 394.249), the TIC of as a receivedrelatively fromcomplex the mixture, LC–qTOF-MS in accordance analysis, with revealed what had a clear been and observed signiﬁcant in the peak. LSD (m/z 394.249), as received from the LC–qTOF-MS analysis, revealed a clear and significant peak. The analyticalGC–MS analysis system (Figure allowed S2). forThe aextraction chromatographic of the ion chromatogram separation to (XIC) be performed with the [M whilst + H]+ producingof 1B- The analytical system allowed for a chromatographic separation to be performed whilst producing LSD (m/z 394.249), as received2 from the LC–qTOF-MS analysis, revealed a clear and significant peak. untargeteduntargeted (all-ion (all-ion mode) mode) MS MSspectra2 spectra (Figure (Figure3 3).). The analytical system allowed for a chromatographic separation to be performed whilst producing untargeted (all-ion mode) MS2 spectra (Figure 3).

2 + FigureFigure 3. U(H)PLC–qTOF-MS 3. U(H)PLC–qTOF-MS (A ()A full-scan) full-scan MS MS ( (BB)) untargeteduntargeted MS MS 2spectrumspectrum of ofm/zm 394.249/z 394.249 ([M ([M+ H]+) H]+) of the extract. of theFigure extract. 3. U(H)PLC–qTOF-MS (A) full-scan MS (B) untargeted MS2 spectrum of m/z 394.249 ([M + H]+) of the extract. For theFor Orbitrap-MSthe Orbitrap-MS experiments, experiments, thethe samplesample extr extractact was was directly directly infused infused into into the system the system for for analysis. Full-scan MS was performed initially, with a multitude of ions being found, as the blotter analysis.For Full-scan the Orbitrap-MS MS was performedexperiments, initially, the sample with extr aact multitude was directly of ions infused being into found, the system as the for blotter paper methanol extract, containing several components, was injected directly. In fact, the lack of a analysis. Full-scan MS was performed initially, with a multitude of ions being found, as the blotter paperchromatographic methanol extract, technique containing coupled several to the components, Orbitrap, increased was injected the complexity directly. In of fact, the theresults. lack of a paper methanol extract, containing several components, was injected directly. In fact, the lack of a chromatographicHowever, the technique previous results, coupled indicating to the Orbitrap, the possible increased presence the complexityof 1B-LSD, allowed of the results. an oriented However, chromatographic technique coupled to the Orbitrap, increased the complexity of the results. the previousextraction results, of its protonat indicatinged molecular the possible ion [M presence+ H]+, which of was 1B-LSD, found allowedand confirmed an oriented as m/z 394.25004. extraction of However, the previous results, indicating the possible presence of 1B-LSD, allowed an oriented its protonatedAfter this analysis, molecular the ionion was [M + isolatedH]+, which and fragmented was found successively and conﬁrmed (MS2, MS as3 m and/z 394.25004.MS4), and Figure After this extraction of its protonated molecular ion [M + H]+, which was found and confirmed as m/z 394.25004. analysis,4 shows the the ion fragmentation was isolated spectrum and fragmented of this ion successively (MS2). (MS2, MS3 and MS4), and Figure4 shows After this analysis, the ion was isolated and fragmented successively (MS2, MS3 and MS4), and Figure 2 the fragmentation4 shows the fragmentation spectrum of spectrum this ion of (MS this ).ion (MS2).

2 Figure 4. MS spectrum from Infusion to Orbitrap MS of the extract [selected molecular ion m/z was 394.25004 assigned to the protonated molecular mass [M + H]+. Figure 4. 2MS2 spectrum from Infusion to Orbitrap MS of the extract [selected molecular ion m/z was Figure 4. MS spectrum from Infusion to Orbitrap MS of the extract [selected molecular ion m/z was + 394.25004394.25004 assigned assigned to theto the protonated protonated molecular molecular massmass [M [M ++ H]H]. +.

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All of asresults 1B-LSD, received were as from reported consistent the chemo-informaticsby Brandtwith bothet al. [9].the tool qTOF-MS for the fragmentationOrbitrap-MS,Table 2.andare Orbitrap-MS-identified thepresented fragmentation in Table patternsfragments 2. All of forresults 1B-LSD, 1B-LSD were as(ACD/Spectrus reported consistent by ProcessorBrandt with bothet 2017.2.1). al. [9].the qTOF-MS fragmentationOrbitrap-MS,TableTable 2. Orbitrap-MS-identified areand2. Orbitrap-MS-identified thepresented fragmentation in fragmentsTable patterns fragments 2. forAll 1B-LSDof forresults 1B-LSD, 1B (ACD-LSD were as /(ACD/SpectrusSpectrus reported consistent Processor by ProcessorBrandtwith 2017.2.1). bothet 2017.2.1). al. [9].the qTOF-MS fragmentationTableTable and 2.2. Orbitrap-MS-identifiedOrbitrap-MS-identified the fragmentation patterns fragmentsfragments of for for1B-LSD, 1B1B-LSD-LSD as (ACD/Spectrus(ACD/Spectrus reported by ProcessorBrandtProcessor et 2017.2.1). 2017.2.1).al. [9]. fragmentationFragmentTable and2. Orbitrap-MS-identified Newthe fragmentation patterns fragments of for 1B-LSD, 1B-LSD as (ACD/Spectrus reported by BrandtProcessor et 2017.2.1).al. [9]. No. Fragment New Formula Label m/z Exp.1 1 RI Exp. (%)2 2 No.No. FragmentTableFragment New2. Orbitrap-MS-identified StructureNew Formula Formula fragments for 1B-LSD LabelLabel (ACD/Spectrus m/z Exp.m/z Processor Exp. 1RI 2017.2.1). Exp.RI Exp. (%) (%) 2 FragmentTableStructure 2. Orbitrap-MS-identified New fragments for 1B-LSD (ACD/Spectrus Processor1 2017.2.1). 2 No. FragmentTableStructure 2. Orbitrap-MS-identified New Formula fragments for 1B-LSDLabel (ACD/Spectrus m/z Processor Exp. 1 2017.2.1).RI Exp. (%) 2 No. Structure O Formula Label m/z Exp. 1 RI Exp. (%) 2 No. Table 2. Orbitrap-MS-identifiedO Formula fragments for 1B-LSDLabel (ACD/Spectrus m/z Processor Exp. 2017.2.1).RI Exp. (%) FragmentO Structure New Structure ON 1 2 No. FragmentO N New Formula Label m/z Exp. RI Exp. (%) 1 1 Fragment NewO N C24H31N3O2(+)C24H31N3O2( +) MM 394.24890394.24890 1 3.5 3.5 2 No. O StructureN + O Formula Label m/z Exp. RI Exp. (%) 1 O N N C24H31N3O2(+) M 394.248901 3.5 2 No. Fragment New+ Formula Label m/z Exp. RI Exp. (%) StructureN N N 1 2 1 O N O C24H31N3O2(+) M 394.24890 3.5 No.1 Structure+ N C24H31N3O2(+)Formula LabelM 394.24890m/z Exp. RI Exp.3.5 (%) N N+ O 1 O StructureON C24H31N3O2(+) M 394.24890 3.5 + O N O N NO 1 N O N C24H31N3O2(+) M 394.24890 3.5 + O N +O 1 C N NO N N C24H31N3O2(+) M 394.24890 3.5 2 + O N + C21H26N3O2(+) M + H-C3H6 352.20195 3.6 C N N N 21 O N O N C21H26N3O2(+)C24H31N3O2(+) M + H-C3H6M 352.20195394.24890 3.63.5 2 + N + C21H26N3O2(+) M + H-C3H6 352.20195 3.6 1 O C N N N N C24H31N3O2(+) M 394.24890 3.5 2 C+ N O+ C21H26N3O2(+) M + H-C3H6 352.20195 3.6 N N 2 O + O N C21H26N3O2(+) M + H-C3H6 352.20195 3.6 2 O C N N O C21H26N3O2(+) M + H-C3H6 352.20195 3.6 O O N O C+ N N 2 ON O N C21H26N3O2(+) M + H-C3H6 352.20195 3.6 + O 2 O C N N N N C21H26N3O2(+) M + H-C3H6 352.20195 3.6 C+ N O 32 O N N N C19H21N2O(+)C21H26N3O2(+) M + M H-C5H11NO + H-C3H6 293.16454 352.20195 52.7 3.6 C+ N N 32 O O N C21H26N3O2(+)C19H21N2O(+) M + M H-C5H11NOM + +H-C3H6 293.16454 352.20195 52.7 3.6 N N 3 3 O O N C19H21N2O(+)C19H21N2O(+ ) M + H-C5H11NO293.16454 293.16454 52.7 52.7 + N 3 N C19H21N2O(+) MH-C5H11NO + H-C5H11NO 293.16454 52.7 O + N 3 O N C19H21N2O(+) M + H-C5H11NO 293.16454 52.7 +N N + 3 N+ C19H21N2O(+) M + H-C5H11NO 293.16454 52.7 N CH+ 3 HN + C19H21N2O(+) M + H-C5H11NO 293.16454 52.7 4 CHN C15H15N2(+) M-C9H16NO2 223.12265 27.5 3 HN + C19H21N2O(+) M + H-C5H11NO 293.16454 52.7 4 N N + C15H15N2(+) M-C9H16NO2 223.12265 27.5 3 HN CH+ C19H21N2O(+) M + H-C5H11NO 293.16454 52.7 N CH+ N 4 HN + C15H15N2(+) M-C9H16NO2 223.12265 27.5 4 + C15H15N2(+) M-C9H16NO2 223.12265 27.5 4 HN N CHN C15H15N2(+) M-C9H16NO2 223.12265 27.5 4 N + + C15H15N2(+) M-C9H16NO2 223.12265 27.5 CH NH N + + 4 N CHCH C15H15N2(+) M-C9H16NO2 223.12265 27.5 HN + + 54 N NCHCH C12H20N2O(+)C15H15N2(+) M-C12H12NOM-C9H16NO2 208.07528223.12265 69.327.5 5 HN N ++ C12H20N2O(+) M-C12H12NO 208.07528 69.3 4 N N CHCH+ C15H15N2(+) M-C9H16NO2 223.12265 27.5 HN HN CH 45 N N + C12H20N2O(+)C15H15N2(+) M-C9H16NO2M-C12H12NO 223.12265208.07528 27.569.3 5 N HNNCH C12H20N2O(+) M-C12H12NO 208.07528 69.3 5 5 N + C12H20N2O(+)C12H20N2O(+ ) M-C12H12NOM-C12H12NO 208.07528208.07528 69.369.3 H CH N HNN + 5 CH C12H20N2O(+) M-C12H12NO 208.07528 69.3 N + HN + 5 N CH C12H20N2O(+) M-C12H12NO 208.07528 69.3 6 N C + NO + C12H19N2O(+) M + H-C12H13NO 207.14919 13.5 5 HNN C12H20N2O(+) M-C12H12NO 208.07528 69.3 6 N C CHO C12H19N2O(+) M + H-C12H13NO 207.14919 13.5 5 + HNN C12H20N2O(+) M-C12H12NO 208.07528 69.3 H2C C+N O 6 NH C12H19N2O(+) M + H-C12H13NO 207.14919 13.5 6 H C C+N O C12H19N2O(+) M + H-C12H13NO 207.14919 13.5 2 HN 6 H C C N O C12H19N2O(+) M + H-C12H13NO 207.14919 13.5 2 +N N M + H2C C O 6 6 + N C12H19N2O(+)C12H19N2O(+ ) M + H-C12H13NO207.14919 207.14919 13.5 13.5 H2C N + 6 C+ N O C12H19N2O(+) MH-C12H13NO + H-C12H13NO 207.14919 13.5 7 HN CH N C + C12H8N2(+) M-C12H11NO 180.06820 36.0 6 2 C+ O C12H19N2O(+) M + H-C12H13NO 207.14919 13.5 7 HN C + C12H8N2(+) M-C12H11NO 180.06820 36.0 6 H2C C N NO C12H19N2O(+) M + H-C12H13NO 207.14919 13.5 7 HN N C+ N C12H8N2(+) M-C12H11NO 180.06820 36.0 7 H2C C C12H8N2(+) M-C12H11NO 180.06820 36.0 HHNC N + N 7 HN2 C C12H8N2(+) M-C12H11NO 180.06820 36.0 + N 7 C N C12H8N2(+) M-C12H11NO 180.06820 36.0 8 NH + C11H6N2(+) M-C11H9NO 166.05255 5.1 7 7 HNHN C+N C12H8N2(+)C12H8N2(+ ) M-C12H11NOM-C12H11NO 180.06820180.06820 36.036.0 8 HN + C C11H6N2(+) M-C11H9NO 166.05255 5.1 7 HN C + C +NN C12H8N2(+) M-C12H11NO 180.06820 36.0 78 HN C CC N C12H8N2(+)C11H6N2(+) M-C12H11NOM-C11H9NO 180.06820166.05255 36.05.1 8 HHNN + O N C11H6N2(+) M-C11H9NO 166.05255 5.1 + 8 HN C + CO NN C11H6N2(+) M-C11H9NO 166.05255 5.1 NC+ C C N 9 + O C5H10NO(+) M-C19H22N2O 100.07569 4.5 8 NH CN CCO N C11H6N2(+) M-C11H9NO 166.05255 5.1 9 + + C5H10NO(+) M-C19H22N2O 100.07569 4.5 8 HN CN C+O NC C11H6N2(+) M-C11H9NO 166.05255 5.1 8 89 N+ C C5H10NO(+)C11H6N2(+)C11H6N2(+ ) M-C11H9NOM-C19H22N2OM-C11H9NO 166.05255166.05255100.07569 5.1 5.1 4.5 9 1 Hm/zN Exp.:C C+ experimentalN C5H10NO(+) m/z; 2 RI Exp. (%): RelativeM-C19H22N2O Intensity (%) of 100.07569experimental m/z. 4.5 98 1HN N+ C O C5H10NO(+)C11H6N2(+)2 M-C19H22N2OM-C11H9NO 100.07569166.05255 4.55.1 m/z CExp.:+ +C experimentalN m/z; RI Exp. (%): Relative Intensity (%) of experimental m/z. 1 CN CCO N 2 9 1 m/z Exp.:+O experimentalC5H10NO(+) m/z; 2 RI Exp. (%): RelativeM-C19H22N2O Intensity (%) of100.07569 experimental m/z. 4.5 m/z Exp.:N C experimental m/z; RI Exp. (%): Relative Intensity (%) of experimental m/z. 9 1 +O C5H10NO(+)2 M-C19H22N2O 100.07569 4.5 m/z Exp.:N C experimental m/z; RI Exp. (%): Relative Intensity (%) of experimental m/z. 2.3. NMR9 + C5H10NO(+) M-C19H22N2O 100.07569 4.5 9 2.3. NMR9 1 m/z Exp.:N C experimentalC5H10NO(+) C5H10NO(m/z; 2 RI Exp.+ ) (%):M-C19H22N2O RelativeM-C19H22N2O Intensity (%) 100.07569 of100.07569 experimental m/z 4.5. 4.5 1 m/z Exp.: experimental m/z; 2 RI Exp. (%): Relative Intensity (%) of experimental m/z. 2.3.2.3. NMRNMR 1 m/z Exp.: experimental m/z; 2 RI Exp. (%): Relative Intensity (%) of experimental m/z. Despite 1the fact that the sample aliquot2 was so small (the amount of LSD in a blotter is usually 2.3. NMRDespite 1 m/ thezm/zExp.: Exp.:fact experimental that experimental the samplem/z; 2 m/zRI Exp.aliquot; RI(%): Exp. was Relative (%): so Relative Intensitysmall (theIntensity (%) ofamount experimental (%) of of experimental LSDm/z .in a blotter m/z. is usually 2.3. NMRDespite the fact that the1 sample aliquot was so small (the amount of LSD in a blotter is usually in2.3. the NMRDespite range 30–100 the fact μg), that the the 1H-NMR sample spectrum aliquot was revealed so small a complex (the amount mixture of LSD (Figure in a 5) blotter in which is usually many in2.3. the NMRDespite range 30–100 the fact μ g),that the the 1H-NMR sample spectrumaliquot was revealed so small a complex (the amount mixture of LSD (Figure in a 5)blotter in which is usually many signalsin2.3.in thethe NMRDespite range rangecould 30–100 30–100 thebe identifiedfact μμ g),thatg), thethe the as 1H-NMRH-NMR sampletypical spectrumaliquotspectrumof LSD-li was kerevealedrevealed sosubstances, small aa complexcomplex(the and amount mixturewhichmixture of shareLSD (Figure(Figure in the a 5) 5)blottersame inin whichwhich backboneis usually manymany signalsin theDespite range could 30–100 thebe factidentified μ g),that the the as1H-NMR sample typical spectrumaliquot of LSD-li was revealedke so substances, small a complex (the andamount mixturewhich of LSDshare (Figure in the a 5)blotter same in which backboneis usually many 2.3. NMRstructure.signalsinsignals theDespite range couldcould Nevertheless, 30–100 the bebe factidentifiedidentified μ g),that thethe the most1asasH-NMR sample typicaltypical noteworthy spectrumaliquot ofof LSD-liLSD-li wassignal revealedkeke so substances,substances,s smallwere a complex those(the andamountand which mixture whichwhich allowedof LSDshare share(Figure in the thethe a 5) blotteridentification same samein which backboneisbackbone usually many of structure.signalsin theDespite range could Nevertheless, 30–100 the be factidentified μ g),that the the as1mostH-NMR sample typical noteworthy spectrumaliquot of LSD-li was signal revealedke so substances,s small were a complex (thethose andamount which mixturewhich ofallowed LSDshare (Figure in thethe a 5) blotter identificationsame in which backboneis usually many of 1B-LSDstructure.instructure. the range in Nevertheless,thisNevertheless, 30–100 mixture, μg), i.e., thethe those 1mostmostH-NMR noteworthyassignednoteworthy spectrum to thesignalsignal revealed butylss werewere (CH a complex thosethose3 and whichwhichCH mixture2) part allowedallowed (Figureof the thethe molecule. 5) identificationidentification in which On many the ofof 1B-LSDsignalsin the range couldin this 30–100 be mixture, identified μg), i.e.,the as1thoseH-NMR typical assigned spectrum of LSD-li to the revealedke butylsubstances, (CHa complex3 and and CH mixturewhich2) part share (Figure of the the molecule.5) same in which backbone On many the Despitestructure.signals thecould Nevertheless, fact be that identified the sample13 the asmost typical aliquot noteworthy wasof LSD-li so smallsignalke substances, (thes were amount those3 and of which LSDwhich2 inallowed ashare blotter thethe is identificationsame usually backbone in of othersignals1B-LSD hand, couldin thisa direct be mixture, identified 1D i.e.,C NMR asthose typical measurement assigned of LSD-li to the waske butylsubstances, not possible(CH3 and and due CH which to2 ) thepart share reduced of the the molecule. quantitysame backbone Onof the the other1B-LSDstructure. hand, in Nevertheless, this a direct mixture, 1D1 13i.e.,theC NMR mostthose measurementnoteworthy assigned to signalthe was butyl snot were (CHpossible those and whichdue CH to) partallowed the reducedof the the molecule. identification quantity On of the of the range1B-LSDstructure.signals 30–100 couldin Nevertheless,thisµg), be mixture, theidentifiedH-NMR 13i.e.,the asmostthose spectrumtypical noteworthyassigned of revealedLSD-li to signaltheke a butylsubstances, complexs were (CH those3 mixture and and which CH which (Figure2) partallowed share of5) inthe thethe which molecule. identificationsame many backbone On the of samplestructure.other hand, and Nevertheless, the a direct presence 1D 13 theofC relatively NMRmost noteworthymeasurement large quantiti signal wasess notofwere other possible those interfering whichdue to allowed thesubstances reduced the identificationextracted quantity fromof the of other1B-LSD hand, in this a direct mixture, 1D 13i.e.,C NMR those measurementassigned to the was butyl not (CHpossible3 and due CH 2to) part the reducedof the molecule. quantity On of the signalssampleother1B-LSDstructure. could hand, and in be Nevertheless,this identifiedthea direct mixture,presence 1D as i.e.,the typicalofC NMR relativelymostthose of measurementnoteworthy assigned LSD-like large quantitito substances, signalthe was butyles snot wereof (CHpossibleother and those3 and whichinterfering whichdue CH share 2to) part allowedthe substances the reducedof samethe the molecule. identification backbonequantityextracted On of from the of thesampleother1B-LSDsample original hand, and andin this matrix. theathe direct mixture, presencepresence The 1D bond 13i.e., Cofof NMR relatively relativelythose connection measurement assigned largelarge between quantitiquantitito the was thebutyleses not ofbutylof (CH possibleotherother proton3 and interferinginterfering due CH resonances 2to) partthe substancessubstances reducedof wasthe molecule. confirmed quantityextractedextracted On of with fromfrom thethe structure.thesampleother1B-LSD original Nevertheless,hand, and in this matrix.thea direct mixture,presence The the1D most13bondi.e., ofC NMR relativelythose noteworthyconnection measurementassigned large between signals quantitito the was were butylthees not ofbutyl those (CHpossibleother 3proton whichand interfering due CH resonances allowed 2to) part the substances reducedof the thewas identification molecule. confirmed quantityextracted On of fromwith the theotherthe 2D original hand,DQF-COSY amatrix. direct experiment The1D 13bondC NMR connection(Figure measurement S4). betweenThe 2D was HSQC the not butyl possible confirmed proton due resonancesthe to presencethe reduced was of the confirmedquantity methyl of andwith the thesample 2Doriginal DQF-COSY and thematrix. presence experiment The 13bond of relatively connection (Figure large S4). between Thequantiti 2D theHSQCes ofbutyl other confirmed proton interfering resonances the presence substances was of confirmed theextracted methyl fromwith and of 1B-LSDthesampleother original inhand, and this matrix.the mixture,a direct presence The1D i.e., bond Cof those NMRrelatively connection assigned measurement large tobetween thequantiti butylwas thees not (CH ofbutyl possibleother3 and proton interfering CH due2 resonances) partto the substances of reduced the was molecule. confirmed quantityextracted On of fromwith the methylenethesamplethe 2Doriginal2D DQF-COSY DQF-COSYand groups matrix.the presence and experiment experimentThe HMBC bond of relatively connection showed (Figure(Figure large S4).S4).the between TheconnectionThequantiti 2D2D the HSQCHSQCes of butylof other theconfirmedconfirmed proton methylene interfering resonances thethe presencepresencegroups substances was with ofof confirmed the theextracted a methylsignalmethyl withfromof andand a the othermethylenethethesample 2Doriginal hand, DQF-COSY and agroups directmatrix.the presence 1D andexperiment The13 HMBCC bond of NMR relatively connection (Figureshowed measurement large S4). the between Thequantiticonnection was 2D the notHSQCes ofbutyl possibleof other confirmedthe proton methyleneinterfering due resonances to the the presence groupssubstances reduced was withof quantity confirmedthe extracted a methylsignal of fromwith ofand a quaternarymethylenethethemethylene 2Doriginal DQF-COSY carbongroupsgroups matrix. at andexperimentand The171.6 HMBC HMBCbond ppm connection in (Figureshowedshowed line with S4). thethe between the The connectionconnection C=O 2D HSQCthereported butyl ofof confirmed thethe byproton methylene methyleneBrandt resonances the et al.presence groupsgroups [9]. All was withofwith our confirmedthe NMR aa methyl signalsignal results withofandof aa the samplequaternarymethylenethe 2Doriginal and DQF-COSY thecarbongroups matrix. presence at andexperiment The 171.6 HMBC ofbond ppm relatively connection (Figureinshowed line large with S4). the between quantities the Theconnection C=O 2D the HSQCreported of butyl of other confirmedthe byproton interferingmethylene Brandt resonances the et presence al. substancesgroups [9]. Allwas withof our confirmedthe extracted NMRa methylsignal results with ofand a arequaternarymethylenethequaternary consistent 2D DQF-COSY groups carboncarbon with atthose atandexperiment 171.6171.6 HMBC for ppmppm 1B-LSD (Figure showedinin lineline as withwith reportedS4). the the Thetheconnection C=OC=O 2Dby Brandt HSQC reportedreported of confirmedetthe byal.by methylene Brandt[9].Brandt the etet presence al.al.groups [9].[9]. AllAll with of ourour the NMRaNMR methylsignal resultsresults ofand a arequaternarymethylenethe consistent2D DQF-COSY carbongroups with at thoseandexperiment 171.6 HMBC for ppm 1B-LSD (Figureinshowed line as with reportedS4). the the Theconnection C=O 2D by HSQC Brandtreported of confirmed theet byal. methylene Brandt[9]. the et presence al.groups [9]. All withof our the NMRa methylsignal results ofand a arequaternarymethyleneare consistentconsistent carbongroups withwith at thoseandthose 171.6 HMBC forfor ppm 1B-LSD1B-LSD inshowed line asas with reported reportedthe the connection C=O byby BrandtreportedBrandt of the etet by al.al. methylene Brandt [9].[9]. et al. groups [9]. All with our NMRa signal results of a arequaternarymethylene consistent groupscarbon with atandthose 171.6 HMBC for ppm 1B-LSD showedin line as with reported the the connection C=O by Brandtreported of theet byal. methylene Brandt[9]. et al.groups [9]. All with our NMRa signal results of a arequaternary consistent carbon with atthose 171.6 for ppm 1B-LSD in line as with reported the C=O by Brandtreported et byal. Brandt[9]. et al. [9]. All our NMR results arequaternary consistent carbon with at those 171.6 for ppm 1B-LSD in line as with reported the C=O by Brandtreported et by al. Brandt [9]. et al. [9]. All our NMR results are consistent with those for 1B-LSD as reported by Brandt et al. [9]. are consistent with those for 1B-LSD as reported by Brandt et al. [9].

Molecules 2020, 25, 712 6 of 10 from the original matrix. The bond connection between the butyl proton resonances was conﬁrmed with the 2D DQF-COSY experiment (Figure S4). The 2D HSQC conﬁrmed the presence of the methyl and methylene groups and HMBC showed the connection of the methylene groups with a signal of a quaternary carbon at 171.6 ppm in line with the C=O reported by Brandt et al. [9]. All our NMR results are consistent with those for 1B-LSD as reported by Brandt et al. [9]. Molecules 2020, 25, x FOR PEER REVIEW 6 of 10

FigureFigure 5. 15.H 1 NMRH NMR spectrum spectrum 1B-LSD 1B-LSD (a) ( fulla) full spectrum, spectrum, (b) ( expansionb) expansion of theof the aromatic aromatic region region (c) (c) expansionexpansion of the of aliphaticthe aliphatic region region showing showing the regionthe region of interest of interest for thefor identiﬁcationthe identification of the of 1-butylthe 1-butyl group.group. The The peaks peaks labelled labelled with with * at * 3.1 at and3.1 and 3.4 ppm3.4 ppm correspond correspond to the to 13theC satellite13C satellite signals signals of the of the residualresidual protonated protonated NMR NMR solvent solvent (methanol-d4 (methanol-d499.8 ≥ 99.8 atom atom % D). % D). ≥

Molecules 2020, 25, 712 7 of 10

3. Discussion

3.1. GC–MS and HR-MS From the GC–MS spectrum (Figure1), the 1B-LSD molecular ion ( m/z 393) could be identified, as well as some characteristic clusters of these types of substances. More precisely, the clusters at m/z 293, m/z 292 and m/z 291, were consistent with the additional CH2 substituent of 1B-LSD compared to that reported by Brandt et al. [9], when compared with 1P-LSD, which presents a fragment cluster at m/z 277, m/z 278 and m/z 279, as also previously reported [5]. In the case of LSD, with no substituent at the indole nitrogen, this fragment cluster of ions is shifted towards the cluster at m/z 221, m/z 222 and m/z 223 [5,9]. With the exception of m/z 322, which represents the loss of the acyl radical from 1B-LSD, the remaining three species might have represented the butyl-substituted counterparts of those species, previously described as LSD, detected at m/z 280 (retro-Diels Alder), m/z 223 and m/z 196 (loss of N,N-diethylacrylamide). A neutral loss of N,N-diethylformamide from the M + might • have led to the formation of the m/z 291 ion [5]. The EI+ characterization of 1B-LSD, 1P-LSD and LSD, presented by Brandt and other authors, was perfectly consistent with the results of the present study, and allows the unequivocal identification of 1B-LSD [5–9]. In the case of UHPLC–qTOF-MS analysis, the extraction of the ion chromatogram (XIC) with the [M + H]+ of 1B-LSD (m/z 394.249) revealed a clear and abundant peak. From the respective MS2 fragmentation pattern, five abundant ions were identified which related to the suspected molecular structure, namely the m/z 293.165 (loss of [(diethylamino)methylidyne]oxidanium ion), 223.124 (loss of but-1-en-1-one), 208.075 (loss of a methyl), 192.091 (loss of methylamine) and 180.081 (internal rearrangement of tetrahydropyridine with loss of a methyl). The obtained results were in accordance with previously reported works on the ESI+ analysis of 1B-LSD itself, and also 1P-LSD [5,9,11–14]. Regarding the Orbitrap-MS, the MS2 fragmentation pattern was in full accordance with the qTOF-MS2 results, with the four most abundant ions being m/z 293.16454 (loss of [(diethylamino)methylidyne]oxidanium ion), 223.12265 (loss of but-1-en-1-one), 208.07528 (loss of a methyl) and fragments 180.08114 and 181.08117 (internal rearrangement of tetrahydropyridine with loss of a methyl). Once again, these fragments are typical of 1B-LSD and 1-P-LSD [5,9,11–14]. For both the qTOF and Orbitrap-MS analysis, some of the fragments identified with the Orbitrap-MS analysis were also consistent with those of the GC–MS analysis, even though different ionization modes were used (ESI+ vs. EI+).

3.2. NMR Despite the fact that the sample aliquot was so small, the 1H-NMR spectrum still revealed the identity of a complex mixture (Figure4) in which many signals could be identified (like protons 14 to 16 of the aromatic ring) as typical of LSD-like substances which share the same backbone structure. However, the signals that are most noteworthy are those which allow the identification of 1B-LSD in this mixture, i.e., those assigned to the butyl (CH3 and CH2) part of the molecule with δH (MeOD-d4): 1.09 ppm [4] (3H, t, J = 7.4 Hz), 1.86 ppm [3] (2H, m, J = 7.4 Hz) and 2.98 ppm [2] (2H, t, J = 7.4 Hz). These results are consistent with a recent study by Brandt et al. on this substance [9]. On the other hand, the direct 1D 13C NMR measurement was not possible due to the reduced quantity of the sample and the presence of relatively large quantities of other substances extracted from the original matrix. However, with 2D HSQC, it was possible to confirm the presence of the methyl and methylene groups. The bond connections between the protons in this butyl group were confirmed by the 2D DQF-COSY. Furthermore, the long-range 1H-13C HMBC showed the correlation of these two methylene groups with a C=O at 171.6 ppm. These results are consistent with a recent work of Brandt et al. on this substance [9]. Molecules 2020, 25, 712 8 of 10

4. Materials and Methods

4.1. Chemical and Reagents All solvents for the NMR, HR-MS/MS and GC–MS, were obtained from Sigma-Aldrich (Steinheim, Germany) and all LC-MS solvents were ChromaSolv grade, obtained from Fluka Analytical. Ultrapure water (18.2 MΩ) was obtained from a Milli-Q system (Millipore, Bedford, MA, USA).

4.2. Seized Blotter Sample A plastic bag containing one blotter paper labeled as “1B-LSD Blotters 25 MCG” was seized at Stockholm Arlanda Airport, Sweden, together with one plastic bag containing 10 ﬂualprazolam tablets and one plastic bag containing 0.5 g of 2-ﬂuorodeschloroketamine (both bags were correctly labelled). No sub-sample was available for any of the seized materials.

4.3. Sample Preparation The material was extracted at the Swedish Customs Laboratory with 0.5 mL of MeOH, followed by ﬁltration. After GC–MS analysis, aliquots of the samples were shipped to the JRC for further analysis. A direct injection of the methanol sample was carried out for the UHPLC–qTOF-MS analysis, while for the HR-Orbitrap-MS, the extract was diluted 1:50 with acetonitrile.

4.4. Instrumental Analysis

4.4.1. GC–MS An Agilent 7890 Gas Chromatograph equipped with a 5977 MS (Agilent, Santa Clara, CA, USA) was used for the analysis of the sample. The GC-column was a HP5-MS UI (Agilent Technologies, 30 m, 0.250 mm, 0.25 mm) with helium as the carrier gas, at a ﬂow of 0.8 mL/min. An initial oven temperature of 100 ◦C was set with a 5 min isothermal period followed by heating up to 300 ◦C at a rate of 30 ◦C/min and held for 13 min. The total run time was 25 min. The injection volume was 1 µL in split mode (20:1), and the injector temperature was set at 250 ◦C. The GC–MS operated at scan mode m/z ranging from 30–550. GC–MS analysis was performed by the Swedish Customs Laboratory, as well as at the JRC. Data have been processed with an ACD/labs spectrus processor.

4.4.2. NMR For the acquisition of a 1H NMR spectrum, the remaining methanol extract was mixed with deuterated methanol MeOD-d4 (up to a ﬁnal volume of 600 µL), which was used as an internal lock and chemical shift reference at δH = 3.3 ppm. The 1H NMR experiments were performed at 300 K on a Bruker (Rheinstetten, Germany) spectrometer Avance III HD 600 (nominal proton frequency 600.13 MHz) equipped with a 5 mm QCI cryo-probe (1H, 13C, 15N and 19F). The 1H and 13C chemical shifts 1 are expressed in ppm, referenced to the proton signal of the MeOD-d4 (3.3 ppm for H and 47.6 for 13C). Compounds were characterized by one-dimensional 1H, as well as two-dimensional 1H/1H COSY, 1H/1H TOCSY, 1H/13C HSQC, 1H/13C HMBC and 1H/15N HMBC experiments.

4.4.3. HR-MS/MS For UHPLC–qTOF-MS analysis, a UHPLC system (Agilent 1290) with a quadrupole Time-Of-Flight (TOF) mass spectrometer (Agilent 6540 UHD Accurate-Mass, Agilent, Waldbronn, Germany), using an ESI interface, operating in positive ionization mode with a 4 kV capillary voltage, was used. The source operated at 325 ◦C and nitrogen was used as both the drying (40 psi) and nebulizing gas (10 L min-1). The injection volume was 5 µL. The TOF-MS detector was set to acquire MS data over an m/z range of 100–1600. All-Ions MS/MS experiments were performed to screen and quantify constituents of the sample in a single analysis. Under the aforementioned conditions, fragmentation collision energies ranged from 5–60 eV. Molecules 2020, 25, 712 9 of 10

The analytical column was a Waters BEH C18 100 2.1 mm, 1.7 µm particle size (Waters, Milford, × MA, USA), temperature-controlled at 40 ◦C. The mobile phase consisted of water with 0.1% formic 1 acid (A) and methanol with 0.1% formic acid (B) at a flow rate of 200 µL min− . The gradient program changed linearly from 50% to 95% (B) in 25 min, followed by an isocratic elution for 4 min. An equilibration time of 1 min was set for the mobile phase to reach initial conditions again. For Direct infusion Orbitrap-MS, and in correlation with the UHPLC–qTOF-MS analysis, additional measurements were performed using an Orbitrap MS system, to confirm the molar mass (M) and the mass fragmentation patterns of the suspected NPS. This analysis was performed in positive-ion mode with Electro Spray Ionization (ESI) using a Thermo LTQ Orbitrap MS (Thermo Scientific, Bremen, Germany), and operated with mass resolution of 140,000 at m/z 200. The sample was infused at a flow rate of 5 µL/min on the system.

4.5. Subsection Chemoinformatics Tools The ACD/Labs platform (ACD/Labs, Toronto, Canada) was used in combination with Agilent’s MassHunter (Agilent Technologies) and XCalibur (Thermo Scientiﬁc) for the assessment and evaluation of the obtained data, including all the obtained chromatographic and MS data. This platform allows the conﬁrmation and checking of the consistency of any suggested chemical structures with NMR experimental data, as well as assigning experimental spectra to structures, enabling the creation of a central, fully searchable repository of the assigned NMR spectra. The software was also used to project fragmentation paths, by comparing experimental MS and MS/MS data with theoretical data.

5. Conclusions The identification of NPS in seized samples still remains a challenge for many laboratories. In cases where a novel unreported substance is found, or when a sample is seized, either in very small amounts or in a complex mixture, routine analytical techniques are often not sufficient. In these cases, an analytical workflow that combines hyphenated techniques with HR-MS, NMR and chemo-informatics tools is the most effective approach to identify and/or confirm the presence of an NPS with sufficient precision. By following such an analytical workflow, it was possible, in this study, to identify 1B-LSD in the methanol extract of a single blotter paper by using four different techniques and chemo-informatics tools. Although previously characterized at analytical-standard levels, it is the first time that this substance was found and identified in samples circulating in the street market. This work can also be seen as proof that scientific cooperation between modern forensic laboratories can lead to the correct and reliable identification of NPS, even if present in trace amounts.

Supplementary Materials: The following are available online, Figure S1: GC chromatogram analysis (Full scan mode) of the blotter paper methanol extract, Figure S2: UHPLC-qTOF-MS analysis of the blotter paper methanol extract (A) and the extracted ion chromatogram (XIC) of the 1B-LSD [M + H]+, Figure S3: 2D COSY NMR spectra of the blotter paper methanol extract; Figure S4: HMBC NMR spectra of the blotter paper methanol extract. Author Contributions: Investigation and experimental analysis, E.T., J.A.L., M.H., F.R., J.Å. and C.G.; Writing—original draft, E.T. and J.A.L.; Revision and experimental follow-ups: E.T., J.A.L., M.H., F.R., J.Å. and C.G.; Visualization and final revision: E.T., J.A.L., M.H., F.R. and C.G. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by EUROPEAN COMMISSION DIRECTORATE GENERAL FOR TAXATION AND CUSTOMS UNIONS (DG TAXUD) for the administrative and financial support provided to the JRC through the Administrative Arrangement CLEN2SAND II (JRC-Nr 34606-DG TAXUD-Nr TAXUD/2016/DE/331). Acknowledgments: Hendrik Emons (JRC) is acknowledged for reviewing this manuscript. Conflicts of Interest: The authors declare no conflict of interest. Molecules 2020, 25, 712 10 of 10

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Sample Availability: No sample is available.

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