Mapping intracellular NAD content in entire human brain using phosphorus-31 MR spectroscopic imaging at 7 Tesla.

Introduction: Nicotinamide adenine dinucleotide (NAD) is a crucial molecule in cellular metabolism and signaling. Mapping intracellular NAD content of human brain has long been of interest. However, the sub-millimolar level of cerebral NAD concentration poses significant challenges for in vivo measurement and imaging. Methods: In this study, we demonstrated the feasibility of non-invasively mapping NAD contents in entire human brain by employing a phosphorus-31 magnetic resonance spectroscopic imaging (31P-MRSI)-based NAD assay at ultrahigh field (7 Tesla), in combination with a probabilistic subspace-based processing method. Results: The processing method achieved about a 10-fold reduction in noise over raw measurements, resulting in remarkably reduced estimation errors of NAD. Quantified NAD levels, observed at approximately 0.4 mM, exhibited good reproducibility within repeated scans on the same subject and good consistency across subjects in group data (2.3 cc nominal resolution). One set of higher-resolution data (1.0 cc nominal resolution) unveiled potential for assessing tissue metabolic heterogeneity, showing similar NAD distributions in white and gray matter. Preliminary analysis of age dependence suggested that the NAD level decreases with age. Discussion: These results illustrate favorable outcomes of our first attempt to use ultrahigh field 31P-MRSI and advanced processing techniques to generate a whole-brain map of low-concentration intracellular NAD content in the human brain.

The interplay of social rank perceptions of Trump and Biden and emotions following the U.S. presidential election 2020.

The outcome of the 2020 U.S. election between Trump and Biden evoked strong emotions. In U.S. American (Study 1; N = 405) and German (Study 2; N = 123) samples, we investigated how observers' group membership (i.e. political orientation) and the social rank attainment of both candidates (i.e. dominance vs. prestige) predicted emotional reactions. Trump was generally perceived as more dominant, and Biden as more prestigious. However, perceptions of social rank attainment differed depending on the observers' political orientation, either matching or not matching with the leaders (i.e. Republicans and Democrats, respectively). The candidate who did not share the participants' political orientation was perceived as less prestigious and more dominant and elicited stronger contrastive emotions (i.e. schadenfreude, malicious envy) and weaker assimilative emotions (i.e. happy-for-ness, sympathy, anger), and vice versa. Crucially, dominance and prestige perceptions explained variance in the emotional reactions of more conservative and more liberal participants. Prestige positively predicted assimilative emotions and dominance contrastive emotions. Our work advances theorising by providing evidence that dominance and prestige perceptions contribute to the elicitation of various emotions. Furthermore, it suggests that prestige and dominance are not fixed characteristics of liberal and conservative leaders but depend on the observers' group membership.

The three-dimensional structure of the Vint domain from Tetrahymena thermophila suggests a ligand-regulated cleavage mechanism by the HINT fold.

Vint proteins have been identified in unicellular metazoans as a novel hedgehog-related gene family, merging the von Willebrand factor type A domain and the Hedgehog/INTein (HINT) domains. We present the first three-dimensional structure of the Vint domain from Tetrahymena thermophila corresponding to the auto-processing domain of hedgehog proteins, shedding light on the unique features, including an adduct recognition region (ARR). Our results suggest a potential binding between the ARR and sulfated glycosaminoglycans like heparin sulfate. Moreover, we uncover a possible regulatory role of the ARR in the auto-processing by Vint domains, expanding our understanding of the HINT domain evolution and their use in biotechnological applications. Vint domains might have played a crucial role in the transition from unicellular to multicellular organisms.

GMOCU: Digital Documentation, Management, and Biological Risk Assessment of Genetic Parts.

The continuous evolution of molecular biology and gene synthesis methods paired with an ever-increasing potential of synthetic biology approaches and genome engineering toolkits enables the rapid design of genetic bioparts and genetically modified organisms. Although various software solutions assist with specific design tasks and challenges, lab internal documentation and ensuring compliance with governmental regulations on biosafety assessment of the generated organisms remain the responsibility of individual academic researchers. This results in inconsistent and redundant documentation regimes and a significant time and labor burden. GMOCU (GMO documentation) is a standardized semi-automatic user-oriented software approach -written in Python and freely available- that unifies lab internal data documentation on genetic parts and genetically modified organisms (GMOs). It automatizes biological risk evaluations and maintains a shared up-to-date inventory of bioparts for team-wide data navigation and sharing. GMOCU further enables data export into customizable formats suitable for scientific publications, official biosafety documents, and the research community.

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.

Protocol for the induction of innate immune memory in human smooth muscle cells and endothelial cells in vitro.

Non-immune cells, like innate immune cells, can develop a memory-like phenotype in response to priming with microbial compounds or certain metabolites, which enables an enhanced response to a secondary unspecific stimulus. This paper describes a step-by-step protocol for the induction and analysis of trained immunity in human endothelial and smooth muscle cells. We then describe steps for cell culture with cryopreserved vascular cells, subcultivation, and induction of trained immunity. We then provide detailed procedures for downstream analysis using ELISA and qPCR. For complete details on the use and execution of this protocol, please refer to Sohrabi et al. (2020)1 and Shcnack et al.2.

Understanding the first-offer conundrum: How buyer offers impact sale price and impasse risk in 26 million eBay negotiations.

How low is the ideal first offer? Prior to any negotiation, decision-makers must balance a crucial tradeoff between two opposing effects. While lower first offers benefit buyers by anchoring the price in their favor, an overly ambitious offer increases the impasse risk, thus potentially precluding an agreement altogether. Past research with simulated laboratory or classroom exercises has demonstrated either a first offer's anchoring benefits or its impasse risk detriments, while largely ignoring the other effect. In short, there is no empirical answer to the conundrum of how low an ideal first offer should be. Our results from over 26 million incentivized real-world negotiations on eBay document (a) a linear anchoring effect of buyer offers on sales price, (b) a nonlinear, quartic effect on impasse risk, and (c) specific offer values with particularly low impasse risks but high anchoring benefits. Integrating these findings suggests that the ideal buyer offer lies at 80% of the seller's list price across all products-although this value ranges from 33% to 95% depending on the type of product, demand, and buyers' weighting of price versus impasse risk. We empirically amend the well-known midpoint bias, the assumption that buyer and seller eventually meet in the middle of their opening offers, and find evidence for a "buyer bias." Product demand moderates the (non)linear effects, the ideal buyer offer, and the buyer bias. Finally, we apply machine learning analyses to predict impasses and present a website with customizable first-offer advice configured to different products, prices, and buyers' risk preferences.

Drug Regulatory-Compliant Validation of a qPCR Assay for Bioanalysis Studies of a Cell Therapy Product with a Special Focus on Matrix Interferences in a Wide Range of Organ Tissues.

Quantitative polymerase chain reaction (qPCR) has emerged as an important bioanalytical method for assessing the pharmacokinetics of human-cell-based medicinal products after xenotransplantation into immunodeficient mice. A particular challenge in bioanalytical qPCR studies is that the different tissues of the host organism can affect amplification efficiency and amplicon detection to varying degrees, and ignoring these matrix effects can easily cause a significant underestimation of the true number of target cells in a sample. Here, we describe the development and drug regulatory-compliant validation of a TaqMan® qPCR assay for the quantification of mesenchymal stromal cells in the range of 125 to 20,000 cells/200 µL lysate via the amplification of a human-specific, highly repetitive α-satellite DNA sequence of the chromosome 17 centromere region HSSATA17. An assessment of matrix effects in 14 different mouse tissues and blood revealed a wide range of spike recovery rates across the different tissue types, from 11 to 174%. Based on these observations, we propose performing systematic spike-and-recovery experiments during assay validation and correcting for the effects of the different tissue matrices on cell quantification in subsequent bioanalytical studies by multiplying the back-calculated cell number by tissue-specific factors derived from the inverse of the validated percent recovery rate.

NERNST: a genetically-encoded ratiometric non-destructive sensing tool to estimate NADP(H) redox status in bacterial, plant and animal systems.

NADP(H) is a central metabolic hub providing reducing equivalents to multiple biosynthetic, regulatory and antioxidative pathways in all living organisms. While biosensors are available to determine NADP+ or NADPH levels in vivo, no probe exists to estimate the NADP(H) redox status, a determinant of the cell energy availability. We describe herein the design and characterization of a genetically-encoded ratiometric biosensor, termed NERNST, able to interact with NADP(H) and estimate ENADP(H). NERNST consists of a redox-sensitive green fluorescent protein (roGFP2) fused to an NADPH-thioredoxin reductase C module which selectively monitors NADP(H) redox states via oxido-reduction of the roGFP2 moiety. NERNST is functional in bacterial, plant and animal cells, and organelles such as chloroplasts and mitochondria. Using NERNST, we monitor NADP(H) dynamics during bacterial growth, environmental stresses in plants, metabolic challenges to mammalian cells, and wounding in zebrafish. NERNST estimates the NADP(H) redox poise in living organisms, with various potential applications in biochemical, biotechnological and biomedical research.

Revisiting the role of attention in the "weapon focus effect": Do weapons draw gaze away from the perpetrator under naturalistic viewing conditions?

The presence of a weapon in a scene has been found to attract observers' attention and to impair their memory of the person holding the weapon. Here, we examined the role of attention in this weapon focus effect (WFE) under different viewing conditions. German participants viewed stimuli in which a man committed a robbery while holding a gun or a cell phone. The stimuli were based on material used in a recent U.S. study reporting large memory effects. Recording eye movements allowed us to test whether observers' attention in the gun condition shifted away from the perpetrator towards the gun, compared with the phone condition. When using videos (Experiment 1), weapon presence did not appear to modulate the viewing time for the perpetrator, whereas the evidence concerning the critical object remained inconclusive. When using slide shows (Experiment 2), the gun attracted more gaze than the phone, replicating previous research. However, the attentional shift towards the weapon did not come at a cost of viewing time on the perpetrator. In both experiments, observers focused their attention predominantly on the depicted people and much less on the gun or phone. The presence of a weapon did not cause participants to recall fewer details about the perpetrator's appearance in either experiment. This null effect was replicated in an online study using the original videos and testing more participants. The results seem at odds with the attention-shift explanation of the WFE. Moreover, the results indicate that the WFE is not a universal phenomenon.

Engineering of bidirectional, cyanobacteriochrome-based light-inducible dimers (BICYCL)s.

Optogenetic tools for controlling protein-protein interactions (PPIs) have been developed from a small number of photosensory modules that respond to a limited selection of wavelengths. Cyanobacteriochrome (CBCR) GAF domain variants respond to an unmatched array of colors; however, their natural molecular mechanisms of action cannot easily be exploited for optogenetic control of PPIs. Here we developed bidirectional, cyanobacteriochrome-based light-inducible dimers (BICYCL)s by engineering synthetic light-dependent interactors for a red/green GAF domain. The systematic approach enables the future engineering of the broad chromatic palette of CBCRs for optogenetics use. BICYCLs are among the smallest optogenetic tools for controlling PPIs and enable either green-ON/red-OFF (BICYCL-Red) or red-ON/green-OFF (BICYCL-Green) control with up to 800-fold state selectivity. The access to green wavelengths creates new opportunities for multiplexing with existing tools. We demonstrate the utility of BICYCLs for controlling protein subcellular localization and transcriptional processes in mammalian cells and for multiplexing with existing blue-light tools.

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.

Toxicological Investigation of a Case Series Involving the Synthetic Cathinone α-Pyrrolidinohexiophenone (α-PHP) and Identification of Phase I and II Metabolites in Human Urine.

α-Pyrrolidinohexiophenone (α-PHP) is a derivative of the class of α-pyrrolidinophenones, a subgroup of synthetic cathinones. These substances are the second most abused drugs of new psychoactive substances. Here, we report the toxicological investigation of a series of 29 authentic forensic and clinical cases with analytically confirmed intake of α-PHP including two cases of drug testing in newborns using meconium. The age range of subjects where serum samples were available was 23-51 years (median 39.5), and 90% were male. Serum α-PHP concentrations, determined by a validated LC-MS-MS method, showed a high variability ranging from 1 to 83 ng/mL (mean, 40 ng/mL; median, 36 ng/mL). Comprehensive toxicological analysis revealed co-consumption of other psychotropic drugs in almost all cases with frequent occurrence of opiates (60%), benzodiazepines (35%), cannabinoids (30%), and cocaine (20%). Hence, forensic and clinical symptoms like aggressive behavior, sweating, delayed physical response, and impaired balance could not be explained by the abuse of α-PHP alone but rather by poly-intoxications. Liquid chromatography-quadrupole time-of-flight mass spectrometry and gas chromatography-mass spectrometry were used to investigate the metabolism of α-PHP in vivo using authentic human urine samples. Altogether, 11 phase I metabolites and 5 phase II glucuronides could be identified by this approach. Apart from the parent drug, most abundant findings in urine were the metabolites dihydroxy-pyrrolidinyl-α-PHP and dihydro-α-PHP and, to a lesser extent, 2'-oxo-dihydro-α-PHP and 2'-oxo-α-PHP. Monitoring of these metabolites along with the parent drug in forensic and clinical toxicology could unambiguously prove the abuse of the novel designer drug α-PHP.

Translational development of ABCB5+ dermal mesenchymal stem cells for therapeutic induction of angiogenesis in non-healing diabetic foot ulcers.

BACKGROUND: While rapid healing of diabetic foot ulcers (DFUs) is highly desirable to avoid infections, amputations and life-threatening complications, DFUs often respond poorly to standard treatment. GMP-manufactured skin-derived ABCB5+ mesenchymal stem cells (MSCs) might provide a new adjunctive DFU treatment, based on their remarkable skin wound homing and engraftment potential, their ability to adaptively respond to inflammatory signals, and their wound healing-promoting efficacy in mouse wound models and human chronic venous ulcers. METHODS: The angiogenic potential of ABCB5+ MSCs was characterized with respect to angiogenic factor expression at the mRNA and protein level, in vitro endothelial trans-differentiation and tube formation potential, and perfusion-restoring capacity in a mouse hindlimb ischemia model. Finally, the efficacy and safety of ABCB5+ MSCs for topical adjunctive treatment of chronic, standard therapy-refractory, neuropathic plantar DFUs were assessed in an open-label single-arm clinical trial. RESULTS: Hypoxic incubation of ABCB5+ MSCs led to posttranslational stabilization of the hypoxia-inducible transcription factor 1α (HIF-1α) and upregulation of HIF-1α mRNA levels. HIF-1α pathway activation was accompanied by upregulation of vascular endothelial growth factor (VEGF) transcription and increase in VEGF protein secretion. Upon culture in growth factor-supplemented medium, ABCB5+ MSCs expressed the endothelial-lineage marker CD31, and after seeding on gel matrix, ABCB5+ MSCs demonstrated formation of capillary-like structures comparable with human umbilical vein endothelial cells. Intramuscularly injected ABCB5+ MSCs to mice with surgically induced hindlimb ischemia accelerated perfusion recovery as measured by laser Doppler blood perfusion imaging and enhanced capillary proliferation and vascularization in the ischemic muscles. Adjunctive topical application of ABCB5+ MSCs onto therapy-refractory DFUs elicited median wound surface area reductions from baseline of 59% (full analysis set, n = 23), 64% (per-protocol set, n = 20) and 67% (subgroup of responders, n = 17) at week 12, while no treatment-related adverse events were observed. CONCLUSIONS: The present observations identify GMP-manufactured ABCB5+ dermal MSCs as a potential, safe candidate for adjunctive therapy of otherwise incurable DFUs and justify the conduct of a larger, randomized controlled trial to validate the clinical efficacy. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03267784, Registered 30 August 2017, https://clinicaltrials.gov/ct2/show/NCT03267784.

LXRα Regulates oxLDL-Induced Trained Immunity in Macrophages.

Reprogramming of metabolic pathways in monocytes and macrophages can induce a proatherosclerotic inflammatory memory called trained innate immunity. Here, we have analyzed the role of the Liver X receptor (LXR), a crucial regulator of metabolism and inflammation, in oxidized low-density lipoprotein (oxLDL)-induced trained innate immunity. Human monocytes were incubated with LXR agonists, antagonists, and oxLDL for 24 h. After five days of resting time, cells were restimulated with the TLR-2 agonist Pam3cys. OxLDL priming induced the expression of LXRα but not LXRß. Pharmacologic LXR activation was enhanced, while LXR inhibition prevented the oxLDL-induced inflammatory response. Furthermore, LXR inhibition blocked the metabolic changes necessary for epigenetic reprogramming associated with trained immunity. In fact, enrichment of activating histone marks at the IL-6 and TNFα promotor was reduced following LXR inhibition. Based on the differential expression of the LXR isoforms, we inhibited LXRα and LXRß genes using siRNA in THP1 cells. As expected, siRNA-mediated knock-down of LXRα blocked the oxLDL-induced inflammatory response, while knock-down of LXRß had no effect. We demonstrate a specific and novel role of the LXRα isoform in the regulation of oxLDL-induced trained immunity. Our data reveal important aspects of LXR signaling in innate immunity with relevance to atherosclerosis formation.

Analytical profile, in vitro metabolism and behavioral properties of the lysergamide 1P-AL-LAD.

Lysergic acid diethylamide (LSD) is known to induce powerful psychoactive effects in humans, which cemented its status as an important tool for clinical research. A range of analogues and derivatives has been investigated over the years, including those classified as new psychoactive substances. This study presents the characterization of the novel lysergamide N,N-diethyl-1-propanoyl-6-(prop-2-en-1-yl)-9,10-didehydroergoline-8ß-carboxamide (1P-AL-LAD) using various mass spectrometric, gas- and liquid chromatographic and spectroscopic methods. In vitro metabolism studies using pooled human liver microsomes (pHLM) confirmed that 1P-AL-LAD converted to AL-LAD as the most abundant metabolite consistent with the hypothesis that 1P-AL-LAD may act as a prodrug. Fourteen metabolites were detected in total; metabolic reactions included hydroxylation of the core lysergamide ring structure or the N6 -allyl group, formation of dihydrodiol metabolites, N-dealkylation, N1 -deacylation, dehydrogenation, and combinations thereof. The in vivo behavioral activity of 1P-AL-LAD was evaluated using the mouse head twitch response (HTR), a 5-HT2A -mediated head movement that serves as a behavioral proxy in rodents for human hallucinogenic effects. 1P-AL-LAD induced a dose-dependent increase in HTR counts with an inverted U-shaped dose-response function, similar to lysergic acid diethylamide (LSD), psilocybin, and other psychedelics. Following intraperitoneal injection, the median effective dose (ED50 ) for 1P-AL-LAD was 491 nmol/kg, making it almost three times less potent than AL-LAD (174.9 nmol/kg). Previous studies have shown that N1 -substitution disrupts the ability of lysergamides to activate the 5-HT2A receptor; based on the in vitro metabolism data, 1P-AL-LAD may induce the HTR because it acts as a prodrug and is metabolized to AL-LAD after administration to mice.

Structural Basis for the Propagation of Homing Endonuclease-Associated Inteins.

Inteins catalyze their removal from a host protein through protein splicing. Inteins that contain an additional site-specific endonuclease domain display genetic mobility via a process termed "homing" and thereby act as selfish DNA elements. We elucidated the crystal structures of two archaeal inteins associated with an active or inactive homing endonuclease domain. This analysis illustrated structural diversity in the accessory domains (ACDs) associated with the homing endonuclease domain. To augment homing endonucleases with highly specific DNA cleaving activity using the intein scaffold, we engineered the ACDs and characterized their homing site recognition. Protein engineering of the ACDs in the inteins illuminated a possible strategy for how inteins could avoid their extinction but spread via the acquisition of a diverse accessory domain.

Allogeneic ABCB5+ mesenchymal stem cells for treatment-refractory chronic venous ulcers: a phase I/IIa clinical trial.

A significant number of chronic venous ulcers (CVUs) fail to heal despite of guideline-conform standard of care. Skin-derived ABCB5+ mesenchymal stem cells (MSCs) can dampen the sustained IL-1ß-driven inflammation present in chronic wounds. Based on their wound healing-facilitating effects in a mouse CVU model and an autologous first-in-human study, ABCB5+ MSCs have emerged as a potential candidate for cell-based advanced therapy of non-healing CVUs. In the present interventional, multicenter, single-arm, phase I/IIa clinical trial, subjects whose CVU had emerged as standard therapy-resistant received one or two topical applications of 1×106 allogeneic ABCB5+ MSCs/cm2 wound area in addition to standard treatment. Out of 83 treatment-emergent adverse events, only three were judged related to the cell product; they were mild or moderate and recovered without sequelae. Wound size markedly decreased from baseline to week 12, resulting in a median wound size reduction of 76% (full analysis set, N=31), 78% (per-protocol set, N=27) and 87% (subset of responders; n=21). In conclusion, the study treatment was well tolerated and safe. The treatment elicited a profound wound size reduction within 12 weeks, identifying ABCB5+ MSCs as a potential candidate for adjunctive therapy of otherwise incurable CVUs. These results justify the conduct of a larger, randomized, controlled trial to confirm clinical efficacy.

Separating the wheat from the chaff: Observations on the analysis of lysergamides LSD, MIPLA, and LAMPA.

Lysergic acid diethylamide (LSD) is a potent psychoactive substance that has attracted great interest in clinical research. As the pharmacological exploration of LSD analogs continues to grow, some of those analogs have appeared on the street market. Given that LSD analogs are uncontrolled in many jurisdictions, it is important that these analogs be differentiated from LSD. This report presents the analysis of blotters found to contain the N-methyl-N-isopropyl isomer of LSD (MIPLA), and techniques to differentiate it from LSD and the N-methyl-N-propyl isomer (LAMPA) under routine conditions. Gas chromatography (GC)-solid phase infrared spectroscopy was particularly helpful. GC-electron ionization-tandem mass spectrometry of the m/z 72 iminium ion also provided sufficient information to distinguish the three isomers on mass spectral grounds alone, where chromatographic separation proved challenging. Derivatization with 2,2,2-trifluoro-N,N-bis (trimethylsilyl)acetamide (BSTFA) also led to improved GC separation. Liquid chromatography single quadrupole mass spectrometry (LC-Q-MS) and in-source collision-induced dissociation allowed for the differentiation between MIPLA and LAMPA based on distinct m/z 239 ion ratios when co-eluting. An alternative LC-MS/MS method improved the separation between all three lysergamides, but LSD was found to co-elute with iso-LSD. However, a comparison of ion ratios recorded for transitions at m/z 324.2 > 223.2 and m/z 324.2 > 208.2 facilitated their differentiation. The analysis of two blotters by LC-Q-MS revealed the presence of 180 and 186 µg MIPLA per blotter. These procedures may be used to avoid inadvertent misidentification of MIPLA or LAMPA as LSD.