Pharmaceutical Screening Research Articles

O25: HPLC-HRMS vs UPLC-MS/MS for a systematic toxicological screening in hair

Aims The aim of the present work was to compare the performances of HPLC-High Resolution Mass Spectrometry (HPLC-HRMS) in full scan and UPLC- Multiple mass spectrometry (UPLC-MS/MS) in multiple reaction monitoring (MRM) mode for the systematic, general unknown screening of drugs and pharmaceuticals in hair samples collected for clinical toxicology purposes at the local University Hospital. Methods 10 mg hair were washed and extracted by a rapid, micropulverized method already published. Extracts were analyzed in two different instruments, the former consisting of an Orbitrap coupled to a Finnigan Surveyor HPLC, the latter of a Xevo TQD triple quadrupole coupled to an Acquity UPLC. For HPLC, an Atlantis T3 (150 × 2,1 mm, 3 μm) was used while the UPLC employed a HSS C18 (150 × 2.1 × 1,8 μm), with a gradient of ammonium acetate and acetonitrile (formic acid 0.1%) at 300 μL/min and 400 μL/min respectively. The Orbitrap was operated in positive ion electrospray mode at a resolution of 60000 with mass locking, in full scan mode, with a in-house developed library of 600 compounds; the identificatiion criteria were 2 ppm accuracy of the exact mass of the protonated molecule, ± 0.1 min of the retention time, a minimum area count of 10000 arbitrary units; the Xevo TQD worked in switching ion polarity electrospray mode, and in MRM using a Waters toxicology library of 800 (untargeted screening) or 180 compounds (targeted screening), a recognition window of ±0.2 min and a minimum area count of 600 arbitraty units. Results Compounds belonging to drugs of abuse and/or to pharmaceutical drugs could be identified with both analytical approaches in the same hair samples. In more than 100 hair samples collected in the clinical settings in a year, drugs belonging to the following therapeutic categories were identified: anaesthetics, anti-epileptics, antidepressants, benzodiazepines, sedatives, hypnotics, opioids, anti-psychotics, neuroleptics, barbiturates, stimulants, non steroidal analgesics, steroids, beta-blockers, diuretics, antibiotics, antiarrhythmic agents, antihypertensive drugs, anti-coagulants, hypoglycaemic agents. The screening results were in every case confirmed by injection of the tentatively identified analyte(s) in methanol standard mixtures. When possible, the compliance with prescribed therapies or alleged use/abuse was verified. Conclusions The UPLC-MRM method turned to be more rapid than the HPLC-HRMS (14 min vs 30 min total run time); the possibility of polarity switching could be applied in routine only in the Xevo TQD allowing to easily identify acidic drugs such as non steroidal analgesics. The HPLC-Orbitrap, working in full scan, resulted superior for implementing the library with new molecules, since no adjustment of the acquisition method was needed, and for the postacquisition, retrospective screening of the raw files. In the UPLCMRM method, limits of identification with targeted analytes were in the order 0.1 – 0.2 ng/mg for basic drugs, with 10 mg hair samples, whereas in the HPLC-HRMS limits were generally lower.

Small-Angle X-ray Scattering Screening Complements Conventional Biophysical Analysis: Comparative Structural and Biophysical Analysis of Monoclonal Antibodies IgG1, IgG2, and IgG4

A crucial step in the development of therapeutic monoclonal antibodies is the selection of robust pharmaceutical candidates and screening of efficacious protein formulations to increase the resistance toward physicochemical degradation and aggregation during processing and storage. Here, we introduce small-angle X-ray scattering (SAXS) to characterize antibody solution behavior, which strongly complements conventional biophysical analysis. First, we apply a variety of conventional biophysical techniques for the evaluation of structural, conformational, and colloidal stability and report a systematic comparison between designed humanized IgG1, IgG2, and IgG4 with identical variable regions. Then, the high information content of SAXS data enables sensitive detection of structural differences between three IgG subclasses at neutral pH and rapid formation of dimers of IgG2 and IgG4 at low pH. We reveal subclass-specific variation in intermolecular repulsion already at low and medium protein concentrations, which explains the observed improved stability of IgG1 with respect to aggregation. We show how excipients dramatically influence such repulsive effects, hence demonstrating the potential application of extensive SAXS screening in antibody selection, eventual engineering, and formulation development.

A practical guide to pharmaceutical polymorph screening & selection

Prevalence and importance of polymorphism occurring in pharmaceutical compounds are well recognized. It is of great importance to prepare and select the right form from the beginning during drug discovery and development. This review introduces the basic concepts of “What is polymorphism?”, addresses a fundamental question of “Why do polymorphs form?”, and provides practical guidelines of “How to prepare polymorphs?” “How to evaluate the relative thermodynamic stability between polymorphs?”, and “How to analyze polymorphs?”. Moreover, case studies of pharmaceutically important polymorphs are provided.

Integrated chip‐based physiometer for automated fish embryo toxicity biotests in pharmaceutical screening and ecotoxicology

Transgenic zebrafish (Danio rerio) models of human diseases have recently emerged as innovative experimental systems in drug discovery and molecular pathology. None of the currently available technologies, however, allow for automated immobilization and treatment of large numbers of spatially encoded transgenic embryos during real-time developmental analysis. This work describes the proof-of-concept design and validation of an integrated 3D microfluidic chip-based system fabricated directly in the poly(methyl methacrylate) transparent thermoplastic using infrared laser micromachining. At its core, the device utilizes an array of 3D micromechanical traps to actively capture and immobilize single embryos using a low-pressure suction. It also features built-in piezoelectric microdiaphragm pumps, embryo-trapping suction manifold, drug delivery manifold, and optically transparent indium tin oxide heating element to provide optimal temperature during embryo development. Furthermore, we present design of the proof-of-concept off-chip electronic interface equipped with robotic servo actuator driven stage, innovative servomotor-actuated pinch valves, and embedded miniaturized fluorescent USB microscope. Our results showed that the innovative device has 100% embryo-trapping efficiency while supporting normal embryo development for up to 72 hr in a confined microfluidic environment. We also showed data that this microfluidic system can be readily applied to kinetic analysis of a panel of investigational antiangiogenic agents in transgenic zebrafish lines. The optical transparency and embryo immobilization allow for convenient visualization of developing vasculature patterns in response to drug treatment without the need for specimen re-positioning. The integrated electronic interfaces bring the lab-on-a-chip systems a step closer to realization of complete analytical automation.

Suspect screening and target quantification of multi-class pharmaceuticals in surface water based on large-volume injection liquid chromatography and time-of-flight mass spectrometry

The ever-growing number of emerging micropollutants such as pharmaceuticals requests rapid and sensitive full-spectrum analytical techniques. Time-of-flight high-resolution mass spectrometry (TOF-HRMS) is a promising alternative for the state-of-the-art tandem mass spectrometry instruments because of its ability to simultaneously screen for a virtually unlimited number of suspect analytes and to perform target quantification. The challenge for such suspect screening is to develop a strategy, which minimizes the false-negative rate without restraining numerous false-positives. At the same time, omitting laborious sample enrichment through large-volume injection ultra-performance liquid chromatography (LVI-UPLC) avoids selective preconcentration. A suspect screening strategy was developed using LVI-UPLC-TOF-MS aiming the detection of 69 multi-class pharmaceuticals in surface water without the a priori availability of analytical standards. As a novel approach, the screening takes into account the signal-intensity-dependent accurate mass error of TOF-MS, hereby restraining 95 % of the measured suspect pharmaceuticals present in surface water. Application on five Belgian river water samples showed the potential of the suspect screening approach, as exemplified by a false-positive rate not higher than 15 % and given that 30 out of 37 restrained suspect compounds were confirmed by the retention time of analytical standards. Subsequently, this paper discusses the validation and applicability of the LVI-UPLC full-spectrum HRMS method for target quantification of the 69 pharmaceuticals in surface water. Analysis of five Belgian river water samples revealed the occurrence of 17 pharmaceuticals in a concentration range of 17 ng L(-1) up to 3.1 μg L(-1).

Systematic screening of common wastewater-marking pharmaceuticals in urban aquatic environments: implications for environmental risk control

In this report, we refer to pharmaceuticals that are widespread in the urban aquatic environment and that mainly originate from wastewater treatment plants or non-point source sewage as "wastewater-marking pharmaceuticals" (WWMPs). To some extent, they reflect the condition or trend of water contamination and also contribute to aquatic environmental risk assessment. The method reported here for screening typical WWMPs was proposed based on academic concerns about them and their concentrations present in the urban aquatic environment, as well as their properties of accumulation, persistence, eco-toxicity and related environmental risks caused by them. The screening system consisted of an initial screening system and a further screening system. In the former, pharmaceuticals were categorised into different evaluation levels, and in the latter, each pharmaceutical was given a normalised final evaluation score, which was the sum of every score for its properties of accumulation, persistence, eco-toxicity and environmental risk in the aquatic environment. The system was applied to 126 pharmaceuticals frequently detected in the aquatic environment. In the initial screening procedure, five pharmaceuticals were classified into the "high" category, 16 pharmaceuticals into the "medium" category, 15 pharmaceuticals into the "low" category and 90 pharmaceuticals into the "very low" category. Subsequently, further screening were conducted on 36 pharmaceuticals considered as being of "high", "medium" and "low" categories in the former system. We identified 7 pharmaceuticals with final evaluation scores of 1-10, 10 pharmaceuticals with scores of 11-15, 15 pharmaceuticals with scores from 16 to 20 and 4 pharmaceuticals with scores above 21. The results showed that this screening system could contribute to the effective selection of target WWMPs, which would be important for spatial-temporal dynamics, transference and pollution control of pharmaceuticals in the urban aquatic environment. However, there remains a number of pharmaceutical parameters with measured data gaps, such as organic carbon adsorption coefficients and bioconcentration factors, which, if filled, would improve the accuracy of the screening system.

Electrochemical screening of biomembrane-active compounds in water

Interactions of biomembrane-active compounds with phospholipid monolayers on microfabricated Pt/Hg electrodes in an on-line high throughput flow system are demonstrated by recording capacitance current peak changes as rapid cyclic voltammograms (RCV). Detection limits of the compounds’ effects on the layer have been estimated from the data. Compounds studied include steroids, polycyclic aromatic hydrocarbons, tricyclic antidepressants and tricyclic phenothiazines. The results show that the extent and type of interaction depends on the—(a) presence and number of aromatic rings and substituents, (b) presence and composition of side chains and, (c) molecular shape. Interaction is only indirectly related to compound hydrophobicity. For a selection of tricyclic antidepressants and tricyclic phenothiazines the detection limit in water is related to their therapeutic normal threshold. The sensing assay has been tested in the presence of humic acid as a potential interferent and in a tap water matrix. The system can be applied to the screening of putative hazardous substances and pharmaceuticals allowing for early detection thereof in the water supply. The measurements are made in real time which means that potentially toxic compounds are detected rapidly within <10min per assay. This technology will contribute greatly to environment safety and health.

Parallel Reconstitution of Bacterial Toxins, Porins and Ion Channels into Suspended Lipid Membrane Microarrays for High-Throughput Electrophysiology

High-throughput electrophysiological recordings in artificial membranes have a great potential for pharmaceutical screening and nanopore analytics. In addition to parallelization and automation of lipid bilayers formation, different protein reconstitution protocols are required depending on the concrete application.We report on functional reconstitutions of three classes of pore-forming proteins into suspended lipid bilayers on Micro Electrode Cavity Array (MECA). Three reconstitution strategies have been employed and optimized depending on the protein structure: self-insertion for pore-forming toxins, transfer from detergent micelles for porins and vesicle fusion for ion channels.By optimizing protein concentration and facilitating spontaneous insertion with short voltage pulses just below the electroporation threshold, a controlled insertion of alpha-hemolysin was achieved. Exactly one pore per bilayer was inserted in over 50% of bilayers in the array, which is substantially higher than the 37% predicted by the Poisson distribution.We investigated and optimized insertion of outer membrane porins OmpF, MspA and OccK1 into bilayer arrays via detergent dilution. Best strategy was to use mild nonionic detergents and sequentially dilute protein stock solution to minimize influence of the detergent on the membrane. Protein injection in the immediate vicinity of a bilayer array and thorough mixing of the solution were decisive factors for homogeneous distribution of functional porins.Reconstitution via detergent micelles was found to be problematic for ion channels, but proteovesicle fusion was successful. The tetrameric potassium channel KcsA was expressed in vitro with cotranslational insertion into lipid vesicles or nanodiscs. Transfer from nanodiscs into suspended bilayers was shown to be inefficient. Additionally, the presence of nanodiscs substantially destabilized the membranes. For proteovesicles best fusion rates were achieved upon addition of negatively charged phospholipids followed by extrusion of the recovered proteovesicles through a polycarbonate filter.

OpenSource Lab‐on‐a‐Chip Physiometer for Accelerated Zebrafish Embryo Biotests

Zebrafish (Danio rerio) embryo assays have recently come into the spotlight as convenient experimental models in both biomedicine and ecotoxicology. As a small aquatic model organism, zebrafish embryo assays allow for rapid physiological, embryo-, and genotoxic tests of drugs and environmental toxins that can be simply dissolved in water. This protocol describes prototyping and application of an innovative, miniaturized, and polymeric chip-based device capable of immobilizing a large number of living fish embryos for real-time and/or time-lapse microscopic examination. The device provides a physical address designation to each embryo during analysis, continuous perfusion of medium, and post-analysis specimen recovery. Miniaturized embryo array is a new concept of immobilization and real-time drug perfusion of multiple individual and developing zebrafish embryos inside the mesofluidic device. The OpenSource device presented in this protocol is particularly suitable to perform accelerated fish embryo biotests in ecotoxicology and phenotype-based pharmaceutical screening.

Combining Computational Methods for Hit to Lead Optimization in Mycobacterium Tuberculosis Drug Discovery

Tuberculosis treatments need to be shorter and overcome drug resistance. Our previous large scale phenotypic high-throughput screening against Mycobacterium tuberculosis (Mtb) has identified 737 active compounds and thousands that are inactive. We have used this data for building computational models as an approach to minimize the number of compounds tested. A cheminformatics clustering approach followed by Bayesian machine learning models (based on publicly available Mtb screening data) was used to illustrate that application of these models for screening set selections can enrich the hit rate. In order to explore chemical diversity around active cluster scaffolds of the dose-response hits obtained from our previous Mtb screens a set of 1924 commercially available molecules have been selected and evaluated for antitubercular activity and cytotoxicity using Vero, THP-1 and HepG2 cell lines with 4.3%, 4.2% and 2.7% hit rates, respectively. We demonstrate that models incorporating antitubercular and cytotoxicity data in Vero cells can significantly enrich the selection of non-toxic actives compared to random selection. Across all cell lines, the Molecular Libraries Small Molecule Repository (MLSMR) and cytotoxicity model identified ~10% of the hits in the top 1% screened (>10 fold enrichment). We also showed that seven out of nine Mtb active compounds from different academic published studies and eight out of eleven Mtb active compounds from a pharmaceutical screen (GSK) would have been identified by these Bayesian models. Combining clustering and Bayesian models represents a useful strategy for compound prioritization and hit-to lead optimization of antitubercular agents.

Inquisitive Microbiological Analysis of Pharmaceutical Effluents and Screening for a Potential Strain Capable of Utilizing Acetaminophen and Ethylacetoacetate as Sole Carbon and Energy Source

Inquisitive Microbiological Analysis of Pharmaceutical Effluents and Screening for a Potential Strain Capable of Utilizing Acetaminophen and Ethylacetoacetate as Sole Carbon and Energy Source

A hydrophilic polymer based microfluidic system with planar patch clamp electrode array for electrophysiological measurement from cells

This paper presents a microfluidic planar patch clamp system based on a hydrophilic polymer poly(ethylene glycol) diacrylate (PEGDA) for whole cell current recording. The whole chip is fabricated by UV-assisted molding method for both microfluidic channel structure and planar electrode partition. This hydrophilic patch clamp chip has demonstrated a relatively high gigaseal success rate of 44% without surface modification compared with PDMS based patch clamp devices. This chip also shows a capability of rapid intracellular and extracellular solution exchange with high stability of gigaseals. The capillary flow kinetic experiments demonstrate that the flow rates of PEGDA microfluidic channels are around two orders of magnitude greater than those for PDMS-glass channels with the same channel dimensions. This hydrophilic polymer based patch clamp chips have significant advantages over current PDMS elastomer based systems such as no need for surface modification, much higher success rate of cell gigaseals and rapid solution exchange with stable cell gigaseals. Our results indicate the potential of these devices to serve as useful tools for pharmaceutical screening and biosensing tasks.

Review on diabetes, synthetic drugs and glycemic effects of medicinal plants

There has been increased scientific interest in medicinal plants that have been reported to be used traditionally to treat diabetes in humans. This is due to increased efficacy of new plant-derived drugs, growing interests in natural products and the presence of serious side effects of conventional medicines. The development of drugs from plants by drug companies also encourages large pharmaceutical screening of herbs. Accordingly, large numbers of plant and plant products have been scientifically tested and reported to possess the ability to decrease blood glucose levels of normal and/or diabetic animals. Among these are Coccinia indica, Tragia involucrate, Gymnema sylvestre, Pterocarpus marsupium, Trignonella foenum-graecum, Moringa oleifera, Eugenia jambolana, Tinospora cordifolia, Swertia chirayita, Momordica charantia, Ficus benghalensis, Vinca rosea, Premna integrifolia, Macuna prurita, Terminalia bellirica, Sesbinia aegytiaca, Azadirachta indica, Dendrocamalus hamittonni, Zingiber officinale, Aegle marmelos, Cinnamomum tamala, Trichosanthes cucumerina,Hyphaene thebaica, Leptadania hastata, Anisopus mannii and Opium sanctum. Therefore, the aim of this review is to discuss diabetes, past and recent trend with respect to application of medicinal plants in the management of diabetes and provide readers and researchers with the basic concepts of understanding the condition/disorder of diabetes and the glycemic effects of medicinal plants. Key words: Diabetes mellitus, hypoglycemia, hyperglycemia, medicinal plants.

Human Skin-Derived Stem Cells as a Novel Cell Source for In Vitro Hepatotoxicity Screening of Pharmaceuticals

Human skin-derived precursors (hSKP) are postnatal stem cells with neural crest properties that reside in the dermis of human skin. These cells can be easily isolated from small (fore) skin segments and have the capacity to differentiate into multiple cell types. In this study, we show that upon exposure to hepatogenic growth factors and cytokines, hSKP acquire sufficient hepatic features that could make these cells suitable in vitro tools for hepatotoxicity screening of new chemical entities and already existing pharmaceutical compounds. Indeed, hepatic differentiated hSKP [hSKP-derived hepatic progenitor cells (hSKP-HPC)] express hepatic progenitor cell markers (EPCAM, NCAM2, PROM1) and adult hepatocyte markers (ALB), as well as key biotransformation enzymes (CYP1B1, FMO1, GSTA4, GSTM3) and influx and efflux drug transporters (ABCC4, ABCA1, SLC2A5). Using a toxicogenomics approach, we could demonstrate that hSKP-HPC respond to acetaminophen exposure in a comparable way to primary human hepatocytes in culture. The toxicological responses "liver damage", "liver proliferation", "liver necrosis" and "liver steatosis" were found to be significantly enriched in both in vitro models. Also genes associated with either cytotoxic responses or induction of apoptosis (BCL2L11, FOS, HMOX1, TIMP3, and AHR) were commonly upregulated and might represent future molecular biomarkers for hepatotoxicity. In conclusion, our data gives a first indication that hSKP-HPC might represent a suitable preclinical model for in vitro screening of hepatotoxicity. To the best of our knowledge, this is the first report in which human postnatal stem cells derived from skin are described as a potentially relevant cell source for in vitro hepatotoxicity testing of pharmaceutical compounds.

Monitoring by LC-MS/MS of 48 compounds of sildenafil, tadalafil, vardenafil and their analogues in illicit health food products in the Korean market advertised as enhancing male sexual performance

More than 46 phosphodiesterase type 5 (PDE5) inhibitor analogues have been found to be present as illegal adulterants in various forms of health food products (powder, tablet, capsule, etc.), thereby placing the health of consumers at risk through product intake. In this study, 164 samples advertised to be effective at enhancing male sexual performance were collected over a 4-year period (2009–2012) from the Korean on-line or off-line market and screened. An LC-MS/MS method was employed to screen for the presence of 48 compounds including sildenafil, tadalafil, vardenafil and their analogues. Method validation established LOQs (0.30–10.00 ng ml−1 or ng g−1) and recoveries (spiked in liquid sample, 84–112%; spiked in solid sample, 83–110%). Most of the illicit products screened were adulterated with 14 of the PDE5 derivatives under examination, including considerable amounts of sildenafil and tadalafil; of the 48 compounds, tadalafil was the most frequent adulterant (42.6%), followed by sildenafil (27.9%). Specifically, tadalafil concentration ranges (mg g−1) in the samples collected over the 4-year period were determined as follows: 2.91–52.20 (2009), 4.50–108.10 (2010), 0.37–101.40 (2011), and 0.08–138.69 mg g−1 (2012). The concentration ranges (mg g−1) of sildenafil were also at high levels: 4.90–117.96 (2009), 1.30–369.93 (2010), 0.03–241.77 (2011), and 18.34–297.91 mg g−1 (2012). The results of screening for PDE5 inhibitor pharmaceuticals as adulterants in illicit health food products are of great significance with respect to the protection of public health and consumer safety.

Experimental and computational approaches towards solid-form screening of pharmaceuticals

Experimental and computational approaches towards solid-form screening of pharmaceuticals

Real time in vivo investigation of superoxide dynamics in zebrafish liver using a single-fiber fluorescent probe

Superoxide anion is the key radical that causes intracellular oxidative stress. The lack of a method to directly monitor superoxide concentration in vivo in real time has severely hindered our understanding on its pathophysiology. We made transgenic zebrafish to specifically express yellow fluorescent proteins, a reversible superoxide-specific indicator, in the liver and used a fiber-optic fluorescent probe to noninvasively monitor the superoxide concentration in real time. Several superoxide-inducing and scavenging reagents were administrated onto the fish to alter superoxide concentrations. The distinct biochemical pathways of the reagents can be discerned from the transient behaviors of fluorescence time courses. These results demonstrate the feasibility of this method for analyzing superoxide dynamics and its potential as an in vivo pharmaceutical screening platform.

Integrated RNA extraction and RT-PCR for semi-quantitative gene expression studies on a microfluidic device

This paper describes the development of a microfluidic methodology, using RNA extraction and reverse transcription PCR, for investigating expression levels of cytochrome P450 genes. Cytochrome P450 enzymes are involved in the metabolism of xenobiotics, including many commonly prescribed drugs, therefore information on their expression is useful in both pharmaceutical and clinical settings. RNA extraction, from rat liver tissue or primary rat hepatocytes, was performed using a silica-based solid-phase extraction technique. Following elution of the purified RNA, amplification of target sequences for the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the cytochrome P450 gene CYP1A2, was carried out using a one-step reverse transcription PCR. Once the microfluidic methodology had been optimized, analysis of control and 3-methylcholanthrene-induced primary rat hepatocytes were used to evaluate the system. As expected, GAPDH was consistently expressed, whereas CYP1A2 levels were found to be raised in the drug-treated samples. The proposed system offers an initial platform for development of both rapid throughput analyzers for pharmaceutical drug screening and point-of-care diagnostic tests to aid provision of drug regimens, which can be tailor-made to the individual patient.

Assessment of the Integrity of Compounds Stored in Assay-Ready Plates Using a Kinase Sentinel Assay

Sentinel assays are a convenient adjunct to LC-MS purity assessment to monitor the integrity of compounds in pharmaceutical screening collections over time. To assess the stability of compounds stored both at room temperature and at -20°C in assay-ready plates that were either vacuum pack-sealed using a convenient industrial vacuum sealing method or individually sealed using conventional foil seals, a diverse collection of &#126; 5,000 compounds was assayed using a robust biochemical kinase assay at intervals over a one year period. Assay results at each time point were compared to those of initial assay using a series of correlations of compound Percent of Control (POC) values as well as IC50 values of a subset of compounds in 200 nL or 500 nL plates. The fraction of hits in common between initial assays and assays at later time points ranged from 82% to 95% over one year and remained relatively constant over time with all storage temperatures or sealing methods tested. A majority of the hits that exhibited a consistent gradual trend to lower potency over one year storage were shifted to lower potency upon the rapid removal of DMSO solvent. Compound precipitation rather than compound decomposition is the likely reason for trends to lower potency for most such compounds over the storage period. Plates stored at room temperature featured a significantly higher fraction of hits that exhibited a trend to lower apparent potency than those stored at -20°C suggesting that this lower temperature is preferable for longer-term storage.

A Cluster of Deaths Involving 5-(2-Aminopropyl)Indole (5-IT)

During 2012, the designer drug 5-(2-aminopropyl)indole emerged in Sweden, and became available at different web sites under the name 5-IT or 5-API. This compound is an indole derivative and a positional isomer of alpha-methyltryptamine. In this paper, we report the pathology and toxicology from 15 deaths involving 5-IT. Routine postmortem toxicology was performed in femoral blood, using a targeted screening for pharmaceuticals and drugs of abuse with liquid chromatography time-of-flight technology, and positive results were quantified using chromatographic techniques. For 5-IT, a new method was developed using ultra-high-performance liquid chromatography and tandem mass spectrometry. In 11 cases, intoxication was the cause of death. Two cases were signed out as causa ignota, and they were considered to be natural deaths. All determinations of 5-IT were performed in femoral blood and the concentrations ranged from 0.7 to 18.6 µg/g. Two cases had 5-IT as the only drug identified, while the others presented with other psychotropic drugs or medications in the blood as well. Shortly after this series of deaths, 5-IT was scheduled as a hazardous substance according to the regulation Certain Goods Dangerous to Health on 18 September 2012 prohibiting the handling and selling of the drug. Since then, no positive cases have been found.