In this paper we present a new, versatile workflow for a synthesis impurity profiling concept, using the combination of flash chromatography (F-LC), liquid chromatography coupled to mass spectrometry ...(LC-MS), and multivariate data analysis. For three highly pure, structurally different synthetic cannabinoids, we demonstrate that via F-LC more than 99% of the main component (API) can be removed from a sample to enrich present impurities and yield combined fractions of targeted synthesis impurities with reproducible chromatographic signatures via LC-MS. The maximum overall relative standard deviation (RSD) of the complete experimental procedure for isolation and measurement of the impurity profiles (FL-C + LC-MS) was found to be 13.8% on average. The impurity signatures of 40 1 kg samples of MDMB-CHMICA (methyl (S)-2-(1-(cyclohexylmethyl)-1H-indole-3-carboxamido)-3,3-dimethylbutanoate) from one large seizure by Luxembourg customs were assessed via UHPLC-MS and compared via principle component analysis (PCA) to possibly discriminate between individual synthesis pathways or production batches and to deduce batch sizes. Three of these 40 samples could be identified as outliers, i. a., as a result of a highly abundant impurity with m/z 498, isolated via F-LC and identified as methyl 2-(2-(1-(cyclohexylmethyl)-1H-indole-3-carboxamido)-3,3-dimethylbutanamido)-3,3-dimethylbutanoate, most probably manufactured with a varying synthesis pathway. The remaining 37 samples were subdivided via PCA and hierarchical cluster analysis into five clusters between five and ten samples, representing a maximum possible batch size of 10 kg of pure MDMB-CHMICA. Furthermore, the profiling concept was successfully applied to self-produced and seized “spice-products” to extract impurity profiles of MDMB-CHMICA without any ion suppression or chemical interference.
NPHP4 mutations cause nephronophthisis, an autosomal recessive cystic kidney disease associated with renal fibrosis and kidney failure. The NPHP4 gene product nephrocystin-4 interacts with other ...nephrocystins, cytoskeletal and ciliary proteins; however, the molecular and cellular functions of nephrocystin-4 have remained elusive. Here we demonstrate that nephrocystin-4 is required for normal cloaca formation during zebrafish embryogenesis. Time-lapse imaging of the developing zebrafish pronephros revealed that tubular epithelial cells at the distal pronephros actively migrate between the yolk sac extension and the blood island towards the ventral fin fold to join the proctodeum and to form the cloaca. Nphp4-deficient pronephric duct cells failed to connect with their ectodermal counterparts, and instead formed a vesicle at the obstructed end of the pronephric duct. Nephrocystin-4 interacts with nephrocystin-1 and Par6. Depletion of zebrafish NPHP1 (nphp1) increased the incidence of cyst formation and randomization of the normal body axis, but did not augment cloaca malformation in nphp4-deficient zebrafish embryos. However, simultaneous depletion of zebrafish Par6 (pard6) aggravated cloaca formation defects in nphp4-depleted embryos, suggesting that nphp4 orchestrates directed cell migration and cloaca formation through interaction with the Par protein complex.
The synthetic cannabinoid MDMB-CHMCZCA was characterized by various spectroscopic techniques including NMR spectroscopy and tandem mass spectrometry. The synthetic sample was found to be of
...-configuration by VCD spectroscopy and comparison of the data with DFT calculations, while ECD spectroscopy was found to be inconclusive in this case. The enantiomeric purity of samples from test purchases and police seizures was assessed by a self-developed chiral HPLC method.
•Assessing stability of specific substances related to clandestine production of ATS.•Tentative identification of novel transformation products from APAA and ephedrine.•Tracking of ATS synthesis ...markers supports wastewater monitoring studies.
Environmental impact of toxic and corrosive synthesis waste generated by the clandestine production of amphetamine-type stimulants (ATS) is a known problem, which can even result in malfunction of wastewater treatment plants (WWTPs), e. g. in case of illegal discharge of large amounts of highly acidic chemical waste into the sewage system, which is generated in clandestine labs converting pre-precursors to the most prevalent ATS precursor benzyl methyl ketone (BMK). ATS synthesis-specific substances, precursor chemicals, intermediates and route-specific by-products may also support wastewater-based epidemiology (WBE) studies to explain abnormally high loads of drugs in wastewater by distinguishing whether these high loads were caused by consumption or disposal of synthesis waste into the sewage system. Although some of these synthesis-specific substances can be detected in traces in the final form of consumption of the product, these substances are removed from the drug product to a large extent during cleaning steps, e.g. the frequently applied steam distillation step to purify the amphetamine raw base after clandestine Leuckart synthesis. In contrast, these synthesis-specific by-products are very prominent in chemical synthesis wastes, whereby their detection in wastewater would prove a disposal of synthesis wastes instead of excretion after drug product consumption. As a prerequisite, such substances need to exhibit a certain chemical and biological stability in wastewater and, therefore, lab-scale experiments were performed in a mixture of WWTP effluent and activated sludge. Fourteen selected synthesis-specific substances, all related to the production of ATS, comprised pre-precursors (e.g. α-phenylacetoacetonitrile (APAAN) or α-phenylacetoacetamide (APAA)), precursors (e.g. BMK), intermediates (e.g. N-formylamphetamine (NFA)), synthesis by-products (e.g. N,N-di-(β-phenylisopropyl)amine (DPIA)) and final products (e.g. amphetamine (AMPH)). Stability of test substances was evaluated by targeted HPLC-MS/MS analysis, while HPLC-HRMS techniques were used for the identification of transformation products (TPs) of substances that have undergone primary degradation. All substances were detectable for five days minimum and seven out of 14 substances underwent at least primary degradation. A total of three TPs were identified: TP164 was formed by oxidation of ephedrine (EPHE) and was further transformed after maximum formation, while TP180-1 and TP180-2 were formed by reduction of APAA and both remained stable. This is the first study investigating the stability of ATS synthesis-specific substances in wastewater demonstrating sufficient stability for wastewater monitoring studies.
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An in-house-built ion trap mass spectrometer combined with a soft ionization source has been set up and tested. As ionization source, an electron beam pumped vacuum UV (VUV) excimer lamp (EBEL) was ...used for single-photon ionization. It was shown that soft ionization allows the reduction of fragmentation of the target analytes and the suppression of most matrix components. Therefore, the combination of photon ionization with the tandem mass spectrometry (MS/MS) capability of an ion trap yields a powerful tool for molecular ion peak detection and identification of organic trace compounds in complex matrixes. This setup was successfully tested for two different applications. The first one is the detection of security-relevant substances like explosives, narcotics, and chemical warfare agents. One test substance from each of these groups was chosen and detected successfully with single photon ionization ion trap mass spectrometry (SPI-ITMS) MS/MS measurements. Additionally, first tests were performed, demonstrating that this method is not influenced by matrix compounds. The second field of application is the detection of process gases. Here, exhaust gas from coffee roasting was analyzed in real time, and some of its compounds were identified using MS/MS studies.
Fast and reliable information is crucial for first responders to draw correct conclusions at crime scenes. An ambient pressure laser desorption (APLD) mass spectrometer is introduced for this ...scenario, which enables detecting substances on surfaces without sample pretreatment. It is especially useful for substances with low vapor pressure and thermolabile ones. The APLD allows for the separation of desorption and ionization into two steps and, therefore, both can be optimized separately. Within this work, an improved version of the developed system is shown that achieves limits of detection (LOD) down to 500 pg while remaining fast and flexible. Furthermore, realistic scenarios are applied to prove the usability of this system in real-world issues. For this purpose, post-blast residues of a bomb from the Second World War were analyzed, and the presence of PETN was proven without sample pretreatment. In addition, the analyzable substance range could be expanded by various drugs and drug precursors. Thus, the presented instrumentation can be utilized for an increased number of forensically important compound classes without changing the setup. Drug precursors revealed a LOD ranging from 6 to 100 ng. Drugs such as cocaine hydrochloride, heroin, (3,4-methylendioxy-methamphetamine) hydrochloride (MDMA) hydrochloride, and others exhibit a LOD between 10 to 200 ng.
Synthetic cannabimimetics (SC) are a diverse group of new psychoactive substances with varying potency and harm potential. New SCs appear on the drug market every year, and reliable and correct ...identification of these new derivatives independent from the matrix relies on the availability of verified spectra. Three new synthetic cannabimimetics featuring a norbornyl methyl side chain and varying core structure elements were identified in different seizures and forms. Cumyl-BC2.2.1HpMeGaClone and Cumyl-BC2.2.1HpMINACA were laced onto herbal blends, whereas Cumyl-BC2.2.1HpMICA was seized as a pure solid powder. The data collection process involves a comprehensive set of orthogonal analytical techniques allowing for the unambiguous identification of the respective endo- and exo-isomers.
Furthermore, the diversity of analytical techniques allows a greater number of laboratories working in the field of forensic chemistry to confidently identify the substances described in our original research article 1. Structure elucidation and analytical characterisation were performed within the EU-project ADEBAR plus using gas chromatography-mass spectrometry (GC-MS), gas chromatography-solid state infrared spectroscopy (GC-sIR), as well as solid and neat IR spectroscopy, Raman spectroscopy, liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS), and high resolution (HR)-LC-ESI-MS, and nuclear magnetic resonance (NMR) spectroscopy. The raw analytical data files are included in the Mendeley repository alongside the individual spectra in a universally importable format. The use of the universal JCAMP format for storage of the spectra facilitates database maintenance and enables seamless integration of the verified spectra. Thus, the dataset enables other researchers worldwide to identify these three new SCs confidently.
Nephronophthisis (NPHP) is an autosomal recessive cystic kidney disease, caused by mutations of at least nine different genes. Several extrarenal manifestations characterize this disorder, including ...cerebellar defects, situs inversus and retinitis pigmentosa. While the clinical manifestations vary significantly in NPHP, mutations of NPHP5 and NPHP6 are always associated with progressive blindness. This clinical finding suggests that the gene products, nephrocystin-5 and nephrocystin-6, participate in overlapping signaling pathways to maintain photoreceptor homeostasis. To analyze the genetic interaction between these two proteins in more detail, we studied zebrafish embryos after depletion of NPHP5 and NPHP6. Knockdown of zebrafish zNPHP5 and zNPHP6 produced similar phenotypes, and synergistic effects were observed after the combined knockdown of zNPHP5 and zNPHP6. The N-terminal domain of nephrocystin-6-bound nephrocystin-5, and mapping studies delineated the interacting site from amino acid 696 to 896 of NPHP6. In Xenopus laevis, knockdown of NPHP5 caused substantial neural tube closure defects. This phenotype was copied by expression of the nephrocystin-5-binding fragment of nephrocystin-6, and rescued by co-expression of nephrocystin-5, supporting a physical interaction between both gene products in vivo. Since the N- and C-terminal fragments of nephrocystin-6 engage in the formation of homo- and heteromeric protein complexes, conformational changes seem to regulate the interaction of nephrocystin-6 with its binding partners.
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