Low resolution and high resolution MS for studies on the metabolism and toxicological detection of the new psychoactive substance methoxypiperamide (MeOP)

In 2013, the new psychoactive substance methoxypiperamide (MeOP) was first reported to the European Monitoring Centre for Drug and Drug Addiction. Its structural similarity to already controlled piperazine designer drugs might have contributed to the decision to offer MeOP for online purchase. The aims of this work were to identify the phase I/II metabolites of MeOP in rat urine and the human cytochrome P450 (CYP) isoenzymes responsible for the initial metabolic steps. Finally, the detectability of MeOP in rat urine by gas chromatography–mass spectrometry (GC‐MS) and liquid chromatography coupled with multistage mass spectrometry (LC‐MSⁿ) standard urine screening approaches (SUSAs) was evaluated. After sample preparation by cleavage of conjugates followed by extraction for elucidating phase I metabolites, the analytes were separated and identified by GC‐MS as well as liquid chromatography‐high resolution‐tandem mass spectrometry (LC‐HR‐MS/MS). For detection of phase II metabolites, the analytes were separated and identified after urine precipitation followed by LC‐HR‐MS/MS. The following metabolic steps could be postulated: hydrolysis of the amide, N‐oxide formation, N‐ and/or O‐demethylation, oxidation of the piperazine ring to the corresponding keto‐piperazine, piperazine ring opening followed by oxidation of a methylene group to the corresponding imide, and hydroxylation of the phenyl group. Furthermore, N‐acetylation, glucuronidation and sulfation were observed. Using human CYPs, CYP1A2, CYP2C19, CYP2D6, and/or CYP3A4 were found to catalyze N‐oxide formation and N‐, O‐demethylation and/or oxidation. Mostly MeOP and N‐oxide‐MeOP but to a minor degree also other metabolites could be detected in the GC‐MS and LC‐MSⁿ SUSAs. Copyright © 2015 John Wiley & Sons, Ltd.     

Quantitative analysis of amino acids by HPLC in dried blood and urine in the neonatal period: Establishment of reference values

Quantitative analysis of amino acids in blood and urine is primarily indicated for the diagnosis of amino acid disorders. The high-performance liquid chromatography (HPLC) technique is frequently used for this detection. The frequency of sample collection on filter paper has been increasing exponentially, and there are many advantages attributed to processing biological samples in this way. The aim of this study was to validate a quantitative analysis of amino acids by HPLC in blood and urine collected on filter paper and to establish reference values in the neonatal period. Dried blood and dried urine samples of respectively 58 and 45 healthy newborns (2–9 days) were collected. Pre-treatment and extraction of samples were done according to the literature. Separation and analysis of amino acids were carried out by HPLC with fluorescence detection. The developed method demonstrated excellent separation, linearity, limits of detection and quantification, repeatability and recovery. The reference values for 17 amino acids were defined in dried blood and urine samples of newborns. This work presents a simple, fast and effective method for the simultaneous analysis of 17 amino acids in blood and urine collected on filter paper in a single run. The reference values were established and validated.     

Zeolite as an efficient and recyclable activation surface for the synthesis of bis-thiazolidinones: theoretical screening owing to experimental biology

Abstract

Zeolite 5Å has been used as an efficient and cost effective activating catalyst for the synthesis of bis-thiazolidinones 4 (a–h) starting from bis-imines 3 (a–h) and thioacetic acid. The reactions were performed under microwave irradiation in solventless condition. The catalyst was recycled and used for several times. This reaction is scalable to multigram scale and the methodology has resulted in an efficient synthesis. A benign, environmentally friendly, efficient, and extremely fast procedure for synthesis of bis-thiazolidinones has been demonstrated. The produced bis-thiazolidinone molecules were characterized on the basis of elemental analysis, IR, mass, and ¹H-NMR spectral data. The synthesized moieties were screened virtually using some of bioinformatical softwares and discussed.     

Analysis of Acylcarnitine Ester for Neonatal Screening of Inborn Errors of Metabolism Using Tandem Mass-spectrometry

Summary. Tandem mass-spectrometry has been introduced worldwide into neonatal screening programs for the quantitative analysis of acylcarnitine species and amino acids for the diagnosis of organic acidurias, defects of fatty acid oxidation, and amino acidopathies, respectively. Since April 2002 more than 200000 newborn infants have been screened in Austria using tandem mass-spectrometry. In this cohort 37 infants with amino acidopathies and 38 infants with fatty acid oxidation defects or organic acidurias have been diagnosed. The overall incidence of these disorders is 0.036%. The analysis of acylcarnitine species using tandem mass-spectrometry has enabled the diagnosis of infants with inborn errors of metabolism prior to clinical presentation and to initiate therapy early enough to prevent long-term sequelae and to reduce mortality in these disorders.     

Virtual fragment screening: an exploration of various docking and scoring protocols for fragments using Glide

Fragment-based drug discovery approaches allow for a greater coverage of chemical space and generally produce high efficiency ligands. As such, virtual and experimental fragment screening are increasingly being coupled in an effort to identify new leads for specific therapeutic targets. Fragment docking is employed to create target-focussed subset of compounds for testing along side generic fragment libraries. The utility of the program Glide with various scoring schemes for fragment docking is discussed. Fragment docking results for two test cases, prostaglandin D2 synthase and DNA ligase, are presented and compared to experimental screening data. Self-docking, cross-docking, and enrichment studies are performed. For the enrichment runs, experimental data exists indicating that the docking decoys in fact do not inhibit the corresponding enzyme being examined. Results indicate that even for difficult test cases fragment docking can yield enrichments significantly better than random. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

妊娠晚期羊水中微量元素测定及其与胎儿生长发育关系的研究

对１０２名足月正常妊娠第一胎的产妇妊娠晚期羊水中６种微量元素（锌、铜、锰、铁、钙、镁）首次进行了测定，并通过对出生新生儿生长发育的监测探讨其与胎儿生长发育的关系。妊娠晚期羊水中６种微量元素含量（±Ｓ／×１０－５）分别为：Ｚｎ２．２９±１．７７，Ｃｕ０．１４±０．１４，Ｍｎ０．１３±０．２３，Ｆｅ３．６９±２．７０，Ｃａ８２．５４±７４．１８，Ｍｇ１８．７１±１１．０９．男女新生儿其母羊水中微量元素含量比较无显著差异，Ｐ≥０．０５．与文献报道中期妊娠羊水中所含微量元素比较有增加趋势．６种微量元素相互关系中．与锌、钙呈正相关外，锌与铜、锰、铁、镁呈负相关．低体重组与低身长组新生儿孕母羊水中锌含量明显低于高体重组和高身长组，有显著差异，Ｐ≤０．０１．铁在高身长组含量增高，与低身长组比较有显著差异，Ｐ≤０．０５．锰于低体重组含量升高，与高体重组比较有显著差异，Ｐ≤０．０１．镁与铜于低身长组含量升高，与高身长组比较有显著差异，Ｐ分别≤０．０５与０．０１．可见高铜、高锰、高镁可致胎儿生长发育迟缓．     

Simulation of chemical metabolism for fate and hazard assessment. I. Approach for simulating metabolism

Information regarding the metabolism of xenobiotic chemicals plays a central role in regulatory risk assessments. In regulatory programmes where metabolism studies are required, the studies of metabolic pathways are often incomplete and the identification of activated metabolites and important degradation products are limited by analytical methods. Because so many more new chemicals are being produced than can be assessed for potential hazards, setting assessment priorities among the thousands of untested chemicals requires methods for predictive hazard identification which can be derived directly from chemical structure and their likely metabolites. In a series of papers we are sharing our experience in the computerized management of metabolic data and the development of simulators of metabolism for predicting the environmental fate and (eco)toxicity of chemicals. The first paper of the series presents a knowledge-based formalism for the computer simulation of non-intermediary metabolism for untested chemicals, with an emphasis on qualitative and quantitative aspects of modelling metabolism.     

细胞不同代谢类型的热化学研究 I.生长、非生长及内源代谢水平的量热测定

Using microcalorimetric method, the thermally measured curves of different types of metabolism of E. coli were determined. From these thermal measured curves, we can obtain the heat output of E. coli at growth metabolism to be 1 .74+10.63 pW.cell- 1; and0.14+0.04 pW.cell-1 at the non-growth metabolism while at the endogenous metabolismit is 0.045+0.001 pW﹒cell-1     

Determination of the spectrum of low molecular mass organic acids in urine by capillary electrophoresis with contactless conductivity and ultraviolet photometric detection--an efficient tool for monitoring of inborn metabolic disorders

A mixture of 29 organic acids (OAs) occurring in urine was analyzed by capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C⁴D) and UV photometric detection. The optimized analytical system involved a 100 cm long polyacrylamide-coated capillary (50 μm i.d.) and the background electrolyte of 20 mM 2-morpholinoethanesulfonic acid (MES)/NaOH + 10% (v/v) methanol, pH 6.0 (pH is related to the 20 mM MES/NaOH buffer in water). The LOD values obtained by C⁴D for the OAs which do not absorb UV radiation range from 0.6 μM (oxalic acid) to 6.8 μM (pyruvic acid); those obtained by UV photometry for the remaining OAs range from 2.9 μM (5-hydroxy-3-indoleacetic acid) to 10.2 μM (uric acid). The repeatability of the procedure developed is characterized by the coefficients of variation, which vary between 0.3% (tartaric acid) and 0.6% (5-hydroxy-3-indoleacetic acid) for the migration time and between 1.3% (tartaric acid) and 3.5% (lactic acid) for the peak area. The procedure permitted quantitation of 20 OAs in a real urine sample and was applied to monitoring of the occurrence of the inborn metabolic fault of methylmalonic aciduria.     

Shapelets: possibilities and limitations of shape-based virtual screening

Complementarity of molecular surfaces is crucial for molecular recognition. A method for representation of molecular shape is presented. We decompose the molecular surface into commensurate patches with defined shape by fitting hyperbolical paraboloids onto a triangulated isosurface of the Gaussian model of a molecule. As a result of this decomposition we obtain a 3D graph representation of the molecular shape, which can be used for complete and partial shape matching and isosteric group searching. To point out the possibilities and limitations of shape-only models, we challenged our method by three scenarios in a virtual screening contest: rigid body alignment, consensus shape filtering, and target-specific screening.     

Aroma WaterLOGSY: a fast and sensitive screening tool for drug discovery

One-dimensional NMR spectroscopy has proven to be a powerful technique for screening compound libraries in drug discovery. We report a novel water ligand-observed gradient spectroscopy (WaterLOGSY) pulse sequence, named Aroma WaterLOGSY, that selectively detects aromatic WaterLOGSY signals from compounds or ligands. In the Aroma WaterLOGSY, water magnetization is untouched after water excitation and utilizes the whole period of the remaining pulse sequence to relax back to the +z direction. Due to the phase cycling design, the water magnetization is allowed to relax for the period of two full scans before it gets inverted again. Therefore, the recycle delay can be significantly shortened. Within similar experimental time, Aroma WaterLOGSY shows approximately two times higher sensitivity than the standard scheme. This method also allows the use of non-deuterated reagents, thereby accelerating experimental set-up time for ligand-binding studies.     

A novel phosphorus oxide quantum dots as an emissive nanomaterial for inorganic ions screening and bioimaging

In this work, a novel blue-green fluorescence phosphorous oxide quantum dots (PO QDs) was synthesized by solvothermal method in N-methyl-2-pyrrolidone (NMP) solution without any protection treatment during synthesis. Upon excitation at 400 nm, PO QDs emitted blue-green fluorescence with quantum yield of 0.28. PO QDs exhibited the high inertness to air or moisture, the excellent water solubility, and stable emission intensity in a wide pH range and in high ionic strength solution. Interestingly, PO QDs could give the positive optical response to iron ions (Fe³⁺) and iodine ion (I⁻). The photoluminescence (PL) of PO QDs could be directly quenched by Fe³⁺. While I⁻ quenched the PO QDs PL by means of Ag⁺-mediated PO QDs system via the internal filtration effects (IFE) induced by the formation of AgI. Moreover, the biocompatibility and low toxicity of PO QDs verified in bean sprout and Hela cells indicated the promising application of PO QDs in medicine related fields. Furthermore, PO QDs could also be utilized in luminescent composite film for various application scenarios     

Intrinsic Fluorescence of Metabolite Amyloids Allows Label‐Free Monitoring of Their Formation and Dynamics in Live Cells

The formation of apoptosis‐inducing amyloidal structures by metabolites has significantly extended the “amyloid hypothesis” to include non‐proteinaceous, single metabolite building blocks. However, detection of metabolite assemblies is restricted compared to their larger protein‐based counterparts owing to the hindrance of external labelling and limited immunohistochemical detection tools. Herein, we present the detection of the formation, dynamics, and cellular distribution of metabolite amyloid‐like structures and provide mechanistic insights into the generation of supramolecular chromophores. Moreover, the intrinsic fluorescence properties allow the detection of metabolite assemblies in living cells without the use of external dyes. Altogether, this intrinsic fluorescence of metabolite assemblies further verifies their amyloidal nature, while providing an important tool for further investigation of their pathological role in inborn error of metabolism disorders.     

Novel strategy for quality consistency evaluation of Chinese medicine “YIQING” tablet that combines the simultaneous quantification and screening of ten bioactive constituents

The UV characteristics for different categories compounds in complex traditional Chinese medicines and herbal preparations usually vary. Thus, to achieve the integral analysis of multiwavelength fingerprint characteristics, we introduced a novel strategy of multiwavelength total fusion profiling. The simultaneous separation and quantification of multiple components by reversed‐phase high‐performance liquid chromatography coupled with diode array detection was developed in an effective, accurate, and reliable way. Furthermore, a 2,2‐diphenyl‐1‐picrylhydrazyl radical (DPPH) scavenging assay was set up to detect and screen the bioactivity of similar‐structure constituents (aloe‐emodin, rhein, emodin, chrysophanol, baicalein, wogonin, baicalin, wogonoside, berberine hydrochloride, and jatrorrhizine hydrochloride). Moreover, the high‐performance liquid chromatography DPPH assay was developed to monitor the relationship between the biological activity and the spatial structure, the number of hydroxyl groups, the concentration of the analytes in samples. The result of qualitative classification for 15 batches of “YIQING” tablets using principle component analysis was consistent with the quantitative fingerprint assessment using the average linear quantitative fingerprint method. Therefore, chemometrics, multiwavelength total fusion profiling in conjunction with average linear quantitative fingerprint method and antioxidant activity can control the quality of traditional Chinese medicines/herbal preparations comprehensively and practically.     

Lessons for fragment library design: analysis of output from multiple screening campaigns

Over the past 8 years, we have developed, refined and applied a fragment based discovery approach to a range of protein targets. Here we report computational analyses of various aspects of our fragment library and the results obtained for fragment screening. We reinforce the finding of others that the experimentally observed hit rate for screening fragments can be related to a computationally defined druggability index for the target. In general, the physicochemical properties of the fragment hits display the same profile as the library, as is expected for a truly diverse library which probes the relevant chemical space. An analysis of the fragment hits against various protein classes has shown that the physicochemical properties of the fragments are complementary to the properties of the target binding site. The effectiveness of some fragments appears to be achieved by an appropriate mix of pharmacophore features and enhanced aromaticity, with hydrophobic interactions playing an important role. The analysis emphasizes that it is possible to identify small fragments that are specific for different binding sites. To conclude, we discuss how the results could inform further development and improvement of our fragment library. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Rapid screening of bioactive compounds from natural products by integrating 5-channel parallel chromatography coupled with on-line mass spectrometry and microplate based assays

A high throughput method was developed for rapid screening and identification of bioactive compounds from traditional Chinese medicine, marine products and other natural products. The system, integrated with five-channel chromatographic separation and dual UV–MS detection, is compatible with in vitro 96-well microplate based bioassays. The stability and applicability of the proposed method was validated by testing radical scavenging capability of a mixture of seven known compounds (rutin, dihydroquercetin, salvianolic acid A, salvianolic acid B, glycyrrhizic acid, rubescensin A and tangeretin). Moreover, the proposed method was successfully applied to the crude extracts of traditional Chinese medicine and a marine sponge from which 12 bioactive compounds were screened and characterized based on their anti-oxidative or anti-tumor activities. In particular, two diterpenoid derivatives, agelasine B and (−)-agelasine D, were identified for the first time as anti-tumor compounds from the sponge Agelas mauritiana, showing a considerable activity toward MCF-7 cells (IC₅₀ values of 7.84 ± 0.65 and 10.48 ± 0.84 μM, respectively). Our findings suggested that the integrated system of 5-channel parallel chromatography coupled with on-line mass spectrometry and microplate based assays can be a versatile and high efficient approach for the discovery of active compounds from natural products.     

血脑屏障药物筛选模型的建立及丹参活性成分筛选分析

ECV304细胞在C6细胞的诱导下生长,通过细胞培养时间优化、渗透系数测定和细胞形态学观察等,建立ECV304/C6共培养血脑屏障(BBB)药物筛选模型.将该模型应用于从丹参提取液中筛选可能作用于中枢神经系统(CNS)的活性成分,结合液相色谱-质谱联用技术(LC-MS)分析,发现丹参提取液中至少有16种成分能够穿越BBB模型,其中4种成分被确认为隐丹参酮、丹参酮Ⅰ、丹参素和原儿茶酸.通过定量构效关系(QASR)分析,进一步从理论上证明所确认的4种化合物均符合CNS靶向药物的特征.研究结果表明,ECV304/C6共培养BBB模型能够在模拟生理状态下从中药复杂体系中筛选分离跨越BBB的活性成分组,可用于CNS药物开发的早期快速筛选,服务于中药现代化研究.