43 research outputs found

    Technological advances for analyzing the content of organ-on-a-chip by mass spectrometry

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    Three-dimensional (3D) cell cultures, including organ-on-a-chip (OOC) devices, offer the possibility to mimic human physiology conditions better than 2D models. The organ-on-a-chip devices have a wide range of applications, including mechanical studies, functional validation, and toxicology investigations. Despite many advances in this field, the major challenge with the use of organ-on-a-chips relies on the lack of online analysis methods preventing the real-time observation of cultured cells. Mass spectrometry is a promising analytical technique for real-time analysis of cell excretes from organ-on-a-chip models. This is due to its high sensitivity, selectivity, and ability to tentatively identify a large variety of unknown compounds, ranging from metabolites, lipids, and peptides to proteins. However, the hyphenation of organ-on-a-chip with MS is largely hampered by the nature of the media used, and the presence of nonvolatile buffers. This in turn stalls the straightforward and online connection of organ-on-a-chip outlet to MS. To overcome this challenge, multiple advances have been made to pre-treat samples right after organ-on-a-chip and just before MS. In this review, we summarised these technological advances and exhaustively evaluated their benefits and shortcomings for successful hyphenation of organ-on-a-chip with MS

    On-line electrochemistry–bioaffinity screening with parallel HR-LC-MS for the generation and characterization of modified p38α kinase inhibitors

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    In this study, an integrated approach is developed for the formation, identification and biological characterization of electrochemical conversion products of p38α mitogen-activated protein kinase inhibitors. This work demonstrates the hyphenation of an electrochemical reaction cell with a continuous-flow bioaffinity assay and parallel LC-HR-MS. Competition of the formed products with a tracer (SKF-86002) that shows fluorescence enhancement in the orthosteric binding site of the p38α kinase is the readout for bioaffinity. Parallel HR-MSn experiments provided information on the identity of binders and non-binders. Finally, the data produced with this on-line system were compared to electrochemical conversion products generated off-line. The electrochemical conversion of 1-{6-chloro-5-[(2R,5S)-4-(4-fluorobenzyl)-2,5-dimethylpiperazine-1-carbonyl]-3aH-indol-3-yl}-2-morpholinoethane-1,2-dione resulted in eight products, three of which showed bioaffinity in the continuous-flow p38α bioaffinity assay used. Electrochemical conversion of BIRB796 resulted, amongst others, in the formation of the reactive quinoneimine structure and its corresponding hydroquinone. Both products were detected in the p38α bioaffinity assay, which indicates binding to the p38α kinase

    Development of an online p38α mitogen-activated protein kinase binding assay and integration of LC–HR-MS

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    A high-resolution screening method was developed for the p38α mitogen-activated protein kinase to detect and identify small-molecule binders. Its central role in inflammatory diseases makes this enzyme a very important drug target. The setup integrates separation by high-performance liquid chromatography with two parallel detection techniques. High-resolution mass spectrometry gives structural information to identify small molecules while an online enzyme binding detection method provides data on p38α binding. The separation step allows the individual assessment of compounds in a mixture and links affinity and structure information via the retention time. Enzyme binding detection was achieved with a competitive binding assay based on fluorescence enhancement which has a simple principle, is inexpensive, and is easy to interpret. The concentrations of p38α and the fluorescence tracer SK&F86002 were optimized as well as incubation temperature, formic acid content of the LC eluents, and the material of the incubation tubing. The latter notably improved the screening of highly lipophilic compounds. For optimization and validation purposes, the known kinase inhibitors BIRB796, TAK715, and MAPKI1 were used among others. The result is a high-quality assay with Z′ factors around 0.8, which is suitable for semi-quantitative affinity measurements and applicable to various binding modes. Furthermore, the integrated approach gives affinity data on individual compounds instead of averaged ones for mixtures

    Arterial blood pressure in anesthetized neonates and infants: a retrospective analysis of 1091 cases

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    BackgroundHypotension during general anesthesia in neonates and infants is considered to contribute to poor neurological outcome. AimThe aim of this retrospective analysis was to determine the incidence of hypotension after induction of anesthesia and sustained hypotension (>10 min) during the anesthesia, and to determine factors contributing to the development of (sustained) hypotension. MethodWe performed a retrospective analysis of 1091 electronic anesthesia records from children <1 year. Patients were stratified for age (group 1: <1 month, group 2: 1-3 months, group 3: 4-6 months, group 4: >6 months). Hypotension was defined as a mean arterial pressure (MAP) <35 mmHg in patients 6 months and <43 mmHg in patients >6 months. ResultsThe incidence of hypotension after induction was highest in group 1 (25.5%) [P = 0.009 vs group 2 (13.3%), P < 0.0001 vs groups 3 (3.4%) and 4 (1.0%)], in group 2, it was higher than in groups 3 and 4 (P < 0.0001), and in group 3, it was higher than in group 4 (P = 0.033). The incidence of sustained hypotension was highest in group 1 (43.6%) (P < 0.0001 vs groups 2-4), followed by group 2 (15.7%) [P < 0.0001 vs group 3 (3.4%) and P = 0.006 vs group 4 (8.8%)] and group 4 (P = 0.004 vs group 3). Hypotension after induction occurred more often in emergency procedures than in elective procedures in groups 1 (P = 0.002), 2 (P = 0.029), and 3 (P = 0.037). ConclusionHypotension, both postinduction and sustained during surgery, is a common phenomenon in anesthetized children under 1 year, peaking in neonates. Generally accepted lower limits of MAP in anesthetized infants urgently need to be defined, enabling us to develop anesthesia strategies avoiding cerebral hypoperfusion

    Uncovering the behaviour of ions in the gas-phase to predict the ion mobility separation of isomeric steroid compounds

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    Bile acids are steroid compounds involved in biological mechanisms of neurodegenerative diseases making them potential biomarkers for diagnosis or treatment. These compounds exist as structural and conformational isomers, which hinder distinguishing them in physiological processes. We aimed to develop tandem mass spectrometry-ion mobility spectrometry (MS/MS-IMS) methodologies to explore and understand the behaviour of isomeric steroids in the gas-phase and rapidly separate them. Unlike previously published ion mobility data, various isomers were investigated in mixtures to better mimic complex (pre-) clinical samples. The experimental collision cross sections (CCS)s were compared to the theoretical CCS values for an in-depth analysis of isomeric ions' behaviour in the gas-phase. Based on density-functional theory, we identified the impact of adduct positioning on the 3D conformation of enantiomers, diastereomers and structural isomers. The curling of the large side chains hedged the small differences among the isomers and lowered the CCS values. On the other hand, fragmenting off the identical side branches as well as imposing the bending of the steroid ring resulted in ion mobility differentiation. Careful data evaluation revealed the tendency of isomers to form homo-cluster in the mixture solutions and assist the separation. Our fundamental and experimental findings enable the ion mobility separation of isomeric steroids to be predicted. The introduced rapid and optimal MS/MS-IMS analytical methodology can be applied to distinguish isomeric bile acids both in a solution and potentially in patients' tissue samples, and consequently, reveal their molecular pathways

    Quality-In(Process)Line (QuIProLi) process intensification for a micro-flow UV-photo synthesis enabled by online UHPLC analysis

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    Process intensification commonly enables reaction acceleration and therefore continuous-flow/PAT is urgently needed. The low volumes typical for micro-flow pose challenges for online sampling operations in analytics. In this paper, a very fast process is combined with a modified ultra-high-performance liquid chromatography (UHPLC) system allowing for very fast sampling and analysis. Low-volume online sampling according to the needs posed by PAT for pharma quality control, is introduced here for UHPLC analysis of the photo-Claisen rearrangement in micro-flow. Chances and challenges are critically reviewed, including the reproducibility and robustness of the sampling. Furthermore, the ability and speed of the chosen set-up in order to capture process changes and adjust the process parameters properly is investigated. With the applied online sampling system, it was possible to perform, almost unattended and spending 12 times less sampling volume, a full factorial analysis of all relevant reaction conditions (243 experiments) in three days. Such quality-in-the-process-line (QuIProLi) online sampling avoided random errors due to automation. (C) 2018 Elsevier Ltd. All rights reserved

    Racemic and Enantiopure Camphene and Pinene Studied by the Crystalline Sponge Method

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    Contains fulltext : 187716.pdf (publisher's version ) (Open Access

    Quality-in(Process)Line (QuIProLi) process intensification for a micro-flow UV-photo synthesis enabled by online UHPLC analysis

    Get PDF
    Process intensification commonly enables reaction acceleration and therefore continuous-flow/PAT is urgently needed. The low volumes typical for micro-flow pose challenges for online sampling operations in analytics. In this paper, a very fast process is combined with a modified ultra-high-performance liquid chromatography (UHPLC) system allowing for very fast sampling and analysis. Low-volume online sampling according to the needs posed by PAT for pharma quality control, is introduced here for UHPLC analysis of the photo-Claisen rearrangement in micro-flow. Chances and challenges are critically reviewed, including the reproducibility and robustness of the sampling. Furthermore, the ability and speed of the chosen set-up in order to capture process changes and adjust the process parameters properly is investigated. With the applied online sampling system, it was possible to perform, almost unattended and spending 12 times less sampling volume, a full factorial analysis of all relevant reaction conditions (243 experiments) in three days. Such quality-in-the-process-line (QuIProLi) online sampling avoided random errors due to automation
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