Workflow automation in liquid chromatography mass spectrometry

We describe the fully automated workflow path developed for the ingest and analysis of liquid chromatography mass spectrometry (LCMS) data. With the help of this computational workflow, we were able to replace two human work days to analyze data with two hours of unsupervised computation time. In addition, this tool also can compute confidence intervals for all its results, based on the noise level present in the data. We leverage only open source tools and libraries in this workflow

Automated processing of whole blood samples for the determination of immunosuppressants by liquid chromatography tandem-mass spectrometry

Background: Liquid chromatography tandem-mass spectrometry (LC-MS/MS) is an efficient technology for routine determination of immunosuppressants in whole blood; however, time-consuming manual sample preparation remains a significant limitation of this technique. Methods: Using a commercially available robotic pipetting system (Tecan Freedom EVO), we developed an automated sample-preparation protocol for quantification of tacrolimus in whole blood by LC-MS/MS. Barcode reading, sample resuspension, transfer of whole blood aliquots into a deep-well plate, addition of internal standard solution, mixing, and protein precipitation by addition of an organic solvent is performed by the robotic system. After centrifugation of the plate, the deproteinized supernatants are submitted to on-line solid phase extraction, using column switching prior to LC-MS/MS analysis. The only manual actions within the entire process are decapping of the tubes, and transfer of the deep-well plate from the robotic system to a centrifuge and finally to the HPLC autosampler. Whole blood pools were used to assess the reproducibility of the entire analytical system for measuring tacrolimus concentrations. Results: A total coefficient of variation of 1.7% was found for the entire automated analytical process (n=40; mean tacrolimus concentration, 5.3 mu g/L). Close agreement between tacrolimus results obtained after manual and automated sample preparation was observed. Conclusions: The analytical system described here, comprising automated protein precipitation, on-line solid phase extraction and LC-MS/MS analysis, is convenient and precise, and minimizes hands-on time and the risk of mistakes in the quantification of whole blood immunosuppressant concentrations compared to conventional methods

Updates in metabolomics tools and resources: 2014-2015

Data processing and interpretation represent the most challenging and time-consuming steps in high-throughput metabolomic experiments, regardless of the analytical platforms (MS or NMR spectroscopy based) used for data acquisition. Improved machinery in metabolomics generates increasingly complex datasets that create the need for more and better processing and analysis software and in silico approaches to understand the resulting data. However, a comprehensive source of information describing the utility of the most recently developed and released metabolomics resources—in the form of tools, software, and databases—is currently lacking. Thus, here we provide an overview of freely-available, and open-source, tools, algorithms, and frameworks to make both upcoming and established metabolomics researchers aware of the recent developments in an attempt to advance and facilitate data processing workflows in their metabolomics research. The major topics include tools and researches for data processing, data annotation, and data visualization in MS and NMR-based metabolomics. Most in this review described tools are dedicated to untargeted metabolomics workflows; however, some more specialist tools are described as well. All tools and resources described including their analytical and computational platform dependencies are summarized in an overview Table

Application of a Rapid and Integrated Analysis System (RIAS) as a High-Throughput Processing Tool for In Vitro ADME Samples by Liquid Chromatography/Tandem Mass Spectrometry

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.Over the past decade, drug discovery programs have started to address the optimization of key ADME properties already at an early stage of the process. Hence, analytical chemists have been confronted with tremendously rising sample numbers and have had to develop methodologies accelerating quantitative liquid chromatography/tandem mass spectrometry (LC/MS/MS). This article focuses on the application of a generic and fully automated LC/MS/MS, named Rapid and Integrated Analysis System (RIAS), as a high-throughput platform for the rapid quantification of drug-like compounds in various in vitro ADME assays. Previous efforts were dedicated to the setup and feasibility study of a workflow-integrated platform combining a modified high-throughput liquid handling LC/MS/MS system controlled by a customized software interface and a customized data-processing and reporting tool. Herein the authors present an extension of this previously developed basic application to a broad set of ADME screening campaigns, covering CYP inhibition, Caco-2, and PAMPA assays. The platform is capable of switching automatically between various ADME assays, performs MS compound optimization if required, and provides a speed of 8 s from sample to sample, independently of the type of ADME assay. Quantification and peak review are adopted to the high-throughput environment and tested against a standard HPLC-ESI technology

A Unique Automation Platform for Measuring Low Level Radioactivity in Metabolite Identification Studies

Generation and interpretation of biotransformation data on drugs, i.e. identification of physiologically relevant metabolites, defining metabolic pathways and elucidation of metabolite structures, have become increasingly important to the drug development process. Profiling using 14C or 3H radiolabel is defined as the chromatographic separation and quantification of drug-related material in a given biological sample derived from an in vitro, preclinical in vivo or clinical study. Metabolite profiling is a very time intensive activity, particularly for preclinical in vivo or clinical studies which have defined limitations on radiation burden and exposure levels. A clear gap exists for certain studies which do not require specialized high volume automation technologies, yet these studies would still clearly benefit from automation. Use of radiolabeled compounds in preclinical and clinical ADME studies, specifically for metabolite profiling and identification are a very good example. The current lack of automation for measuring low level radioactivity in metabolite profiling requires substantial capacity, personal attention and resources from laboratory scientists. To help address these challenges and improve efficiency, we have innovated, developed and implemented a novel and flexible automation platform that integrates a robotic plate handling platform, HPLC or UPLC system, mass spectrometer and an automated fraction collector

Towards a comprehensive characterisation of the human internal chemical exposome: Challenges and perspectives

The holistic characterisation of the human internal chemical exposome using high-resolution mass spectrometry (HRMS) would be a step forward to investigate the environmental AE tiology of chronic diseases with an unprecedented precision. HRMS-based methods are currently operational to reproducibly profile thousands of endogenous metabolites as well as externally-derived chemicals and their biotransformation products in a large number of biological samples from human cohorts. These approaches provide a solid ground for the discovery of unrecognised biomarkers of exposure and metabolic effects associated with many chronic diseases. Nevertheless, some limitations remain and have to be overcome so that chemical exposomics can provide unbiased detection of chemical exposures affecting disease susceptibility in epidemiological studies. Some of these limitations include (i) the lack of versatility of analytical techniques to capture the wide diversity of chemicals; (ii) the lack of analytical sensitivity that prevents the detection of exogenous (and endogenous) chemicals occurring at (ultra) trace levels from restricted sample amounts, and (iii) the lack of automation of the annotation/identification process. In this article, we discuss a number of technological and methodological limitations hindering applications of HRMS-based methods and propose initial steps to push towards a more comprehensive characterisation of the internal chemical exposome. We also discuss other challenges including the need for harmonisation and the difficulty inherent in assessing the dynamic nature of the internal chemical exposome, as well as the need for establishing a strong international collaboration, high level networking, and sustainable research infrastructure. A great amount of research, technological development and innovative bio-informatics tools are still needed to profile and characterise the "invisible" (not profiled), "hidden" (not detected) and "dark" (not annotated) components of the internal chemical exposome and concerted efforts across numerous research fields are paramount

Mass Spectrometry Application Strategies of Dried Blood Spots Analysis

El análisis de manchas de sangre seca (DBS) es una tecnología de análisis bien conocida, cuyas primeras aplicaciones de rutina se remontan a la década de 1960. Los avances en los instrumentos de espectrometría de masas durante el siglo pasado, permitieron alcanzar la sensibilidad necesaria para abrir nuevos mercados. Sin embargo, algunas incertidumbres y la falta de comprensión de los métodos han impedido hasta el momento que la tecnología tenga una amplia aceptación en el mercado.En esta tesis doctoral se han desarrollado y validado diversas técnicas, condiciones y flujos de trabajo de análisis DBS, que demuestran la viabilidad y el potencial de aplicación de la tecnología DBS-LC-MS.Se han desarrollado métodos avanzados para su aplicación en el campo de la pediatría neonatal, en particular la prueba del talón en recién nacidos, donde se ha ampliado y estandarizado el panel de análisis.También se han investigado nuevos campos de aplicación, como la vigilancia de medicamentos terapéuticos y la toxicología forense. Se describe la aplicación de la farmacovigilancia remota de antirretrovirales en regiones de escasos recursos y se presentan nuevos enfoques analíticos para la vigilancia del abuso de alcohol. Finalmente, en esta tesis se ha introducido un método innovador con el que se pueden detectar más de 1.200 drogas ilícitas a partir de una sola gota de sangre.La investigación científica realizada se presenta en forma de compendio de publicaciones (6), que son incluidas en esta Memoria. Se adjuntan a modo de apéndice otros dos trabajos del candidato que no constan oficialmente en dicho compendio. Todos los trabajos constituyen una unidad temática coherente sobre la técnica de la DBS y su acoplamiento a la espectrometría de masas.This doctoral thesis is a compendium of dried blood spot (DBS) applications in the fields of newborn screening, forensic toxicology and therapeutic drug monitoring. DBS is a well-known analysis technology, which first routine applications date back to the 1960s. Advancements in mass spectrometry instruments during the last century, enabled to reach the required sensitivity to open up new markets. Some uncertainties and missing method understanding remain and this is holding back the technology from wide spread market acceptance. For the general scientific acceptance of this technology, several methods have been developed and validated within this work. Advanced methods for the field of newborn screening were developed, where the analysis panel has been extended and standardized. Goal of the first study was to transfer the amino acids and acyl carnitines analysis onto the automated DBS-MS 500 platform. Also, a steroid panel of 17OHP, cortisol and androstenedione was defined to exclude the 17OHP from the immune assay panel and to transfer this as well onto the fully automated DBS-LC-MS/MS. The conventional 17OHP enzyme-linked immunosorbent assay (ELISA) based on a manually punched DBS disc leads to a high percentage of false positive. First, cortisol increases when the babies are stressed, which also leads to elevated 17OHP concentrations and secondly, the ELISA has significant cross reaction potential with steroid sulfate which can be monitored with androstenedione. Both, the amino acid and acyl carnitine detection plus the integration of a steroid panel into the DBS-LC-MS/MS workflow was successfully achieved [1]. Newborn screening panels are not unified across borders and sometimes even differ within countries and laboratories. The implementation of the fully automated DBS platform could be a good starting point of standardization and unification of those programs. Here, a method based on an official newborn screening kit was introduced within this thesis. The analysis takes only 2 minutes per sample; however it is limited to amino acids and acyl carnitines only. The DBS extract is directly guided to the mass spectrometer without any column. This is a well-known procedure in newborn screening and allows speeding up the process to its optimum [2]. Therapeutic drug monitoring, especially in remote and rural areas is another upcoming application field of DBS. Several methods have been supported and co-authored, whereas the first method focuses of the three antiretroviral drugs nevirapine, efavirenz and lopinavir. The study highlights the transportation advantages of DBS, without any biohazard labeling neither requiring cooling chains. DBS were drawn in Tanzania, Africa, and sent to Switzerland where the samples went through different climate zones. Nonetheless, the samples showed very good results and stability of the monitored drugs [3]. We ran a follow up study on some of the samples approximately one year after the publication, where still all analytes could be recovered in the same concentration as published. Also the DBS sampling itself was investigated in a rural area of Tanzania [4] and the technique proved to be suitable. Another therapeutic drug, where a more efficient sampling process is required for mass drug administration (MDA) campaigns, is Ivermectin. This drug lowers the incidence of river blindness and lymphatic filariasis infections. Further, recent studies demonstrated that Ivermectin is also active against several other parasites and even against certain mosquitoes. As one of these mosquitoes is Anopheles gambiae, the major vector of malaria in Africa, Ivermectin could be mass administered to fight malaria. In other words, the drug makes the human blood lethal for the Anopheles gambiae and therefore reduces the number of vectors in an area. Still, more safety data is required from a larger population. Here, an according fully automated DBS method has been developed and validated. In addition, a comparison study to plasma samples, stability and hematocrit impact has been studied [5]. In forensic toxicology, it was shown that Dried Matrix Spot (DMS) is a suitable technology for large scale DBS applications. A fully automated method, where either 1200 drugs can be screened from a DBS and a quantitative follow up method focusing on the 28 most abundant drugs of abuse, was developed. This was documented within two publications, where the first publication was a proof of concept study to determine if the detection limits can be reached using the DBS-LC-MS/MS methodology [6]. The second, follow up publication was a specific method development and implementation. More than 1200 illicit drugs can be screened from a single DBS or Dried Urine Spots (DUS) within 20 minutes per sample using a Forensic Toxicology Database. The criteria of bio-analytical method validation guidelines were fulfilled, and the method was transferred into a routine laboratory successfully [7]. Furthermore, the discovery of phosphatidylethanol (PEth) as direct alcohol marker prolonged the window of detection for alcohol consumption to several weeks. PEth proved to be instable during storage of liquid blood samples. By using DBS sampling, this biomarker can be stabilized due to the inactivation of enzymatic activity. Also, for PEth, a fully automated DBS-LC-MS/MS was established for the determination of the two most abundant PEth homologs in a range from 20–1500 ng/mL. Automated DBS card handling and online solid phase extraction LC-MS/MS permits baseline separation and detection of PEth 16:0/18:1 and PEth 16:0/18:2 within 7 minutes per sample [8]. The methods from the various fields of application were presented at several conferences as oral presentations and posters, showing their interest from the scientific blood analysis community.<br /

Immobilized-Enzyme Reactors Integrated into Analytical Platforms: Recent Advances and Challenges

Immobilized-enzyme reactors (IMERs) are flow-through devices containing enzymes that are physically confined or localized with retention of their catalytic activities. IMERs can be used repeatedly and continuously and have been applied for (bio)polymer degradation, proteomics, biomarker discovery, inhibitor screening, and detection. Online integration of IMERs with analytical instrumentation, such as high-performance liquid chromatography (HPLC) systems, reduces the time needed for multi-step workflows, reduces the need for sample handling, and enables automation. However, online integration can also be challenging, as reaching its full potential requires complex instrumental setups and experienced users. This review aims to provide an assessment of recent advances and challenges in online IMER-based (analytical) LC platforms, covering publications from 2014-2021. A critical discussion of challenges often encountered in IMER fabrication, sample preparation, integration into the analytical workflow, long-term usage, and of potential ways to overcome these is provided. Finally, the obstacles preventing the proliferation of IMERs as efficient tools for high-throughput pharmacological, industrial, and biological studies are discussed

Mol. Cell. Proteomics

Chemical cross-linking in combination with mass spectrometric analysis offers the potential to obtain low-resolution structural information from proteins and protein complexes. Identification of peptides connected by a cross-link provides direct evidence for the physical interaction of amino acid side chains, information that can be used for computational modeling purposes. Despite impressive advances that were made in recent years, the number of experimentally observed cross-links still falls below the number of possible contacts of cross-linkable side chains within the span of the cross-linker. Here, we propose two complementary experimental strategies to expand cross-linking data sets. First, enrichment of cross-linked peptides by size exclusion chromatography selects cross-linked peptides based on their higher molecular mass, thereby depleting the majority of unmodified peptides present in proteolytic digests of cross-linked samples. Second, we demonstrate that the use of proteases in addition to trypsin, such as Asp-N, can additionally boost the number of observable cross-linking sites. The benefits of both SEC enrichment and multiprotease digests are demonstrated on a set of model proteins and the improved workflow is applied to the characterization of the 20S proteasome from rabbit and Schizosaccharomyces pombe