Synthesis of 4-Deoxy-4-Fluoro-d-Sedoheptulose: A Promising New Sugar to Apply the Principle of Metabolic Trapping.

Fluorinated carbohydrates are important tools for understanding the deregulation of metabolic fluxes and pathways. Fluorinating specific positions within the sugar scaffold can lead to enhanced metabolic stability and subsequent metabolic trapping in cells. This principle has, however, never been applied to study the metabolism of the rare sugars of the pentose phosphate pathway (PPP). In this study, two fluorinated derivatives of d-sedoheptulose were designed and synthesized: 4-deoxy-4-fluoro-d-sedoheptulose (4DFS) and 3-deoxy-3-fluoro-d-sedoheptulose (3DFS). Both sugars are taken up by human fibroblasts but only 4DFS is phosphorylated. Fluorination of d-sedoheptulose at C-4 effectively halts the enzymatic degradation by transaldolase and transketolase. 4DFS thus has a high potential as a new PPP imaging probe based on the principle of metabolic trapping. Therefore, the synthesis of potential radiolabeling precursors for 4DFS for future radiofluorinations with fluorine-18 is presented.

Scrutinizing different ionization responses of polar lipids in a reversed-phase gradient by implementing a counter-gradient.

Lipidomics studies strive for a comprehensive identification and quantification of lipids. While reversed phase (RP) liquid chromatography (LC) coupled to high resolution mass spectrometry (MS) offers unrivalled selectivity and thus is the preferred method for lipid identification, accurate lipid quantification remains challenging. The widely adopted one-point lipid class specific quantification (one internal standard per lipid class) suffers from the fact that ionization of internal standard and target lipid occurs under different solvent composition as a consequence of chromatographic separation. To address this issue, we established a dual flow injection and chromatography setup that allows to control solvent conditions during ionization enabling isocratic ionization while running a RP gradient through the use of a counter-gradient. Using this dual LC pump platform, we investigated the impact of solvent conditions within a RP gradient on ionization response and arising quantification biases. Our results confirmed that changing solvent composition significantly influences ionization response. Quantification of human plasma (SRM 1950) lipids under gradient and isocratic ionization conditions further confirmed these findings as significant differences between the two conditions were found for the majority of lipids. While the quantity of sphingomyelins with >40 C atoms was consistently overestimated under gradient ionization, isocratic ionization improved their recovery compared to consensus values. However, the limitation of consensus values was demonstrated as overall only small changes in z-score were observed because of high uncertainties of the consensus values. Furthermore, we observed a trueness bias between gradient and isocratic ionization when quantifying a panel of lipid species standards which is highly dependent on lipid class and ionization mode. Uncertainty calculations under consideration of the trueness bias as RP gradient uncertainty revealed that especially ceramides with >40 C atoms had a high bias leading to total combined uncertainties of up to 54%. The assumption of isocratic ionization significantly decreases total measurement uncertainty and highlights the importance of studying the trueness bias introduced by a RP gradient to reduce quantification uncertainty.

Evaluation of Sustainable Recycled Products to Increase the Production of Nutraceutical and Antibacterial Molecules in Basil Plants by a Combined Metabolomic Approach.

BACKGROUND: An important goal of modern medicine is the development of products deriving from natural sources to improve environmental sustainability. In this study, humic substances (HS) and compost teas (CTs) extracted from artichoke (ART) and coffee grounds (COF) as recycled biomasses were employed on Ocimum basilicum plants to optimize the yield of specific metabolites with nutraceutical and antibacterial features by applying sustainable strategies. METHODS: The molecular characteristics of compost derivates were elucidated by Nuclear Magnetic Resonance spectroscopy to investigate the structure-activity relationship between organic extracts and their bioactive potential. Additionally, combined untargeted and targeted metabolomics workflows were applied to plants treated with different concentrations of compost extracts. RESULTS: The substances HS-ART and CT-COF improved both antioxidant activity (TEAC values between 39 and 55 µmol g-1) and the antimicrobial efficacy (MIC value between 3.7 and 1.3 µg mL-1) of basil metabolites. The metabolomic approach identified about 149 metabolites related to the applied treatments. Targeted metabolite quantification further highlighted the eliciting effect of HS-ART and CT-COF on the synthesis of aromatic amino acids and phenolic compounds for nutraceutical application. CONCLUSIONS: The combination of molecular characterization, biological assays, and an advanced metabolomic approach, provided innovative insight into the valorization of recycled biomass to increase the availability of natural compounds employed in the medical field.

Decoding Distinct Ganglioside Patterns of Native and Differentiated Mesenchymal Stem Cells by a Novel Glycolipidomics Profiling Strategy.

Gangliosides are an indispensable glycolipid class concentrated on cell surfaces with a critical role in stem cell differentiation. Nonetheless, owing to the lack of suitable methods for scalable analysis covering the full scope of ganglioside molecular diversity, their mechanistic properties in signaling and differentiation remain undiscovered to a large extent. This work introduces a sensitive and comprehensive ganglioside assay based on liquid chromatography, high-resolution mass spectrometry, and multistage fragmentation. Complemented by an open-source data evaluation workflow, we provide automated in-depth lipid species-level and molecular species-level annotation based on decision rule sets for all major ganglioside classes. Compared to conventional state-of-the-art methods, the presented ganglioside assay offers (1) increased sensitivity, (2) superior structural elucidation, and (3) the possibility to detect novel ganglioside species. A major reason for the highly improved sensitivity is the optimized spectral readout based on the unique capability of two parallelizable mass analyzers for multistage fragmentation. We demonstrated the high-throughput universal capability of our novel analytical strategy by identifying 254 ganglioside species. As a proof of concept, 137 unique gangliosides were annotated in native and differentiated human mesenchymal stem cells including 78 potential cell-state-specific markers and 38 previously unreported gangliosides. A general increase of the ganglioside numbers upon differentiation was observed as well as cell-state-specific clustering based on the ganglioside species patterns. The combination of the developed glycolipidomics assay with the extended automated annotation tool enables comprehensive in-depth ganglioside characterization as shown on biological samples of interest. Our results suggest ganglioside patterns as a promising quality control tool for stem cells and their differentiation products. Additionally, we believe that our analytical workflow paves the way for probing glycolipid-based biochemical processes shedding light on the enigmatic processes of gangliosides and glycolipids in general.

Integrated Exposomics/Metabolomics for Rapid Exposure and Effect Analyses.

The totality of environmental exposures and lifestyle factors, commonly referred to as the exposome, is poorly understood. Measuring the myriad of chemicals that humans are exposed to is immensely challenging, and identifying disrupted metabolic pathways is even more complex. Here, we present a novel technological approach for the comprehensive, rapid, and integrated analysis of the endogenous human metabolome and the chemical exposome. By combining reverse-phase and hydrophilic interaction liquid chromatography (HILIC) and fast polarity-switching, molecules with highly diverse chemical structures can be analyzed in 15 min with a single analytical run as both column's effluents are combined before analysis. Standard reference materials and authentic standards were evaluated to critically benchmark performance. Highly sensitive median limits of detection (LODs) with 0.04 µM for >140 quantitatively assessed endogenous metabolites and 0.08 ng/mL for the >100 model xenobiotics and human estrogens in solvent were obtained. In matrix, the median LOD values were higher with 0.7 ng/mL (urine) and 0.5 ng/mL (plasma) for exogenous chemicals. To prove the dual-column approach's applicability, real-life urine samples from sub-Saharan Africa (high-exposure scenario) and Europe (low-exposure scenario) were assessed in a targeted and nontargeted manner. Our liquid chromatography high-resolution mass spectrometry (LC-HRMS) approach demonstrates the feasibility of quantitatively and simultaneously assessing the endogenous metabolome and the chemical exposome for the high-throughput measurement of environmental drivers of diseases.

The Anticancer Ruthenium Compound BOLD-100 Targets Glycolysis and Generates a Metabolic Vulnerability towards Glucose Deprivation.

Cellular energy metabolism is reprogrammed in cancer to fuel proliferation. In oncological therapy, treatment resistance remains an obstacle and is frequently linked to metabolic perturbations. Identifying metabolic changes as vulnerabilities opens up novel approaches for the prevention or targeting of acquired therapy resistance. Insights into metabolic alterations underlying ruthenium-based chemotherapy resistance remain widely elusive. In this study, colon cancer HCT116 and pancreatic cancer Capan-1 cells were selected for resistance against the clinically evaluated ruthenium complex sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (BOLD-100). Gene expression profiling identified transcriptional deregulation of carbohydrate metabolism as a response to BOLD-100 and in resistance against the drug. Mechanistically, acquired BOLD-100 resistance is linked to elevated glucose uptake and an increased lysosomal compartment, based on a defect in downstream autophagy execution. Congruently, metabolomics suggested stronger glycolytic activity, in agreement with the distinct hypersensitivity of BOLD-100-resistant cells to 2-deoxy-d-glucose (2-DG). In resistant cells, 2-DG induced stronger metabolic perturbations associated with ER stress induction and cytoplasmic lysosome deregulation. The combination with 2-DG enhanced BOLD-100 activity against HCT116 and Capan-1 cells and reverted acquired BOLD-100 resistance by synergistic cell death induction and autophagy disturbance. This newly identified enhanced glycolytic activity as a metabolic vulnerability in BOLD-100 resistance suggests the targeting of glycolysis as a promising strategy to support BOLD-100 anticancer activity.

Achieving Absolute Molar Lipid Concentrations: A Phospholipidomics Cross-Validation Study.

Standardization is essential in lipidomics and part of a huge community effort. However, with the still ongoing lack of reference materials, benchmarking quantification is hampered. Here, we propose traceable lipid class quantification as an important layer for the validation of quantitative lipidomics workflows. 31P nuclear magnetic resonance (NMR) and inductively coupled plasma (ICP)-mass spectrometry (MS) can use certified species-unspecific standards to validate shotgun or liquid chromatography (LC)-MS-based lipidomics approaches. We further introduce a novel lipid class quantification strategy based on lipid class separation and mass spectrometry using an all ion fragmentation (AIF) approach. Class-specific fragments, measured over a mass range typical for the lipid classes, are integrated to assess the lipid class concentration. The concept proved particularly interesting as low absolute limits of detection in the fmol range were achieved and LC-MS platforms are widely used in the field of lipidomics, while the accessibility of NMR and ICP-MS is limited. Using completely independent calibration strategies, the introduced validation scheme comprised the quantitative assessment of the complete phospholipid sub-ome, next to the individual lipid classes. Komagataella phaffii served as a prime example, showcasing mass balances and supporting the value of benchmarks for quantification at the lipid species level.

Structure-Activity Relationships of Triple-Action Platinum(IV) Prodrugs with Albumin-Binding Properties and Immunomodulating Ligands.

Chemotherapy with platinum complexes is essential for clinical anticancer therapy. However, due to side effects and drug resistance, further drug improvement is urgently needed. Herein, we report on triple-action platinum(IV) prodrugs, which, in addition to tumor targeting via maleimide-mediated albumin binding, release the immunomodulatory ligand 1-methyl-d-tryptophan (1-MDT). Unexpectedly, structure-activity relationship analysis showed that the mode of 1-MDT conjugation distinctly impacts the reducibility and thus activation of the prodrugs. This in turn affected ligand release, pharmacokinetic properties, efficiency of immunomodulation, and the anticancer activity in vitro and in a mouse model in vivo. Moreover, we could demonstrate that the design of albumin-targeted multi-modal prodrugs using platinum(IV) is a promising strategy to enhance the cellular uptake of bioactive ligands with low cell permeability (1-MDT) and to improve their selective delivery into the malignant tissue. This will allow tumor-specific anticancer therapy supported by a favorably tuned immune microenvironment.

Benchmarking Non-Targeted Metabolomics Using Yeast-Derived Libraries.

Non-targeted analysis by high-resolution mass spectrometry (HRMS) is an essential discovery tool in metabolomics. To date, standardization and validation remain a challenge. Community-wide accepted cost-effective benchmark materials are lacking. In this work, we propose yeast (Pichia pastoris) extracts derived from fully controlled fermentations for this purpose. We established an open-source metabolite library of >200 identified metabolites based on compound identification by accurate mass, matching retention times, and MS/MS, as well as a comprehensive literature search. The library includes metabolites from the classes of (1) organic acids and derivatives (2) nucleosides, nucleotides, and analogs, (3) lipids and lipid-like molecules, (4) organic oxygen compounds, (5) organoheterocyclic compounds, (6) organic nitrogen compounds, and (7) benzoids at expected concentrations ranges of sub-nM to µM. As yeast is a eukaryotic organism, key regulatory elements are highly conserved between yeast and all annotated metabolites were also reported in the human metabolome database (HMDB). Orthogonal state-of-the-art reversed-phase (RP-) and hydrophilic interaction chromatography mass spectrometry (HILIC-MS) non-targeted analysis and authentic standards revealed that 104 out of the 206 confirmed metabolites were reproducibly recovered and stable over the course of three years when stored at -80 °C. Overall, 67 out of these 104 metabolites were identified with comparably stable areas over all three yeast fermentation and are the ideal starting point for benchmarking experiments. The provided yeast benchmark material enabled not only to test for the chemical space and coverage upon method implementation and developments but also allowed in-house routines for instrumental performance tests. Transferring the quality control strategy of proteomics workflows based on the number of protein identification in HeLa extracts, metabolite IDs in the yeast benchmarking material can be used as metabolomics quality control. Finally, the benchmark material opens new avenues for batch-to-batch corrections in large-scale non-targeted metabolomics studies.

A combined flow injection/reversed-phase chromatography-high-resolution mass spectrometry workflow for accurate absolute lipid quantification with 13C internal standards.

We propose a fully automated novel workflow for lipidomics based on flow injection, followed by liquid chromatography-high-resolution mass spectrometry (FI/LC-HRMS). The workflow combined in-depth characterization of the lipidome achieved via reversed-phase LC-HRMS with absolute quantification by using a large number of lipid species-specific and/or retention time (RT)-matched/class-specific calibrants. The lipidome of 13C-labelled yeast (LILY) provided a large panel of cost-effective internal standards (ISTDs) covering triacylglycerols (TG), steryl esters (SE), free fatty acids (FA), diacylglycerols (DG), sterols (ST), ceramides (Cer), hexosyl ceramides (HexCer), phosphatidylglycerols (PG), phosphatidylethanolamines (PE), phosphatidic acids (PA), cardiolipins (CL), phosphatidylinositols (PI), phosphatidylserines (PS), phosphatidylcholines (PC), lysophosphatidylcholines (LPC) and lysophosphatidylethanolamines (LPE). The workflow in combination with the LILY lipid panel enables simultaneous quantification via (1) external multi-point calibration with internal standardization and (2) internal one-point calibration with LILY as a surrogate ISTD, increasing the coverage while keeping the accuracy and throughput high. Extensive measures on quality control allowed us to rank the calibration strategies and to automatically select the calibration strategy of the highest metrological order for the respective lipid species. Overall, the workflow enabled a streamlined analysis, with a limit of detection in the low femtomolar range, and provided validation tools together with absolute concentration values for >350 lipids in human plasma on a species level. Based on the selected standard panel, lipids from 7 classes (LPC, LPE, PC, PE, PI, DG, TG) passed stringent quality filters, which included QC accuracy, a precision and recovery bias of <30% and concentrations within the 99% confidence interval of the international laboratory comparison of SRM 1950, NIST, USA. The quantitative values are independent of common deuterated or non-endogenous ISTDs, thus offering cross-validation of different lipid methods and further standardizing lipidomics.

Chasing the Major Sphingolipids on Earth: Automated Annotation of Plant Glycosyl Inositol Phospho Ceramides by Glycolipidomics.

Glycosyl inositol phospho ceramides (GIPCs) are the major sphingolipids on earth, as they account for a considerable fraction of the total lipids in plants and fungi, which in turn represent a large portion of the biomass on earth. Despite their obvious importance, GIPC analysis remains challenging due to the lack of commercial standards and automated annotation software. In this work, we introduce a novel GIPC glycolipidomics workflow based on reversed-phase ultra-high pressure liquid chromatography coupled to high-resolution mass spectrometry. For the first time, automated GIPC assignment was performed using the open-source software Lipid Data Analyzer (LDA), based on platform-independent decision rules. Four different plant samples (salad, spinach, raspberry, and strawberry) were analyzed and the results revealed 64 GIPCs based on accurate mass, characteristic MS2 fragments and matching retention times. Relative quantification using lactosyl ceramide for internal standardization revealed GIPC t18:1/h24:0 as the most abundant species in all plants. Depending on the plant sample, GIPCs contained mainly amine, N-acetylamine or hydroxyl residues. Most GIPCs revealed a Hex-HexA-IPC core and contained a ceramide part with a trihydroxylated t18:0 or a t18:1 long chain base and hydroxylated fatty acid chains ranging from 16 to 26 carbon atoms in length (h16:0-h26:0). Interestingly, four GIPCs containing t18:2 were observed in the raspberry sample, which was not reported so far. The presented workflow supports the characterization of different plant samples by automatic GIPC assignment, potentially leading to the identification of new GIPCs. For the first time, automated high-throughput profiling of these complex glycolipids is possible by liquid chromatography-high-resolution tandem mass spectrometry and subsequent automated glycolipid annotation based on decision rules.

Preparative supercritical fluid chromatography for lipid class fractionation-a novel strategy in high-resolution mass spectrometry based lipidomics.

In this work, a lipidomics workflow based on offline semi-preparative lipid class-specific fractionation by supercritical fluid chromatography (SFC) followed by high-resolution mass spectrometry was introduced. The powerful SFC approach offered separation of a wide polarity range for lipids, enabled enrichment (up to 3 orders of magnitude) of lipids, selective fractionation of 14 lipid classes/subclasses, and increased dynamic range enabling in-depth characterization. A significantly increased coverage of low abundant lipids improving lipid identification by numbers and degree (species and molecular level) was obtained in Pichia pastoris when comparing high-resolution mass spectrometry based lipidomics with and without prior fractionation. Proof-of-principle experiments using a standard reference material (SRM 1950, NIST) for human plasma showed that the proposed strategy enabled quantitative lipidomics. Indeed, for 70 lipids, the consensus values available for this sample could be met. Thus, the novel workflow is ideally suited for lipid class-specific purification/isolation from milligram amounts of sample while not compromising on omics type of analysis (identification and quantification). Finally, compared with established fractionation/pre-concentration approaches, semi-preparative SFC is superior in terms of versatility, as it involved only volatile modifiers and salt additives facilitating any follow-up use such as qualitative or quantitate analysis or further purification down to the single lipid species level. Graphical Abstract.

Robust Analytical Methods for the Accurate Quantification of the Total Biomass Composition of Mammalian Cells.

Biomass composition is an important input for genome-scale metabolic models and has a big impact on their predictive capabilities. However, researchers often rely on generic data for biomass composition, e.g. collected from similar organisms. This leads to inaccurate predictions, because biomass composition varies between different cell lines, conditions, and growth phases. In this chapter we present protocols for the determination of the biomass composition of Chinese Hamster Ovary (CHO) cells. These methods can easily be adapted to other types of mammalian cells. The protocols include the quantification of cell dry mass and of the main biomass components, namely protein, lipid, DNA, RNA, and carbohydrates. Cell dry mass is determined gravimetrically by weighing a defined number of cells. Amino acid composition and protein content are measured by gas chromatography mass spectrometry. Lipids are quantified by shotgun mass spectrometry, which provides quantities for the different lipid classes and also the distribution of fatty acids. RNA is purified and then quantified spectrophotometrically. The methods for DNA and carbohydrates are simple fluorometric and colorimetric assays adapted to a 96-well plate format. To ensure quantitative results, internal standards or spike-in controls are used in all methods, e.g. to account for possible matrix effects or loss of material. Finally, the last section provides a guide on how to convert the measured data into biomass equations, which can then be integrated into a metabolic model.

Single Spheroid Metabolomics: Optimizing Sample Preparation of Three-Dimensional Multicellular Tumor Spheroids.

Tumor spheroids are important model systems due to the capability of capturing in vivo tumor complexity. In this work, the experimental design of metabolomics workflows using three-dimensional multicellular tumor spheroid (3D MTS) models is addressed. Non-scaffold based cultures of the HCT116 colon carcinoma cell line delivered highly reproducible MTSs with regard to size and other key parameters (such as protein content and fraction of viable cells) as a prerequisite. Carefully optimizing the multiple steps of sample preparation, the developed procedure enabled us to probe the metabolome of single MTSs (diameter range 790 ± 22 µm) in a highly repeatable manner at a considerable throughput. The final protocol consisted of rapid washing of the spheroids on the cultivation plate, followed by cold methanol extraction. 13C enriched internal standards, added upon extraction, were key to obtaining the excellent analytical figures of merit. Targeted metabolomics provided absolute concentrations with average biological repeatabilities of <20% probing MTSs individually. In a proof of principle study, MTSs were exposed to two metal-based anticancer drugs, oxaliplatin and the investigational anticancer drug KP1339 (sodium trans-[tetrachloridobis(1H-indazole)ruthenate(III)]), which exhibit distinctly different modes of action. This difference could be recapitulated in individual metabolic shifts observed from replicate single MTSs. Therefore, biological variation among single spheroids can be assessed using the presented analytical strategy, applicable for in-depth anticancer drug metabolite profiling.

The Power of LC-MS Based Multiomics: Exploring Adipogenic Differentiation of Human Mesenchymal Stem/Stromal Cells.

The molecular study of fat cell development in the human body is essential for our understanding of obesity and related diseases. Mesenchymal stem/stromal cells (MSC) are the ideal source to study fat formation as they are the progenitors of adipocytes. In this work, we used human MSCs, received from surgery waste, and differentiated them into fat adipocytes. The combination of several layers of information coming from lipidomics, metabolomics and proteomics enabled network analysis of the biochemical pathways in adipogenesis. Simultaneous analysis of metabolites, lipids, and proteins in cell culture is challenging due to the compound's chemical difference, so most studies involve separate analysis with unimolecular strategies. In this study, we employed a multimolecular approach using a two-phase extraction to monitor the crosstalk between lipid metabolism and protein-based signaling in a single sample (~105 cells). We developed an innovative analytical workflow including standardization with in-house produced 13C isotopically labeled compounds, hyphenated high-end mass spectrometry (high-resolution Orbitrap MS), and chromatography (HILIC, RP) for simultaneous untargeted screening and targeted quantification. Metabolite and lipid concentrations ranged over three to four orders of magnitude and were detected down to the low fmol (absolute on column) level. Biological validation and data interpretation of the multiomics workflow was performed based on proteomics network reconstruction, metabolic modelling (MetaboAnalyst 4.0), and pathway analysis (OmicsNet). Comparing MSCs and adipocytes, we observed significant regulation of different metabolites and lipids such as triglycerides, gangliosides, and carnitine with 113 fully reprogrammed pathways. The observed changes are in accordance with literature findings dealing with adipogenic differentiation of MSC. These results are a proof of principle for the power of multimolecular extraction combined with orthogonal LC-MS assays and network construction. Considering the analytical and biological validation performed in this study, we conclude that the proposed multiomics workflow is ideally suited for comprehensive follow-up studies on adipogenesis and is fit for purpose for different applications with a high potential to understand the complex pathophysiology of diseases.

Proposing a validation scheme for 13C metabolite tracer studies in high-resolution mass spectrometry.

13C metabolite tracer and metabolic flux analyses require upfront experimental planning and validation tools. Here, we present a validation scheme including a comparison of different LC methods that allow for customization of analytical strategies for tracer studies with regard to the targeted metabolites. As the measurement of significant changes in labeling patterns depends on the spectral accuracy, we investigate this aspect comprehensively for high-resolution orbitrap mass spectrometry combined with reversed-phase chromatography, hydrophilic interaction liquid chromatography, or anion-exchange chromatography. Moreover, we propose a quality control protocol based on (1) a metabolite containing selenium to assess the instrument performance and on (2) in vivo synthesized isotopically enriched Pichia pastoris to validate the accuracy of carbon isotopologue distributions (CIDs), in this case considering each isotopologue of a targeted metabolite panel. Finally, validation involved a thorough assessment of procedural blanks and matrix interferences. We compared the analytical figures of merit regarding CID determination for over 40 metabolites between the three methods. Excellent precisions of less than 1% and trueness bias as small as 0.01-1% were found for the majority of compounds, whereas the CID determination of a small fraction was affected by contaminants. For most compounds, changes of labeling pattern as low as 1% could be measured. Graphical abstract.

Merging metabolomics and lipidomics into one analytical run.

A novel integrated metabolomics/lipidomics workflow is introduced enabling high coverage of polar metabolites and non-polar lipids within one analytical run. Dual HILIC and RP chromatography were combined to high-resolution mass spectrometry. As a major advantage, only one data file per sample was obtained by fully automated simultaneous analysis of two extracts per sample. Hence, the unprecedented high coverage without compromise on analytical throughput was not only obtained by the orthogonality of the chromatographic separations, but also by the implementation of dedicated sample preparation procedures resulting in optimum extraction efficiency for both sub-omes. Thus, the method addressed completely hydrophilic sugars and organic acids next to water-insoluble triglycerides. As for the timing of the dual chromatography setup, HILIC and RP separation were performed consecutively. However, re-equilibration of the HILIC column during elution of RP compounds and vice versa reduced the overall analysis time by one third to 32 min. Application to the Standard Reference Material SRM 1950 - Metabolites in Frozen Human Plasma resulted in >100 metabolite and >380 lipid identifications based on accurate mass implementing fast polarity switching and acquiring data dependent MS2 spectra with the use of automated exclusion lists. Targeted quantification based on external calibrations and 13C labeled yeast internal standards was successfully accomplished for 59 metabolites. Moreover, the potential for lipid quantification was shown integrating non-endogenous lipids as internal standards. In human plasma, concentrations ranging over 4 orders of magnitude (low nM to high µM) were assessed.