bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2019‒11‒17
twenty papers selected by
Sofia Costa
Cold Spring Harbor Laboratory

  1. J Pharm Biomed Anal. 2019 Nov 05. pii: S0731-7085(19)32054-0. [Epub ahead of print] 112968
      Linezolid (LZD) is a widely used antimicrobial that is active against a broad range of disease-causing bacteria. Myelosuppression is major treatment-limiting toxicity of LZD that occurs more frequently in patients with renal insufficiency. Quantification of LZD and its two primary metabolites (PNU-142300 and PNU-142586), which undergo significant renal elimination, may support design of improved dosing strategies to mitigate the risk of myelosuppression. In this study, we established the first liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of LZD and its main metabolites in human serum. Proteins in serum samples were precipitated with acetonitrile containing a deuterated internal standard. Chromatographic separation of analytes was performed with Waters X-bridge column (C18, 150 × 4.6 mm, 3.5 μm) at 25 °C and subjected to mass analysis using positive electro-spray ionization. The mobile phase A was water with 0.1% formic acid, and mobile phase B was acetonitrile with 0.1% formic acid at a flow rate of 0.6 mL/min, within a 15 min run time. Standard curves were linear and correlation coefficients (r2) were ≥0.99 for concentration ranges of 0.1-50 μg/mL for LZD and PNU-142300, and 0.1-25 μg/mL for PNU-142586. The inter- and intra-assay precisions were <15% for all analytes in quality control samples, and the accuracies ranged from 97 to 112%. Extraction recoveries ranged from 78 to 103% for all analytes, and there was no significant matrix effect. Samples from 10 patients (5 with renal impairment) were assayed. Mean (SD) LZD, PNU-142300 and PNU-142586 trough concentrations were 19.4(6.8), 11.6(6.8), 25.7(16.4) μg/mL, respectively, in patients with renal impairment. These values were 2.5-, 5.8-, and 6.8-fold higher for LZD, PNU-142300 and PNU-142586, respectively compared to patients without renal impairment. The method was effectively applied in the determination of LZD and its main metabolites in human serum.
    Keywords:  Infectious diseases; Linezolid; Myelosuppression; Oxazolidinone; PNU-142300; PNU-142586
  2. Front Plant Sci. 2019 ;10 1366
      Untargeted approaches and thus biological interpretation of metabolomics results are still hampered by the reliable assignment of the global metabolome as well as classification and (putative) identification of metabolites. In this work we present an liquid chromatography-mass spectrometry (LC-MS)-based stable isotope assisted approach that combines global metabolome and tracer based isotope labeling for improved characterization of (unknown) metabolites and their classification into tracer derived submetabolomes. To this end, wheat plants were cultivated in a customized growth chamber, which was kept at 400 ± 50 ppm 13CO2 to produce highly enriched uniformly 13C-labeled sample material. Additionally, native plants were grown in the greenhouse and treated with either 13C9-labeled phenylalanine (Phe) or 13C11-labeled tryptophan (Trp) to study their metabolism and biochemical pathways. After sample preparation, liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis and automated data evaluation, the results of the global metabolome- and tracer-labeling approaches were combined. A total of 1,729 plant metabolites were detected out of which 122 respective 58 metabolites account for the Phe- and Trp-derived submetabolomes. Besides m/z and retention time, also the total number of carbon atoms as well as those of the incorporated tracer moieties were obtained for the detected metabolite ions. With this information at hand characterization of unknown compounds was improved as the additional knowledge from the tracer approaches considerably reduced the number of plausible sum formulas and structures of the detected metabolites. Finally, the number of putative structure formulas was further reduced by isotope-assisted annotation tandem mass spectrometry (MS/MS) derived product ion spectra of the detected metabolites. A major innovation of this paper is the classification of the metabolites into submetabolomes which turned out to be valuable information for effective filtering of database hits based on characteristic structural subparts. This allows the generation of a final list of true plant metabolites, which can be characterized at different levels of specificity.
    Keywords:  LC-HRMS; Triticum aestivum; phenylalanine; tryptophan; wheat
  3. Methods Mol Biol. 2020 ;2084 55-78
      Mass spectrometry-based metabolomics is being increasingly applied to a number of applications, including the fields of clinical, industrial, plant, and nutritional science. Several improvements have advanced the field considerably over the past decade, including ultra-high performance liquid chromatography (uHPLC), column chemistries, instruments, software, and molecular databases. However, challenges remain, including how to separate small molecules that are part of highly complex samples; this can be accomplished using chromatographic techniques or through improved resolution in the gas phase. Ion mobility-mass spectrometry (IM-MS) provides an extra dimension of gas phase separation that can result in improvements to both quantitation and compound identification. Here we describe a typical drift tube IM-MS metabolomics workflow, which includes the following steps: (1) Data acquisition, (2) Data preprocessing, (3) Molecular feature finding, and (4) Differential analysis and Molecular annotation. Overall, these methods can help investigators from a variety of scientific fields use IM-MS metabolomics as part of their own workflow.
    Keywords:  Acquisition parameters; Differential analysis; Drift tube ion mobility; Four-dimensional feature finding; Mass spectrometry; Metabolomics
  4. Plant Methods. 2019 ;15 127
      Background: Sugar phosphates are important intermediates of central carbon metabolism in biological systems, with roles in glycolysis, the pentose-phosphate pathway, tricarboxylic acid (TCA) cycle, and many other biosynthesis pathways. Understanding central carbon metabolism requires a simple, robust and comprehensive analytical method. However, sugar phosphates are notoriously difficult to analyze by traditional reversed phase liquid chromatography.Results: Here, we show a two-step derivatization of sugar phosphates by methoxylamine and propionic acid anhydride after chloroform/methanol (3:7) extraction from Populus leaf and developing wood that improves separation, identification and quantification of sugar phosphates by ultra high performance liquid chromatography-electrospray ionization-mass spectrometry (UHPLC-ESI-MS). Standard curves of authentic sugar phosphates were generated for concentrations from pg to ng/μl with a correlation coefficient R 2 > 0.99. The method showed high sensitivity and repeatability with relative standard deviation (RSD) < 20% based on repeated extraction, derivatization and detection. The analytical accuracy for Populus leaf extracts, determined by a two-level spiking approach of selected metabolites, was 79-107%.
    Conclusion: The results show the reliability of combined reversed phase liquid chromatography-tandem mass spectrometry for sugar phosphate analysis and demonstrate the presence of two unknown sugar phosphates in Populus extracts.
    Keywords:  Extraction; Q-TOF; QqQ-MS; Sugar phosphates; Two-step derivatization; UHPLC–ESI–MS
  5. BMC Res Notes. 2019 Nov 14. 12(1): 743
      OBJECTIVE: Silibinin is an antioxidant agent and is shown to have anticancer effects in different cancers including lung, breast, colorectal, liver, prostate, and kidney. There are challenges in the clinical use of silibinin. The main limitation is low solubility, poor oral absorption, and extensive hepatic metabolism. We aim to develop a High-Performance Liquid Chromatography (HPLC) sensitive method for quantification of silibinin in aqueous samples to quantify its concentration in new formulations. A reverse-phase high-performance liquid chromatography (RP-HPLC) composed of C18 column as stationary phase and the mixture of methanol (90%) and water (10%) as mobile phase. The developed method was validated based on the established guidelines.RESULTS: The retention time for silibinin was seen in 2.97 min after injection. The calibration curve was drawn and the established method demonstrated a linear ranged from 10 to 100 µg/ml, with a correlation coefficient of 0.996. The sensitivity of the developed method was 10 µg/ml. The accuracy calculated in the range of 88-105.9% and the precision (as relative standard deviation) was between 2.7 and 10.9%. These results demonstrate that the developed method can be a fast and accurate method for quantification of silibinin in aqueous samples.
    Keywords:  Analysis method; HPLC; Method validation; Precision and accuracy; Silibinin
  6. Methods Mol Biol. 2020 ;2084 119-132
      Lipidomics is a rapidly growing field that enables the characterization of the entire lipidome in cells, tissues, or an organism. Changes in lipid metabolism and homeostasis caused by different disease states or drug treatments can be probed by lipidomics experiments, which can aid our understanding of normal physiology and disease pathology at the molecular level. While current technologies using liquid chromatography coupled with high-resolution mass spectrometry have greatly increased coverage of the lipidome, there are still limitations in resolving the large number of lipid species with similar masses in a narrow mass window. We recently reported that two orthogonal separation techniques, hydrophilic interaction liquid chromatography (HILIC) and ion mobility (IM), enhance the resolution of lipid species based on headgroup polarity and gas-phase size and shape, respectively, of various classes of glycerolipids, glycolipids, phospholipids, and sphingolipids. Here we describe the application of our HILIC-IM-MS lipidomics protocol to the analysis of lipid extracts derived from either tissues or cells, to identify significant changes in the lipidome in response to an internal or external stimulus, such as exposure to environmental chemicals.
    Keywords:  Collision cross section; Hydrophilic interaction liquid chromatography; Ion mobility; Lipidomics; Mass spectrometry
  7. Metabolites. 2019 Oct 26. pii: E251. [Epub ahead of print]9(11):
      Accurate metabolite identification remains one of the primary challenges in a metabolomics study. A reliable chemical spectral library increases the confidence in annotation, and the availability of raw and annotated data in public databases facilitates the transfer of Liquid chromatography coupled to mass spectrometry (LC-MS) methods across laboratories. Here, we illustrate how the combination of MS2 spectra, accurate mass, and retention time can improve the confidence of annotation and provide techniques to create a reliable library for all ion fragmentation (AIF) data with a focus on the characterization of the retention time. The resulting spectral library incorporates information on adducts and in-source fragmentation in AIF data, while noise peaks are effectively minimized through multiple deconvolution processes. We also report the development of the Mass Spectral LIbrary MAnager (MS-LIMA) tool to accelerate library sharing and transfer across laboratories. This library construction strategy improves the confidence in annotation for AIF data in LC-MS-based metabolomics and will facilitate the sharing of retention time and mass spectral data in the metabolomics community.
    Keywords:  LC–MS; all ion fragmentation; chemical library; mass spectral deconvolution; metabolomics
  8. Anal Chem. 2019 Nov 13.
      Knowledge of the chemical identity of metabolite molecules is critical for the understanding of the complex biological systems they belong to. Since metabolite identities and their concentrations are often directly linked to the phenotype, such information can be used to map biochemical pathways and understand their role in health and disease. A very large number of metabolites however are still unknown, i.e. their spectroscopic signatures do not match those in existing databases, suggesting unknown molecule identification is both imperative and challenging. Although metabolites are structurally highly diverse, the majority shares a rather limited number of structural motifs, which are defined by sets of 1H, 13C chemical shifts of the same spin system. This allows one to characterize unknown metabolites by a divide-and-conquer strategy that identifies their structural motifs first. Here we present the structural motif-based approach "SUMMIT Motif" for the de novo identification of unknown molecular structures in complex mixtures, without the need for extensive purification, using NMR in tandem with two newly curated NMR molecular structural motif metabolomics databases (MSMMDB). In identifying structural motif(s), first the 1H and 13C chemical shifts of all the individual spin systems are extracted from 2D and 3D NMR spectra of the complex mixture. Next, the molecular structural motifs are identified by querying these chemical shifts against the new MSMMDBs. One database, COLMAR MSMMDB, was derived from experimental NMR chemical shifts of known metabolites taken from the COLMAR metabolomics database, while the other MSMMDB, pNMR MSMMDB, is based on empirically predicted chemical shifts of metabolites of several existing large metabolomics databases. For molecules consisting of multiple spin systems, spin systems are connected via a long-range scalar J-coupling NMR experiment. When this motif-based identification method was applied to the hydrophilic extract of mouse bile fluid, two unknown metabolites could be successfully identified. This approach is both accurate and efficient for the identification of unknown metabolites and hence contribute to the understanding of human health and disease.
  9. J Lipid Res. 2019 Nov 15. pii: jlr.D119000393. [Epub ahead of print]
      Quantitative MS of human plasma lipids is a promising technology for translation into clinical applications. Current MS-based lipidomic methods rely on either direct infusion or chromatographic lipid separation methods (including reversed-phase and hydrophilic interaction liquid chromatography). However, the use of lipid markers in laboratory medicine is limited by the lack of reference values, largely because of considerable differences in the concentrations measured by different laboratories worldwide. These inconsistencies can be explained by the use of different sample preparation protocols, method-specific calibration procedures, and other experimental and data-reporting parameters, even when using identical starting materials. Here, we systematically investigated the roles of some of these variables in multiple approaches to lipid analysis of plasma samples from healthy adults, by  considering (1) different sample introduction methods (separation vs. direct infusion methods), (2) different MS instruments and (3) between-laboratory differences in comparable analytical platforms. Each of these experimental variables resulted in different quantitative results, even with the inclusion of isotope-labelled internal standards for individual lipid classes. We demonstrated that appropriate normalization to commonly available reference samples (i.e., "shared references") can largely correct for these systematic, method-specific quantitative biases. Thus, to harmonize data in the field of lipidomics, in-house, long-term references should be complemented by a commonly available shared reference sample, such as NIST SRM 1950, in the case of human plasma.
    Keywords:  Harmonization; Lipidomics; Lipids; Liquid chromatography; Mass spectrometry; NIST SRM 1950; Phospholipids; Plasma; Quantitation; Sphingolipids
  10. Methods Mol Biol. 2020 ;2084 79-94
      This chapter describes the developments in drift-tube ion mobility-mass spectrometry (DTIM-MS) that have driven application development in 'omics analyses. Harnessing the additional, orthogonal separation that DTIM provides increased confidence in compound identifications as the mass spectral complexity can be reduced and mobility-derived parameters (most prominently the collision cross section, CCS) used to support identity confirmation goals for a variety of 'omics application areas. Presented within this contribution is a methodology for improving the transmission and maintaining accurate determination of drift time-derived CCS (DTCCS) for low molecular weight compounds for a typical nontargeted 'omics (metabolomics) workflow using liquid chromatography in combination with DTIM-MS.
    Keywords:  CCS databases; Collision cross section; Drift-tube ion mobility-mass spectrometry; Ion mobility; Mass spectrometry; Metabolomics; Non-targeted analysis; Small molecules
  11. Methods Mol Biol. 2020 ;2084 269-282
      Untargeted lipidomics aims to comprehensively measure and characterize all lipid species in biological systems. Ion mobility-mass spectrometry (IM-MS) has showed a great potential for untargeted lipidomic analysis. Coupling with liquid chromatography and data-independent tandem MS techniques, acquired IM-MS data set contains four-dimensional information for lipid identification, including m/z of MS1 ion, retention time (RT), collision cross section (CCS), and MS/MS spectra. In this protocol, we introduced a data processing workflow using an integrative web server, namely, LipidIMMS Analyzer, to support accurate lipid identification. The protocol demonstrated the integration of all four dimensional information to achieve unambiguous identifications of lipids in complex biological samples.
    Keywords:  Collision cross section; Ion mobility-mass spectrometry; Lipid identification; LipidIMMS Analyzer; Untargeted lipidomics
  12. Front Chem. 2019 ;7 658
      DNA can be damaged through covalent modifications of the nucleobases by endogenous processes. These modifications, commonly referred to as DNA adducts, can persist and may lead to mutations, and ultimately to the initiation of cancer. A screening methodology for the majority of known endogenous DNA adducts would be a powerful tool for investigating the etiology of cancer and for the identification of individuals at high-risk to the detrimental effects of DNA damage. This idea led to the development of a DNA adductomic approach using high resolution data-dependent scanning, an extensive MS2 fragmentation inclusion list of known endogenous adducts, and neutral loss MS3 triggering to profile all DNA modifications. In this method, the detection of endogenous DNA adducts is performed by observation of their corresponding MS3 neutral loss triggered events and their relative quantitation using the corresponding full scan extracted ion chromatograms. The method's inclusion list consists of the majority of known endogenous DNA adducts, compiled, and reported here, as well as adducts specific to tobacco exposure included to compare the performance of the method with previously developed targeted approaches. The sensitivity of the method was maximized by reduction of extraneous background signal through the purification and minimization of the amount of commercially obtained enzymes used for the DNA hydrolysis. In addition, post-hydrolysis sample purification was performed using off-line HPLC fraction collection to eliminate the highly abundant unmodified bases, and to avoid introduction of plasticizers found in solid-phase extraction cartridges. Also, several instrument parameters were evaluated to optimize the ion signal intensities and fragmentation spectra quality. The method was tested on an animal model of lung carcinogenesis where A/J mice were exposed to the tobacco specific lung carcinogen 4-methylnitrosamino-1-3-pyridyl-1-butanone (NNK) with its effects enhanced by co-exposure to the pro-inflammatory agent lipopolysaccharide (LPS). Lung DNA were screened for endogenous DNA adducts known to result from oxidative stress and LPS-induced lipid peroxidation, as well as for adducts due to NNK exposure. The relative quantitation of the detected DNA adducts was performed using parallel reaction monitoring MS2 analysis, demonstrating a general workflow for analysis of endogenous DNA adducts.
    Keywords:  DNA adductomics; DNA damage; cancer; inflammation; lipid peroxidation; mass spectrometry; tobacco carcinogens
  13. Methods Mol Biol. 2020 ;2084 245-265
      Matrix-Assisted Laser Desorption Ionization (MALDI) and Desorption Electrospray Ionization (DESI) are two complementary ionization techniques that have transformed the field of biomolecular analysis, enabling the measurement of a wide range of biomolecules by mass spectrometry. These techniques have also been applied to imaging mass spectrometry where the spatial localization of molecules is determined. Coupling this with Ion Mobility Spectrometry (IM) allows an additional level of separation and specificity to be obtained. Here, we describe the coupling of the technologies and the practical advantages of these combinations, highlighting specific examples.
    Keywords:  DESI; Imaging MS; Ion mobility; MALDI; Mass spectrometry
  14. Methods Mol Biol. 2020 ;2084 103-117
      Ion mobility has become a valuable tool in mass spectrometry-based lipidomics workflows, thanks to its ability to separate ions in the gas phase based on their size and conformation. Over the last years, it was demonstrated that the comparative analysis of ion mobility data has the potential to highlight the presence of low abundance species in untargeted lipidomics. The present chapter illustrates the background of the topic and guides the reader from the sample preparation to the data analysis of an untargeted lipidomics experiment performed using ion mobility.
    Keywords:  High-resolution LC-MS/MS; Ion mobility-mass spectrometry; Lipidomics
  15. J Lipid Res. 2019 Nov 11. pii: jlr.D119000318. [Epub ahead of print]
      Lipid metabolism plays an important role in the regulation of cellular homeostasis. However, since it is difficult to measure the actual rates of synthesis and degradation of individual lipid species, lipid compositions are used often as a surrogate to evaluate lipid metabolism even though they provide only static snapshots of the lipodome. Here, we designed a simple method to determine the turnover rate of phospholipid and acylglycerol species based on the incorporation of 13C6-glucose combined with LC-MS/MS. We labeled adult Drosophila melanogaster with 13C6-glucose that incorporates into the entire lipidome, derived kinetic parameters from mass spectra, and studied effects of deletion of CG6718, the fly homologue of the calcium-independent phospholipase A2β, on lipid metabolism. Although 13C6-glucose gave rise to a complex pattern of 13C incorporation, we were able to identify discrete isotopomers in which 13C atoms were confined to the glycerol group. With these isotopomers, we calculated turnover rate constants, half-life times, and fluxes of the glycerol backbone of multiple lipid species. To perform these calculations, we estimated the fraction of labeled molecules in glycerol-3-phosphate, the lipid precursor, by mass isotopomer distribution analysis of the spectra of phosphatidylglycerol. When we applied this method to D. melanogaster, we found a range of lipid half-lives from 2 to 200 days, demonstrated tissue-specific fluxes of individual lipid species, and identified a novel function of CG6718 in triacylglycerol metabolism. This method provides fluxomics-type data with significant potential to improve the understanding of complex lipid regulation in a variety of research models.
    Keywords:  Genetics; Glycerolipids; Lipidomics; Mass spectrometry; Phospholipids/Metabolism
  16. J Steroid Biochem Mol Biol. 2019 Nov 08. pii: S0960-0760(19)30420-0. [Epub ahead of print] 105528
      Long-term studies investigating hormone-dependent cancers and reproductive health often require prolonged frozen storage of serum which assumes that the steroid molecules and measurements are stable over that time. Previous studies of reproducibility of circulating steroids have relied upon flawed historical rather than contemporaneous controls. We measured serum testosterone (T), dihydrotestosterone (DHT), estradiol (E2) and estrone (E1) in 150 randomly selected serum samples by liquid chromatography-mass spectrometry (LC-MS) from men 70 years or older (mean age 77 years) in the CHAMP study. The original measurements in 2009 were repeated 10 years later using the identical serum aliquot (having undergone 2-4 freeze-thaw cycles in the interim) in 2019 together with another never-thawed aliquot of the same serum sample. The results of all three sets of measurements were evaluated by Passing-Bablok regression and Bland-Altman difference analysis. Serum androgens (T, DHT) and estrogens (E2, E1) measured by LC-MS display excellent reproducibility when stored for 10 years at -80 C without thawing. Serum T and DHT displayed high level of reproducibility across all three sets of measurements. Multiple freeze-thaw cycles over those storage conditions do not significantly affect serum T, DHT and E1 concentrations but produce a modest increase (21%) in serum E2 measurements.
    Keywords:  dihydrotestosterone; estradiol; estrone; long-term storage; reproducibility; steroid mass spectrometry; testosterone
  17. Angew Chem Int Ed Engl. 2019 Nov 15.
      Current metabolomics approaches which utilise cellular metabolite extracts require high cell numbers. They are cell destructive and thus provide only instantaneous snapshots of metabolism. We introduce here an approach that enables the monitoring of cellular metabolism at significantly lower cell numbers by observing consumption/production of different metabolites over several kinetic data points of up to 48 hours. We establish our new methods on models of acute myeloid leukaemia. Our approach does not influence cellular viability or proliferation capacity, as we optimised the cellular matrix in comparison to other materials used in a variety of in-cell NMR experiments. The investigated cells can respond to any external stimuli or interferences arising from cellular signalling in an unbiased manner. We are able to monitor real-time metabolism of primary patient cells, which are extremely sensitive to external stress. Measurements are set up in an interleaved manner with short acquisition times (∼7 minutes per sample) which allows monitoring of up to 15 patient samples simultaneously. Further, we implemented our approach for performing tracer-based assays to monitor different tracers at the same time. Our approach will be important not only in the metabolomics fields, but also in individualized diagnostics where patients' samples can be profiled and later maintained for further tests.
    Keywords:  Real time NMR, metabolism, personalised medicine, tracer-based assay
  18. J Chromatogr A. 2019 Nov 01. pii: S0021-9673(19)31089-1. [Epub ahead of print] 460667
      This research focuses on retention mechanisms in a LC column with C18 stationary phase when novel eluent additives (HFIP, HFTB and TFE as well as NFTB and perfluoropinacol) are used. The retention factors between novel eluent additives and conventional ones like ammonium acetate and ammonium bicarbonate at different eluent pH values were compared. A simple set of drug-like molecules, widely spread over different logP values, containing protonated and deprotonated acids and bases was selected for this investigation. HFIP, HFTB, NFTB and PP demonstrated strong influence on basic polar analytes in basic medium. These additives drastically increased retention. A decrease in retention was observed for acidic analytes when novel eluent additives were used. Additionally, for the first time, the absolute pH (pHabs) scale was used for expressing the mobile phase pH.
    Keywords:  Eluent additives; HFIP; HFTB; NFTB; PP; Retention mechanisms
  19. Methods Mol Biol. 2020 ;2084 95-101
      Differential mobility spectrometry (DMS) is capable of separating molecules based on their size and shape. When coupled to mass spectrometry (MS), DMS reduces chemical background and enhances signal-to-noise (S/N) ratio. Flow injection analysis (FIA) is a technique used to introduce samples into the source of the DMS-MS platform. Here we describe the application of FIA-DMS-MS/MS for the analysis of urinary acylcarnitine species. More than 20 acylcarnitine species can be detected and quantified during a single FIA-DMS-MS/MS acquisition.
    Keywords:  Acylcarnitine; Differential mobility spectrometry; Mass spectrometry; Solid-phase extraction
  20. Methods Mol Biol. 2020 ;2084 133-144
      The structural identification of phase-I and phase-II metabolites of mycotoxins is a difficult task, mostly due to the lack of standards and because of the large number of isomeric forms. Here, we describe the use of ion mobility-mass spectrometry to analyze cereal extracts and how structural information on newly discovered mycotoxins metabolites could be obtained.
    Keywords:  CCS; Ion mobility-mass spectrometry; Metabolite identification; Mycotoxins; TWIMS