bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2021‒02‒14
seventeen papers selected by
Sofia Costa
Cold Spring Harbor Laboratory

  1. Chem Phys Lipids. 2021 Feb 06. pii: S0009-3084(21)00001-3. [Epub ahead of print] 105048
      Small molecules, including metabolites and lipids, provide information on metabolic pathways and active biological processes in living organisms. They are often diagnostic of disease. Current exploratory methods for metabolomics and lipidomics mostly rely on separation using liquid or gas chromatography (LC or GC) coupled with mass spectrometers capable of acquiring high resolution data to generate an enormous data, but at the cost of lengthy processing and data acquisition. Even though many molecules can be identified and quantified by these methods, the laborious protocols for purification, identification, and validation limit the accessible sample chemical information. To improve the speed and efficiency of exploratory metabolomics and lipidomics, multiple reaction monitoring profiling (MRM profiling) has been developed. This strategy involves a three-stage workflow which starts by considering the metabolome as a collection of functional groups. The Discovery Stage interrogates a representative sample mixture for functional groups using the functional group specific precursor ion (Prec) scans and neutral loss (NL) scans. This experiment usually uses a triple quadrupole mass spectrometer without chromatography, i.e. by direct sample infusion. In the second Screening Stage, the main features seen in the Prec and NL scans are organized into lists of precursor ion/product ion transitions (MRMs) which are then used for the fast, specific, and sensitive interrogation of each individual sample. Data analysis by univariate and multivariate statistical methods is used to identify the most informative MRMs and so classify the individual samples. The compounds (biomarkers) which are responsible for the most informative MRMs in particular sample classes can be investigated in an optional third Identification Stage i.e. in a structural identification study. MRM profiling benefits from the much smaller number of functional groups compared to the number of individual metabolites existing in biological samples (where most metabolites are still unknown), resulting in acquisition of a much smaller data set and a shorter analysis time. The application of MRM Profiling to several biological and clinical problems is used to illustrate its features.
    Keywords:  Lipid profiling; Parkinson’s disease; exploratory lipidomics, oocytes; functional group profiling; microorganisms; tandem mass spectrometry
  2. Anal Chim Acta. 2021 Mar 08. pii: S0003-2670(21)00030-1. [Epub ahead of print]1149 338210
      When using liquid chromatography/mass spectrometry (LC/MS) to perform untargeted metabolomics, it is common to detect thousands of features from a biological extract. Although it is impractical to collect non-chimeric MS/MS data for each in a single chromatographic run, this is generally unnecessary because most features do not correspond to unique metabolites of biological relevance. Here we show that relatively simple data-processing strategies that can be applied on the fly during acquisition of data with an Orbitrap ID-X, such as blank subtraction and well-established adduct or isotope calculations, decrease the number of features to target for MS/MS analysis by up to an order of magnitude for various types of biological matrices. We demonstrate that annotating these non-biological contaminants and redundancies in real time during data acquisition enables comprehensive MS/MS data to be acquired on each remaining feature at a single collision energy. To ensure that an appropriate collision energy is applied, we introduce a method using a series of hidden ion-trap scans in an Orbitrap ID-X to find an optimal value for each feature that can then be applied in a subsequent high-resolution Orbitrap scan. Data from 100 metabolite standards indicate that this real-time optimization of collision energies leads to more informative MS/MS patterns compared to using a single fixed collision energy alone. As a benchmark to evaluate the overall workflow, we manually annotated unique biological features by independently subjecting E. coli samples to a credentialing analysis. While credentialing led to a more rigorous reduction in feature number, on-the-fly annotation with blank subtraction on an Orbitrap ID-X did not inappropriately discard unique biological metabolites. Taken together, our results reveal that optimal fragmentation data can be obtained in a single LC/MS/MS run for >90% of the unique biological metabolites in a sample when features are annotated during acquisition and collision energies are selected by using parallel mass spectrometry detection.
    Keywords:  Credentialing; Liquid chromatography; Mass spectrometry; Metabolite identification; Untargeted metabolomics
  3. J Pharm Biomed Anal. 2021 Feb 01. pii: S0731-7085(21)00051-0. [Epub ahead of print]196 113939
      A high-sensitivity and -selectivity mass spectrometry derivatization reagent, (R)-(5-(3-isothiocyanatopyrrolidin-1-yl)-5-oxopentyl) triphenylphosphonium (NCS-OTPP), was developed for the enantiomeric separation of chiral thiol compounds as prospectively important diagnostic markers for oxidative stress-related diseases. Complete separation of GSH, DL-Cys, and DL-Hcy was achieved. The parent ions of all derivatives had a fragment of m/z 473.18 and a structure of m/z 75.95 (R-S = C-S-R'), conducive to qualitative and quantitative analysis. Good linear relationships were obtained for all analytes (R2≥ 0.9995). The intra-day and inter-day precision were 0.82-5.16 % and 1.02-4.18 % in saliva, and 0.81-3.45 % and 0.99-6.47 % in urine, with mean recoveries of 83.31-105.66 % and 84.09-101.11 %, respectively. The limit of detection (S/N = 3) was 19.20-57.60 nM. Free and total GSH, DL-Cys, and DL-Hcy were detected simultaneously in saliva and urine from 10 volunteers in the normal, stressed, and stable states by UHPLC-Q-Orbitrap HRMS. The thiol compounds were quantitatively related to oxidative stress state changes.
    Keywords:  Chiral resolution; DL-cysteine; DL-homocysteine; NCS-OTPP; Oxidative stress; Thiol; UHPLC-Q-Orbitrap HRMS
  4. Anal Bioanal Chem. 2021 Feb 09.
      Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) is a common molecular imaging modality used to characterise the abundance and spatial distribution of lipids in situ. There are several technical challenges predominantly involving sample pre-treatment and preparation which have complicated the analysis of clinical tissues by MALDI-MSI. Firstly, the common embedding of samples in optimal cutting temperature (O.C.T.), which contains high concentrations of polyethylene glycol (PEG) polymers, causes analyte signal suppression during mass spectrometry (MS) by competing for available ions during ionisation. This suppressive effect has constrained the application of MALDI-MSI for the molecular mapping of clinical tissues. Secondly, the complexity of the mass spectra is obtained by the formation of multiple adduct ions. The process of analyte ion formation during MALDI can generate multiple m/z peaks from a single lipid species due to the presence of alkali salts in tissues, resulting in the suppression of protonated adduct formation and the generation of multiple near isobaric ions which produce overlapping spatial distributions. Presented is a method to simultaneously remove O.C.T. and endogenous salts. This approach was applied to lipid imaging in order to prevent analyte suppression, simplify data interpretation, and improve sensitivity by promoting lipid protonation and reducing the formation of alkali adducts.
    Keywords:  Ammonium formate; Lipids; MALDI imaging; O.C.T.; Washing
  5. J Pharm Biomed Anal. 2021 Jan 28. pii: S0731-7085(21)00047-9. [Epub ahead of print]196 113935
      BACKGROUND: The present COVID-19 pandemic has prompted worldwide repurposing of drugs. The aim of the present work was to develop and validate a two-dimensional isotope-dilution liquid chromatrography tandem mass spectrometry (ID-LC-MS/MS) method for accurate quantification of remdesivir and its active metabolite GS-441524, chloroquine, hydroxychloroquine, lopinavir, ritonavir, favipiravir and azithromycin in serum; drugs that have gained attention for repurposing in the treatment of COVID-19.METHODS: Following protein precipitation, samples were separated with a two-dimensional ultra-high performance liquid chromatography (2D-UHPLC) setup, consisting of an online solid phase extraction (SPE) coupled to an analytical column. For quantification, stable isotope-labelled analogues were used as internal standards for all analytes. The method was validated on the basis of the European Medicines Agency bioanalytical method validation protocol.
    RESULTS: Detuning of lopinavir and ritonavir allowed simultaneous quantification of all analytes with different concentration ranges and sensitivity with a uniform injection volume of 5 μL. The method provided robust validation results with inaccuracy and imprecision values of ≤ 9.59 % and ≤ 11.1 % for all quality controls.
    CONCLUSION: The presented method is suitable for accurate and simultaneous quantification of remdesivir, its metabolite GS-441525, chloroquine, hydroxychloroquine, lopinavir, ritonavir, favipiravir and azithromycin in human serum. The quantitative assay may be an efficient tool for the therapeutic drug monitoring of these potential drug candidates in COVID-19 patients in order to increase treatment efficacy and safety.
    Keywords:  Antiviral therapy; Isotope dilution liquid chromatography tandem mass spectrometry (ID-LC–MS/MS); Therapeutic drug monitoring
  6. J Anal Toxicol. 2021 Feb 12. pii: bkab018. [Epub ahead of print]
      5-(2-aminopropyl)benzofuran (5-APB) and 6-(2-aminopropyl)benzofuran (6-APB) are benzofuran analogues of amphetamine and belong to the category of new psychoactive substances (NPS). Despite already published fatal 5- and 6-APB intoxication - in most cases, a combination of both substances - no sensitive method for the simultaneous detection and quantification of these new psychoactive compounds in human blood samples has yet been developed. Therefore, an easy and fast sample preparation-, as well as specific high-performance liquid chromatography and tandem mass spectrometry (HPLC-MS/MS) method for the determination of both substances in blood, were established and validated. In a fatal intoxication in 2017 at the Institute of Forensic and Traffic Medicine in Heidelberg, Germany, concentrations of 850 ng/mL (5-APB) and 300 ng/mL (6-APB) were determined in peripheral blood. Besides, other body fluids (central blood, urine, bile), hair, and various tissues were examined to verify the presence of both compounds and to gain first insights into their distribution. In this publication, we show a method for the simultaneous determination of 5- and 6-APB in human samples by a chromatographic method and to investigate their distribution in the human body.
    Keywords:  5-APB; 6-APB; HPLC-MS/MS; intoxication; new psychoactive substances (NPS)
  7. Anal Chim Acta. 2021 Mar 08. pii: S0003-2670(21)00016-7. [Epub ahead of print]1149 338204
      Sample preparation is often reported as the main bottleneck of analytical processes. To meet the requirements of both high-throughput and high sensitivity, improved sample-preparation methods capable of fast analyte preconcentration are urgently needed. To this end, a new three-phase electroextraction (EE) method is presented that allows for ultrafast electroextraction hyphenated to flow-injection analysis mass spectrometry (FIA-MS). Four model compounds, i.e., propranolol, amitriptyline, bupivacaine, and oxeladin, were used to optimize and evaluate the method. Within only 30 s extraction time, enrichment factors (EF) of 105-569 and extraction recoveries (ER) of 10.2%-55.7% were achieved for these analytes, with limits of detection (LODs) ranging from 0.36 to 3.21 ng mL-1, good linear response function (R2 > 0.99), low relative standard deviation (0.6%-17.8%) and acceptable accuracy (73-112%). Finally, the optimized three-phase EE method was successfully applied to human urine and plasma samples. Our three-phase electroextraction method is simple to construct and offers ultrafast, online extraction of trace amounts of analytes from biological samples, and therefore has great potential for high-throughput analysis.
    Keywords:  Electroextraction; Human plasma; Human urine; Hyphenation to MS; Sample pretreatment; Ultrafast
  8. Anal Chim Acta. 2021 Mar 08. pii: S0003-2670(21)00034-9. [Epub ahead of print]1149 338214
      Recently, there has been significant interest in the influences of the human gut microbiota on many diseases, such as cardiovascular disease (CVD) and metabolic disorders. Trimethylamine N-oxide (TMAO) is one of the most frequently discussed gut-derived metabolites. Dried blood spot (DBS) sampling has been regarded as an attractive alternative sampling strategy for clinical studies and offers many advantages. For DBS sample processing, whole-spot analysis could minimize hematocrit-related bias, but it requires blood volume calibration. This study developed a method combining matrix-induced ion suppression (MIIS) with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) to estimate blood volume and quantify TMAO and its precursors and derivatives, including choline, carnitine and acetylcarnitine, in DBSs. The MIIS method used an ion suppression indicator (ISI) to measure the extent of ion suppression caused by the blood matrix, which was related to the blood volume. The results showed that the volume estimation accuracy of the MIIS method was within 91.7-109.7%. The combined MIIS and LC-MS/MS method for quantifying TMAO, choline, carnitine and acetylcarnitine was validated in terms of linearity, precision and accuracy. The quantification accuracy was within 91.2-113.2% (with LLOQ <119%), and the imprecision was below 8.0% for all analytes. A stability study showed that the analytes in DBSs were stable at all evaluated temperatures for at least 30 days. The validated method was applied to quantify DBS samples (n = 56). Successful application of the new method demonstrated the potential of this method for real-world DBS samples and to facilitate our understanding of the gut microbiota in human health.
    Keywords:  Blood volume correction; Dried blood spot (DBS); Mass spectrometry; Matrix induced ion suppression (MIIS); Trimethylamine N-Oxide (TMAO)
  9. Anal Bioanal Chem. 2021 Feb 11.
      Lipid identification is one of the current bottlenecks in lipidomics and lipid profiling, especially for novel lipid classes, and requires multidimensional data for correct annotation. We used the combination of chromatographic and ion mobility separation together with data-independent acquisition (DIA) of tandem mass spectrometric data for the analysis of lipids in the biomedical model organism Caenorhabditis elegans. C. elegans reacts to harsh environmental conditions by interrupting its normal life cycle and entering an alternative developmental stage called dauer stage. Dauer larvae show distinct changes in metabolism and morphology to survive unfavorable environmental conditions and are able to survive for a long time without feeding. Only at this developmental stage, dauer larvae produce a specific class of glycolipids called maradolipids. We performed an analysis of maradolipids using ultrahigh performance liquid chromatography-ion mobility spectrometry-quadrupole-time of flight-mass spectrometry (UHPLC-IM-Q-ToFMS) using drift tube ion mobility to showcase how the integration of retention times, collisional cross sections, and DIA fragmentation data can be used for lipid identification. The obtained results show that combination of UHPLC and IM separation together with DIA represents a valuable tool for initial lipid identification. Using this analytical tool, a total of 45 marado- and lysomaradolipids have been putatively identified and 10 confirmed by authentic standards directly from C. elegans dauer larvae lipid extracts without the further need for further purification of glycolipids. Furthermore, we putatively identified two isomers of a lysomaradolipid not known so far.
    Keywords:  Caenorhabditis elegans; Ion mobility spectrometry; Lipid identification; Lipidomics
  10. F1000Res. 2021 ;10 4
      Lipidomics increasingly describes the quantitation using mass spectrometry of all lipids present in a biological sample.  As the power of lipidomics protocols increase, thousands of lipid molecular species from multiple categories can now be profiled in a single experiment.  Observed changes due to biological differences often encompass large numbers of structurally-related lipids, with these being regulated by enzymes from well-known metabolic pathways.  As lipidomics datasets increase in complexity, the interpretation of their results becomes more challenging.  BioPAN addresses this by enabling the researcher to visualise quantitative lipidomics data in the context of known biosynthetic pathways.  BioPAN provides a list of genes, which could be involved in the activation or suppression of enzymes catalysing lipid metabolism in mammalian tissues.
    Keywords:  Biosynthetic pathway analysis; LIPID MAPS; Lipidomics; lipid profiling.; lipids
  11. Anal Chem. 2021 Feb 08.
      Quantitative metabolomics requires the analysis of the same or a very similar amount of samples in order to accurately determine the concentration differences of individual metabolites in comparative samples. Ideally, the total amount or concentration of metabolites in each sample is measured to normalize all the analyzed samples. In this work, we describe a very sensitive method to measure a subclass of metabolites as a surrogate quantifier for normalization of samples with limited amounts. This method starts with low-volume dansyl labeling of all metabolites containing a primary/secondary amine or phenol group in a sample to produce a final solution of 21 μL. The dansyl-labeled metabolites generate fluorescence signals at 520 nm with photoexcitation at 250 nm. To remove the interference of dansyl hydroxyl products (Dns-OH) formed from the labeling reagents used, a fast-gradient liquid chromatography separation is used to elute Dns-OH using aqueous solution, followed by organic solvent elution to produce a chromatographic peak of labeled metabolites, giving a measurement throughput of 6 min per sample. The integrated fluorescence signals of the peak are found to be related to the injection amount of the dansyl-labeled metabolites. A calibration curve using mixtures of dansyl-labeled amino acids is used to determine the total concentration of labeled metabolites in a sample. This concentration is used for normalization of samples in the range from 2 to 120 μM in 21 μL with only 1 μL consumed for fluorescence quantification (i.e., 2-120 pmol). We demonstrate the application of this sensitive sample normalization method in comparative metabolome analysis of human cancer cells, MCF-7 cells, treated with and without resveratrol, using a starting material of as low as 500 cells.
  12. Anal Chem. 2021 Feb 12.
      Matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) is an emerging label-free method for mapping the distribution of diverse molecular species in tissue sections. Despite recent progress in MALDI-MSI analyses of lipids, it is still difficult to visualize minor bioactive lipids including lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P). Here, we have developed a novel on-tissue derivatization method using Phos-tag, a zinc complex that specifically binds to a phosphate monoester group. MALDI-MSI with Phos-tag derivatization made it possible to image LPA and S1P in the murine brain. Furthermore, we were able to visualize other low-abundance lipids containing phosphate monoester, such as phosphatidic acid and ceramide-1-phosphate. Compared with conventional MALDI-MS, this derivatization produced LPA images with high spatial accuracy discriminating LPA artificially produced during MALDI-MS analysis. In mice with deficiencies in enzymes that degrade LPA and S1P, we observed marked S1P and/or LPA accumulation in specific regions of the brain. Thus, the present study provides a simple and optimal way to reveal the spatial localization of potent bioactive lipid phosphates such as LPA and S1P in tissues.
  13. Anal Chem. 2021 Feb 08.
      High-dimensional molecular measurements are transforming the field of pathology into a data-driven discipline. While hematoxylin and eosin (H&E) stainings are still the gold standard to diagnose diseases, the integration of microscopic and molecular information is becoming crucial to advance our understanding of tissue heterogeneity. To this end, we propose a data fusion method that integrates spatial omics and microscopic data obtained from the same tissue slide. Through correspondence-aware manifold learning, we can visualize the biological trends observed in the high-dimensional omics data at microscopic resolution. While data fusion enables the detection of elements that would not be detected taking into account the separate data modalities individually, out-of-sample prediction makes it possible to predict molecular trends outside of the measured tissue area. The proposed dimensionality reduction-based data fusion paradigm will therefore be helpful in deciphering molecular heterogeneity by bringing molecular measurements such as mass spectrometry imaging (MSI) to the cellular resolution.
  14. Steroids. 2021 Feb 05. pii: S0039-128X(21)00012-X. [Epub ahead of print]167 108800
      BACKGROUND: Steroid hormones are essential signalling molecules in prostate cancer (PC). However, many studies focusing on liquid biomarkers fail to take the hormonal status of these patients into account. Steroid measurements are sensitive to bias caused by matrix effects, thus assessing potential matrix effects is an important step in combining circulating tumour DNA (ctDNA) analysis with hormone status.METHODS: We investigated the accuracy of multi-steroid hormone profiling in mechanically-separated plasma (MSP) samples and in plasma from CellSave Preservative (CS) tubes, that are typically used to obtain ctDNA, compared to measurements in serum. We performed multiplex steroid profiling by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in samples obtained from ten healthy controls and ten castration-resistant prostate cancer (CRPC) patients.
    RESULTS: Steroid measurements were comparable between MSP and serum. A small but consistent decrease of 8-21% compared to serum was observed when using CS plasma, which was considered to be within the acceptable margin. The minimal residual testosterone levels of CRPC patients could be sensitively quantified in both MSP and CS samples.
    CONCLUSIONS: We validated the use of MSP and CS samples for multi-steroid profiling by LC-MS/MS. The optimised use of these samples in clinical trials will allow us to gain further insight into the steroid metabolism in PC patients.
    Keywords:  Androgens; CellSave; LC–MS/MS; Steroids; Testosterone
  15. Redox Biol. 2021 Jan 28. pii: S2213-2317(21)00020-3. [Epub ahead of print] 101872
      Oxidatively generated damage to DNA has been implicated in the pathogenesis of a wide variety of diseases. Increasingly, interest is also focusing upon the effects of damage to the other nucleic acids, RNA and the (2'-deoxy-)ribonucleotide pools, and evidence is growing that these too may have an important role in disease. LC-MS/MS has the ability to provide absolute quantification of specific biomarkers, such as 8-oxo-7,8-dihydro-2'-deoxyGuo (8-oxodG), in both nuclear and mitochondrial DNA, and 8-oxoGuo in RNA. However, significant quantities of tissue are needed, limiting its use in human biomonitoring studies. In contrast, the comet assay requires much less material, and as little as 5 μL of blood may be used, offering a minimally invasive means of assessing oxidative stress in vivo, but this is restricted to nuclear DNA damage only. Urine is an ideal matrix in which to non-invasively study nucleic acid-derived biomarkers of oxidative stress, and considerable progress has been made towards robustly validating these measurements, not least through the efforts of the European Standards Committee on Urinary (DNA) Lesion Analysis. For urine, LC-MS/MS is considered the gold standard approach, and although there have been improvements to the ELISA methodology, this is largely limited to 8-oxodG. Emerging DNA adductomics approaches, which either comprehensively assess the totality of adducts in DNA, or map DNA damage across the nuclear and mitochondrial genomes, offer the potential to considerably advance our understanding of the mechanistic role of oxidatively damaged nucleic acids in disease.
    Keywords:  Biomarkers; DNA; DNA repair; Nucleotide pool; Oxidative stress; RNA
  16. Clin Chem Lab Med. 2020 Nov 25. pii: cclm-2020-0410. [Epub ahead of print]
      Objectives: Accurate measurements of serum 17-hydroxyprogesterone (17OHP) are essential for diagnosis and treatment monitoring for congenital adrenal hyperplasia patients. The performance of serum 17OHP routine methods remains highly variable that calls for a candidate reference measurement procedure (cRMP) to improve the standardization of serum 17OHP measurements.Methods: Serum samples spiked with internal standards were extracted with a combination of solid-phase extraction and liquid-liquid extraction. The 17OHP was quantified by the isotope dilution coupled with liquid chromatography/tandem mass spectrometry (ID-LC/MS/MS) with electrospray ionization in positive ion mode. Nine structural analogs of 17OHP were evaluated for interferences. The precision and analytical recovery were assessed. Twenty native and 40 spiked serum for performance evaluation were measured by the cRMP and two clinical LC/MS routine methods.
    Results: No apparent interferences were found with the 17OHP measurement. The within-run, between-run, and total precision for our method were 0.4-0.8%, 0.6-2.0%, and 1.0-2.1% for four pooled serum (2.46-102.72 nmol/L), respectively. The recoveries of added 17OHP were 100.0-100.2%. For the performance of two LC/MS routine methods, they showed relative deviation ranges of -22.1 to 1.1% and -6.7 to 12.8%, respectively.
    Conclusions: We developed and validated a reliable serum 17OHP method using ID-LC/MS/MS. The desirable accuracy and precision of this method enable it to serve as a promising cRMP to improve the standardization for serum 17OHP routine measurements.
    Keywords:  congenital adrenal hyperplasia; liquid chromatography/tandem mass spectrometry; method evaluation; reference measurement procedure; serum 17-hydroxyprogesterone
  17. J Agric Food Chem. 2021 Feb 08.
      In this study, a rapid and reliable method based on ultrahigh-performance liquid chromatography coupled with Q Exactive HF-X mass spectrometry (UHPLC-QE/MS) was established for the simultaneous quantification and validation of acrylamide, 5-hydroxymethylfurfural, and 14 heterocyclic aromatic amines in thermally processed foods. With the optimization of the pretreatment method, all 16 hazardous compounds with different polarities were simultaneously extracted and purified by one-step purification. By studying various acquisition modes in detail, full MS + PRM detection using an electrospray ionization source in the positive mode gives an excellent-shaped chromatographic peak and thereby achieves a better quantitative ability for analytes in the matrix. This method demonstrated good quantification recovery in the range of 68.85-146.42%. The limits of quantification were within the range from 0.1 to 50 ng/mL. With the method proposed, the simultaneous determination of 16 hazardous compounds in different thermally processed foods was successfully applied. The all-fragment-ion approaches at high resolution have the ability to reduce false-positive peak detections arising from peak alignment software in the detection of samples significantly. The proposed isotope dilution UHPLC-QE/MS method was validated and demonstrated to be sensitive, accurate, and precise for the simultaneous quantification of multiple contaminants in one injection.
    Keywords:  Q Exactive HF-X; UHPLC; hazardous compound; thermally processed foods