bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2021‒01‒10
sixteen papers selected by
Sofia Costa
Cold Spring Harbor Laboratory


  1. Biomed Chromatogr. 2021 Jan 06. e5063
    Jia Y, Liu X, Xu L, Ni J, Sun L, Li C.
      A method was established for the simultaneous determination of 20 kinds of bile acids in human serum employing ultra performance liquid chromatography-tandem mass spectrometry. Chromatographic condition and sample preparation were optimized to achieve good separation and maximum sensitivity for these analytes. The linearity, accuracy and repeatability of the development method were validated with a series of experiments. Under the optimum conditions, good linearities were achieved in the quantitative range for each bile acid with the correlation coefficients (r2 ) larger than 0.9901. The limit of detections (S/N = 3) of the method were in the range from 0.02 to 0.57 nmol/L. The recoveries were in the range of 88.1%~109.9%, RSD < 6.12%. This method had been successfully applied for the determination of bile acids in human serum sample with simple operation, high sensitivity and good accuracy, and would provide a reference for the clinical determination of bile acid content.
    Keywords:  UPLC-MS/MS; bile acids; human serum; quantitative determination
    DOI:  https://doi.org/10.1002/bmc.5063
  2. Metabolites. 2020 Dec 30. pii: E19. [Epub ahead of print]11(1):
    Tokuoka SM, Kita Y, Sato M, Shimizu T, Yatomi Y, Oda Y.
      In clinical lipidomics, it is a challenge to measure a large number of samples and to reproduce the quantitative results. We expanded the range of application of the tandem mass tag (TMT) method, which is widely used in proteomics, to lipidomic fields. There are various types of lipid molecule, for example, eicosanoids have a carboxyl group and phosphatidic acid has a phosphate group. We modified these functional groups simultaneously with TMT. This approach allows for a single analysis by mixing six samples and using one of the six samples as a bridging sample; the quantitative data can be easily normalized even if the number of measurements increases. To accommodate a large number of samples, we utilize a pooled serum sample of 300 individuals as a bridging sample. The stability of these lipid molecules in serum was examined as an analytical validation for the simultaneous TMT labeling. It was found that the stability of these lipid molecules in serum differs greatly depending on the lipid species. These findings reaffirmed the importance of proper sample preparation and storage to obtain reliable data. The TMT labeling method is expected to be a useful method for lipidomics with high-throughput and reliable reproducibility.
    Keywords:  high-throughput; lipids with carboxy groups; lipids with phosphate groups; relative quantitation
    DOI:  https://doi.org/10.3390/metabo11010019
  3. Anal Chem. 2021 Jan 08.
    Helmer PO, Nordhorn ID, Korf A, Behrens A, Buchholz R, Zubeil F, Karst U, Hayen H.
      Lipids, such for example the multifaceted category of glycerophospholipids (GP), play a major role in many biological processes. High-resolution mass spectrometry is able to identify these highly diverse lipid species in combination with fragmentation experiments (MS/MS) on the basis of the accurate m/z and fragmentation pattern. However, for the differentiation of isomeric lipids or isobaric interferences, more elaborate separation methods are required. Especially for imaging techniques, such as matrix-assisted laser desorption/ionization (MALDI)-MS imaging, the identification is often exclusively based on the accurate m/z. Fragmentation via MS/MS increases the confidence in lipid annotation in imaging approaches. However, this is sometimes not feasible due to insufficient sensitivity and significantly prolonged analysis time. The use of a separation dimension such as trapped ion mobility spectrometry (TIMS) after ionization strengthens the confidence of the identification based on the collision cross section (CCS). Since CCS libraries are limited, a tissue-specific database was initially generated using hydrophilic interaction liquid chromatography-TIMS-MS. Using this database, the identification of isomeric lipid classes as well as isobaric interferences in a lipid class was performed using a mouse spleen sample in a workflow described in this study. Besides a CCS-based identification as an additional identification criterion for GP in general, the focus was on the distinction of the isomeric GP classes phosphatidylglycerol and bis(monoacylglycero)phosphate, as well as the differentiation of possible isobaric interferences based on the formation of adducts by MALDI-TIMS-MS imaging on a molecular level.
    DOI:  https://doi.org/10.1021/acs.analchem.0c03942
  4. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Dec 30. pii: S1570-0232(20)31386-6. [Epub ahead of print]1163 122510
    Khan W, Wang YH, Nanayakkara NPD, Herath HMTB, Catchings Z, Khan S, Fasinu PS, ElSohly MA, McChesney JD, Khan IA, Chaurasiya ND, Tekwani BL, Walker LA.
      The antimalarial drug primaquine (PQ) causes methemoglobinemia and hemolysis in individuals with a genetic deficiency of glucose 6-phosphate dehydrogenase. Reactive oxygen species (ROS) generated by redox cycling of the metabolite primaquine-5,6-orthoquinone (POQ) in erythrocytes has been attributed to be responsible for the toxicity of PQ. Carboxyprimaquine (CPQ), the major human plasma metabolite of PQ, can also form the analogous carboxyprimaquine-5,6-orthoquinone (CPOQ) metabolite, which can also generate ROS in erythrocytes by redox cycling, thus contributing to the hematotoxicity of this drug. In order to study these pathways and characterize such effects in vivo, methods are needed for characterization and quantification of POQ and CPOQ in human erythrocytes. The purpose of this work was to develop a validated method for the quantitative determination of CPOQ and POQ metabolites in human erythrocytes, suitable for clinical studies of PQ metabolism. Several liquid-liquid extraction methods using different organic solvents had been investigated. The solvent mixture of water-methanol-acetonitrile (9:9:5, v/v) was shown to yield the best results for the two analytes. Chromatographic analysis of POQ and CPOQ in human erythrocytes was achieved on a high strength silica (HSS) column and gradient elution (water and acetonitrile, both containing 0.1% formic acid) by ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). Quantitative estimation of POQ and CPOQ was executed by monitoring ion pairs of m/z 260.23 > 175.03 and m/z 275.19 > 175.04, respectively. The method, which was validated for precision, accuracy, selectivity, and linearity, was successfully applied for the quantitative determination of POQ and CPOQ, the key metabolites of PQ in human erythrocytes in PQ clinical study.
    Keywords:  Carboxyprimaquine-5,6-orthoquinone; Erythrocytes; Method validation; Primaquine; Primaquine-5,6-orthoquinone; Quantification
    DOI:  https://doi.org/10.1016/j.jchromb.2020.122510
  5. Clin Chim Acta. 2020 Dec 31. pii: S0009-8981(20)30596-9. [Epub ahead of print]
    Zhong Z, Huang Y, Huang Q, Zheng S, Huang Z, Deng W, Li T.
      BACKGROUND: Gout is a common kind of inflammatory arthritis with metabolic disorders. However, the detailed pathogenesis of gout is complex and not fully clear. We investigated the serum metabolic profiling of gout patients by ultra-performance liquid chromatograph quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS).METHODS: Serum metabolites were extracted from 31 gout patients and 31 healthy controls. Metabolite extracts were analyzed in negative mode by UPLC-Q-TOF/MS for global metabolomics. Principal components analysis (PCA), orthogonal partial least squares-discriminant analysis (OPLS-DA) and hierarchical clustering analysis were performed to detect different compounds between the 2 groups. Receiver operating characteristic (ROC) curve analysis and pathway analysis of the different metabolites were conducted.
    RESULTS: A total of 9192 compounds were detected, of which 138 significantly different compounds were selected, according to the criteria of (Variable importance in projection (VIP)>3). Hierarchical clustering analysis showed that the relative levels of the differential compounds were different between the 2 groups. Ninety-one reliable metabolites matching the human metabolome database (HMDB) were confirmed. ROC curve results revealed that 4-hydroxytriazolam, urate and bilirubin exerted higher AUC values. Pathway analysis indicated that the significantly different metabolites were mainly involved in primary bile acid biosynthesis, purine metabolism and glycerophospholipid metabolism.
    CONCLUSIONS: The serum metabolic profiling of gout patients was significantly different from healthy subjects based on UPLC-Q-TOF/MS. Bilirubin was the potential biomarker. Primary bile acid biosynthesis may be a novel metabolic pathway of gout.
    Keywords:  Biomarkers; Gout; Metabolomics; UPLC-Q-TOF/MS
    DOI:  https://doi.org/10.1016/j.cca.2020.12.028
  6. Anal Bioanal Chem. 2021 Jan 03.
    Liu C, Li Y, Xiong C, Nie Z.
      We develop a capillary-paper spray (CPS) ion source which allows for sample separation in the capillary and enables rapid and sensitive paper spray (PS) mass spectrometry (MS) analysis of biofluids. The CPS employs a glass capillary to load liquid analytes, vertically standing at the rear of the PS. To further reduce the matrix effect, a nitrocellulose filter membrane is placed between the glass tube and chromatography paper to absorb proteins and other macromolecules, which is beneficial for the detection of the small molecules. Compared with the normal PS method, the CPS method markedly improves spray stability and prolongs analysis duration, and also generates significantly better signal intensities during the analysis of drugs, thus indicating its potential for clinical use. As a proof of concept, quantitative analysis of drugs (metformin hydrochloride and berberine hydrochloride) in serum is performed.
    Keywords:  Bio-sample analysis; Capillary-paper spray; Mass spectrometry; Paper spray
    DOI:  https://doi.org/10.1007/s00216-020-03072-y
  7. Antioxidants (Basel). 2020 Dec 31. pii: E38. [Epub ahead of print]10(1):
    Sambiagio N, Sauvain JJ, Berthet A, Auer R, Schoeni A, Hopf NB.
      Human biomonitoring of oxidative stress relies on urinary effect biomarkers such as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), and 8-iso-prostaglandin F2α (8-isoprostane); however, their levels reported for similar populations are inconsistent in the scientific literature. One of the reasons is the multitude of analytical methods with varying degrees of selectivity used to quantify these biomarkers. Single-analyte methods are often used, requiring multiple injections that increase both time and cost. We developed a rapid ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method to quantify both urinary biomarkers simultaneously. A reversed-phase column using a gradient consisting of 0.1% acetic acid in water and 0.1% acetic acid in methanol/acetonitrile (70:30) was used for separation. The MS detection was by positive (8-oxodG) and negative (8-isoprostane) ion-mode by multiple reaction monitoring. Very low limit of detection (<20 pg/mL), excellent linearity (R2 > 0.999), accuracy (near 100%), and precision (CV < 10%) both for intra-day and inter-day experiments were achieved, as well as high recovery rates (>91%). Matrix effects were observed but were compensated by using internal standards. Our newly developed method is applicable for biomonitoring studies as well as large epidemiological studies investigating the effect of oxidative damage, as it requires only minimal clean up using solid phase extraction.
    Keywords:  8-isoprostane; 8-oxodG; biomarker; biomonitoring; liquid chromatography; mass spectrometry; oxidative stress
    DOI:  https://doi.org/10.3390/antiox10010038
  8. J Sep Sci. 2021 Jan 04.
    Valluri VR, Katari NK, Khatri C, Yadlapalli SSR, Anand K, Gundla R, Polagani SR.
      A rapid, robust, simple, selective, and sensitive liquid chromatography-tandem mass spectrometry method was developed for the simultaneous estimation of obeticholic acid and its two pharmacologically active metabolites, glyco-obeticholic acid and tauro-obeticholic acid in human plasma. The analytes and their heavy stable isotope-labeled internal standards were extracted from 250 μL human plasma by a solid-phase extraction technique. The method linearity was established over a concentration range of 0.410 to 120.466 ng/mL for obeticholic acid, 0.414 to 121.708 ng/mL for glyco-obeticholic acid, and 0.255 to 75.101 ng/mL for tauro-obeticholic acid. The method was fully validated as per current guidelines on bioanalytical method validation of 'united states of food and drug administration' and 'European medicines agency.' The method was successfully applied to study the pharmacokinetics of obeticholic acid, glyco-obeticholic acid and tauro-obeticholic acid following oral administration of obeticholic acid tablets to healthy male volunteers. All the measured concentrations were within calibration curve ranges. This article is protected by copyright. All rights reserved.
    Keywords:  Human plasma; mass spectrometry; metabolites; obeticholic acid; pharmacokinetics
    DOI:  https://doi.org/10.1002/jssc.202001050
  9. Front Microbiol. 2020 ;11 609033
    Starostin KV, Demidov EA, Ershov NI, Bryanskaya AV, Efimov VM, Shlyakhtun VN, Peltek SE.
      Identification of microorganisms by MALDI-TOF mass spectrometry is a very efficient method with high throughput, speed, and accuracy. However, it is significantly limited by the absence of a universal database of reference mass spectra. This problem can be solved by creating an Internet platform for open databases of protein spectra of microorganisms. Choosing the optimal mathematical apparatus is the pivotal issue for this task. In our previous study we proposed the geometric approach for processing mass spectrometry data, which represented a mass spectrum as a vector in a multidimensional Euclidean space. This algorithm was implemented in a Jacob4 stand-alone package. We demonstrated its efficiency in delimiting two closely related species of the Bacillus pumilus group. In this study, the geometric approach was realized as R scripts which allowed us to design a Web-based application. We also studied the possibility of using full spectra analysis (FSA) without calculating mass peaks (PPA), which is the logical development of the method. We used 74 microbial strains from the collections of ICiG SB RAS, UNIQEM, IEGM, KMM, and VGM as the models. We demonstrated that the algorithms based on peak-picking and analysis of complete data have accuracy no less than that of Biotyper 3.1 software. We proposed a method for calculating cut-off thresholds based on averaged intraspecific distances. The resulting database, raw data, and the set of R scripts are available online at https://icg-test.mydisk.nsc.ru/s/qj6cfZg57g6qwzN.
    Keywords:  Geometric approach; MALDI—TOF; MS data processing; biotyping; microorganisms identification
    DOI:  https://doi.org/10.3389/fmicb.2020.609033
  10. Anal Chem. 2021 Jan 05.
    Yu H, Huan T.
      Despite the well-known nonlinear response of electrospray ionization (ESI) in mass spectrometry (MS)-based analysis, its complicated response patterns and negative impact on quantitative comparison are still understudied. We showcase in this work that the patterns of nonlinear ESI response are feature-dependent and can cause significant compression or inflation to signal ratios. In particular, our metabolomics study of serial diluted human urine samples showed that over 72% and 16% metabolic features suffered ratio compression and inflation, respectively, whereas only 12% of the signal ratios represent real metabolic concentration ratios. More importantly, these ratio compression and inflation largely exist in the linear response ranges, suggesting that it cannot be resolved by simply diluting the sample solutions to the linear ESI response ranges. Furthermore, we demonstrated that a polynomial regression model that converts MS signals to sample injection amounts can correct the biased ratios and, surprisingly, outperform the linear regression model in both data fitting and data prediction. Therefore, we proposed a metabolic ratio correction (MRC) strategy to minimize signal ratio bias in untargeted metabolomics for accurate quantitative comparison. In brief, by using the data of serial diluted quality control (QC) samples, we applied a cross-validation strategy to determine the best regression model, between linear and polynomial, for each metabolic feature and to convert the measured MS intensities to QC injection amounts for accurate metabolic ratio calculation. Both the studies of human urine samples and a metabolomics application supported that our MRC approach is very efficient in correcting the biased signal ratios. This novel insight of patterned ESI nonlinear response and MRC workflow can significantly benefit the downstream statistical comparison and biological interpretation for untargeted metabolomics.
    DOI:  https://doi.org/10.1021/acs.analchem.0c04113
  11. J Proteome Res. 2021 Jan 08.
    Klein MS.
      Data from untargeted metabolomics studies employing nuclear magnetic resonance (NMR) spectroscopy oftentimes contain negative values. These negative values hamper data processing and analysis algorithms and prevent the use of such data in multiomics integration settings. New methods to deal with such negative values are thus an urgent need in the metabolomics community. This study presents affine transformation of negative values (ATNV), a novel algorithm for replacement of negative values in NMR data sets. ATNV was implemented in the R package mrbin, which features interactive menus for user-friendly application and is available for free for various operating systems within the free R statistical programming language. The novel algorithms were tested on a set of human urinary NMR spectra and were able to successfully identify relevant metabolites.
    Keywords:  binning; bucketing; data transformation; logarithm; multiomics integration
    DOI:  https://doi.org/10.1021/acs.jproteome.0c00684
  12. Bioinformatics. 2021 Jan 08. pii: btaa1093. [Epub ahead of print]
    Li C, Gao M, Yang W, Zhong C, Yu R.
      MOTIVATION: We developed Diamond, a Nextflow-based, containerized, multi-modal data-independent acquisition (DIA) mass spectrometry (MS) data processing pipeline for peptide identification and quantification. Diamond integrated two mainstream workflows for DIA data analysis, namely, spectrum-centric scoring (SCS) and peptide-centric scoring (PCS), for use cases both with and without assay libraries. This multi-modal pipeline serves as a versatile, easy-to-use, and easily extendable toolbox for large-scale DIA data processing.AVAILABILITY: The Docker image is available at https://hub.docker.com/r/zeroli/diamond and the source codes are freely accessible at https://github.com/xmuyulab/Diamond.
    DOI:  https://doi.org/10.1093/bioinformatics/btaa1093
  13. Metabolites. 2020 Dec 31. pii: E26. [Epub ahead of print]11(1):
    Loef M, von Hegedus JH, Ghorasaini M, Kroon FPB, Giera M, Ioan-Facsinay A, Kloppenburg M.
      It is essential to measure lipid biomarkers with a high reproducibility to prevent biased results. We compared the lipid composition and inter-day reproducibility of lipid measurements in plasma and erythrocytes. Samples from 42 individuals (77% women, mean age 65 years, mean body mass index (BMI) 27 kg/m2), obtained non-fasted at baseline and after 6 weeks, were used for quantification of up to 1000 lipid species across 13 lipid classes with the Lipidyzer platform. Intraclass correlation coefficients (ICCs) were calculated to investigate the variability of lipid concentrations between timepoints. The ICC distribution of lipids in plasma and erythrocytes were compared using Wilcoxon tests. After data processing, the analyses included 630 lipids in plasma and 286 in erythrocytes. From these, 230 lipids overlapped between sample types. In plasma, 78% of lipid measurements were reproduced well to excellently, compared to 37% in erythrocytes. The ICC score distribution in plasma (median ICC 0.69) was significantly better than in erythrocytes (median ICC 0.51) (p-value < 0.001). At the class level, reproducibility in plasma was superior for triacylglycerols and cholesteryl esters while ceramides, diacylglycerols, (lyso)phosphatidylethanolamines, and sphingomyelins showed better reproducibility in erythrocytes. Although in plasma overall reproducibility was superior, differences at individual and class levels may favor the use of erythrocytes.
    Keywords:  erythrocytes; lipids; metabolomics; plasma; reproducibility
    DOI:  https://doi.org/10.3390/metabo11010026
  14. J Anal Toxicol. 2021 Jan 07. pii: bkab003. [Epub ahead of print]
    Cabarcos-Fernández P, Tabernero-Duque MJ, Álvarez-Freire I, Bermejo-Barrera AM.
      Although blood is often used to detect and quantify the presence of drugs, there are some instances where samples obtained from other biological matrices, like pericardial fluid (PF), are necessary since adequate blood samples may not be available. PF is an epicardial transudate, which contains plasma components that include toxicological substances making this sample useful when blood samples are not available. This fluid is a well preserved postmortem sample and can easily be collected in larger amounts without significant contamination, compared with other body fluids. Although studies involving PF began around the 1980's, the adequacy of such fluid as a biological matrix has been poorly investigated. Antidepressants are frequently detected in postmortem samples from forensic cases. Nowadays, they constitute some of the most commonly prescribed drugs worldwide. A total of seven antidepressants (venlafaxine, mirtazapine, olanzapine, paroxetine, sertraline, fluoxetine and citalopram) were evaluated in this study. A new extraction method involving dispersive liquid-liquid microextraction (DLLME) is presented in which chloroform and acetonitrile are determined to be the best extraction and dispersing solvents. The experimental design was achieved using StatGraphics 18. The Response Surface Methodology enabled us to know the optimal volume for the two solvents used in the DLLME. The detection technique used was gas chromatography-mass spectrometry (GC-MS) with electron impact ionization as ionization source. A temperature gradient has been used and the total chromatographic separation time was 19.43 min. Validation results met the international validation guidances (FDA). Under the optimal condition, the method offered good validation parameters showing a new efficient, simple, rapid, and sensitive method. The analytical method was applied to thirty-one pericardial fluid samples. Twenty-one samples were positive with concentrations between 0.19 and 8.48 µg/mL. Venlafaxine and olanzapine were the antidepressants most frequently found.
    Keywords:  Pericardial fluid; antidepressants; dispersive liquid-liquid microextraction; forensic toxicology, depression; postmortem samples
    DOI:  https://doi.org/10.1093/jat/bkab003
  15. J Pharm Biomed Anal. 2020 Dec 19. pii: S0731-7085(20)31742-8. [Epub ahead of print]195 113855
    Chutvirasakul B, Joseph JF, Parr MK, Suntornsuk L.
      The objective of this work was to develop a high-performance liquid chromatographic method coupled with a mass spectrometer (LC-MS) for the simultaneous analysis of artemisinin-based drugs (e.g. artemisinin, dihydroartemisinin, artesunate, artemether) and piperaquine in formulations. Simultaneous separation of the investigated drugs was achieved in 14 min on a C18 column (2.1 mm x 100 mm, particle size 1.8 μm) using a gradient elution of 0.05 % v/v formic acid in water and acetonitrile. MS detection was done in a positive ionization mode using electrospray ionization with acquisition in a single ion monitoring mode. Proper diluent and storage time in an autosampler played significant roles on the quantitation accuracy since the target analytes possessed varied solubility and stability in aqueous and organic solvents. The method was fully validated according to ICH guideline and data showed good linearity (R > 0.999, precision (RSD < 3.89 %) and accuracy (%recovery between 98.5 and 103.7) with low limits of detection (LOD < 24.7 ng/mL) and quantitation (LOQ < 82.40 ng/mL). Validation data indicated that the developed LC-MS method is fit for the intended purpose and was successfully applied to evaluate the drug contents in formulations. Among the tested samples, the percent labeled amounts found were between 93.1 and 105.0 % and one supplement capsule contained 0.039 %w/w of artemisinin. The newly developed method could benefit both the quality control departments in pharmaceutical industries and the authorities working on falsified drug problems since official methods for the analysis of these drugs are not available in pharmacopoeias. The method is fast and environmentally friendly due to the requirement of less chemicals and production of less wastes.
    Keywords:  Artemether; Artemisinin; Artesunate; Dihydroartemisinin; Liquid chromatography-mass spectrometry; Piperaquine
    DOI:  https://doi.org/10.1016/j.jpba.2020.113855
  16. Rapid Commun Mass Spectrom. 2021 Jan 07. e9041
    Ranasinghe A, Ciccimaro E, D'Arienzo C, Olah TV, Ponath P, Hnatyshyn S.
      RATIONALE: Gangliosides (GS) are attractive targets in biomarker discovery because of their physiological significance in numerous human diseases including certain cancers and developmental and metabolic disorders. The robust strategy described here enables the profiling of numerous GS while obtaining quantitative data of exploratory biomarkers present in human plasma and whole blood.METHOD: The gangliosides from human blood, human plasma and several cell lines were extracted using a mixture of methanol and isopropanol/0.1% formic acid followed by direct analysis of the supernatant. The simultaneous Qualitative and Quantitative (Qual/Quan) approach involves micro flow (20 μL/min) HPLC/high resolution mass spectrometry (HRMS) and post-acquisition data processing with Skyline software for profiling numerous GS in biological matrices. The quantitative assay involves reverse-phase liquid chromatography/HRMS and calibration curves utilizing commercially available gangliosides.
    RESULTS: Protein precipitation resulted in ~60-80% GS recovery from biological matrices. Direct injection of the extract allowed for quantification of targeted GS in human blood, plasma and cancer cell lines. The lower limit of detection for the target analytes, GM1, GT1, GD1, spiked into 1% BSA/PBS, ranged from 1 to 10 ng/mL. Human lung cancer cell lines contained variable amounts (1-130 ng/mL) of soluble Fuc-GM1 analogs, potential biomarkers of lung cancer.
    CONCLUSIONS: A combination of simple extraction and Micro-HPLC/HRMS allowed for quantification of gangliosides in human serum and whole blood. Integration of HRMS with Skyline allowed for GS profiling in the same samples using post-acquisition HRMS data without the need for reanalysis. The strategy presented here is expected to play an important role in profiling exploratory GS biomarkers in discovery bioanalytical research.
    DOI:  https://doi.org/10.1002/rcm.9041