bims-metlip 2021-08-22 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2021–08–22
23 papers selected by
Sofia Costa, Cold Spring Harbor Laboratory

Improving data quality in liquid chromatography-mass spectrometry metabolomics of human urine.
Lipidomics in Biomarker Research.
Evaluation of multiple hydrophilic interaction chromatography columns and surrogate matrix for arginine quantification in saliva by high resolution mass spectrometry.
A liquid chromatography-tandem mass spectrometry method for the analysis of primary aromatic amines in human urine.
A comprehensive UHPLC-MS/MS screening method for the analysis of 98 New Psychoactive Substances and related compounds in human hair.
Untargeted UHPLC-ESI-QTOF-MS/MS analysis with targeted feature extraction at precursor and fragment level for profiling of the platelet lipidome with ex vivo thrombin-activation.
A Cross-Platform Metabolomics Comparison Identifies Serum Metabolite Signatures of Liver Fibrosis Progression in Chronic Hepatitis C Patients.
gQuant, an Automated Tool for Quantitative Glycomic Data Analysis.
Selective ESI-MS detection of carbonyl containing compounds by aminooxyacetic acid immobilized on a resin.
A simulated strategy for analysis of Short- to Long- chain fatty acids in mouse serum beyond chemical standards.
CFM-ID 4.0: More Accurate ESI-MS/MS Spectral Prediction and Compound Identification.
Mass spectrometry based metabolomics approach on the elucidation of volatile metabolites formation in fermented foods: A mini review.
Large-scale Multi-omics Genome-wide Association Studies (Mo-GWAS): Guidelines for Sample Preparation and Normalization.
High-throughput quantitation of trace level melatonin in human milk by on-line enrichment liquid chromatography-tandem mass spectrometry.
Variable Data Independent Acquisition and Data Mining Exploring Feature-Based Molecular Networking Analysis for Untargeted Screening of Synthetic Cannabinoids in Oral Fluid.
Applicable and cost-efficient microplastic analysis by quantitative 1 H-NMR spectroscopy using benchtop NMR and NoD methods.
Ambient electric arc ionization for versatile sample analysis using mass spectrometry.
A simple and accurate HFCF-UF method for the analysis of homocysteine, cysteine, cysteinyl-glycine, and glutathione in human blood.
Desorption electrospray ionization (DESI) source coupling ion mobility mass spectrometry for imaging fluoropezil (DC20) distribution in rat brain.
Simultaneous determination of eight catechins and four theaflavins in bottled tea by liquid chromatography-tandem mass spectrometry for forensic analysis.
A hierarchical approach to removal of unwanted variation for large-scale metabolomics data.
Using a stable isotope-labeled internal standard for liquid chromatography-tandem mass spectrometry quantitation of meloxicam in human plasma.
Comprehensive profile of DNA adducts as both tissue and urinary biomarkers of exposure to acrylamide and chemo-preventive effect of catechins in rats.

J Chromatogr A. 2021 Aug 08. pii: S0021-9673(21)00581-1. [Epub ahead of print]1654 462457

Improving data quality in liquid chromatography-mass spectrometry metabolomics of human urine.

Rosilene Cristina Rossetto Burgos, Adriana Nori de Macedo, Pedro Luis Rocha da Cruz, Hélio Tedesco-Silva Júnior, Karina Helena Morais Cardozo, Valdemir Melechco Carvalho, Marina Franco Maggi Tavares.

  Signal variation is a common drawback in untargeted metabolomics using liquid chromatography-mass spectrometry (LC-MS), mainly due to the complexity of biological matrices and reduced sample preparation, which results in the accumulation of sample components in the column and the ion source. Here we propose a simple, easy to implement approach to improve data quality in untargeted metabolomics by LC-MS. This approach involves the use of a divert valve to direct the column effluent to waste at the beginning of the chromatographic run and during column cleanup and equilibration, in combination with longer column cleanups in between injections. Our approach was tested using urine samples collected from patients after renal transplantation. Analytical responses were contrasted before and after introducing these modifications by analyzing a batch of untargeted metabolomics data. A significant improvement in peak area repeatability was observed for the quality controls, with relative standard deviations (RSDs) for several metabolites decreasing from ∼60% to ∼10% when our approach was introduced. Similarly, RSDs of peak areas for internal standards improved from ∼40% to ∼10%. Furthermore, calibrant solutions were more consistent after introducing these modifications when comparing peak areas of solutions injected at the beginning and the end of each analytical sequence. Therefore, we recommend the use of a divert valve and extended column cleanup as a powerful strategy to improve data quality in untargeted metabolomics, especially for very complex types of samples where minimum sample preparation is required, such as in this untargeted metabolomics study with urine from renal transplanted patients.

Keywords:  Column cleanup; Divert valve; Liquid chromatography; Quality control; Untargeted metabolomics

DOI:  https://doi.org/10.1016/j.chroma.2021.462457
Handb Exp Pharmacol. 2021 Aug 19.

Lipidomics in Biomarker Research.

Th Hornemann.

  Lipids are natural substances found in all living organisms and involved in many biological functions. Imbalances in the lipid metabolism are linked to various diseases such as obesity, diabetes, or cardiovascular disease. Lipids comprise thousands of chemically distinct species making them a challenge to analyze because of their great structural diversity.Thanks to the technological improvements in the fields of chromatography, high-resolution mass spectrometry, and bioinformatics over the last years, it is now possible to perform global lipidomics analyses, allowing the concomitant detection, identification, and relative quantification of hundreds of lipid species. This review shall provide an insight into a general lipidomics workflow and its application in metabolic biomarker research.

Keywords:  Biomarkers; Data analysis; Dyslipidemia; LC/MS; Lipids; Metabolic disease; Metabolites

DOI:  https://doi.org/10.1007/164_2021_517
J Sep Sci. 2021 Aug 18.

Evaluation of multiple hydrophilic interaction chromatography columns and surrogate matrix for arginine quantification in saliva by high resolution mass spectrometry.

Yu Wang, Zhigang Hao, Long Pan.

  Arginine, a pivotal ingredient in many biochemical synthetic pathways, can be used as a biomarker for many oral care clinical applications. It is still a challenge to develop a sensitive and reliable chromatographic method to quantify the arginine as a biomarker in saliva, with or without arginine product pretreatment. The current method solved two critical issues for arginine quantitation in human saliva. The first issue was how to optimize arginine peak shape. A hydrophilic interaction chromatography method based on the column selection, pH and pKa relationship, mobile phase ionic strength, organic solvent consideration, and temperature effects was developed. An optimized chromatographic condition for arginine quantitation in the saliva matrix was obtained. The second issue was how to build confidence in the use of a simple surrogate matrix methodology to replace the more complex traditional standard addition methodology. The surrogate matrix methodology we developed is applicable to the measurement of arginine as a potential non-invasive biomarker in human saliva. The method detection and quantification limit reached 2 and 6 ng/mL. The tailing factor was within the 0.9∼1.1 range even though arginine had three pKa values at 2.18, 9.09 and 13.2. This article is protected by copyright. All rights reserved.

Keywords:  arginine; hydrophilic interaction chromatography; retention mechanism; saliva; surrogate matrix method

DOI:  https://doi.org/10.1002/jssc.202100361
J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Aug 05. pii: S1570-0232(21)00369-X. [Epub ahead of print]1180 122888

A liquid chromatography-tandem mass spectrometry method for the analysis of primary aromatic amines in human urine.

Sridhar Chinthakindi, Kurunthachalam Kannan.

  Aromatic amines are widely used in personal care products and human exposure to this class of chemicals is widespread. Bioanalytical methods to determine trace levels of aromatic amines in human urine are scarce. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to determine 39 primary aromatic amines (AAs) along with nicotine and cotinine in human urine. Chromatographic separation of the 41 analytes was achieved on an Ultra Biphenyl (100 mm × 2.1 mm, 5 µm) column. Mass spectrometry was operated in electrospray ionization positive ion multiple reaction monitoring (MRM) mode. The method exhibited excellent linear dynamic range (0.1-50 ng/mL) with correlation coefficients (r) > 0.999 for all analytes. Urine samples (2 mL) were hydrolyzed using 10 M NaOH at 95 °C for 15 h and target analytes were extracted using methyl-tert-butyl ether (MTBE). Addition of 15 µL of 0.25 M HCl to the sample extracts improved the recoveries of several target analytes. The method was validated through the analysis of fortified quality control (QC) samples and a certified standard reference material (SRM). Relative recoveries (%) of target analytes fortified in QC samples were in the range of 75-114% for 37 of the 41 analytes while the other analytes exhibited lower recoveries (16-74%). The limits of detection (LOD) and limits of quantification (LOQ) of target analytes were in the range of 0.025-0.20 ng/mL and 0.1-1.0 ng/mL, respectively. Intra-day and inter-day precision of the method assessed through the analysis of fortified urine QC samples at three different concentrations were < 11.7% and < 15.9% (measured as RSD), respectively. The method was applied in the analysis of urine samples from the general population and known smokers; aniline, para-anisidine, para-toluidine, ortho/meta-toluidine, 3-chloroaniline, 4-chloroaniline, 3,4-dichloroaniline, and 4,4'-methylenedianiline were found in all smoker's urine at sum concentrations ranging from 0.04 to 9.16 ng/mL.

Keywords:  Aniline; Aromatic amines; Biomonitoring; LC-MS/MS; Urine

DOI:  https://doi.org/10.1016/j.jchromb.2021.122888
J Pharm Biomed Anal. 2021 Aug 08. pii: S0731-7085(21)00421-0. [Epub ahead of print]205 114310

A comprehensive UHPLC-MS/MS screening method for the analysis of 98 New Psychoactive Substances and related compounds in human hair.

Franks Kamgang Nzekoue, Marco Agostini, Michele Verboni, Caterina Renzoni, Lucia Alfieri, Simone Barocci, Massimo Ricciutelli, Giovanni Caprioli, Simone Lucarini.

  In this study, a rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the targeted analysis of 98 New Psychoactive Substances (NPS) from the hair matrix. The monitored compounds included various chemical classes (7 phenethylamines, 10 tryptamines, 18 cathinones, 24 synthetic opioids, and 38 synthetic cannabinoids) with emphasis given to newly emerged NPS. The method employed a direct extraction process through the incubation of hair samples (25 mg) and internal standards with M3® reagent at 100 °C for 60 min, followed by extract purification through acid and basic liquid-liquid micro-extraction (LLME). Extracted compounds were analyzed through LC-MS/MS system operating in multiple reaction monitoring mode. NPS were separated in 9.5 min with a Poroshell 120 EC-C18 column (2.7 μm, 4.6 × 50 mm) using a gradient eluting mobile phase composed of water and acetonitrile/water (95:5) both containing 0.1 % of formic acid. The developed and validated method shows a good precision (≤ 15 %), linearity (R2 between 0.993 and 0.999), selectivity, and sensitivity (LOD: 0.6-10.3 pg mg-1 and LOQ: 2.1-34.4 pg mg-1). The method showed also reduced matrix effect and acceptable recovery for most of the targeted compounds. Our results showed that this method is suitable for quantifying NPS in hair matrix and could be employed in the context of routine analyses in analytical laboratories.

Keywords:  Abuse drugs; Cannabinoids; Hair analysis; Novel psychoactive substances (NPS); Opioids; UHPLC–MS/MS

DOI:  https://doi.org/10.1016/j.jpba.2021.114310
J Pharm Biomed Anal. 2021 Aug 04. pii: S0731-7085(21)00412-X. [Epub ahead of print]205 114301

Untargeted UHPLC-ESI-QTOF-MS/MS analysis with targeted feature extraction at precursor and fragment level for profiling of the platelet lipidome with ex vivo thrombin-activation.

Malgorzata Cebo, Carlos Calderón Castro, Jörg Schlotterbeck, Meinrad Gawaz, Madhumita Chatterjee, Michael Lämmerhofer.

  Lipids play a major role in platelet signaling and activation. In this study, we analyzed the platelet lipidome in an untargeted manner by reversed-phase UHPLC for lipid species separation coupled to high-resolution QTOF-MS/MS in data-independent acquisition (DIA) mode with sequential window acquisition of all theoretical fragment ion mass spectra (SWATH) for compound detection. Lipid identification and peak picking was supported by the characteristic regular elution pattern of lipids differing in carbon and double bond numbers. It was primarily based on post-acquisition targeted feature extraction from the SWATH data. Multiple extracted ion chromatograms (EICs) from SWATH data of diagnostic ions on MS1 and MS2 level from both positive and negative ion mode allowed to distinguish between poorly resolved isomeric lipids based on their distinct fragment ions, which were used for relative quantification at a molecular lipid species level. It supports assay specificity for relative lipid quantitation via multiple quantifiably ions unlike to data-dependent acquisition methods which rely on precursor ions only. This approach was used to analyze human platelet samples. 457 lipids were annotated. Concentrations of lipids were estimated by stable isotope-labelled lipid class-specific internal standards as surrogate calibrants. Heatmaps of lipid concentrations in dependence on carbon and double bond numbers for the distinct lipid classes revealed a snapshot of the platelet lipidome in the resting state with lipid species distributions within classes supporting some functional interpretations. As expected, activation of the platelets by thrombin has led to significant alterations in the platelet lipidome as proven by univariate (volcano plot) and multivariate (PLS-DA) statistics. Several lipids were significantly up-regulated (lysophosphatidylinositols, oxylipins such as thromboxane B2 (TXB2), hydroxyheptadecatrienoic acid (HHT), hydroxyeicosatetraenoic acid (HETE), hydroxyoctadecadienoic acid (HODE), sphingoid-bases, (very) long chain saturated fatty acids) or down-regulated (lysophosphatidylethanolamines, polyunsaturated fatty acids, phosphatidylinositols). Several of them are well known as biomarkers of platelet activation while others may provide some further insights into pathways of platelet activation and platelet metabolism.

Keywords:  Biomarker; Lipid identification; Lipidomics; Platelet activation; Platelet lipidome; Sequential window acquisition of all theoretical fragment ion mass spectra

DOI:  https://doi.org/10.1016/j.jpba.2021.114301
Front Mol Biosci. 2021 ;8 676349

A Cross-Platform Metabolomics Comparison Identifies Serum Metabolite Signatures of Liver Fibrosis Progression in Chronic Hepatitis C Patients.

Meera Shanmuganathan, Mohammad Omair Sarfaraz, Zachary Kroezen, Holly Philbrick, Richel Poon, Andrew Don-Wauchope, Marco Puglia, David Wishart, Philip Britz-McKibbin.

  Metabolomics offers new insights into disease mechanisms that is enhanced when adopting orthogonal instrumental platforms to expand metabolome coverage, while also reducing false discoveries by independent replication. Herein, we report the first inter-method comparison when using multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS) and nuclear magnetic resonance (NMR) spectroscopy for characterizing the serum metabolome of patients with liver fibrosis in chronic hepatitis C virus (HCV) infection (n = 20) and non-HCV controls (n = 14). In this study, 60 and 30 serum metabolites were detected frequently (>75%) with good technical precision (median CV < 10%) from serum filtrate samples (n = 34) when using standardized protocols for MSI-CE-MS and NMR, respectively. Also, 20 serum metabolite concentrations were consistently measured by both methods over a 500-fold concentration range with an overall mean bias of 9.5% (n = 660). Multivariate and univariate statistical analyses independently confirmed that serum choline and histidine were consistently elevated (p < 0.05) in HCV patients with late-stage (F2-F4) as compared to early-stage (F0-F1) liver fibrosis. Overall, the ratio of serum choline to uric acid provided optimal differentiation of liver disease severity (AUC = 0.848, p = 0.00766) using a receiver operating characteristic curve, which was positively correlated with liver stiffness measurements by ultrasound imaging (r = 0.606, p = 0.0047). Moreover, serum 5-oxo-proline concentrations were higher in HCV patients as compared to non-HCV controls (F = 4.29, p = 0.0240) after adjustment for covariates (age, sex, BMI), indicative of elevated oxidative stress from glutathione depletion with the onset and progression of liver fibrosis. Both instrumental techniques enable rapid yet reliable quantification of serum metabolites in large-scale metabolomic studies with good overlap for biomarker replication. Advantages of MSI-CE-MS include greater metabolome coverage, lower operating costs, and smaller sample volume requirements, whereas NMR offers a robust platform supported by automated spectral and data processing software.

Keywords:  biomarkers; capillary electrophoresis-mass spectrometry; hepatitis C virus infection; liver fibrosis; metabolomics (OMICS); nuclear magnetic resonance; serum

DOI:  https://doi.org/10.3389/fmolb.2021.676349
Front Chem. 2021 ;9 707738

gQuant, an Automated Tool for Quantitative Glycomic Data Analysis.

Jiangming Huang, Biyun Jiang, Mingqi Liu, Pengyuan Yang, Weiqian Cao.

  MALDI-MS-based glycan isotope labeling methods have been effectively and widely used for quantitative glycomics. However, interpretation of the data produced by MALDI-MS is inaccurate and tedious because the bioinformatic tools are inadequate. In this work, we present gQuant, an automated tool for MALDI-MS-based glycan isotope labeling data processing. gQuant was designed with a set of dedicated algorithms to improve the efficiency, accuracy and convenience of quantitation data processing. When tested on the reference data set, gQuant showed a fast processing speed, as it was able to search the glycan data of model glycoproteins in a few minutes and reported more results than the manual analysis did. The reported quantitation ratios matched well with the experimental glycan mixture ratios ranging from 1:10 to 10:1. In addition, gQuant is fully open-source and is coded in Python, which is supported by most operating systems, and it has a user-friendly interface. gQuant can be easily adapted by users for specific experimental designs, such as specific glycan databases, different derivatization types and relative quantitation designs and can thus facilitate fast glycomic quantitation for clinical sample analysis using MALDI-MS-based stable isotope labeling.

Keywords:  MALDI-MS analysis; automated processing; glycomic quantitation; quantitative tool; stable isotope labeling

DOI:  https://doi.org/10.3389/fchem.2021.707738
Anal Chim Acta. 2021 Sep 01. pii: S0003-2670(21)00593-6. [Epub ahead of print]1176 338767

Selective ESI-MS detection of carbonyl containing compounds by aminooxyacetic acid immobilized on a resin.

Monika Kijewska, Tomasz Koch, Mateusz Waliczek, Andrzej Konieczny, Piotr Stefanowicz, Zbigniew Szewczuk.

  There are numerous examples of bioactive compounds containing carbonyl groups including modified proteins with oxidation of side chain of amino acid residues to aldehyde/ketone groups which are frequently considered as markers of oxidative stress. The carbonyl unit can be also distinguished as a substructure in many illegal drugs including anabolic steroids as well as cations derivatives. Based on chemoselective formation of oximes by solid phase immobilized hydroxylamine derivatives we proposed the protocol for derivatization and selective detection of carbonylated compounds in human serum albumin hydrolysate as a complex peptide mixture and of testosterone in urine samples. This allowed for the removal of the matrix and the qualitative and quantitative analysis of the derivatized analyte by LC-MS/MS (or LC-MRM). Herein we report the preparation and chemical characterization of a novel, ChemMatrix - based resin functionalized with aminooxyacetic acid (AOA). The hydroxylamine moiety in this resin is combined with a peptide linker (GRG) containing an arginine residue to enhance the ionization efficiency. Application of an isotopically labeled carbonylated peptide ((H-Leu-Val-Thr(O)-Asp-Leu-Thr-Lys [13C6,15N2]-OH and testosterone-d3 allowed us to carry out quantitative analyses of detected compounds. Our method is general and may be applied for analysis of carbonylated compounds in biological samples. Our method based on application of functionalized resin allowed to quantify the level of free testosterone in small sample (0.5 mL) of urine, while the non-derivatized testosterone from urine sample was not detected during direct LC-MRM analysis.

Keywords:  Aminooxyacetic acid; Carbonylated peptides; ChemMatrix rink resin; Derivative of testosterone; LC-MS analysis; Oxime; Selective detection

DOI:  https://doi.org/10.1016/j.aca.2021.338767
J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Aug 12. pii: S1570-0232(21)00376-7. [Epub ahead of print]1180 122895

A simulated strategy for analysis of Short- to Long- chain fatty acids in mouse serum beyond chemical standards.

Xueying Wang, Huabin Ruan, Zhaoyun Zong, Fengfeng Mao, Yusong Wang, Yupei Jiao, Lina Xu, Tao Yang, Wenhui Li, Xiaohui Liu.

  Broadening coverage in fatty acid (FA) analysis benefits the understanding of metabolic regulation in biological system. However, the limited access of chemical standards makes it challenging. In this work, we introduced a simulation assisted strategy to analyze short-, medium-, long- and very-long-chain fatty acids beyond the use of chemical standards. This targeted analysis in selected reaction monitoring (SRM) mode incorporated 3-nitrophenylhydrazine derivatization and mathematical simulation of ion transitions, collision energies, RF values and retention times to identify and quantify the fatty acids without chemical standards. Serum analysis using high resolution mass spectrometry coupled with paired labeling was employed to refine the computational retention times. Based on the simulation, 116 free fatty acids from C1 to C24 were covered in a single analysis on use of 34 standard chemicals. Background interference is commonly observed in fatty acid analysis. For certain fatty acids, e.g. acetic acid or palmitic acid, reliable quantitation is largely restricted by contamination level instead of detection limit. Therefore, the background interference and quantifiable serum volume required for each fatty acid were also evaluated. At least 20 µL serum was suggested to cover most molecules. Using this approach, a total of 66 free fatty acids with various chain lengths and saturations were detected in NTCP knockout mice serum, of which 34 FAs were confirmed by chemical standards and 32 FAs were potentially assigned based on the simulation. Gender dependent fatty acid regulation was observed by NTCP knockout. This work provides a unique strategy that enables to broaden the fatty acid coverage with the absence of chemical standards and is applicable to other derivatizations.

Keywords:  Free Fatty Acids; LC-MS/MS; Mathematical Simulation; Quantitation; Short- to Long- Chain

DOI:  https://doi.org/10.1016/j.jchromb.2021.122895
Anal Chem. 2021 Aug 17.

CFM-ID 4.0: More Accurate ESI-MS/MS Spectral Prediction and Compound Identification.

Fei Wang, Jaanus Liigand, Siyang Tian, David Arndt, Russell Greiner, David S Wishart.

In the field of metabolomics, mass spectrometry (MS) is the method most commonly used for identifying and annotating metabolites. As this typically involves matching a given MS spectrum against an experimentally acquired reference spectral library, this approach is limited by the coverage and size of such libraries (which typically number in the thousands). These experimental libraries can be greatly extended by predicting the MS spectra of known chemical structures (which number in the millions) to create computational reference spectral libraries. To facilitate the generation of predicted spectral reference libraries, we developed CFM-ID, a computer program that can accurately predict ESI-MS/MS spectrum for a given compound structure. CFM-ID is one of the best-performing methods for compound-to-mass-spectrum prediction and also one of the top tools for in silico mass-spectrum-to-compound identification. This work improves CFM-ID's ability to predict ESI-MS/MS spectra from compounds by (1) learning parameters from features based on the molecular topology, (2) adding a new approach to ring cleavage that models such cleavage as a sequence of simple chemical bond dissociations, and (3) expanding its hand-written rule-based predictor to cover more chemical classes, including acylcarnitines, acylcholines, flavonols, flavones, flavanones, and flavonoid glycosides. We demonstrate that this new version of CFM-ID (version 4.0) is significantly more accurate than previous CFM-ID versions in terms of both EI-MS/MS spectral prediction and compound identification. CFM-ID 4.0 is available at http://cfmid4.wishartlab.com/ as a web server and docker images can be downloaded at https://hub.docker.com/r/wishartlab/cfmid.

DOI: https://doi.org/10.1021/acs.analchem.1c01465
Food Sci Biotechnol. 2021 Jul;30(7): 881-890

Mass spectrometry based metabolomics approach on the elucidation of volatile metabolites formation in fermented foods: A mini review.

Min Kyung Park, Young-Suk Kim.

  Metabolomics can be applied for comparative and quantitative analyses of the metabolic changes induced by microorganisms during fermentation. In particular, mass spectrometry (MS) is a powerful tool for metabolomics that is widely used for elucidating biomarkers and patterns of metabolic changes. Fermentation involves the production of volatile metabolites via diverse and complex metabolic pathways by the activities of microbial enzymes. These metabolites can greatly affect the organoleptic properties of fermented foods. This review provides an overview of the MS-based metabolomics techniques applied in studies of fermented foods, and the major metabolic pathways and metabolites (e.g., sugars, amino acids, and fatty acids) derived from their metabolism. In addition, we suggest an efficient tool for understanding the metabolic patterns and for identifying novel markers in fermented foods.

Keywords:  Fermentation; Mass spectrometry; Metabolic pathway; Metabolomics; Volatile metabolites

DOI:  https://doi.org/10.1007/s10068-021-00917-9
J Vis Exp. 2021 Jul 27.

Large-scale Multi-omics Genome-wide Association Studies (Mo-GWAS): Guidelines for Sample Preparation and Normalization.

Mustafa Bulut, Alisdair R Fernie, Saleh Alseekh.

Both gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) are widely used metabolomics approaches to detect and quantify hundreds of thousands of metabolite features. However, the application of these techniques to a large number of samples is subject to more complex interactions, particularly for genome-wide association studies (GWAS). This protocol describes an optimized metabolic workflow, which combines an efficient and fast sample preparation with the analysis of a large number of samples for legume crop species. This slightly modified extraction method was initially developed for the analysis of plant and animal tissues and is based on extraction in methyl tert-butyl ether: methanol solvent to allow the capture of polar and lipid metabolites. In addition, we provide a step-by-step guide for reducing analytical variations, which are essential for the high-throughput evaluation of metabolic variance in GWAS.

DOI: https://doi.org/10.3791/62732
Anal Chim Acta. 2021 Sep 01. pii: S0003-2670(21)00590-0. [Epub ahead of print]1176 338764

High-throughput quantitation of trace level melatonin in human milk by on-line enrichment liquid chromatography-tandem mass spectrometry.

Weiqi Jin, Juan Gui, Guipu Li, Fan Jiang, Dongmei Han.

  The level of melatonin in human milk might be closely related to infant development and the building up of their circadian rhythms. The large population investigation on this topic would provide insights for the prevention and treatment of diseases related to early development and circadian rhythms. However, it has not been well studied. Trace level endogenous melatonin and difficulties in sample collection are among the challenges limiting the progress. High throughput analytical method with high specificity and sensitivity to determine the endogenous melatonin concentration is highly desired. A newly developed easily operated and high-throughput sensitive on-line enrichment liquid chromatography-tandem mass spectrometry (LC-MS/MS) method would be reported in this paper. Melatonin-d3 (MEL-d3) was used as a surrogate standard for the calibration curve and melatonin-d4 (MEL-d4) was used as an internal standard. Sample preparation was simply performed in 96-well plate by protein precipitation using acetonitrile (ACN). The supernatant was injected directly into the easily configured LC-MS/MS system with an enlarged sample loop and a mixer. Positive mode multiple reaction monitoring (MRM) was adopted for the measurement of melatonin in milk. 100 μL sample was used for analysis and the calibration curve linear range was 1-1000 pg mL-1. In three validation batches, the accuracy was within 11.0% deviation from the relative nominal concentration, whereas the intra- and inter-assay precision was ≤4.1% and ≤6.8% relative standard deviation (RSD), respectively. Although matrix effect was observed in the validation experiments, the stable isotope labeled internal standard (MEL-d4) could correct it and the overall relative matrix effect of MEL-d3/MEL-d4 was close to 100%. The overall spike recovery of the method was 101.7% with 5.1% RSD. Compared to currently reported methods, it could reach 1 pg mL-1 lower limit of quantification (LLOQ) with a smaller sample volume, sample preparation could be easily performed by automated liquid handling system and was more suitable for large population cohort studies on trace level endogenous melatonin determination.

Keywords:  Circadian rhythm; High-throughput; Human milk melatonin; Infant development; Quantification; Tandem mass spectrometry

DOI:  https://doi.org/10.1016/j.aca.2021.338764
J Am Soc Mass Spectrom. 2021 Aug 16.

Variable Data Independent Acquisition and Data Mining Exploring Feature-Based Molecular Networking Analysis for Untargeted Screening of Synthetic Cannabinoids in Oral Fluid.

Gustavo de Albuquerque Cavalcanti, Ricardo Moreira Borges, Gabriel Reis Alves Carneiro, Monica Costa Padilha, Henrique Marcelo Gualberto Pereira.

Novel psychoactive substances (NPS) are constantly emerging in the drug market, and synthetic cannabinoids (SCs) are included in this NPS family. Forensic laboratories often struggle with these continually emerging SCs, forcing them to develop an untargeted workflow to incorporate these psychoactive drugs in their procedures. Usually, forensic laboratories select analytical methods based on targeted mass spectrometry (MS) technologies for strictly tracking already known NPS. The appropriate way to tackle unknown substances is to develop pipelines for untargeted analysis that include LC-HRMS analytical methods and data analysis. Once established, this strategy would allow drug testing laboratories to be always one step ahead of the new trends concerning the "designer drugs" market. To address this challenge an untargeted workflow based on mass spectrometry data acquisition and data analysis was developed to detect SCs in oral fluid (OF) samples at a low concentration range. The samples were extracted by mixed-mode solid-phase extraction and analyzed by Liquid Chromatography - High-Resolution Mass Spectrometry (LC-HRMS). Tandem mass spectra (MS2) were recorded performing a variable isolation width across a mass range of all theoretical precursor ions (vDIA) after the chromatographic separation. After raw data processing with the MSDial software, the deconvoluted features were sent to GNPS for Feature-Based Molecular Networking (FBMN) construction for nontargeted data mining. The FBMN analysis created a unique integrated network for most of the SCs assessed in the OF at a low level (20 ng/mL). These results demonstrate the potential of an untargeted approach to detect different derivatives of SCs at trace levels for forensic applications.

DOI: https://doi.org/10.1021/jasms.1c00124
Magn Reson Chem. 2021 Aug 20.

Applicable and cost-efficient microplastic analysis by quantitative 1 H-NMR spectroscopy using benchtop NMR and NoD methods.

Nadine Peez, Torsten Rinesch, Jürgen Kolz, Wolfgang Imhof.

In continuation of our work on the proof-of-concept that quantitative NMR spectroscopy may be a valuable tool in microplastic (MP) analysis and quantification we present here investigations using low-field NMR-spectrometers and non-deuterated solvents for the analysis of solutions of MP particles in suitable solvents. The use of low-field NMR-spectrometers (benchtop NMR) which are considerably more cost-effective in terms of purchase and operating costs compared to high-field NMR-spectrometers and the use of non-deuterated solvents (NoD method) lead to an applicable and cost-efficient method for mass-based MP analysis. For benchtop 80 MHz NMR, limits of detection for PVC, PET, and PS are in the same range as if a high-field 500 MHz NMR-spectrometer was used for quantification (500 MHz: PET 1 μg/mL, PVC 42 μg/mL, PS 9 μg/mL; 80 MHz: PET 4 μg/mL, PVC 19 μg/mL, PS 21 μg/mL) for polymers being dissolved in deuterated solvents. The same is true for the corresponding limits of quantification. Moreover, it is shown for the first time that quantitative determination of the mass concentration of PET, PVC and PS is also possible using NoD methods by evaluating the integrals of polymer-specific signals relative to an internal or external standard. Detection limits for NoD methods are in a similar range as if deuterated solvents were used (PET 2 μg/mL, PVC 39 μg/mL, PS 8 μg/mL) using a high-field 500 MHz spectrometer or the 80 MHz spectrometer (PET 5 μg/mL).

DOI: https://doi.org/10.1002/mrc.5210
Analyst. 2021 Aug 16.

Ambient electric arc ionization for versatile sample analysis using mass spectrometry.

Yuanji Gao, Yuan Li, Binpeng Zhan, Quan He, Heping Zhu, Weiwei Chen, Qi Yin, Hongru Feng, Yuanjiang Pan.

A novel, convenient ambient electric arc ionization (AEAI) device was developed as a mass spectrometry ion source for versatile sample analysis. AEAI could be considered as a soft ionization technique in which the protonated ion ([M + H]+) is the main ion species with little or no in-source fragmentation for most analytes. Coupled with a high-resolution Orbitrap mass spectrometer, AEAI could be applied to the analysis of a variety of organic compounds having a wide range of polarities, ranging from non-polar species such as polybenzenoid aromatic hydrocarbons (PAHs) to highly polar species such as amino acids. With its versatile capabilities in the mass spectrometric analysis of small molecules, AEAI has the potential to be an alternative to traditional ionization methods such as electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and electron impact (EI) ionization. The limitations of AEAI are also discussed.

DOI: https://doi.org/10.1039/d1an00872b
Anal Bioanal Chem. 2021 Aug 18.

A simple and accurate HFCF-UF method for the analysis of homocysteine, cysteine, cysteinyl-glycine, and glutathione in human blood.

Wei-Chong Dong, Jia-Liang Guo, Meng-Qiang Zhao, Xi-Kun Wu, Yi-Xuan Cui, Jing-Ying Feng, Chen-Xiao Zhang, Ye Jiang, Zhi-Qing Zhang.

  The presence of reduced aminothiols, including homocysteine (Hcy), cysteine (Cys), cysteinyl-glycine (CG), and glutathione (GSH), is significantly increased in the pathological state. However, there have been no reports on the relationship between reduced aminothiols (Hcy, Cys, CG, and GSH) and different genders, ages, and drug combinations in human blood. The accurate quantification of these reduced thiols in biological fluids is important for monitoring some special pathological conditions of humans. However, the published methods typically not only require cumbersome and technically challenging processing procedures to ensure reliable measurements, but are also laborious and time-consuming, which may disturb the initial physiological balance and lead to inaccurate results. We developed a hollow fiber centrifugal ultrafiltration (HFCF-UF) method for sample preparation coupled with a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method and used it to determine four reduced aminothiols (Hcy, Cys, CG, and GSH) in human blood for the first time. A total of 96 clinical patients were enrolled in our study. The influence of different genders, ages, and drug combinations on the levels of four reduced thiols in human blood was also discussed by SPSS 24.0. The sample preparation was simplified to a single 5 min centrifugation step in a sealed system that did not disturb the physiological environment. The validation parameters for the methodological results were excellent. The procedure was successfully applied to monitoring the concentrations of four reduced aminothiols (Hcy, Cys, CG, and GSH) in 96 clinical blood samples. There were no significant differences in Hcy, Cys, CG, or GSH for the different genders, ages, or combinations with methotrexate or vancomycin (P > 0.05). However, there was a significant increase in Hcy concentration in patients treated with valproic acid who were diagnosed with epilepsy (p=0.0007). It is advisable to measure reduced Hcy level in patients taking valproic acid. The developed HFCF-UF method was simple and accurate. It can be easily applied in clinical research to evaluate oxidative stress in further study.

Keywords:  Cysteine; Cysteinyl-glycine; Glutathione; HFCF-UF; Homocysteine; Valproic acid

DOI:  https://doi.org/10.1007/s00216-021-03578-z
Anal Bioanal Chem. 2021 Aug 18.

Desorption electrospray ionization (DESI) source coupling ion mobility mass spectrometry for imaging fluoropezil (DC20) distribution in rat brain.

Runcong Guo, Lei Zhou, Xiaoyan Chen.

  Fluoropezil (DC20) is a new selective acetylcholinesterase inhibitor, and it was developed for the treatment of Alzheimer's disease patients. In this study, a desorption electrospray ionization source coupling ion mobility mass spectrometry imaging (DESI/IMS-MSI) method was developed to explore the distribution of DC20 in brain tissue following oral administration. Rat brain coronal slices obtained 1 h and 3 h following drug dosing were used in the study. D6-DC20 was used as internal standard and sprayed by matrix sprayer on the brain slices to calibrate the matrix effect. Ion mobility separation was used to reduce the interference from background noise and the biological matrix. By optimizing DESI-MSI parameters for improved sensitivity, the limit of quantitation of the method was 1.45 pg/mm2 with a linear range from 1.45 to 72.7 pg/mm2. DESI-MSI data showed that DC20 could quickly enter and diffuse across whole brain and tended to be much more enriched in striatum than cerebral cortex and hippocampus, which was consistent with quantitative analysis using high-performance liquid chromatography-electrospray tandem mass spectrometry to measure DC20 concentration in each homogenized brain sub-region. The workflow of tissue imaging method optimization and strategy were established, and for the first time, the DESI-MSI technique and optimized method were used to explore the distribution characteristics of DC20 in rat brain, which could help elucidate pharmacological effect mechanisms and improve clinical outcomes.

Keywords:  Brain distribution; DESI; Fluoropezil (DC20); Mass spectrometry imaging; Optimization strategy

DOI:  https://doi.org/10.1007/s00216-021-03563-6
J Forensic Sci. 2021 Aug 18.

Simultaneous determination of eight catechins and four theaflavins in bottled tea by liquid chromatography-tandem mass spectrometry for forensic analysis.

Hitomi S Kikkawa, Manami Kobayashi, Ayaka Minamimoto, Hiromi Ono, Kouichiro Tsuge.

  Tea, and particularly bottled tea, is widely consumed worldwide and is often encountered at crime scenes in poisoning cases or used in place of urine in drug abuse monitoring. Tea is a rich source of polyphenols, such as catechins and theaflavins, and these compounds are useful for identification of trace quantities of tea samples. However, information on the contents of catechins and theaflavins in bottled tea is limited. In this study, a method was developed for simultaneous analysis of eight catechins and four theaflavins in tea using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The concentrations of these polyphenols were determined in bottled black, oolong, and green teas after a simple pretreatment process by the standard addition method. The developed LC-MS/MS method was rapid and all tested polyphenol compounds were separated within ~14 min. All tea types contained all the catechins, at varying concentrations, but not all the theaflavins were present in all the tea types. This indicates that the theaflavin composition reflects the degree of the fermentation and could be used for discrimination among different types of tea. All the green tea samples contained all eight catechins; however, the concentrations of these compounds varied among the tea samples. Principal component analysis and hierarchical cluster analysis were useful for discrimination of samples. It has been unclear whether the variations of chemical components are useful for forensic discrimination. Our results demonstrate that, in addition to identification of tea varieties, catechins and theaflavins can be used for the discrimination of bottled tea samples.

Keywords:  LC-MS/MS; bottled tea; catechin; hierarchical cluster analysis; principal component analysis; theaflavin

DOI:  https://doi.org/10.1111/1556-4029.14864
Nat Commun. 2021 08 17. 12(1): 4992

A hierarchical approach to removal of unwanted variation for large-scale metabolomics data.

Taiyun Kim, Owen Tang, Stephen T Vernon, Katharine A Kott, Yen Chin Koay, John Park, David E James, Stuart M Grieve, Terence P Speed, Pengyi Yang, Gemma A Figtree, John F O'Sullivan, Jean Yee Hwa Yang.

Liquid chromatography-mass spectrometry-based metabolomics studies are increasingly applied to large population cohorts, which run for several weeks or even years in data acquisition. This inevitably introduces unwanted intra- and inter-batch variations over time that can overshadow true biological signals and thus hinder potential biological discoveries. To date, normalisation approaches have struggled to mitigate the variability introduced by technical factors whilst preserving biological variance, especially for protracted acquisitions. Here, we propose a study design framework with an arrangement for embedding biological sample replicates to quantify variance within and between batches and a workflow that uses these replicates to remove unwanted variation in a hierarchical manner (hRUV). We use this design to produce a dataset of more than 1000 human plasma samples run over an extended period of time. We demonstrate significant improvement of hRUV over existing methods in preserving biological signals whilst removing unwanted variation for large scale metabolomics studies. Our tools not only provide a strategy for large scale data normalisation, but also provides guidance on the design strategy for large omics studies.

DOI: https://doi.org/10.1038/s41467-021-25210-5
Biomed Chromatogr. 2021 Jul 19. e5217

Using a stable isotope-labeled internal standard for liquid chromatography-tandem mass spectrometry quantitation of meloxicam in human plasma.

Cuijiao Zhan, Changmao Wang, Yaqin Wang, Haitang Xie, Jiru Chu, Rong Zhang, Rongfeng Hu, Jie Shen, Yuanwei Jia.

  A sensitive and highly efficient LC-ESI-MS/MS method using a stable isotope-labeled internal standard (SIL IS) to detect meloxicam in human plasma was developed and validated. Sample preparation used only 50 μL human plasma with one-step methanol protein precipitation. A gradient mobile phase system was adopted for chromatographic separation on a Poroshell 120 SB-C18 column (2.1 × 50 mm, 2.7 μm). Positive ion pattern was chosen for quantification under multiple reaction monitoring. Ion pairs were [M + H]+ m/z 352.1 → 115.1 for meloxicam and [M + H]+ m/z 355.1 → 187.1 for meloxicam-d3 (SIL IS). Total run time was 4.0 min. Standard curve was linear over a concentration range from 8.00 to 1600 ng mL-1 . This method was fully validated to evaluate its performance, including specificity, carryover, sensitivity, linearity, accuracy, precision, recovery, matrix effects, stability, dilution reliability and incurred sample reanalysis, which provided a reliable basis for pharmacokinetic studies of meloxicam in 28 healthy Chinese volunteers. After a single-dose oral administration of 7.5 mg meloxicam, the main pharmacokinetic parameters were as follows: Cmax , 814.79 ± 201.37 ng mL-1 ; Tmax , 4.54 ± 1.42 h; AUC0-t , 24,572.04 ± 5766.93 ng·h mL-1 ; AUC0-∞ , 25,810.89 ± 6796.60 ng·h mL-1 and t1/2 , 21.11 ± 5.35 h.

Keywords:  LC-ESI-MS/MS; isotope-labeled internal standard; meloxicam; pharmacokinetics

DOI:  https://doi.org/10.1002/bmc.5217
Chemosphere. 2021 Aug 09. pii: S0045-6535(21)02324-9. [Epub ahead of print]286(Pt 3): 131852

Comprehensive profile of DNA adducts as both tissue and urinary biomarkers of exposure to acrylamide and chemo-preventive effect of catechins in rats.

Yiju Zhang, Qiao Wang, Yaoran Li, Jun Cheng, Xinyu Chen, Yu Zhang.

  Two representative DNA adducts from acrylamide exposure, N7-(2-carbamoyl-2-hydroxyethyl) guanine (N7-GA-Gua) and N3-(2-carbamoyl-2-hydroxyethyl) adenine (N3-GA-Ade), are important long-term exposure biomarkers for evaluating genotoxicity of acrylamide. Catechins as natural antioxidants present in tea possess multiple health benefits, and may also have the potential to protect against acrylamide-induced DNA damage. The current study developed an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous analysis of N7-GA-Gua and N3-GA-Ade in tissues and urine. The validated UHPLC-MS/MS method showed high sensitivity, with limit of detection and limit of quantification ranging 0.2-0.8 and 0.5-1.5 ng/mL, respectively, and achieved qualified precision (RSD<14.0%) and spiking recovery (87.2%-110.0%) with elution within 6 min, which was suitable for the analysis of the two DNA adducts in different matrices. The levels of N7-GA-Gua and N3-GA-Ade ranged 0.9-11.9 and 0.6-3.5 μg/g creatinine in human urine samples, respectively. To investigate the interventional effects of catechins on the two DNA adducts from acrylamide exposure, rats were supplemented with three types of catechins (tea polyphenols, epigallocatechin gallate, and epicatechin) 30 min before administration with acrylamide. Our results showed that catechins effectively inhibited the formation of DNA adducts from acrylamide exposure in both urine and tissues of rats. Among three catechins, epicatechin performed the best inhibitory effect. The current study provided evidence for the chemo-preventive effect of catechins, indicating that dietary supplement of catechins may contribute to health protection against exposure to acrylamide.

Keywords:  Acrylamide; Biomarker; Catechins; Chemo-preventive effect; DNA adducts

DOI:  https://doi.org/10.1016/j.chemosphere.2021.131852