bims-metlip 2021-05-02 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2021–05–02
seventeen papers selected by
Sofia Costa, Cold Spring Harbor Laboratory

In Vivo Estimation of Ketogenesis Using Metabolic Flux Analysis-Technical Aspects and Model Interpretation.
Development and validation of a fast ultra high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for determining carbonic anhydrase inhibitors and their metabolites in urine and hair.
Metabolomics, Lipidomics, and Immunometabolism.
Validation of a novel UPLC-MS/MS method for estimation of metformin and empagliflozin simultaneously in human plasma using freezing lipid precipitation approach and its application to pharmacokinetic study.
Targeted and Untargeted Metabolic Profiling to Discover Bioactive Compounds in Seaweeds and Hemp Using Gas and Liquid Chromatography-Mass Spectrometry.
Application of Lipid Class Ratios for Sample Stability Monitoring-Evaluation of Murine Tissue Homogenates and SDS as a Stabilizer.
Online In-Tube Solid-Phase Microextraction Coupled to Liquid Chromatography-Tandem Mass Spectrometry for the Determination of Tobacco-Specific Nitrosamines in Hair Samples.
Determination of Antiviral Drugs and Their Metabolites Using Micro-Solid Phase Extraction and UHPLC-MS/MS in Reversed-Phase and Hydrophilic Interaction Chromatography Modes.
Metabolic Evaluation of Urine from Patients Diagnosed with High Grade (HG) Bladder Cancer by SPME-LC-MS Method.
A method for the analysis of 121 multi-class environmental chemicals in urine by high-performance liquid chromatography-tandem mass spectrometry.
Human Brain Lipidomics: Utilities of Chloride Adducts in Flow Injection Analysis.
Quantification of Intracellular Thiols by HPLC-Fluorescence Detection.
nLossFinder-A Graphical User Interface Program for the Nontargeted Detection of DNA Adducts.
A normalized signal calibration with a long-term reference improves the robustness of RPLC-MRM/MS lipidomics in plasma.
Clinical Evaluation of Assays for Plasma Renin Activity and Aldosterone Measurement by Liquid Chromatography-Tandem Mass Spectrometry.
Simultaneous determination of triclosan, triclocarban, triclocarban metabolites and byproducts in urine and serum by UHPLC-ESI-MS/MS.
An Analytical Toolbox for Fast and Straightforward Structural Characterisation of Commercially Available Tannins.

Metabolites. 2021 Apr 28. pii: 279. [Epub ahead of print]11(5):

In Vivo Estimation of Ketogenesis Using Metabolic Flux Analysis-Technical Aspects and Model Interpretation.

Stanislaw Deja, Blanka Kucejova, Xiaorong Fu, Jeffrey D Browning, Jamey D Young, Shawn Burgess.

  Ketogenesis occurs in liver mitochondria where acetyl-CoA molecules, derived from lipid oxidation, are condensed into acetoacetate (AcAc) and reduced to β-hydroxybutyrate (BHB). During carbohydrate scarcity, these two ketones are released into circulation at high rates and used as oxidative fuels in peripheral tissues. Despite their physiological relevance and emerging roles in a variety of diseases, endogenous ketone production is rarely measured in vivo using tracer approaches. Accurate determination of this flux requires a two-pool model, simultaneous BHB and AcAc tracers, and special consideration for the stability of the AcAc tracer and analyte. We describe the implementation of a two-pool model using a metabolic flux analysis (MFA) approach that simultaneously regresses liquid chromatography-tandem mass spectrometry (LC-MS/MS) ketone isotopologues and tracer infusion rates. Additionally, 1H NMR real-time reaction monitoring was used to evaluate AcAc tracer and analyte stability during infusion and sample analysis, which were critical for accurate flux calculations. The approach quantifies AcAc and BHB pool sizes and their rates of appearance, disposal, and exchange. Regression analysis provides confidence intervals and detects potential errors in experimental data. Complications for the physiological interpretation of individual ketone fluxes are discussed.

Keywords:  13C MFA; 1H NMR; BHB; LC-MS/MS; acetoacetate; flux; in vivo; ketogenesis; liver; metabolism; metabolomics; stable isotope

DOI:  https://doi.org/10.3390/metabo11050279
Drug Test Anal. 2021 Apr 27.

Development and validation of a fast ultra high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for determining carbonic anhydrase inhibitors and their metabolites in urine and hair.

Alfredo Fabrizio Lo Faro, Anastasio Tini, Massimo Gottardi, Filippo Pirani, Ascanio Sirignano, Raffaele Giorgetti, Francesco Paolo Busardò.

  A new, rapid, sensitive and comprehensive ultra-high-performance liquid chromatographic-mass spectrometry (UHPLC-MS/MS) method for quantifying diuretics (acetazolamide, brinzolamide, dorzolamide and their metabolites) in human urine and hair was developed and fully validated. Twenty-five milligrams of hair, were incubated with 500 μL M3® buffer reagent at 100°C for 1 h for complete digestion. After cooling, 1 μL supernatant was injected onto chromatography system. Urine samples were simply diluted before injection. The chromatographic run time was short (8 min) through a column with a mobile phase gradient. The method was linear (determination coefficients always higher than 0.99) from limit of quantification (LOQ)-500 ng/mL in urine and from LOQ-10 ng/mg in hair. LOQs ranged from 0.07-1.16 ng/mL in urine and from 0.02-0.15 ng/mg in hair. No significant ion suppression due to matrix effect was observed and process efficiency was always higher than 80%. Intra- and inter-assay precision was lower than 15%. The suitability of the methods was tested with six urine and hair specimens from patients treated with acetazolamide, dorzolamide or brinzolamide for ocular diseases or systemic hypertension. Average urine concentrations were 266.32 ng/mL for dorzolamide and 47.61 ng/mL for N-deethyl-dorzolamide (n=3), 109.27 ng/mL for brinzolamide) and 1.02 ng/mL for O-desmethyl-brinzolamide (n=2) and finally 12.63 ng/mL for acetazolamide. Average hair concentrations were 5.94 ng/mg for dorzolamide and 0.048 ng/mg for N-deethyl-dorzolamide (n=3); 3.26 ng/mg for brinzolamide (n=2) and 2.3 ng/mg for acetazolamide (n=1). The developed method was simple and fast both in the extraction procedure making it eligible in high-throughput analysis for clinical forensic and doping purposes.

Keywords:  UHPLC-MS/MS; carbonic anhydrase inhibitors; diuretics; hair; urine

DOI:  https://doi.org/10.1002/dta.3055
Methods Mol Biol. 2021 ;2285 319-328

Metabolomics, Lipidomics, and Immunometabolism.

Fortunata Carbone, Sara Bruzzaniti, Clorinda Fusco, Alessandra Colamatteo, Teresa Micillo, Paola De Candia, Fabrizia Bonacina, Giuseppe Danilo Norata, Giuseppe Matarese.

  Metabolomics, lipidomics, and the study of cellular metabolism are gaining increasing interest particularly in the field of immunology, since the activation and effector functions of immune cells are profoundly controlled by changes in cellular metabolic asset. Among the different techniques that can be used for the evaluation of cellular metabolism, the Seahorse Extracellular Flux Analyzer allows the real time measurement of both glycolytic and mitochondrial respiration pathways in cells of interest, through the assessment of extracellular acidification and oxygen consumption rate. Metabolomics, on the other hand, is the high-throughput analysis of metabolites, i.e., the substrates, intermediates, and products of cellular metabolism, starting from biofluids, cells or tissues. The metabolome does not include lipids as their properties are different from water-soluble metabolites and are classified under the lipidome. Lipidomics analysis allows the identification and quantification of lipid species. Metabolomics and lipidomics are currently performed with mass-spectrometry coupled with liquid or gas chromatography (LC-MS or GC-MS) and/or nuclear-magnetic resonance (NMR). Here we describe the protocol for the evaluation of metabolic rate, metabolomics, and lipidomics in T cells, examining the detailed experimental approaches.

Keywords:  Bioenergetics; Glycolysis; Lipidomics; Metabolism; Metabolomics; Mitochondrial respiration; T cells

DOI:  https://doi.org/10.1007/978-1-0716-1311-5_24
J Pharm Biomed Anal. 2021 Apr 18. pii: S0731-7085(21)00189-8. [Epub ahead of print]200 114078

Validation of a novel UPLC-MS/MS method for estimation of metformin and empagliflozin simultaneously in human plasma using freezing lipid precipitation approach and its application to pharmacokinetic study.

M N Abou-Omar, M Kenawy, A O Youssef, Sarah Alharthi, M S Attia, Ekram H Mohamed.

  A fast, sensitive one step UPLC ESI-MS/MS method was successfully applied for the simultaneous estimation of two concurrently administrated antidiabetic drugs, Metformin (MET) and Empagliflozin (EMPA) in human plasma. Metformin-d6 (MET-d6) and Empagliflozin-d4 (EMPA-d4) were utilized as internal standards. Extraction of the analytes from the human plasma was performed through acetonitrile precipitation technique followed by freezing the precipitated plasma proteins and lipids to minimize the matrix effect. Chromatographic analysis was developed on Acquity UPLC BEH C18 column (1.7 μm, 2.1 × 50 mm) using isocratic elution mode. A mobile phase of formic acid (0.01 %): acetonitrile (70:30 v/v) with a flow rate of 0.3 mL/min achieved optimum separation. Multiple reaction monitoring (MRM) in positive ion mode, with transitions at (m/z) 130.14 →71.08 for (MET), 451.72 →71.29 for (EMPA), 136.03 →77.02 for (MET-d6), and 455.43 → 75.05 for (EMPA-d4) was used for quantification. The obtained linearity covered the concentration ranges of 10-1500 ng/mL and 2.0-250.0 ng/mL for MET and EMPA, respectively. The run time of the proposed Method didn't exceed 3.0 min allowing faster analysis and determination of larger number of samples per day without affecting accuracy and sensitivity. The presented chromatographic method could be successfully applied in pharmacokinetics studies and therapeutic monitoring of MET and EMPA in patients' plasma administrating fixed dose combination of both drug with high reproducibility and ruggedness.

Keywords:  Antidiabetic drugs; Empagliflozin; Matrix effect; Metformin; Pharmacokinetic; UPLC–MS/MS

DOI:  https://doi.org/10.1016/j.jpba.2021.114078
Metabolites. 2021 Apr 22. pii: 259. [Epub ahead of print]11(5):

Targeted and Untargeted Metabolic Profiling to Discover Bioactive Compounds in Seaweeds and Hemp Using Gas and Liquid Chromatography-Mass Spectrometry.

Natalja P Nørskov, Annette Bruhn, Andrew Cole, Mette Olaf Nielsen.

  Greenhouse gas emissions are a global problem facing the dairy/beef industry. Novel feed additives consisting of seaweeds and hemp containing bioactive compounds are theorized to reduce enteric methane emissions. In this study we aimed to investigate the metabolic profiles of brown, red and green seaweeds and hemp using gas chromatography and liquid chromatography mass spectrometry. We used targeted and untargeted approaches, quantifying known halomethanes and phenolics, as well as identifying potentially novel bioactive compounds with anti-methanogenic properties. The main findings were: (a) Asparagopsis taxiformis contained halomethanes, with high concentrations of bromoform (4200 µg/g DW), six volatile halocarbons were tentatively identified; (b) no halomethanes were detected in the other studied seaweeds nor in hemp; (c) high concentrations of lignans were measured in hemp; (d) a high numbers of sulfated phenolic acids and unidentified sulfuric acid-containing compounds were detected in all seaweeds; (e) flavonoid glucosides and glucuronides were mainly identified in hemp; and (f) the condensed tannin gallocatechin was tentatively identified in Fucus sp. Using the combined metabolomics approach, an overview and in-depth information on secondary metabolites were provided. Halomethanes of Asparagopsis sp. have already been shown to be anti-methanogenic; however, metabolic profiles of seaweeds such as Dictyota and Sargassum have also been shown to contain compounds that may have anti-methanogenic potential.

Keywords:  Gas Chromatography-Mass Spectrometry (GC-MS); Liquid Chromatography-Mass Spectrometry (LC-MS); greenhouse gasses; halomethanes; hemp; metabolomic profiling; methane; phenolics; seaweeds

DOI:  https://doi.org/10.3390/metabo11050259
Metabolites. 2021 Apr 27. pii: 277. [Epub ahead of print]11(5):

Application of Lipid Class Ratios for Sample Stability Monitoring-Evaluation of Murine Tissue Homogenates and SDS as a Stabilizer.

Sabrina Krautbauer, Raquel Blazquez, Gerhard Liebisch, Marcus Hoering, Philip Neubert, Tobias Pukrop, Ralph Burkhardt, Alexander Sigruener.

  Lipids are a ubiquitous class of structurally complex molecules involved in various biological processes. In the fast-growing field of lipidomics, preanalytical issues are frequently neglected. Here, we investigated the stability of lipid profiles of murine liver, brain, lung, heart, and spleen homogenates by quantitative flow injection analysis using tandem mass spectrometry and high-resolution mass spectrometry. Storage of tissue homogenates at room temperature showed substantial alterations of the lipid profiles reflecting lipolytic action. Therefore, ratios of ceramide to sphingomyelin, lysophosphatidylethanolamine to phosphatidylethanolamine, lysophosphatidylcholine to phosphatidylcholine, and diglyceride to triglyceride were applied to monitor sample stability and the effect of sodium dodecyl sulfate (SDS) as a potential stabilizing agent. The addition of SDS led to a concentration-dependent stabilization of lipid profiles in liver, brain, and heart homogenates, while in lung and spleen homogenates, in particular, the lysophosphatidylethanolamine to phosphatidylethanolamine ratio increased upon addition of SDS. In conclusion, we demonstrated that lipid class ratios reflecting lipolytic activity could be applied to evaluate both the stability of samples and the influence of stabilizers.

Keywords:  lipid class ratio; lipidomics; lipolytic ratios; mass spectrometry; sodium dodecyl sulfate; stabilization; tissue

DOI:  https://doi.org/10.3390/metabo11050277
Molecules. 2021 Apr 03. pii: 2056. [Epub ahead of print]26(7):

Online In-Tube Solid-Phase Microextraction Coupled to Liquid Chromatography-Tandem Mass Spectrometry for the Determination of Tobacco-Specific Nitrosamines in Hair Samples.

Atsushi Ishizaki, Hiroyuki Kataoka.

  Active and passive smoking are serious public health concerns Assessment of tobacco smoke exposure using effective biomarkers is needed. In this study, we developed a simultaneous determination method of five tobacco-specific nitrosamines (TSNAs) in hair by online in-tube solid-phase microextraction (SPME) coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS). TSNAs were extracted and concentrated on Supel-Q PLOT capillary by in-tube SPME and separated and detected within 5 min by LC-MS/MS using Capcell Pak C18 MGIII column and positive ion mode multiple reaction monitoring systems. These operations were fully automated by an online program. The calibration curves of TSNAs showed good linearity in the range of 0.5-1000 pg mL-1 using their stable isotope-labeled internal standards. Moreover, the limits of detection (S/N = 3) of TSNAs were in the range of 0.02-1.14 pg mL-1, and intra-day and inter-day precisions were below 7.3% and 9.2% (n = 5), respectively. The developed method is highly sensitive and specific and can easily measure TSNA levels using 5 mg hair samples. This method was used to assess long-term exposure levels to tobacco smoke in smokers and non-smokers.

Keywords:  exposure biomarkers; hair; in-tube solid-phase microextraction (SPME); liquid chromatography-tandem mass spectrometry (LC–MS/MS); tobacco-specific nitrosamines

DOI:  https://doi.org/10.3390/molecules26072056
Molecules. 2021 Apr 07. pii: 2123. [Epub ahead of print]26(8):

Determination of Antiviral Drugs and Their Metabolites Using Micro-Solid Phase Extraction and UHPLC-MS/MS in Reversed-Phase and Hydrophilic Interaction Chromatography Modes.

Luboš Fical, Maria Khalikova, Hana Kočová Vlčková, Ivona Lhotská, Zuzana Hadysová, Ivan Vokřál, Lukáš Červený, František Švec, Lucie Nováková.

  Two new ultra-high performance liquid chromatography (UHPLC) methods for analyzing 21 selected antivirals and their metabolites were optimized, including sample preparation step, LC separation conditions, and tandem mass spectrometry detection. Micro-solid phase extraction in pipette tips was used to extract antivirals from the biological material of Hanks balanced salt medium of pH 7.4 and 6.5. These media were used in experiments to evaluate the membrane transport of antiviral drugs. Challenging diversity of physicochemical properties was overcome using combined sorbent composed of C18 and ion exchange moiety, which finally allowed to cover the whole range of tested antivirals. For separation, reversed-phase (RP) chromatography and hydrophilic interaction liquid chromatography (HILIC), were optimized using extensive screening of stationary and mobile phase combinations. Optimized RP-UHPLC separation was carried out using BEH Shield RP18 stationary phase and gradient elution with 25 mmol/L formic acid in acetonitrile and in water. HILIC separation was accomplished with a Cortecs HILIC column and gradient elution with 25 mmol/L ammonium formate pH 3 and acetonitrile. Tandem mass spectrometry (MS/MS) conditions were optimized in both chromatographic modes, but obtained results revealed only a little difference in parameters of capillary voltage and cone voltage. While RP-UHPLC-MS/MS exhibited superior separation selectivity, HILIC-UHPLC-MS/MS has shown substantially higher sensitivity of two orders of magnitude for many compounds. Method validation results indicated that HILIC mode was more suitable for multianalyte methods. Despite better separation selectivity achieved in RP-UHPLC-MS/MS, the matrix effects were noticed while using both chromatographic modes leading to signal enhancement in RP and signal suppression in HILIC.

Keywords:  UHPLC-MS/MS; antiviral drug; hydrophilic interaction chromatography; microextraction; reversed phase; solid phase extraction

DOI:  https://doi.org/10.3390/molecules26082123
Molecules. 2021 Apr 11. pii: 2194. [Epub ahead of print]26(8):

Metabolic Evaluation of Urine from Patients Diagnosed with High Grade (HG) Bladder Cancer by SPME-LC-MS Method.

Kamil Łuczykowski, Natalia Warmuzińska, Sylwia Operacz, Iga Stryjak, Joanna Bogusiewicz, Julia Jacyna, Renata Wawrzyniak, Wiktoria Struck-Lewicka, Michał J Markuszewski, Barbara Bojko.

  Bladder cancer (BC) is a common malignancy of the urinary system and a leading cause of death worldwide. In this work, untargeted metabolomic profiling of biological fluids is presented as a non-invasive tool for bladder cancer biomarker discovery as a first step towards developing superior methods for detection, treatment, and prevention well as to further our current understanding of this disease. In this study, urine samples from 24 healthy volunteers and 24 BC patients were subjected to metabolomic profiling using high throughput solid-phase microextraction (SPME) in thin-film format and reversed-phase high-performance liquid chromatography coupled with a Q Exactive Focus Orbitrap mass spectrometer. The chemometric analysis enabled the selection of metabolites contributing to the observed separation of BC patients from the control group. Relevant differences were demonstrated for phenylalanine metabolism compounds, i.e., benzoic acid, hippuric acid, and 4-hydroxycinnamic acid. Furthermore, compounds involved in the metabolism of histidine, beta-alanine, and glycerophospholipids were also identified. Thin-film SPME can be efficiently used as an alternative approach to other traditional urine sample preparation methods, demonstrating the SPME technique as a simple and efficient tool for urinary metabolomics research. Moreover, this study's results may support a better understanding of bladder cancer development and progression mechanisms.

Keywords:  bladder cancer (BC); liquid chromatography; mass spectrometry; metabolomics; solid phase microextraction (SPME); urine

DOI:  https://doi.org/10.3390/molecules26082194
J Chromatogr A. 2021 Apr 09. pii: S0021-9673(21)00270-3. [Epub ahead of print]1646 462146

A method for the analysis of 121 multi-class environmental chemicals in urine by high-performance liquid chromatography-tandem mass spectrometry.

Hongkai Zhu, Sridhar Chinthakindi, Kurunthachalam Kannan.

  Biomonitoring of human exposure to environmental chemicals has gained momentum in recent years. Biomonitoring methods often include analysis of a single class of chemicals with similar chemical properties. In this study, we describe a method that involves solid-phase extraction (SPE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and capable of measuring 121 environmental chemicals comprising plasticizers (PMs; n = 45), environmental phenols (EPs; n = 45), and pesticides (n = 31) through a single extraction of urine. Urine samples were incubated with 20 µL of β-glucuronidase/arylsulfatase (4000 units/mL urine) (from Helix pomatia) buffered at pH 5.5 for 2 h at 37 °C for optimal deconjugation conditions. We compared two extraction methods, namely liquid-liquid extraction and SPE, and the latter with ABS Elut NEXUS® cartridges was optimized to yield best extraction efficiencies. For increased resolution and chromatographic separation, two methods involving Ultra AQ C18® and Betasil™ C18® columns were used. The MS/MS analyses were performed under both negative and positive ionization modes. The optimized method yielded excellent intra- and inter-day variabilities (relative standard deviation: 0.40-11%) and satisfactory recoveries (80-120%) for >95% of the analytes. The limits of detection were ≤ 0.1 ng/mL for 101 analytes and between 0.1 and 1.0 ng/mL for 18 analytes. The optimized SPE LC-MS/MS method was validated through the analysis of standard reference materials and proficiency test urine samples and further applied in the analysis of 21 real urine samples to demonstrate simultaneous determination of 121 environmental chemicals in urine samples.

Keywords:  Endocrine disruptors; Exposome; Human biomonitoring; LC-MS/MS; Multi-class method; Urine

DOI:  https://doi.org/10.1016/j.chroma.2021.462146
Life (Basel). 2021 Apr 28. pii: 403. [Epub ahead of print]11(5):

Human Brain Lipidomics: Utilities of Chloride Adducts in Flow Injection Analysis.

Paul L Wood, Kathleen A Hauther, Jon H Scarborough, Dustin J Craney, Beatrix Dudzik, John E Cebak, Randall L Woltjer.

  Ceramides have been implicated in a number of disease processes. However, current means of evaluation with flow infusion analysis (FIA) have been limited primarily due to poor sensitivity within our high-resolution mass spectrometry lipidomics analytical platform. To circumvent this deficiency, we investigated the potential of chloride adducts as an alternative method to improve sensitivity with electrospray ionization. Chloride adducts of ceramides and ceramide subfamilies provided 2- to 50-fold increases in sensitivity both with analytical standards and biological samples. Chloride adducts of a number of other lipids with reactive hydroxy groups were also enhanced. For example, monogalactosyl diacylglycerols (MGDGs), extracted from frontal lobe cortical gray and subcortical white matter of cognitively intact subjects, were not detected as ammonium adducts but were readily detected as chloride adducts. Hydroxy lipids demonstrate a high level of specificity in that phosphoglycerols and phosphoinositols do not form chloride adducts. In the case of choline glycerophospholipids, the fatty acid substituents of these lipids could be monitored by MS2 of the chloride adducts. Monitoring the chloride adducts of a number of key lipids offers enhanced sensitivity and specificity with FIA. In the case of glycerophosphocholines, the chloride adducts also allow determination of fatty acid substituents. The chloride adducts of lipids possessing electrophilic hydrogens of hydroxyl groups provide significant increases in sensitivity. In the case of glycerophosphocholines, chloride attachment to the quaternary ammonium group generates a dominant anion, which provides the identities of the fatty acid substituents under MS2 conditions.

Keywords:  ceramides; chloride adducts; flow infusion analysis; glycerophosphocholines; human brain; sphingolipids

DOI:  https://doi.org/10.3390/life11050403
Molecules. 2021 Apr 19. pii: 2365. [Epub ahead of print]26(8):

Quantification of Intracellular Thiols by HPLC-Fluorescence Detection.

Hiroki Yamamoto, Takuya Fujiwara, Takashi Funatsu, Makoto Tsunoda.

  Biothiols, such as cysteine and glutathione, play important roles in various intracellular reactions represented by the redox equilibrium against oxidative stress. In this study, a method for intracellular thiol quantification using HPLC-fluorescence detection was developed. Thiols were derivatized with a thiol-specific fluorescence derivatization reagent, viz. ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F), followed by reversed-phase separation on an InertSustain AQ-C18 column. Six different SBD-thiols (homocysteine, cysteine, cysteinylglycine, γ-glutamylcysteine, glutathione, and N-acetylcysteine as an internal standard) were separated within 30 min using a citric buffer (pH 3.0)/MeOH mobile phase. The calibration curves of all the SBD-thiols had strong linearity (R2 > 0.999). Using this developed method, the thiol concentrations of human chronic myelogenous leukemia K562 cell samples were found to be 5.5-153 pmol/1 × 106 cells. The time-dependent effect of a thiol scavenger, viz. N-ethyl maleimide, on intracellular thiol concentrations was also quantified. This method is useful for elucidating the role of intracellular sulfur metabolism.

Keywords:  cysteine; derivatization; glutathione; oxidative stress

DOI:  https://doi.org/10.3390/molecules26082365
Toxics. 2021 Apr 07. pii: 78. [Epub ahead of print]9(4):

nLossFinder-A Graphical User Interface Program for the Nontargeted Detection of DNA Adducts.

Pedro F M Sousa, Giulia Martella, K Magnus Åberg, Bahare Esfahani, Hitesh V Motwani.

  DNA adductomics is a relatively new omics approach aiming to measure known and unknown DNA modifications, called DNA adducts. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the most common method for analyzing DNA adducts. Recent advances in the field of mass spectrometry have allowed the possibility to perform a comprehensive analysis of adducts, for instance, by using a nontargeted data-independent acquisition method, with multiple precursor m/z windows as an inclusion list. However, the generated data are large and complex, and there is a need to develop algorithms to simplify and automate the time-consuming manual analysis that has hitherto been used. Here, a graphical user interface (GUI) program was developed, with the purpose of tracking a characteristic neutral loss reaction from tandem mass spectrometry of the nucleoside adducts. This program, called nLossFinder, was developed in the MATLAB platform, available as open-source code. Calf thymus DNA was used as a model for method optimization, and the overall adductomics approach was applied to DNA from amphipods (Monoporeia affinis) collected within the Swedish National Marine Monitoring Program. In the amphipod DNA, over 150 putative adducts were found in comparison to 18 using a manual approach in a previous study. The developed program can improve the processing time for large MS data, as it processes each sample in a few seconds, and hence can be applicable for high-throughput screening of adducts.

Keywords:  DNA adducts; MATLAB; data-independent acquisition; environmental monitoring; high-resolution mass spectrometry

DOI:  https://doi.org/10.3390/toxics9040078
Anal Bioanal Chem. 2021 Apr 28.

A normalized signal calibration with a long-term reference improves the robustness of RPLC-MRM/MS lipidomics in plasma.

Bohdan Babiy, Rebeca Busto, Óscar Pastor.

  Improving the reliability of quantification in lipidomic analyses is crucial for its successful application in the discovery of new biomarkers or in clinical practice. In this study, we propose a workflow to improve the accuracy and precision of lipidomic results issued by the laboratory. Lipid species from 11 classes were analyzed by a targeted RPLC-MRM/MS method. The peak areas of species were used to estimate concentrations by an internal standard calibration approach (IS-calibration) and by an alternative normalization signal calibration schema (NS-calibration). The latter uses a long-term reference plasma material as a matrix-matched external calibrator whose accuracy was compared to the NIST SRM-1950 mean consensus values reported by the Interlaboratory Lipidomics Comparison Exercise. The bias of lipid concentrations showed a good accuracy for 69 of 89 quantified lipids. The quantitation of species by the NS-calibration schema improved the within- and between-batch reproducibility in quality control samples, in comparison to the usual IS-calibration approach. Moreover, the NS-calibration workflow improved the robustness of the lipidomics measurements reducing the between-batch variability (relative standard deviation <10% for 95% of lipid species) in real conditions tested throughout the analysis of 120 plasma samples. In addition, we provide a free access web tool to obtain the concentration of lipid species by the two previously mentioned quantitative approaches, providing an easy follow-up of quality control tasks related to lipidomics.

Keywords:  Lipidomics; Mass spectrometry; Molar concentration; NIST SRM-1950; Quality assurance; Software

DOI:  https://doi.org/10.1007/s00216-021-03364-x
J Appl Lab Med. 2021 Apr 29. 6(3): 668-678

Clinical Evaluation of Assays for Plasma Renin Activity and Aldosterone Measurement by Liquid Chromatography-Tandem Mass Spectrometry.

Stephen M Gibbons, Helen P Field, April Fairhurst, Anne Fleming, Clive Ford, Emma L Williams, Sophie C Barnes, Julian H Barth.

BACKGROUND: Aldosterone and renin are pivotal hormones in the regulation of salt and water homeostasis and blood pressure. Measurement of renin and aldosterone in serum/plasma is essential for the investigation of primary hyperaldosteronism (PA) and monitoring of glucocorticoid replacement therapy.
METHODS: We report 2 LC-MS/MS methods developed to measure aldosterone and plasma renin activity (PRA). PRA was determined by endogenous enzymatic generation of angiotensin I using 150 µL of sample. Generated angiotensin I was purified by solid phase extraction prior to chromatographic separation and mass spectrometry. Aldosterone measurement required 300 μL of sample extracted with MTBE prior to LC-MS/MS analysis.
RESULTS: The PRA method was linear (1.2-193 nmol/L), sensitive (LLOQ = 1.2 nmol/L), precise (CV = 4.1%), and specific (no cross reactivity for a number of structurally similar steroids). Dilutional linearity and recovery (84%) were acceptable. Accuracy was confirmed by comparison against our current RIA method. The aldosterone method had equally acceptable performance characteristics. Reference ranges in 110 healthy normotensive subjects were: PRA 0.2-3.7 nmol/L/h and aldosterone 50-950 pmol/L. Consecutive patients (n = 62) with adrenal incidentalomas shown to have no functional adrenal disease; their post overnight 1 mg dexamethasone test values were: PRA 0.2-2.6 nmol/L/h and aldosterone 55-480 pmol/L. Serum aldosterone values after 2 liter saline suppression were-normal subjects (n = 17): 78-238 pmol/L and confirmed primary hyperaldosteronism (n = 25): 131-1080 pmol/L.
CONCLUSIONS: We have developed robust assays for PRA and aldosterone with appropriate clinical evaluation. These assays are now in routine practice in the UK.

DOI: https://doi.org/10.1093/jalm/jfaa177
Rapid Commun Mass Spectrom. 2021 Apr 29. e9117

Simultaneous determination of triclosan, triclocarban, triclocarban metabolites and byproducts in urine and serum by UHPLC-ESI-MS/MS.

Qiong Luo, Hongna Zhang, Yanqiu Zhou, Zehua Liu, Zongwei Cai.

RATIONAL: Triclosan (TCS) and triclocarban (TCC) are ubiquitous antimicrobial agents incorporated in consumer and personal care products. Due to their human health risks, it is essential to develop a sensitive and accurate analytical method to simultaneously quantify TCS, TCC, as well as their metabolites and byproducts in urine and serum samples.
METHODS: The quantitative parameters of TCS, TCC, TCC metabolites and byproducts (2'-OH-TCC, 3'-OH-TCC, 6-OH-TCC, DHC, DCC, NCC) were optimized by using the ultra-high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS). Enzymatic hydrolysis of the samples was optimized based on enzyme dosage and incubation time. The efficiencies of solid phase extraction (SPE) and liquid-liquid extraction (LLE) were compared. The effectiveness of the established method was evaluated, and method application was validated using real urine and serum samples.
RESULTS: The conjugates were sufficiently hydrolyzed under 500 U/mL β-glucuronidase and 80 U/mL sulfatase at 37°C for 4 h. Compared with LLE method, SPE achieved higher extraction efficiency in both urine and serum samples. The optimized SPE-UHPLC-ESI-MS/MS method showed low limits of detection (LODs) ranging 0.001-0.3 ng/mL and good linearity (R2 >0.99) at 0.01-150 ng/mL in both matrices. Excellent recoveries of 82.0%-120.7% (urine) and 76.7%-113.9% (serum) were obtained with low relative standard deviation (RSD, <7.6%) for inter-day and intra-day injections. This method was applicable to quantify target compounds in multiple biological urine and serum samples. Notably, TCS and TCC were detected with average concentrations of 8.37 and 10.46 ng/mL, respectively, in 15 Chinese female urines, with the simultaneous detection of TCC metabolites and byproducts.
CONCLUSION: A reliable method was established to simultaneously determine TCS, TCC, TCC metabolites and byproducts in urine and serum samples by using UHPLC-ESI-MS/MS. This sensitive methodology provides basis for the evaluation of TCS and TCC exposure at the metabolic level.

DOI: https://doi.org/10.1002/rcm.9117
Molecules. 2021 Apr 26. pii: 2532. [Epub ahead of print]26(9):

An Analytical Toolbox for Fast and Straightforward Structural Characterisation of Commercially Available Tannins.

Lili Zhen, Heiko Lange, Claudia Crestini.

  Both condensed and hydrolysable tannins represent versatile natural polyphenolic structures exhibiting a broad range of activities that could be exploited in various fields including nutraceutics, cosmesis, consumer care, household and pharmaceutical applications. Various tannins are commercially available nowadays for use in such application fields. We have analysed a representative selection of commercially available condensed and hydrolysable tannins for structural features and purity. Using a combination of quantitative 31P NMR spectroscopy, HSQC measurements, MALDI-ToF analyses, gel permeation chromatography and wet chemical analysis, detailed structural characterisations and descriptions were possible, allowing for verification and falsification of claimed structural features.

Keywords:  31P NMR; HSQC; MALDI-ToF; condensed tannins; hydrolysable tannins

DOI:  https://doi.org/10.3390/molecules26092532