bims-metlip 2021-10-24 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2021–10–24
twenty papers selected by
Sofia Costa, Icahn School of Medicine at Mount Sinai

Recommendations for Good Practice in Mass Spectrometry-Based Lipidomics.
Progress and Challenges in Quantifying Carbonyl-Metabolomic Phenomes with LC-MS/MS.
MSCAT: A Machine Learning Assisted Catalog of Metabolomics Software Tools.
Nontargeted Serum Lipid Profiling of Nonalcoholic Steatohepatitis by Multisegment Injection-Nonaqueous Capillary Electrophoresis-Mass Spectrometry: A Multiplexed Separation Platform for Resolving Ionic Lipids.
Automated Trimethyl Sulfonium Hydroxide Derivatization Method for High-Throughput Fatty Acid Profiling by Gas Chromatography-Mass Spectrometry.
Comparison of Kit-Based Metabolomics with Other Methodologies in a Large Cohort, towards Establishing Reference Values.
Development of a high-throughput method for the comprehensive lipid analysis in milk using ultra-high performance supercritical fluid chromatography combined with quadrupole time-of-flight mass spectrometry.
Isotope dilution LC-MS/MS quantification of the cystic fibrosis transmembrane conductance regulator (CFTR) modulators ivacaftor, lumacaftor, tezacaftor, elexacaftor, and their major metabolites in human serum.
Lipid Profiling in Cancer Diagnosis with Hand-Held Ambient Mass Spectrometry Probes: Addressing the Late-Stage Performance Concerns.
A UHPLC-MS/MS Method for Therapeutic Drug Monitoring of Aciclovir and Ganciclovir in Plasma and Dried Plasma Spots.
Analysis of urinary organic acids by gas chromatography tandem mass spectrometry method for metabolic profiling applications.
UPLC-TOF-MS Method for Simultaneous Quantification of Steroid Hormones in Tissue Homogenates of Zebrafish with Solid-Phase Extraction.
Metabolomics of Chlorophylls and Carotenoids: Analytical Methods and Metabolome-Based Studies.
Multicenter Evaluation of a New, Fully Automated Androstenedione Electrochemiluminescence Immunoassay: Precision Analysis, Method Comparison, and Determination of Reference Ranges.
Analytical Methodology for a Metabolome Atlas of Goat's Plasma, Milk and Feces Using 1H-NMR and UHPLC-HRMS.
Rapid simultaneous determination of ketamine and midazolam in biological samples using ion mobility spectrometry combined by headspace solid-phase microextraction.
New Advances in Tissue Metabolomics: A Review.
Simultaneous determination of multiple isomeric hydroxylated polycyclic aromatic hydrocarbons in urine by using ultra-high performance liquid chromatography tandem mass spectrometry.
Robot-Assisted SpiderMass for In Vivo Real-Time Topography Mass Spectrometry Imaging.
Targeted volatolomics of human monocytes: Comparison of 2D-GC/TOF-MS and 1D-GC/Orbitrap-MS methods.

J Lipid Res. 2021 Oct 15. pii: S0022-2275(21)00120-6. [Epub ahead of print] 100138

Recommendations for Good Practice in Mass Spectrometry-Based Lipidomics.

Harald C Köfeler, Robert Ahrends, Erin S Baker, Kim Ekroos, Xianlin Han, Nils Hoffmann, Michal Holčapek, Markus R Wenk, Gerhard Liebisch.

  In the last two decades, lipidomics has become one of the fastest expanding scientific disciplines in biomedical research. With an increasing number of new research groups to the field, it is even more important to design guidelines for assuring high standards of data quality. The Lipidomics Standards Initiative (LSI) is a community-based endeavor for the coordination of development of these best practice guidelines in lipidomics, and is embedded within the International Lipidomics Society (ILS). It is the intention of this review to highlight the most quality-relevant aspects of the lipidomics workflow, including preanalytics, sample preparation, mass spectrometry, and lipid species identification and quantitation. Furthermore, this review does not just highlight examples of best practice, but also sheds light on strengths, drawbacks, and pitfalls in the lipidomic analysis workflow. While this review is not designed to be a step-by-step protocol by itself, nor to be dedicated to a specific application of lipidomics, it should nevertheless provide the interested reader with links and original publications to obtain a comprehensive overview concerning the state of the art practices in the field.

Keywords:  Chromatography; Ion Mobility Spectrometry; LC-MS; Lipid Identification; Lipidomics; Mass Spectrometry; Metabolomics; Phospholipids; Sphingolipids

DOI:  https://doi.org/10.1016/j.jlr.2021.100138
Molecules. 2021 Oct 12. pii: 6147. [Epub ahead of print]26(20):

Progress and Challenges in Quantifying Carbonyl-Metabolomic Phenomes with LC-MS/MS.

Yuting Sun, Huiru Tang, Yulan Wang.

  Carbonyl-containing metabolites widely exist in biological samples and have important physiological functions. Thus, accurate and sensitive quantitative analysis of carbonyl-containing metabolites is crucial to provide insight into metabolic pathways as well as disease mechanisms. Although reversed phase liquid chromatography electrospray ionization mass spectrometry (RPLC-ESI-MS) is widely used due to the powerful separation capability of RPLC and high specificity and sensitivity of MS, but it is often challenging to directly analyze carbonyl-containing metabolites using RPLC-ESI-MS due to the poor ionization efficiency of neutral carbonyl groups in ESI. Modification of carbonyl-containing metabolites by a chemical derivatization strategy can overcome the obstacle of sensitivity; however, it is insufficient to achieve accurate quantification due to instrument drift and matrix effects. The emergence of stable isotope-coded derivatization (ICD) provides a good solution to the problems encountered above. Thus, LC-MS methods that utilize ICD have been applied in metabolomics including quantitative targeted analysis and untargeted profiling analysis. In addition, ICD makes multiplex or multichannel submetabolome analysis possible, which not only reduces instrument running time but also avoids the variation of MS response. In this review, representative derivatization reagents and typical applications in absolute quantification and submetabolome profiling are discussed to highlight the superiority of the ICD strategy for detection of carbonyl-containing metabolites.

Keywords:  RPLC-ESI-MS; absolute quantification; carbonyl-containing metabolites; stable isotope-coded derivatization; submetabolome profiling

DOI:  https://doi.org/10.3390/molecules26206147
Metabolites. 2021 Oct 02. pii: 678. [Epub ahead of print]11(10):

MSCAT: A Machine Learning Assisted Catalog of Metabolomics Software Tools.

Jonathan Dekermanjian, Wladimir Labeikovsky, Debashis Ghosh, Katerina Kechris.

  The bottleneck for taking full advantage of metabolomics data is often the availability, awareness, and usability of analysis tools. Software tools specifically designed for metabolomics data are being developed at an increasing rate, with hundreds of available tools already in the literature. Many of these tools are open-source and freely available but are very diverse with respect to language, data formats, and stages in the metabolomics pipeline. To help mitigate the challenges of meeting the increasing demand for guidance in choosing analytical tools and coordinating the adoption of best practices for reproducibility, we have designed and built the MSCAT (Metabolomics Software CATalog) database of metabolomics software tools that can be sustainably and continuously updated. This database provides a survey of the landscape of available tools and can assist researchers in their selection of data analysis workflows for metabolomics studies according to their specific needs. We used machine learning (ML) methodology for the purpose of semi-automating the identification of metabolomics software tool names within abstracts. MSCAT searches the literature to find new software tools by implementing a Named Entity Recognition (NER) model based on a neural network model at the sentence level composed of a character-level convolutional neural network (CNN) combined with a bidirectional long-short-term memory (LSTM) layer and a conditional random fields (CRF) layer. The list of potential new tools (and their associated publication) is then forwarded to the database maintainer for the curation of the database entry corresponding to the tool. The end-user interface allows for filtering of tools by multiple characteristics as well as plotting of the aggregate tool data to monitor the metabolomics software landscape.

Keywords:  database; metabolomics; open-source software; text mining; workflows

DOI:  https://doi.org/10.3390/metabo11100678
J Proteome Res. 2021 Oct 22.

Nontargeted Serum Lipid Profiling of Nonalcoholic Steatohepatitis by Multisegment Injection-Nonaqueous Capillary Electrophoresis-Mass Spectrometry: A Multiplexed Separation Platform for Resolving Ionic Lipids.

Ritchie Ly, Nicholas Ly, Kazunori Sasaki, Makoto Suzuki, Kenjiro Kami, Yoshiaki Ohashi, Philip Britz-McKibbin.

  New methods are needed for global lipid profiling due to the complex chemical structures and diverse physicochemical properties of lipids. Herein we introduce a robust data workflow to unambiguously select lipid features from serum ether extracts by multisegment injection-nonaqueous capillary electrophoresis-mass spectrometry (MSI-NACE-MS). An iterative three-stage screening strategy is developed for nontargeted lipid analyses when using multiplexed electrophoretic separations coupled to an Orbitrap mass analyzer under negative ion mode. This approach enables the credentialing of 270 serum lipid features annotated based on their accurate mass and relative migration time, including 128 ionic lipids reliably measured (median CV ≈ 13%) in most serum samples (>75%) from nonalcoholic steatohepatitis (NASH) patients (n = 85). A mobility map is introduced to classify charged lipid classes over a wide polarity range with selectivity complementary to chromatographic separations, including lysophosphatidic acids, phosphatidylcholines, phosphatidylinositols, phosphatidylethanolamines, and nonesterified fatty acids (NEFAs). Serum lipidome profiles were also used to differentiate high- from low-risk NASH patients using a k-means clustering algorithm, where elevated circulating NEFAs (e.g., palmitic acid) were associated with increased glucose intolerance, more severe liver fibrosis, and greater disease burden. MSI-NACE-MS greatly expands the metabolome coverage of conventional aqueous-based CE-MS protocols and is a promising platform for large-scale lipidomic studies.

Keywords:  biomarker discovery; human serum; lipidomics; nonalcoholic steatohepatitis; nonaqueous capillary electrophoresis−mass spectrometry

DOI:  https://doi.org/10.1021/acs.jproteome.1c00682
Molecules. 2021 Oct 15. pii: 6246. [Epub ahead of print]26(20):

Automated Trimethyl Sulfonium Hydroxide Derivatization Method for High-Throughput Fatty Acid Profiling by Gas Chromatography-Mass Spectrometry.

Paul Gries, Atul Singh Rathore, Xiyuan Lu, Jennifer Chiou, Yen Bao Huynh, Alessia Lodi, Stefano Tiziani.

  Fatty acid profiling on gas chromatography-mass spectrometry (GC-MS) platforms is typically performed offline by manually derivatizing and analyzing small batches of samples. A GC-MS system with a fully integrated robotic autosampler can significantly improve sample handling, standardize data collection, and reduce the total hands-on time required for sample analysis. In this study, we report an optimized high-throughput GC-MS-based methodology that utilizes trimethyl sulfonium hydroxide (TMSH) as a derivatization reagent to convert fatty acids into fatty acid methyl esters. An automated online derivatization method was developed, in which the robotic autosampler derivatizes each sample individually and injects it into the GC-MS system in a high-throughput manner. This study investigated the robustness of automated TMSH derivatization by comparing fatty acid standards and lipid extracts, derivatized manually in batches and online automatically from four biological matrices. Automated derivatization improved reproducibility in 19 of 33 fatty acid standards, with nearly half of the 33 confirmed fatty acids in biological samples demonstrating improved reproducibility when compared to manually derivatized samples. In summary, we show that the online TMSH-based derivatization methodology is ideal for high-throughput fatty acid analysis, allowing rapid and efficient fatty acid profiling, with reduced sample handling, faster data acquisition, and, ultimately, improved data reproducibility.

Keywords:  GC–MS; fatty acid profiling; online automated derivatization; trimethyl sulfonium hydroxide

DOI:  https://doi.org/10.3390/molecules26206246
Metabolites. 2021 Sep 24. pii: 652. [Epub ahead of print]11(10):

Comparison of Kit-Based Metabolomics with Other Methodologies in a Large Cohort, towards Establishing Reference Values.

Daisuke Saigusa, Eiji Hishinuma, Naomi Matsukawa, Masatomo Takahashi, Jin Inoue, Shu Tadaka, Ikuko N Motoike, Atsushi Hozawa, Yoshihiro Izumi, Takeshi Bamba, Kengo Kinoshita, Kim Ekroos, Seizo Koshiba, Masayuki Yamamoto.

  Metabolic profiling is an omics approach that can be used to observe phenotypic changes, making it particularly attractive for biomarker discovery. Although several candidate metabolites biomarkers for disease expression have been identified in recent clinical studies, the reference values of healthy subjects have not been established. In particular, the accuracy of concentrations measured by mass spectrometry (MS) is unclear. Therefore, comprehensive metabolic profiling in large-scale cohorts by MS to create a database with reference ranges is essential for evaluating the quality of the discovered biomarkers. In this study, we tested 8700 plasma samples by commercial kit-based metabolomics and separated them into two groups of 6159 and 2541 analyses based on the different ultra-high-performance tandem mass spectrometry (UHPLC-MS/MS) systems. We evaluated the quality of the quantified values of the detected metabolites from the reference materials in the group of 2541 compared with the quantified values from other platforms, such as nuclear magnetic resonance (NMR), supercritical fluid chromatography tandem mass spectrometry (SFC-MS/MS) and UHPLC-Fourier transform mass spectrometry (FTMS). The values of the amino acids were highly correlated with the NMR results, and lipid species such as phosphatidylcholines and ceramides showed good correlation, while the values of triglycerides and cholesterol esters correlated less to the lipidomics analyses performed using SFC-MS/MS and UHPLC-FTMS. The evaluation of the quantified values by MS-based techniques is essential for metabolic profiling in a large-scale cohort.

Keywords:  SFC-MS/MS; UHPLC-FT/MS; UHPLC-MS/MS; large-scale cohort; lipidomics; mass spectrometry; metabolic profiling; widely-targeted metabolomics

DOI:  https://doi.org/10.3390/metabo11100652
J Chromatogr A. 2021 Oct 07. pii: S0021-9673(21)00730-5. [Epub ahead of print]1658 462606

Development of a high-throughput method for the comprehensive lipid analysis in milk using ultra-high performance supercritical fluid chromatography combined with quadrupole time-of-flight mass spectrometry.

Yan Tang, Muhammad Mujahid Ali, Xuechun Sun, Augustine Atta Debrah, Mengyu Wang, Haiyue Hou, Qiaozhen Guo, Zhenxia Du.

  Milk lipids are one of the most complex materials in nature and are associated with many physiological functions, hence it is important to comprehensively characterize lipids profiles to evaluate the nutritional value of milk. A quick method was developed by ultra-high performance supercritical fluid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry (UHPSFC-ESI-QTOF-MS) to analyze the non-polar and polar lipids profiles of cow, goat, buffalo, human milk, and infant formulas in 7 min. All chromatographic conditions were carefully optimized and their effect on the chromatographic behavior of lipid classes and species was discussed. Under optimized conditions, 12 lipid classes (triacylglycerols, diacylglycerols, monoglyceride, fatty acids, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, sphingomyelin, lyso-phosphatidylcholine, and lyso-phosphatidylethanolamine) were separated and each class was further separated in single analysis to facilitate the identification. 250 lipid species in real samples were characterized and quantified. This result demonstrates the applicability of the UHPSFC-ESI-QTOF-MS method in the high-throughput and comprehensive lipid analysis of milk, and will hopefully help to provide nutritionists with the lipid distribution in different types of milk, as well as help in the design of more suitable infant formula for babies.

Keywords:  Milk; Phospholipids; Sphingomyelin; Triacylglycerols; UHPSFC-ESI-QTOF-MS

DOI:  https://doi.org/10.1016/j.chroma.2021.462606
Clin Chem Lab Med. 2021 Oct 19.

Isotope dilution LC-MS/MS quantification of the cystic fibrosis transmembrane conductance regulator (CFTR) modulators ivacaftor, lumacaftor, tezacaftor, elexacaftor, and their major metabolites in human serum.

Katharina Habler, Anne-Sophie Kalla, Michael Rychlik, Mathias Bruegel, Daniel Teupser, Susanne Nährig, Michael Vogeser, Michael Paal.

   OBJECTIVES: Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) modulators have revolutionized the therapeutic landscape in CF treatment. These vital drugs are extensively metabolized via CYP3A, so caution must be exercised in multimodal CF therapy because of the risk of adverse drug interactions. Our goal was to develop a highly sensitive assay for the purpose of therapeutic drug monitoring (TDM) in diagnostic laboratories.
METHODS: After protein precipitation, the CFTR modulators ivacaftor, lumacaftor, tezacaftor, elexacaftor, and their metabolites ivacaftor-M1, ivacaftor-M6, and tezacaftor-M1 were separated with a two-dimensional chromatography setup within 5 min, and quantified with stable isotope-labeled internal standards. The method was validated according to the European Medicines Agency (EMA) guideline on bioanalytical method validation and applied to CF patient samples.
RESULTS: Inaccuracy was ≤7.0% and the imprecision coefficient of variation (CV) was ≤8.3% for all quality controls (QCs). The method consistently compensated for matrix effects, recovery, and process efficiency were 105-115 and 96.5-103%, respectively. Analysis of CF serum samples provided concentrations comparable to the pharmacokinetic profile data reported in the EMA assessment report for the triple combination therapy Kaftrio.
CONCLUSIONS: We hereby present a robust and highly selective isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) assay for the simultaneous quantification of the so far approved CFTR modulators and their metabolites in human serum. The assay is suitable for state-of-the-art pharmacovigilance of CFTR modulator therapy in CF patients, in order to maximize safety and efficacy, and also to establish dose-response relationships in clinical trials.

Keywords:  cystic fibrosis (CF); cystic fibrosis transmembrane conductance regulator (CFTR) modulators; isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS); therapeutic drug monitoring (TDM)

DOI:  https://doi.org/10.1515/cclm-2021-0724
Metabolites. 2021 Sep 28. pii: 660. [Epub ahead of print]11(10):

Lipid Profiling in Cancer Diagnosis with Hand-Held Ambient Mass Spectrometry Probes: Addressing the Late-Stage Performance Concerns.

Lauren Katz, Alessandra Tata, Michael Woolman, Arash Zarrine-Afsar.

  Untargeted lipid fingerprinting with hand-held ambient mass spectrometry (MS) probes without chromatographic separation has shown promise in the rapid characterization of cancers. As human cancers present significant molecular heterogeneities, careful molecular modeling and data validation strategies are required to minimize late-stage performance variations of these models across a large population. This review utilizes parallels from the pitfalls of conventional protein biomarkers in reaching bedside utility and provides recommendations for robust modeling as well as validation strategies that could enable the next logical steps in large scale assessment of the utility of ambient MS profiling for cancer diagnosis. Six recommendations are provided that range from careful initial determination of clinical added value to moving beyond just statistical associations to validate lipid involvements in disease processes mechanistically. Further guidelines for careful selection of suitable samples to capture expected and unexpected intragroup variance are provided and discussed in the context of demographic heterogeneities in the lipidome, further influenced by lifestyle factors, diet, and potential intersect with cancer lipid pathways probed in ambient mass spectrometry profiling studies.

Keywords:  ambient mass spectrometry; cancer diagnosis with ambient mass spectrometry; lipid profiling; untargeted lipidomics; untargeted metabolomics

DOI:  https://doi.org/10.3390/metabo11100660
Biomedicines. 2021 Oct 02. pii: 1379. [Epub ahead of print]9(10):

A UHPLC-MS/MS Method for Therapeutic Drug Monitoring of Aciclovir and Ganciclovir in Plasma and Dried Plasma Spots.

Federica Pigliasco, Alessia Cafaro, Raffaele Simeoli, Sebastiano Barco, Alberto Magnasco, Maura Faraci, Gino Tripodi, Bianca Maria Goffredo, Giuliana Cangemi.

  The role of therapeutic drug monitoring (TDM) of valaciclovir (VA)/aciclovir (A) and valganciclovir/ganciclovir (VG/G) in critically ill patients is still a matter of debate. More data on the dose-concentration relationship might therefore be useful, especially in pediatrics where clinical practice is not adequately supported by robust PK studies. We developed and validated a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) micro-method to simultaneously quantify A and G from plasma and dried plasma spots (DPS). The method was based on rapid organic extraction from DPS and separation on a reversed-phase C-18 UHPLC column after addition of deuterated internal standards. Accurate analyte quantification using SRM detection was then obtained using a Thermo Fisher Quantiva triple-quadrupole MS coupled to an Ultimate 3000 UHPLC. It was validated following international (EMA) guidelines for bioanalytical method validation and was tested on samples from pediatric patients treated with A, VG, or G for cytomegalovirus infection following solid organ or hematopoietic stem cell transplantation. Concentrations obtained from plasma and DPS were compared using Passing-Bablok and Bland-Altman statistical tests. The assay was linear over wide concentration ranges (0.01-20 mg/L) in both plasma and DPS for A and G, suitable for the expected therapeutic ranges for both Cmin and Cmax, accurate, and reproducible in the absence of matrix effects. The results obtained from plasma and DPS were comparable. Using an LC-MS/MS method allowed us to obtain a very specific, sensitive, and rapid quantification of these antiviral drugs starting from very low volumes (50 μL) of plasma samples and DPS. The stability of analytes for at least 30 days allows for cost-effective shipment and storage at room temperature. Our method is suitable for TDM and could be helpful for improving knowledge on PK/PD targets of antivirals in critically ill pediatric patients.

Keywords:  LC-MS/MS; aciclovir; dried plasma spot; ganciclovir; therapeutic drug monitoring; valaciclovir; valganciclovir

DOI:  https://doi.org/10.3390/biomedicines9101379
J Chromatogr A. 2021 Oct 06. pii: S0021-9673(21)00714-7. [Epub ahead of print]1658 462590

Analysis of urinary organic acids by gas chromatography tandem mass spectrometry method for metabolic profiling applications.

T Mouskeftara, C Virgiliou, G Theodoridis, H Gika.

  A sensitive, accurate and precise method was developed for the quantification of a large number of organic acids in human urine by GC-MS/MS. The analytes were selected based on their role as key metabolic intermediates; intermediates of Krebs cycle, fatty acid oxidation, glycolysis, down-stream metabolites of neurotransmitter synthesis and degradation, metabolites indicative of nutritional deficiencies, byproducts of microbial activity in the gastrointestinal tract (GI) etc. The most efficient sample preparation protocol was selected based on tests for extraction with different solvents such as MTBE and ethyl acetate under acidic conditions, whereas finally a more general protocol was applied with methanol. Regarding derivatization, methoxyamine with MSTFA, 1% TMCS was applied. The method was extensively validated, including stability study, ensuring accurate determination of the studied organic acids in human urine. Proof of its utility was exhibited in a set of samples from human volunteers. The method can find wide applicability in the context of metabolomics for clinical or nutritional studies.

Keywords:  Biomarker; Derivatization; Gas chromatography; Metabolic profiling; Tandem mass spectrometry; Urine; Validation

DOI:  https://doi.org/10.1016/j.chroma.2021.462590
Molecules. 2021 Oct 14. pii: 6213. [Epub ahead of print]26(20):

UPLC-TOF-MS Method for Simultaneous Quantification of Steroid Hormones in Tissue Homogenates of Zebrafish with Solid-Phase Extraction.

Yaxi Li, Zhi Yan, Xiaodong Li, Xiuli Yin, Ke Li.

  The quantification of steroid hormones of individual zebrafish (Danio rerio) provides perspective to understand endogenous hormone function. A UPLC-TOF-MS method was developed to provide a reproducible, sensitive, and efficient assay to determine the concentration of steroid hormones, including cortisol, testosterone, androstenedione, 11-deoxycortisol, 11-deoxycorticosterone, and 17-hydroxyprogesterone in whole-body homogenates of each zebrafish. Solid-phase extraction was used to sample matrix clean-up and acquired a recovery from 89.7% to 107.9%. The analytes were separated on an Aquity BEH C18 column using gradient elution. Mass spectrometric analysis was performed by single reaction monitoring (SRM) using positive electrospray ionization mode. The total running time was 6 min, which was greatly shortened compared with a previously reported method. The developed method exhibited excellent linearity for all the analytes, with regression coefficients higher than 0.99. The limit of detection varied between 0.1 and 0.5 ng/L and the limit of quantification was 0.5-1.7 ng/L for all analytes. The precision of the method was assessed on replicate measurements and was found to be in the ranges of 1.9 % to 6.6% and 4.3% to 8.6%, for intra- and inter-day analysis, respectively. This method was validated according to FDA guidance and applied to determine steroid hormone levels in the tissue homogenate of zebrafish acutely treated with caffeine and ethanol.

Keywords:  behavioral ecology; quantitative analysis; steroid hormone; stressor; validation

DOI:  https://doi.org/10.3390/molecules26206213
Antioxidants (Basel). 2021 Oct 15. pii: 1622. [Epub ahead of print]10(10):

Metabolomics of Chlorophylls and Carotenoids: Analytical Methods and Metabolome-Based Studies.

María Roca, Antonio Pérez-Gálvez.

  Chlorophylls and carotenoids are two families of antioxidants present in daily ingested foods, whose recognition as added-value ingredients runs in parallel with the increasing number of demonstrated functional properties. Both groups include a complex and vast number of compounds, and extraction and analysis methods evolved recently to a modern protocol. New methodologies are more potent, precise, and accurate, but their application requires a better understanding of the technical and biological context. Therefore, the present review compiles the basic knowledge and recent advances of the metabolomics of chlorophylls and carotenoids, including the interrelation with the primary metabolism. The study includes material preparation and extraction protocols, the instrumental techniques for the acquisition of spectroscopic and spectrometric properties, the workflows and software tools for data pre-processing and analysis, and the application of mass spectrometry to pigment metabolomics. In addition, the review encompasses a critical description of studies where metabolomics analyses of chlorophylls and carotenoids were developed as an approach to analyzing the effects of biotic and abiotic stressors on living organisms.

Keywords:  antioxidants; carotenoids; chlorophylls; extraction methods; mass spectrometry; metabolism; metabolomics; novel analytical technologies; pathways; pigments

DOI:  https://doi.org/10.3390/antiox10101622
J Appl Lab Med. 2021 Oct 18. pii: jfab107. [Epub ahead of print]

Multicenter Evaluation of a New, Fully Automated Androstenedione Electrochemiluminescence Immunoassay: Precision Analysis, Method Comparison, and Determination of Reference Ranges.

Barbara Obermayer-Pietsch, Marta de Ramon, Claudia Reichmuth, Garnet Bendig, Stefan Hutzler, Judith Taibon, Christopher M Rank, Peter Findeisen.

   BACKGROUND: Androstenedione (ASD) levels can aid diagnosis of hyperandrogenism together with other clinical/laboratory findings. We evaluated performance of the new, automated Elecsys® ASD assay vs an ASD isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) reference measurement procedure and determined reference ranges.
METHODS: Repeatability/intermediate precision were assessed using 3 control levels and 5 human serum pools (n = 75 each; Clinical and Laboratory Standards Institute EP05-A3). Method comparisons vs commercially available immunoassays [IMMULITE ASD (Siemens) and LIAISON ASD (DiaSorin)] and an ID-LC-MS/MS measurement procedure method were conducted using 421 serum samples; Passing-Bablok regression and Pearson's correlation coefficients were calculated. Reference ranges and distribution of values associated with polycystic ovary syndrome (PCOS) were determined in five clinical cohorts using samples from several sites/vendors.
RESULTS: Repeatability/intermediate precision coefficients of variation across all sites were 2.01% to 3.91% and 2.43% to 4.30%, respectively (mean ASD: 7.80-34.7 nmol/L). The Elecsys ASD assay showed poor agreement with IMMULITE ASD (slope = 0.459; r = 0.856; n = 320), fair agreement with LIAISON ASD (slope = 0.625; r = 0.984; n = 327), and very good agreement with ID-LC-MS/MS (slope = 1.040; r = 0.996; n = 332). Reference ranges (2.5th-97.5th percentiles) were: children (≤8 years; n = 140), <0.525 to 1.81 nmol/L; males (≥18 years; n = 138), 0.979 to 5.32 nmol/L; and postmenopausal females (n = 140), 0.654 to 3.74 nmol/L. Reference range (5th-95th percentiles) for females with fertile cycle (≥18 years; n = 84) was 1.71 to 4.58 nmol/L. The distribution of values (2.5th-97.5th percentiles) in females with PCOS (n = 125) was 2.26 to 12.1 nmol/L.
CONCLUSIONS: Elecsys ASD assay demonstrated excellent precision and very good agreement with ID-LC-MS/MS. Reference ranges were established to support results interpretation in routine practice.

Keywords:  ASD quantification; PCOS; androstenedione; hyperandrogenism; immunoassay; polycystic ovary syndrome

DOI:  https://doi.org/10.1093/jalm/jfab107
Metabolites. 2021 Oct 04. pii: 681. [Epub ahead of print]11(10):

Analytical Methodology for a Metabolome Atlas of Goat's Plasma, Milk and Feces Using 1H-NMR and UHPLC-HRMS.

Cécile Martias, Julie Gatien, Léa Roch, Nadine Baroukh, Sylvie Mavel, Antoine Lefèvre, Frédéric Montigny, Laurent Schibler, Patrick Emond, Lydie Nadal-Desbarats.

  Metabolomics has been increasingly used in animal and food sciences. Animal health is one of the most important factor that can also alter animal integrity and welfare. Some studies have already investigated the link between health and metabolic profile of dairy animals. These studies in metabolomics often consider a single type of sample using a single analytical platform (nuclear magnetic resonance or mass spectrometry). Only few studies with multi-platform approaches are also used with a single or a multi type of sample, but they mainly consider dairy cows' metabolome although dairy goats present similar diseases, that it could be interesting to detect early to preserve animal health and milk production. This study aims to create a metabolic atlas of goat plasma, milk and feces, based on healthy animals. Our study describes a standard operating procedure for three goat matrices: blood plasma, milk, and feces using multiple platforms (NMR (1H), UHPLC (RP)-MS and UHPLC (HILIC)-MS) that follows a unique sample preparation procedure for each sample type to be analyzed on multi-platforms basis. Our method was evaluated for its robustness and allowed a better characterization of goat metabolic profile in healthy conditions.

Keywords:  1H-NMR; LC-MS; feces; goat; metabolic fingerprinting; milk; plasma

DOI:  https://doi.org/10.3390/metabo11100681
J Chromatogr A. 2021 Oct 08. pii: S0021-9673(21)00733-0. [Epub ahead of print]1658 462609

Rapid simultaneous determination of ketamine and midazolam in biological samples using ion mobility spectrometry combined by headspace solid-phase microextraction.

Sarah Nakhodchi, Naader Alizadeh.

  Ketamine (Ket) and midazolam (Mdz) are among well-known anesthetic agents which frequently coadministered in surgical procedures and emergency department. Only a few reports have been published for the simultaneous analysis of these compounds. In the present study, we reported a simple, sensitive, and rapid method for simultaneous determination of Ket and Mdz based on headspace solid-phase microextraction coupled with ion mobility spectrometry (HS-SPME-IMS). Ion mobility spectrometer operated under positive mode of a corona discharge ionization source using ammonia as a dopant. The effective parameters on the extraction process consisting of pH of the sample, extraction temperature, extraction time, salt concentration were optimized. The calibration plots exhibited good linearity over the concentration ranges of 10-800 and 100-1500 µg L-1 and detection limit of 8.9 and 52 µg L-1 for Ket and Mdz respectively with correlation coefficients greater than 0.99. The relative standard deviation (RSD) for five replicate measurements was determined to be less than 8%. Finally, the applicability of the proposed method was tested in human plasma and serum samples. These tests showed that the matrix in serum samples interfere with midazolam determination but quantitative recoveries from 85 to 95 % were obtained for both drugs in the human plasma samples. The method herein provides simple and suitable approach while minimizing sample preparation and the overall complexity of the analysis in comparison to existing methodologies.

Keywords:  Anesthetic agents; Headspace solid-phase microextraction; Human plasma and serum samples; Ion mobility spectrometry; Ketamine; Midazolam

DOI:  https://doi.org/10.1016/j.chroma.2021.462609
Metabolites. 2021 Sep 30. pii: 672. [Epub ahead of print]11(10):

New Advances in Tissue Metabolomics: A Review.

Michelle Saoi, Philip Britz-McKibbin.

  Metabolomics offers a hypothesis-generating approach for biomarker discovery in clinical medicine while also providing better understanding of the underlying mechanisms of chronic diseases. Clinical metabolomic studies largely rely on human biofluids (e.g., plasma, urine) as a more convenient specimen type for investigation. However, biofluids are non-organ specific reflecting complex biochemical processes throughout the body, which may complicate biochemical interpretations. For these reasons, tissue metabolomic studies enable deeper insights into aberrant metabolism occurring at the direct site of disease pathogenesis. This review highlights new advances in metabolomics for ex vivo analysis, as well as in situ imaging of tissue specimens, including diverse tissue types from animal models and human participants. Moreover, we discuss key pre-analytical and post-analytical challenges in tissue metabolomics for robust biomarker discovery with a focus on new methodological advances introduced over the past six years, including innovative clinical applications for improved screening, diagnostic testing, and therapeutic interventions for cancer.

Keywords:  cancer; capillary electrophoresis; chromatography; clinical medicine; mass spectrometry; metabolomics; nuclear magnetic resonance; review; sample preparation; tissue biopsies

DOI:  https://doi.org/10.3390/metabo11100672
J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Oct 07. pii: S1570-0232(21)00464-5. [Epub ahead of print]1184 122983

Simultaneous determination of multiple isomeric hydroxylated polycyclic aromatic hydrocarbons in urine by using ultra-high performance liquid chromatography tandem mass spectrometry.

Liting Hua, Sai Guo, Yu Wang, Hongwen Sun, Hongzhi Zhao.

  Monitoring the level of hydroxylated polycyclic aromatic hydrocarbons (OH-PAHs) in urine is the key to exploring human metabolic changes and comprehensive potential toxicity of PAHs. The OH-PAHs with isomeric structure have different biological functions, indicating that their quantification is indispensable. However, the quantitation method is still dissatisfactory due to the poor separation of these isomeric OH-PAHs. The current study established a ultra-high performance liquid chromatography (UHPLC) tandem mass spectrometry (MS) method to complete the simultaneous determination of 17 OH-PAHs, including two naphthalene metabolites (1-hydroxynaphthalene, 2-hydroxynaphthalene), two fluorene metabolites (2-hydroxyfluorene, 3-hydroxyfluorene), five phenanthrene metabolites (1-hydroxyphenanthrene, 2-hydroxyphenanthrene, 3-hydroxyphenanthrene, 4-hydroxyphenanthrene, 9-hydroxyphenanthrene), a pyrene metabolite (1-hydroxypyrene), five chrysene metabolites (1-hydroxychrysene, 2-hydroxychrysene, 3-hydroxychrysene, 4-hydroxychrysene, 6-hydroxychrysene) and two benzo[a]pyrene metabolites (3-hydroxybenzo[a]pyrene, 9-hydroxybenzo[a]pyrene). The method validation results showed good selectivity, linearity (r2 > 0.999), inter-day and intra-day precision (relative standard deviation (RSD) < 5.5% and RSD < 6.3%), stability (RSD < 19.3%), matrix effect (-8.3%-11.5%) and recovery (65.9%-116.2%). This method is convenient, sensitive and efficient, saving expensive materials and complicated derivatization procedures. The practical applicability of developed approach was also tested in urine samples to identify potential biomarkers of PAHs exposure in humans, and a great compromise was obtained between recoveries and extract convenience. The developed approach may be widely utilized for specific determination of OH-PAHs with isomer structure in urine samples. It is expected that the application of this method may provide powerful references for PAHs exposure assessment.

Keywords:  Human biomonitoring; Hydroxylated polycyclic aromatic hydrocarbons; Solid phase extraction; Ultra-high performance liquid chromatography tandem mass spectrometry; Urine

DOI:  https://doi.org/10.1016/j.jchromb.2021.122983
Anal Chem. 2021 Oct 20.

Robot-Assisted SpiderMass for In Vivo Real-Time Topography Mass Spectrometry Imaging.

Nina Ogrinc, Alexandre Kruszewski, Paul Chaillou, Philippe Saudemont, Chann Lagadec, Michel Salzet, Christian Duriez, Isabelle Fournier.

Mass spectrometry imaging (MSI) has shown to bring invaluable information for biological and clinical applications. However, conventional MSI is generally performed ex vivo from tissue sections. Here, we developed a novel MS-based method for in vivo mass spectrometry imaging. By coupling the SpiderMass technology, that provides in vivo minimally invasive analysis-to a robotic arm of high accuracy, we demonstrate that images can be acquired from any surface by moving the laser probe above the surface. By equipping the robotic arm with a sensor, we are also able to both get the topography image of the sample surface and the molecular distribution, and then and plot back the molecular data, directly to the 3D topographical image without the need for image fusion. This is shown for the first time with the 3D topographic MS-based whole-body imaging of a mouse. Enabling fast in vivo MSI bridged to topography paves the way for surgical applications to excision margins.

DOI: https://doi.org/10.1021/acs.analchem.1c01692
J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Oct 05. pii: S1570-0232(21)00456-6. [Epub ahead of print]1184 122975

Targeted volatolomics of human monocytes: Comparison of 2D-GC/TOF-MS and 1D-GC/Orbitrap-MS methods.

Kristýna Zemánková, Kristýna Pavelicová, Antonio Pompeiano, Ludmila Mravcová, Martin Černý, Kamila Bendíčková, Marcela Hortová Kohoutková, Kseniya Dryahina, Markéta Vaculovičová, Jan Frič, Lucie Vaníčková.

  Blood is a complex biological matrix providing valuable information on nutritional, metabolic, and immune status. The detection of blood biomarkers requires sensitive analytical methods because analytes are at very low concentrations. Peripheral blood monocytes play a crucial role in inflammatory processes, and the metabolites released by monocytes during these processes might serve as important signalling molecules and biomarkers of particular physiological states. Headspace solid-phase microextraction (HS-SPME) combined with two different mass spectrometric platforms, two-dimensional (2D) gas chromatography coupled to time-of-flight mass spectrometry (2D-GC/TOF-MS) and one-dimensional gas chromatography coupled to Orbitrap mass spectrometry (GC/Orbitrap-MS), were applied for the investigation of volatile organic compounds (VOCs) produced by human peripheral blood monocytes. An optimized method was subsequently applied for the characterization of changes in VOCs induced by lipopolysaccharides (LPS) and zymosan (ZYM) stimulation. Overall, the 2D-GC/TOF-MS and the 1D-GC/Orbitrap-MS analyses each yielded about 4000 and 400 peaks per sample, respectively. In total, 91 VOCs belonging to eight different chemical classes were identified. The samples were collected in two fractions, conditioned media for monitoring extracellularly secreted molecules and cell pellet samples to determine the intracellular composition of VOCs. Alcohols, ketones, and hydrocarbons were the main chemical classes of the metabolic profile identified in cell fractions. Aldehydes, acids and cyclic compounds were characteristic of the conditioned media fraction. Here we demonstrate that HS-SPME-2D-GC/TOF-MS is more suitable for the identification of specific VOC profiles produced by human monocytes than 1D-GC/Orbitrap-MS. We define the signature of VOCs occurring early after monocyte activation and characterise the signalling compounds released by immune cells into media.

Keywords:  2D gas chromatography; Biomarker; GCxGC-TOF-MS; Monocytes; SPME; Volatile organic compounds

DOI:  https://doi.org/10.1016/j.jchromb.2021.122975