bims-metlip 2022-08-28 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2022‒08‒28
33 papers selected by
Sofia Costa
Matterworks

Addressing big data challenges in mass spectrometry-based metabolomics.
Isotope Ratio Outlier Analysis (IROA) for HPLC-TOFMS-Based Metabolomics of Human Urine.
LipidMS 3.0: an R-package and a web-based tool for LC-MS/MS data processing and lipid annotation.
Mass Spectrometry-Based Nontargeted and Targeted Analytical Approaches in Fingerprinting and Metabolomics of Food and Agricultural Research.
A Selective and Sensitive LC-MS/MS Method for Quantitation of Indole in Mouse Serum and Tissues.
NPvis: An Interactive Visualizer of Peptidic Natural Product-MS/MS Matches.
Metabolomics of Human Semen: A Review of Different Analytical Methods to Unravel Biomarkers for Male Fertility Disorders.
High-Throughput Phospholipidomics of Candida Cells: From Sample Preparation to Data Analysis.
Molecular network strategy in multi-omics and mass spectrometry imaging.
Multi-steroid profiling by UHPLC-MS/MS with post-column infusion of ammonium fluoride.
A Sample Preparation Method for the Simultaneous Profiling of Signaling Lipids and Polar Metabolites in Small Quantities of Muscle Tissues from a Mouse Model for Sarcopenia.
A Micro-Scale Analytical Method for Determining Glycogen Turnover by NMR and FTMS.
An Open-Source Pipeline for Processing Direct Infusion Mass Spectrometry Data of the Human Plasma Metabolome.
Simultaneous Quantification of Steroid Hormones Using hrLC-MS in Endocrine Tissues of Male Rats and Human Samples.
Quality assurance and quality control reporting in untargeted metabolic phenotyping: mQACC recommendations for analytical quality management.
Meta-Analysis Reveals Both the Promises and the Challenges of Clinical Metabolomics.
Analytical Approaches for the Determination of Buprenorphine, Methadone and Their Metabolites in Biological Matrices.
Quantification in bioimaging by LA-ICPMS - Evaluation of isotope dilution and standard addition enabled by micro-droplets.
One-step extraction and determination of 513 psychoactive substances, drugs, and their metabolites from hair by LC-MS/MS.
Development of a Validated UPLC-MS/MS Method for Simultaneous Estimation of Neratinib and Curcumin in Human Plasma: Application to Greenness Assessment and Routine Quantification.
Assessment of creatinine concentration in whole blood spheroids using paper spray ionization-tandem mass spectrometry.
A strategy for accurately and sensitively quantifying free and esterified fatty acids using liquid chromatography mass spectrometry.
Analytical method for the biomonitoring of bisphenols and parabens by liquid chromatography coupled to tandem mass spectrometry in human hair.
Liquid chromatography-tandem mass spectrometry method for simultaneous quantification of neurotransmitters in rat brain tissue exposed to 4'-Fluoro-α-PHP.
Tandem Mass Spectrometry as an Independent Method for Corroborating Fluorine-18 Radioactivity Measurements in Positron Emission Tomography.
Comparison of two azobenzene-based amino acid derivatization reagents for LC-MS/MS analysis in positive and negative ESI modes.
A validated LC-MS/MS method for the determination of L-hyoscyamine in human plasma: Application in clinical study.
Valproic acid determination by liquid chromatography coupled to mass spectrometry (LC-MS/MS) in whole blood for forensic purposes.
Controlled Formation of Protonated and Radical Cation Precursor Ions by Atmospheric Pressure Photoionization with μLC-MS Enables Electron Ionization and MS/MS Library Search.
Quantitation of Enantiomeric Excess in an Achiral Environment Using Trapped Ion Mobility Mass Spectrometry.
Recent Progress in Analysis of Intermediary Metabolism by ex vivo 13 C NMR.
β'-Phenylfentanyl Metabolism in Primary Human Hepatocyte Incubations: Identification of Potential Biomarkers of Exposure in Clinical and Forensic Toxicology.
Natural Deep Eutectic Solvent-Based Dispersive Liquid-Liquid Microextraction Coupled with Direct Analysis in Real Time Mass Spectrometry: A Green Temperature-Mediated Analytical Strategy.

Chem Commun (Camb). 2022 Aug 23.

Addressing big data challenges in mass spectrometry-based metabolomics.

Jian Guo, Huaxu Yu, Shipei Xing, Tao Huan.

Advancements in computer science and software engineering have greatly facilitated mass spectrometry (MS)-based untargeted metabolomics. Nowadays, gigabytes of metabolomics data are routinely generated from MS platforms, containing condensed structural and quantitative information from thousands of metabolites. Manual data processing is almost impossible due to the large data size. Therefore, in the "omics" era, we are faced with new challenges, the big data challenges of how to accurately and efficiently process the raw data, extract the biological information, and visualize the results from the gigantic amount of collected data. Although important, proposing solutions to address these big data challenges requires broad interdisciplinary knowledge, which can be challenging for many metabolomics practitioners. Our laboratory in the Department of Chemistry at the University of British Columbia is committed to combining analytical chemistry, computer science, and statistics to develop bioinformatics tools that address these big data challenges. In this Feature Article, we elaborate on the major big data challenges in metabolomics, including data acquisition, feature extraction, quantitative measurements, statistical analysis, and metabolite annotation. We also introduce our recently developed bioinformatics solutions for these challenges. Notably, all of the bioinformatics tools and source codes are freely available on GitHub (https://www.github.com/HuanLab), along with revised and regularly updated content.

DOI: https://doi.org/10.1039/d2cc03598g
Metabolites. 2022 Aug 12. pii: 741. [Epub ahead of print]12(8):

Isotope Ratio Outlier Analysis (IROA) for HPLC-TOFMS-Based Metabolomics of Human Urine.

Fadi Fadil, Claudia Samol, Raffaela S Berger, Fabian Kellermeier, Wolfram Gronwald, Peter J Oefner, Katja Dettmer.

  Metabolic fingerprinting by mass spectrometry aims at the comprehensive, semiquantitative analysis of metabolites. Isotope dilution, if successfully implemented, may provide a more reliable, relative quantification. Therefore, the 13C labeled yeast extract of the IROA TruQuant kit was added as an internal standard (IS) to human urine samples measured in full-scan mode on a high-performance liquid chromatography-time-of-flight mass spectrometer (HPLC-TOFMS) system. The isotope ratio approach enabled the analysis of 112 metabolites. The correlation with reference data did not improve significantly using 12C/13C ratios compared to absolute 12C peak areas. Moreover, using an intricate 13C-labeled standard increased the complexity of the mass spectra, which made correct signal annotation more challenging. On the positive side, the ratio approach helps to reduce batch effects, but it does not perform better than computational methods such as the "removebatcheffect" function in the R package Limma.

Keywords:  HPLC; IROA; labeled yeast extract; mass spectrometry; metabolic fingerprinting; metabolomics; relative quantification; stable isotope

DOI:  https://doi.org/10.3390/metabo12080741
Bioinformatics. 2022 Aug 25. pii: btac581. [Epub ahead of print]

LipidMS 3.0: an R-package and a web-based tool for LC-MS/MS data processing and lipid annotation.

María Isabel Alcoriza-Balaguer, Juan Carlos García-Cañaveras, Francisco Javier Ripoll-Esteve, Marta Roca, Agustín Lahoz.

MOTIVATION: LipidMS was initially envisioned to use fragmentation rules and data-independent acquisition (DIA) for lipid annotation. However, data-dependent acquisition (DDA) remains the most widespread acquisition mode for untargeted LC-MS/MS-based lipidomics. Here we present LipidMS 3.0, an R package that not only adds DDA and new lipid classes to its pipeline, but also the required functionalities to cover the whole data analysis workflow from pre-processing (i.e., peak-peaking, alignment and grouping) to lipid annotation.RESULTS: We applied the new workflow in the data analysis of a commercial human serum pool spiked with 68 representative lipid standards acquired in full scan, DDA and DIA modes. When focusing on the detected lipid standard features and total identified lipids, LipidMS 3.0 data pre-processing performance is similar to XCMS, whereas it complements the annotations returned by MS-DIAL, providing a higher level of structural information and a lower number of incorrect annotations. To extend and facilitate LipidMS 3.0 usage among less experienced R-programming users, the workflow was also implemented as a web-based application.
AVAILABILITY: The LipidMS R-package is freely available at https://CRAN.R-project.org/package=LipidMS and as a website at http://www.lipidms.com.
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

DOI: https://doi.org/10.1093/bioinformatics/btac581
J Agric Food Chem. 2022 Aug 23.

Mass Spectrometry-Based Nontargeted and Targeted Analytical Approaches in Fingerprinting and Metabolomics of Food and Agricultural Research.

Zhihao Liu, Mengliang Zhang, Pei Chen, James M Harnly, Jianghao Sun.

  Mass spectrometry (MS)-based techniques have been extensively applied in food and agricultural research. This review aims to address the advances and applications of MS-based analytical strategies in nontargeted and targeted analysis and summarizes the recent publications of MS-based techniques, including flow injection MS fingerprinting, chromatography-tandem MS metabolomics, direct analysis using ambient mass spectrometry, as well as development in MS data deconvolution software packages and databases for metabolomic studies. Various nontargeted and targeted approaches are employed in marker compounds identification, material adulteration detection, and the analysis of specific classes of secondary metabolites. In the newly emerged applications, the recent advances in computer tools for the fast deconvolution of MS data in targeted secondary metabolite analysis are highlighted.

Keywords:  ambient mass spectrometry; fingerprinting; flow injection mass spectrometry; food and agriculture; metabolomics; targeted analysis

DOI:  https://doi.org/10.1021/acs.jafc.2c01878
Metabolites. 2022 Aug 02. pii: 716. [Epub ahead of print]12(8):

A Selective and Sensitive LC-MS/MS Method for Quantitation of Indole in Mouse Serum and Tissues.

Vineet Joshi, Yashpal S Chhonker, Dhruvkumar Soni, Kelly C Cunningham, Derrick R Samuelson, Daryl J Murry.

  Indole is an endogenous substance currently being evaluated as a biomarker for ulcerative colitis, irritable bowel syndrome, Crohn's disease and non-alcoholic fatty liver disease. A novel, selective, and sensitive method using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was developed for quantitation of indole concentrations in mouse plasma and tissues. Samples were prepared by protein precipitation using ice-cold acetonitrile (ACN) followed by injecting the extracted analyte to LC-MS/MS system. Indole was separated using Synergi Fusion C18 (4 µm, 250 × 2.0 mm) column with mobile phase 0.1% aqueous formic acid (A) and methanol (B) using gradient flow with run time 12 min. The mass spectrometer was operated in atmospheric pressure chemical ionization (APCI) positive mode at unit resolution in multiple reaction monitoring (MRM) mode, using precursor ion > product ion combinations of 118.1 > 91.1 m/z for indole and 124.15 > 96.1 m/z for internal standard (IS) indole d7. The MS/MS response was linear over the range of indole concentrations (1-500 ng/mL). The validated method was applied for quantitation of indole concentrations range in mouse lungs (4.3-69.4 ng/g), serum (0.8-38.7 ng/mL) and cecum (1043.8-12,124.4 ng/g). This method would help investigate the role of indole as a biomarker and understand its implications in different disease states.

Keywords:  LC-MS/MS; biomarker; indole; indole biodistribution

DOI:  https://doi.org/10.3390/metabo12080716
Metabolites. 2022 Jul 29. pii: 706. [Epub ahead of print]12(8):

NPvis: An Interactive Visualizer of Peptidic Natural Product-MS/MS Matches.

Olga Kunyavskaya, Alla Mikheenko, Alexey Gurevich.

  Peptidic natural products (PNPs) represent a medically important class of secondary metabolites that includes antibiotics, anti-inflammatory and antitumor agents. Advances in tandem mass spectra (MS/MS) acquisition and in silico database search methods have enabled high-throughput PNP discovery. However, the resulting spectra annotations are often error-prone and their validation remains a bottleneck. Here, we present NPvis, a visualizer suitable for the evaluation of PNP-MS/MS matches. The tool interactively maps annotated spectrum peaks to the corresponding PNP fragments and allows researchers to assess the match correctness. NPvis accounts for the wide chemical diversity of PNPs that prevents the use of the existing proteomics visualizers. Moreover, NPvis works even if the exact chemical structure of the matching PNP is unknown. The tool is available online and as a standalone application. We hope that it will benefit the community by streamlining PNP data analysis and validation.

Keywords:  bioinformatics; mass spectrometry; peptidic natural products; software; visualization

DOI:  https://doi.org/10.3390/metabo12080706
Int J Mol Sci. 2022 Aug 12. pii: 9031. [Epub ahead of print]23(16):

Metabolomics of Human Semen: A Review of Different Analytical Methods to Unravel Biomarkers for Male Fertility Disorders.

Janet Blaurock, Sven Baumann, Sonja Grunewald, Jürgen Schiller, Kathrin M Engel.

  BACKGROUND: Human life without sperm is not possible. Therefore, it is alarming that the fertilizing ability of human spermatozoa is continuously decreasing. The reasons for that are widely unknown, but there is hope that metabolomics-based investigations may be able to contribute to overcoming this problem. This review summarizes the attempts made so far.METHODS: We will discuss liquid chromatography-mass spectrometry (LC-MS), gas chromatography (GC), infrared (IR) and Raman as well as nuclear magnetic resonance (NMR) spectroscopy. Almost all available studies apply one of these methods.
RESULTS: Depending on the methodology used, different compounds can be detected, which is (in combination with sophisticated methods of bioinformatics) helpful to estimate the state of the sperm. Often, but not in all cases, there is a correlation with clinical parameters such as the sperm mobility.
CONCLUSIONS: LC-MS detects the highest number of metabolites and can be considered as the method of choice. Unfortunately, the reproducibility of some studies is poor, and, thus, further improvements of the study designs are needed to overcome this problem. Additionally, a stronger focus on the biochemical consequences of the altered metabolite concentrations is also required.

Keywords:  male infertility; metabolome; semen; seminal plasma; spermatozoa

DOI:  https://doi.org/10.3390/ijms23169031
Methods Mol Biol. 2022 ;2542 127-140

High-Throughput Phospholipidomics of Candida Cells: From Sample Preparation to Data Analysis.

Mohit Kumar, Rajendra Prasad, Ashutosh Singh.

  Laboratory identification of Candida species is often complicated by overlapping features. Species specificity is critical to the appropriate use of interventions.Accurate identification and quantification of lipid species in complex lipid mixtures are crucial for assigning biological functions to lipids of fungi. Recently, much has been achieved in the field of mass spectrometry, allowing high-throughput screening of simple and complex lipid structures. The next-generation, high-resolution mass spectrometers allow accurate analysis and a much broader spectrum for lipidomic studies; nonetheless, these are often expensive, and data analysis is complex, which presents a challenge in routine lipid studies. Alternatively, by coupling the ion trap with multiple reaction monitoring (MRM) in an HPLC-ESI-MS/MS (high-performance liquid chromatography-electrospray ionization tandem mass spectrometry) platform, we can achieve rapid, sensitive, and accurate quantification of lipids in Candida extracts. Moreover, the approach is simple and relatively cost-effective. Below, we discuss the key features of ion trap HPLC-ESI-MS/MS-based comparative lipidomics of Candida cells.

Keywords:  Cell membrane; Ion trap-based triple quadrupole mass spectrometry; Lipid profiles; Phospholipids

DOI:  https://doi.org/10.1007/978-1-0716-2549-1_9
Curr Opin Chem Biol. 2022 Aug 23. pii: S1367-5931(22)00084-9. [Epub ahead of print]70 102199

Molecular network strategy in multi-omics and mass spectrometry imaging.

Chao Zhao, Jiyang Dong, Lingli Deng, Yawen Tan, Wei Jiang, Zongwei Cai.

  Human physiological activities and pathological changes arise from the coordinated interactions of multiple molecules. Mass spectrometry (MS)-based multi-omics and MS imaging (MSI)-based spatial omics are powerful methods used to investigate molecular information related to the phenotype of interest from homogenated or sliced samples, including the qualitative, relative quantitative and spatial distributions. Molecular network strategy provides efficient methods to help us understand and mine the biological patterns behind the phenotypic data. It illustrates and combines various relationships between molecules, and further performs the molecule identification and biological interpretation. Here, we describe the recent advances of network-based analysis and its applications for different biological processes, such as, obesity, central nervous system diseases, and environmental toxicology.

Keywords:  Mass spectrometry; Mass spectrometry imaging; Metabolomics; Molecular network strategy; Spatial omics

DOI:  https://doi.org/10.1016/j.cbpa.2022.102199
J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Aug 09. pii: S1570-0232(22)00317-8. [Epub ahead of print]1209 123413

Multi-steroid profiling by UHPLC-MS/MS with post-column infusion of ammonium fluoride.

Lina Schiffer, Fozia Shaheen, Lorna C Gilligan, Karl-Heinz Storbeck, James M Hawley, Brian G Keevil, Wiebke Arlt, Angela E Taylor.

  BACKGROUND: Multi-steroid profiling is a powerful analytical tool that simultaneously quantifies steroids from different biosynthetic pathways. Here we present an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay for the profiling of 23 steroids using post-column infusion of ammonium fluoride.METHODS: Following liquid-liquid extraction, steroids were chromatographically separated over 5 min using a Phenomenex Luna Omega C18 column and a water (0.1 % formic acid) methanol gradient. Quantification was performed on a Waters Acquity UHPLC and Xevo® TQ-XS mass spectrometer. Ammonium fluoride (6 mmol/L, post-column infusion) and formic acid (0.1 % (vol/vol), mobile phase additive) were compared as additives to aid ionisation.
RESULTS: Post-column infusion of ammonium fluoride enhanced ionisation in a steroid structure-dependent fashion compared to formic acid (122-140 % for 3βOH-Δ5 steroids and 477-1274 % for 3-keto-Δ4 steroids). Therefore, we analytically validated post-column infusion of ammonium fluoride. Lower limits of quantification ranged from 0.3 to 3 nmol/L; All analytes were quantifiable with acceptable accuracy (bias range -14 % to 11.9 % for 21/23, -21 % to 11.9 % for all analytes). Average recovery ranged from 91.6 % to 113.6 % and average matrix effects from -29.9 % to 19.9 %. Imprecision ranged from 2.3 % to 23 % for all analytes and was < 15 % for 18/23 analytes. The serum multi-steroid profile of 10 healthy men and 10 healthy women was measured.
CONCLUSIONS: UHPLC-MS/MS with post-column infusion of ammonium fluoride enables comprehensive multi-steroid profiling through enhanced ionisation particularly benefiting the detection of 3-keto-Δ4 steroids.

Keywords:  11-oxygenated androgens; Androgens; Glucocorticoids; Liquid chromatography-tandem mass spectrometry; Mineralocorticoids; Steroid quantification

DOI:  https://doi.org/10.1016/j.jchromb.2022.123413
Metabolites. 2022 Aug 12. pii: 742. [Epub ahead of print]12(8):

A Sample Preparation Method for the Simultaneous Profiling of Signaling Lipids and Polar Metabolites in Small Quantities of Muscle Tissues from a Mouse Model for Sarcopenia.

Yupeng He, Marlien van Mever, Wei Yang, Luojiao Huang, Rawi Ramautar, Yvonne Rijksen, Wilbert P Vermeij, Jan H J Hoeijmakers, Amy C Harms, Peter W Lindenburg, Thomas Hankemeier.

  The metabolic profiling of a wide range of chemical classes relevant to understanding sarcopenia under conditions in which sample availability is limited, e.g., from mouse models, small muscles, or muscle biopsies, is desired. Several existing metabolomics platforms that include diverse classes of signaling lipids, energy metabolites, and amino acids and amines would be informative for suspected biochemical pathways involved in sarcopenia. The sample limitation requires an optimized sample preparation method with minimal losses during isolation and handling and maximal accuracy and reproducibility. Here, two developed sample preparation methods, BuOH-MTBE-Water (BMW) and BuOH-MTBE-More-Water (BMMW), were evaluated and compared with previously reported methods, Bligh-Dyer (BD) and BuOH-MTBE-Citrate (BMC), for their suitability for these classes. The most optimal extraction was found to be the BMMW method, with the highest extraction recovery of 63% for the signaling lipids and 81% for polar metabolites, and an acceptable matrix effect (close to 1.0) for all metabolites of interest. The BMMW method was applied on muscle tissues as small as 5 mg (dry weight) from the well-characterized, prematurely aging, DNA repair-deficient Ercc1∆/- mouse mutant exhibiting multiple-morbidities, including sarcopenia. We successfully detected 109 lipids and 62 polar targeted metabolites. We further investigated whether fast muscle tissue isolation is necessary for mouse sarcopenia studies. A muscle isolation procedure involving 15 min at room temperature revealed a subset of metabolites to be unstable; hence, fast sample isolation is critical, especially for more oxidative muscles. Therefore, BMMW and fast muscle tissue isolation are recommended for future sarcopenia studies. This research provides a sensitive sample preparation method for the simultaneous extraction of non-polar and polar metabolites from limited amounts of muscle tissue, supplies a stable mouse muscle tissue collection method, and methodologically supports future metabolomic mechanistic studies of sarcopenia.

Keywords:  metabolomics extraction; muscle ageing and sarcopenia; muscle tissue; polar metabolites; signaling lipids

DOI:  https://doi.org/10.3390/metabo12080742
Metabolites. 2022 Aug 18. pii: 760. [Epub ahead of print]12(8):

A Micro-Scale Analytical Method for Determining Glycogen Turnover by NMR and FTMS.

Timothy L Scott, Juan Zhu, Teresa A Cassel, Sara Vicente-Muñoz, Penghui Lin, Richard M Higashi, Andrew N Lane, Teresa W-M Fan.

  Glycogen is a readily deployed intracellular energy storage macromolecule composed of branched chains of glucose anchored to the protein glycogenin. Although glycogen primarily occurs in the liver and muscle, it is found in most tissues, and its metabolism has been shown to be important in cancers and immune cells. Robust analysis of glycogen turnover requires stable isotope tracing plus a reliable means of quantifying total and labeled glycogen derived from precursors such as 13C6-glucose. Current methods for analyzing glycogen are time- and sample-consuming, at best semi-quantitative, and unable to measure stable isotope enrichment. Here we describe a microscale method for quantifying both intact and acid-hydrolyzed glycogen by ultra-high-resolution Fourier transform mass spectrometric (UHR-FTMS) and/or NMR analysis in stable isotope resolved metabolomics (SIRM) studies. Polar metabolites, including intact glycogen and their 13C positional isotopomer distributions, are first measured in crude biological extracts by high resolution NMR, followed by rapid and efficient acid hydrolysis to glucose under N2 in a focused beam microwave reactor, with subsequent analysis by UHR-FTMS and/or NMR. We optimized the microwave digestion time, temperature, and oxygen purging in terms of recovery versus degradation and found 10 min at 110-115 °C to give >90% recovery. The method was applied to track the fate of 13C6-glucose in primary human lung BEAS-2B cells, human macrophages, murine liver and patient-derived tumor xenograft (PDTX) in vivo, and the fate of 2H7-glucose in ex vivo lung organotypic tissue cultures of a lung cancer patient. We measured the incorporation of 13C6-glucose into glycogen and its metabolic intermediates, UDP-Glucose and glucose-1-phosphate, to demonstrate the utility of the method in tracing glycogen turnover in cells and tissues. The method offers a quantitative, sensitive, and convenient means to analyze glycogen turnover in mg amounts of complex biological materials.

Keywords:  13C6-glucose; glycogen turnover; microwave-assisted hydrolysis; stable isotope resolved metabolomics (SIRM)

DOI:  https://doi.org/10.3390/metabo12080760
Metabolites. 2022 Aug 21. pii: 768. [Epub ahead of print]12(8):

An Open-Source Pipeline for Processing Direct Infusion Mass Spectrometry Data of the Human Plasma Metabolome.

Anna Kozlova, Timur Shkrigunov, Semyon Gusev, Maria Guseva, Elena Ponomarenko, Andrey Lisitsa.

  Direct infusion mass spectrometry (DIMS) is growing in popularity as an effective method for the screening of biological samples in clinical metabolomics. Being quick to execute, DIMS generally requires special skills when interpreting the results of measurements. By inspecting the similarities between two-dimensional electrospray ionization with quadrupole time-of-flight (ESI-QTOF) and matrix-assisted laser desorption/ionization (MALDI) mass spectra, the pipeline for processing QTOF mass spectra using open-source packages (MALDIquant, MSnbase and MetaboAnalystR) was tested. Previously, all algorithmic workflows have relied on the application of software either provided by a vendor or privately developed by enthusiasts. Here, we computationally examined two ways of interpreting the DIMS results of human blood metabolomic profiling. The studied spectra were acquired using ESI-QTOF maXis Impact II (Bruker Daltonics, Billerica, MA, USA), then pre-processed using COMPASS/DataAnalysis commercial software and mapped onto the metabolites using in-lab-developed MatLab scripts. Alternatively, in this work we used the open-source packages MALDIquant, for spectrum pre-processing, and MetaboAnalystR, for data interpretation, instead of the low-availability commercial and home-made tools. Using a set of 100 plasma samples (20 from volunteers with normal body mass index and 80 from patients at different stages of obesity), we observed a high degree of concordance in annotated metabolic pathways between the proprietary DataAnalysis/MatLab pipeline and our freely available solution.

Keywords:  MALDIquant; MetaboAnalyst; MetaboAnalystR; Mummichog; direct infusion mass-spectrometry; metabolomics

DOI:  https://doi.org/10.3390/metabo12080768
Metabolites. 2022 Jul 30. pii: 714. [Epub ahead of print]12(8):

Simultaneous Quantification of Steroid Hormones Using hrLC-MS in Endocrine Tissues of Male Rats and Human Samples.

Guillermo Bordanaba-Florit, Sebastiaan van Liempd, Diana Cabrera, Félix Royo, Juan Manuel Falcón-Pérez.

  Steroid hormones play a vital role in the regulation of cellular processes, and dysregulation of these metabolites can provoke or aggravate pathological issues, such as autoimmune diseases and cancer. Regulation of steroid hormones involves different organs and biological compartments. Therefore, it is important to accurately determine their levels in tissues and biofluids to monitor changes after challenge or during disease. In this work, we have developed and optimized the extraction and quantification of 11 key members of the different steroid classes, including androgens, estrogens, progestogens and corticoids. The assay consists of a liquid/liquid extraction step and subsequent quantification by high-resolution liquid chromatography coupled time-of-flight mass spectrometry. The recoveries range between 74.2 to 126.9% and 54.9 to 110.7%, using a cell culture or urine as matrix, respectively. In general, the signal intensity loss due to matrix effect is no more than 30%. The method has been tested in relevant steroidogenic tissues in rat models and it has also been tested in human urine samples. Overall, this assay measures 11 analytes simultaneously in 6 min runtime and it has been applied in adrenal gland, testis, prostate, brain and serum from rats, and urine and extracellular vesicles from humans.

Keywords:  androgens; hormone-dependent disease; liquid chromatography–mass spectrometry; metabolomics; steroid hormones; time-of-flight; urinary extracellular vesicles

DOI:  https://doi.org/10.3390/metabo12080714
Metabolomics. 2022 Aug 27. 18(9): 70

Quality assurance and quality control reporting in untargeted metabolic phenotyping: mQACC recommendations for analytical quality management.

metabolomics Quality Assurance and Quality Control Consortium (mQACC)

  BACKGROUND: Demonstrating that the data produced in metabolic phenotyping investigations (metabolomics/metabonomics) is of good quality is increasingly seen as a key factor in gaining acceptance for the results of such studies. The use of established quality control (QC) protocols, including appropriate QC samples, is an important and evolving aspect of this process. However, inadequate or incorrect reporting of the QA/QC procedures followed in the study may lead to misinterpretation or overemphasis of the findings and prevent future metanalysis of the body of work.OBJECTIVE: The aim of this guidance is to provide researchers with a framework that encourages them to describe quality assessment and quality control procedures and outcomes in mass spectrometry and nuclear magnetic resonance spectroscopy-based methods in untargeted metabolomics, with a focus on reporting on QC samples in sufficient detail for them to be understood, trusted and replicated. There is no intent to be proscriptive with regard to analytical best practices; rather, guidance for reporting QA/QC procedures is suggested. A template that can be completed as studies progress to ensure that relevant data is collected, and further documents, are provided as on-line resources.
KEY REPORTING PRACTICES: Multiple topics should be considered when reporting QA/QC protocols and outcomes for metabolic phenotyping data. Coverage should include the role(s), sources, types, preparation and uses of the QC materials and samples generally employed in the generation of metabolomic data. Details such as sample matrices and sample preparation, the use of test mixtures and system suitability tests, blanks and technique-specific factors are considered and methods for reporting are discussed, including the importance of reporting the acceptance criteria for the QCs. To this end, the reporting of the QC samples and results are considered at two levels of detail: "minimal" and "best reporting practice" levels.

Keywords:  Mass spectrometry (MS); Nuclear magnetic resonance (NMR) spectroscopy; Quality assurance (QA); Quality control (QC); Reporting standards; Untargeted metabolomics

DOI:  https://doi.org/10.1007/s11306-022-01926-3
Cancers (Basel). 2022 Aug 18. pii: 3992. [Epub ahead of print]14(16):

Meta-Analysis Reveals Both the Promises and the Challenges of Clinical Metabolomics.

Heidi E Roth, Robert Powers.

  Clinical metabolomics is a rapidly expanding field focused on identifying molecular biomarkers to aid in the efficient diagnosis and treatment of human diseases. Variations in study design, metabolomics methodologies, and investigator protocols raise serious concerns about the accuracy and reproducibility of these potential biomarkers. The explosive growth of the field has led to the recent availability of numerous replicate clinical studies, which permits an evaluation of the consistency of biomarkers identified across multiple metabolomics projects. Pancreatic ductal adenocarcinoma (PDAC) is the third-leading cause of cancer-related death and has the lowest five-year survival rate primarily due to the lack of an early diagnosis and the limited treatment options. Accordingly, PDAC has been a popular target of clinical metabolomics studies. We compiled 24 PDAC metabolomics studies from the scientific literature for a detailed meta-analysis. A consistent identification across these multiple studies allowed for the validation of potential clinical biomarkers of PDAC while also highlighting variations in study protocols that may explain poor reproducibility. Our meta-analysis identified 10 metabolites that may serve as PDAC biomarkers and warrant further investigation. However, 87% of the 655 metabolites identified as potential biomarkers were identified in single studies. Differences in cohort size and demographics, p-value choice, fold-change significance, sample type, handling and storage, data collection, and analysis were all factors that likely contributed to this apparently large false positive rate. Our meta-analysis demonstrated the need for consistent experimental design and normalized practices to accurately leverage clinical metabolomics data for reliable and reproducible biomarker discovery.

Keywords:  NMR; clinical metabolomics; mass spectrometry; meta-analysis; pancreatic cancer biomarkers

DOI:  https://doi.org/10.3390/cancers14163992
Molecules. 2022 Aug 16. pii: 5211. [Epub ahead of print]27(16):

Analytical Approaches for the Determination of Buprenorphine, Methadone and Their Metabolites in Biological Matrices.

Xiaoyue Shan, Chengjian Cao, Bingsheng Yang.

  The abuse of buprenorphine and methadone has grown into a rising worldwide issue. After their consumption, buprenorphine, methadone and their metabolites can be found in the human organism. Due to the difficulty in the assessment of these compounds by routine drug screening, the importance of developing highly sensitive analytical approaches is undeniable. Liquid chromatography tandem mass spectrometry is the preferable technique for the determination of buprenorphine, methadone and their metabolites in biological matrices including urine, plasma, nails or oral fluids. This research aims to review a critical discussion of the latest trends for the monitoring of buprenorphine, methadone and their metabolites in various biological specimens.

Keywords:  analytical methods; biological matrices; buprenorphine; methadone

DOI:  https://doi.org/10.3390/molecules27165211
Anal Chim Acta. 2022 Aug 29. pii: S0003-2670(22)00771-1. [Epub ahead of print]1223 340200

Quantification in bioimaging by LA-ICPMS - Evaluation of isotope dilution and standard addition enabled by micro-droplets.

Andreas Schweikert, Sarah Theiner, Martin Šala, Petra Vician, Walter Berger, Bernhard K Keppler, Gunda Koellensperger.

  This study explores quantitative bioimaging as enabled by laser ablation-inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOFMS), designing standardization methods based on robotic micro-droplet dispensing. The potential of producing controlled and highly precise pL-volume droplets was exploited to establish on-tissue isotope dilution and standard addition. Both strategies eliminate matrix effects and offer high metrological order traceable to SI units. The absolute quantity was obtained for μm-sized regions of interest in tissue samples, as defined by the extension of the deposited pL-volume droplet. While the gold standard isotope dilution (ID) was restricted to the accurate quantification of a single element, i.d. platinum in different tissue samples (mouse liver, spleen and tumor tissue), multiplexed matrix-matched calibration was obtained by on-tissue standard addition by depositing a dilution series of certified multi-element standards. Here, the working range was determined by the heterogeneity of the tissue samples and the background levels of elements intrinsically present and/or artificially introduced during sample preparation. Both methods, ID and standard addition served as reference methods for validation of external calibration using gelatin-based micro-droplet standards. Given full ablation, these external standards revealed a high dynamic range together with an excellent repeatability. Where applicable, the cross-validation revealed consistent quantitative results for the three quantification approaches. The comparable sensitivity obtained for standard addition and external standardization, respectively expressed as slope of the calibration function, provided proof that gelatin-based micro-droplets could serve as matrix-matched calibrations. Therefore, gelatin micro-droplets offer a valid tool for multiplexed matrix-mimicking standardization at high-throughput.

Keywords:  Gelatin micro-droplet standards; Isotope dilution; LA-ICPMS imaging; Quantification; Standard addition

DOI:  https://doi.org/10.1016/j.aca.2022.340200
Arch Toxicol. 2022 Aug 25.

One-step extraction and determination of 513 psychoactive substances, drugs, and their metabolites from hair by LC-MS/MS.

Jadwiga Musiał, Jolanta Powierska-Czarny, Jakub Czarny, Michał Raczkowski, Natalia Galant, Bogusław Buszewski, Renata Gadzała-Kopciuch.

  The emergence of new psychoactive substances on the market is a significant problem on a global scale. This type of substance in society is associated with many negative consequences, such as traffic accidents, accidents at work, rape, homicide, poisoning, or overdose deaths. The analysis of these substances in biological samples is very important for further legal action and saving lives. Therefore, laboratories face a tremendous challenge in tackling the evolving drug market. The paper describes the optimization of the analytical LC-MS/MS method to identify and determine 513 psychoactive substances in hair samples. A method of chromatographic separation was developed, and the working parameters of the mass spectrometer were selected for each analyte. The method has been validated, and the results are as follows: the limit of quantification of the developed method ranges from 0.025 to 1.25 ng/mg hair. The mean recovery of the tested analytes ranges from 80 to 120%. The achieved coefficient of variation in within-run precision ranged from 1.05 to 19.99%. The results achieved for BIAS are in the range of ± 20%.

Keywords:  Drugs; Hair analysis; Liquid chromatography-tandem mass spectrometry (LC–MS/MS); Metabolites; New psychoactive substances (NPS)

DOI:  https://doi.org/10.1007/s00204-022-03343-w
J Chromatogr Sci. 2022 Aug 20. pii: bmac067. [Epub ahead of print]

Development of a Validated UPLC-MS/MS Method for Simultaneous Estimation of Neratinib and Curcumin in Human Plasma: Application to Greenness Assessment and Routine Quantification.

Alhumaidi B Alabbas, Safar M Alqahtani, Sagar Suman Panda, Majed Alrobaian, Ali Altharawi, Waleed H Almalki, Md Abul Barkat, Rehan A Rub, Mahfoozur Rahman, Shehla Nasar Mir Najib Ullah, Sarwar Beg.

A validated ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the first-ever simultaneous analysis of neratinib, curcumin and internal standard (imatinib) using acetonitrile as the liquid-liquid extraction medium. On a BEH C18 (100 mm × 2.1 mm, 1.7 μm) column, the analytes were separated isocratically using acetonitrile (0.1% formic acid):0.002M ammonium acetate. The flow rate was set at 0.5 mL.min-1. The authors utilized multiple reaction monitoring-based transitions for the precursor-to-product ion with m/z 557.099 → 111.928 for neratinib, m/z 369.231 → 176.969 curcumin and m/z 494.526 → 394.141 for imatinib during the study. Validation of the method as per United States Food and Drug Administration requirements for linearity (5-40 ng mL-1), accuracy and precision, stability, matrix effect, etc. were investigated and were observed to be acceptable. Afterward, we evaluated the method for establishing its greenness profile by using two greenness assessment tools and found it green. Overall, a reliable green UPLC-MS/MS method was devised and used to estimate neratinib and curcumin in human plasma simultaneously.

DOI: https://doi.org/10.1093/chromsci/bmac067
Sci Rep. 2022 Aug 22. 12(1): 14308

Assessment of creatinine concentration in whole blood spheroids using paper spray ionization-tandem mass spectrometry.

Tung-Ting Sham, Abraham K Badu-Tawiah, Stephen J McWilliam, Simon Maher.

Accurate quantification of blood creatinine is important to estimate the glomerular filtration rate. Existing techniques using liquid chromatography tandem mass spectrometry (LC-MS/MS) have a high accuracy and eliminate most interferences encountered in routine enzymatic and Jaffé methods. However, they require laborious and time-consuming sample treatment and data acquisition. The aim of this study is to develop a fast and simple method to enable a direct analysis of whole blood creatinine with performance measures that are comparable to conventional LC-MS/MS. 5μL whole blood is formed as a three-dimensional spheroid on hydrophobic silanized paper substrates which then undergoes paper-spray ionization-tandem mass spectrometry (PSI-MS/MS). The method is validated using real human samples and compared with LC-MS/MS. PSI-MS/MS whole blood analysis exhibited a lower limit of quantification of 2.5 μg/mL, precision ≤ 6.3%, recovery in the range of 88-94% and excellent linearity (R2 > 0.99; 2.5-20 μg/mL) covering the normal range for creatinine levels. Creatinine levels were comparable to those measured by LC-MS/MS with small deviations of less than 0.3 μg/mL. This simple, fast and accurate microsampling technique for direct analysis of creatinine from whole blood shows promise for routine clinical screening and monitoring. This approach can be readily extended for other analytes of interest and, due to inherent advantages relating to cost, storability, speed, and simplicity, it can be especially advantageous for use in resource-limited settings.

DOI: https://doi.org/10.1038/s41598-022-18365-8
Front Nutr. 2022 ;9 977076

A strategy for accurately and sensitively quantifying free and esterified fatty acids using liquid chromatography mass spectrometry.

Xiaohui Feng, Juan Wang, Zhonghai Tang, Bingyao Chen, Xinhua Hou, Jing Li, Shengnan Feng, Peng Li, Qingshi Meng.

  Fatty acid (FA) composition of foods dictates a diversity of aspects regarding food quality, ranging from product shelf life, sensory properties to nutrition. There is a challenge to quantitate FAs using liquid chromatography-mass spectrometry due to poor ionization efficiency and matrix effects. Here, an isotopic-tagged derivatization strategy was established to accurately and sensitively quantify free and esterified FAs. After derivatization reaction, the detection sensitivity of FAs was remarkably improved and the limit of quantitation was lower than 100 ng/L. The quantitative errors caused by matrix effects were diminished benefiting from isotope-derivatized internal standards. The established quantitation strategy was successfully applied to verify both free and esterified FA contents in meat after different post-harvest procedures, finding that free polyunsaturated FAs increased significantly during freezing process.

Keywords:  chemical derivatization; fatty acids; isotopic-based derivatization; meat processing; quantification

DOI:  https://doi.org/10.3389/fnut.2022.977076
Ecotoxicol Environ Saf. 2022 Aug 23. pii: S0147-6513(22)00826-0. [Epub ahead of print]243 113986

Analytical method for the biomonitoring of bisphenols and parabens by liquid chromatography coupled to tandem mass spectrometry in human hair.

Julien Robin, Guillaume Binson, Marion Albouy, Alexis Sauvaget, Pascale Pierre-Eugène, Virginie Migeot, Antoine Dupuis, Nicolas Venisse.

  Bisphenols and parabens are endocrine disruptors families widely used in daily life. They are known to be linked to numerous pathologies such as reproductive disorders, obesity, breast cancer, hypertension and asthma. Biomonitoring is an essential tool for assessing population exposure to environmental pollutants. Blood and urine are the main matrices used in human biomonitoring. However, they are not suitable to evaluate long-term exposure to endocrine disruptors with a short elimination half-life such as parabens or phenols. Hair appears to be an interesting alternative matrix allowing a wide window of exposure due to an accumulation of xenobiotics during hair growth. This study presents the development and validation of a high-performance liquid chromatography coupled to tandem mass spectrometry for the simultaneous determination of bisphenol A, its chlorinated derivatives, bisphenol F, bisphenol S and parabens in human hair. An optimised sample preparation based on acidic hydrolysis followed by liquid-liquid extraction was performed, before an analysis by ultra-high performance liquid chromatography coupled to tandem mass spectrometry in multiple reaction monitoring mode. To validate the method, recognized bioanalytical guidelines were used and calibration and quality control samples were prepared in human hair samples. Linearities were over 0.996 in the whole range of concentrations. Trueness and precision were demonstrated for each target analyte with intra-day and inter-day bias values ranging from 86 % to 118 % and relative standard deviation values ranging from 0 % to 19 %. At the same time, limits of quantification were set at 0.25 ng/g for bisphenol A and parabens, 0.05 ng/g for bisphenols F and S and 0.00625 ng/g for the chlorinated derivatives of bisphenol A. This reliable method was applied to hair samples taken from hospital professionals and allowed the quantification of these endocrine disruptors in this population. Chlorinated derivatives of bisphenol A were quantified here in hair for the first time.

Keywords:  Biomonitoring; Bisphenols; Hair; Parabens

DOI:  https://doi.org/10.1016/j.ecoenv.2022.113986
Biomed Chromatogr. 2022 Aug 24. e5487

Liquid chromatography-tandem mass spectrometry method for simultaneous quantification of neurotransmitters in rat brain tissue exposed to 4'-Fluoro-α-PHP.

Roberta Rodrigues Viana, Ana Miguel Fonseca Pego, Tiago Franco de Oliveira, Eliane Dallegrave, Sarah Eller.

  The combination of different advanced analytical techniques makes it possible to determine the concentrations of neurotransmitters in various biological matrices, providing a complex and comprehensive study of the effects of psychoactive substances. The present study aimed to develop and validate a fast and simple analytical method for the determination of acetylcholine, serotonin, γ-aminobutyric acid, glutamate, dopamine, and metabolites in rats brain tissue by liquid chromatography coupled to tandem mass spectrometry. The brain was homogenized and an aliquot of sample, dopamine-d4 , and acetone were added in a tube and then vortexed and centrifuged. The supernatant was collected and dried. The residue was reconstituted and injected. LLOQ ranged from 0.001 to 1 μg/g; intra-run precision from 0.47 to 11.52%; inter-run precision from 0.68 to 17.54%; bias from 89.10 to 109.60%. As proof of concept, the method was applied to animals exposed to the synthetic cathinone 4'-fuoro-α-pyrrolidinohexanophenone (300 mg/kg). In addition, the workflow proved to be simple, rapid, and useful to estimate the concentration of neurotransmitters. This analytical tool can be used to support the investigation of the changes in the neurochemical profile for the characterization of the mechanism of action of psychoactive substances, as well as both neurologic and psychiatric diseases.

Keywords:  4′-Fluoro-α-PHP; LC-MS/MS; Neurotransmitters; quantitative method; rat brain tissue

DOI:  https://doi.org/10.1002/bmc.5487
ACS Meas Sci Au. 2022 Aug 17. 2(4): 370-376

Tandem Mass Spectrometry as an Independent Method for Corroborating Fluorine-18 Radioactivity Measurements in Positron Emission Tomography.

H Umesha Shetty, Cheryl L Morse, Victor W Pike.

Positron emission tomography (PET) uses many tracers labeled with fluorine-18 (t 1/2 = 109.8 min; β+ 97%) for quantitative imaging of biochemical and physiological processes in animal and human subjects. In PET methodology, the radioactivity in a dose of an 18F-labeled tracer to be administered to a living subject is measured with a calibrated ionization chamber. This type of detector measures the radioactivity of a sample relative to those of certified amounts of longer-lived surrogate isotopes that are recommended for detector calibration. No alternative means for corroborating widely varying fluorine-18 radioactivity measurements from calibrated ionization chambers has been available. Here, we describe an independent nonradiometric method for this purpose. In this method, highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) is used to quantify the relative masses of the radioactive isotopologue ([18F]1) and the accompanying nonradioactive counterpart (carrier 1) in an 18F-labeled tracer preparation to give the mole ratio of [18F]1. High-performance liquid chromatography (HPLC) with a mass-calibrated absorbance detection is used alongside to provide a separate measurement of the aggregate mass of all isotopologues. The radioactivity of the radiotracer is then derived in becquerels (Bq) from these two measurements, plus Avogadro's number and the decay constant of fluorine-18. For the chosen example [18F]LSN3316612, the radioactivity values determined nonradiometrically and with a selected ionization chamber were in fair agreement. In addition, LC-MS/MS alone was found to provide an accurate measure of the half-life of fluorine-18.

DOI: https://doi.org/10.1021/acsmeasuresciau.2c00018
Talanta. 2022 Aug 05. pii: S0039-9140(22)00599-9. [Epub ahead of print]252 123803

Comparison of two azobenzene-based amino acid derivatization reagents for LC-MS/MS analysis in positive and negative ESI modes.

Ernesto de Jesus Zapata Flores, Nguyễn Kim Ngân Bùi, Sigrid Selberg, Koit Herodes, Ivo Leito.

  Derivatization reagents based on azobenzene containing different N-hydroxysuccinimidyl moieties- AzoB (carbamate) and AzoC (ester) - are proposed for the LC-ESI-MS analysis of free amino acids in fermented beverages and juices. A dual comparison between LC-MS/MS in positive and negative ESI modes in dynamic Multiple Reaction Monitoring (dMRM) and Neutral Loss Scan was investigated. The results indicate that the studied carbamate derivatization reagent, AzoB, can be employed for targeted analysis (MS/MS) but also for non-targeted analysis of derivatized amino acids thanks to its constant neutral loss (223 Da) that is the same in both ionisation modes. For amines, precursor ion scan can be used as identification tool. The derivatization properties of AzoB and AzoC were compared against other derivatization reagents, and they showed advantages such as fast derivatization reaction and good reactivity with secondary amines. AzoC also displayed a disadvantage -side products were formed that affect the quantitation. Free amino acids profile of Kvass (a fermented beverage from eastern Europe) was determined for first time, proline was found to be the most abundant amino acid.

Keywords:  Amino acids; Derivatization; ESI; LC-MS; MS/MS; Neutral loss scan

DOI:  https://doi.org/10.1016/j.talanta.2022.123803
Biomed Chromatogr. 2022 Aug 22. e5485

A validated LC-MS/MS method for the determination of L-hyoscyamine in human plasma: Application in clinical study.

Yunhai Duan, Zhenglong Zuo, Yinchun Chen, Qingqing Song, Mingming Yu, Yong Yang.

  Atropine is a racemic mixture of D- and L-hyoscyamine, while L-hyoscyamine is the only effective ingredient. In this study, a new sensitive, stable, and selective LC/MS assay was developed for determination of L-hyoscyamine and applied to the clinical study. The parent-product (m/z) transition pair of L-hyoscyamine was 290.1→124.1. Chromatographic separations were performed using a Chiral MZ column (250 mm×4.6 mm, 5.0 μm) by a stepwise gradient elution mode with n-hexane, isopropanol and diethylamine as mobile phases. L-hyoscyamine in human plasma were extracted using liquid-liquid extraction process. This assay displayed a good linearity over a concentration range of 20.0-400 pg/mL for L-hyoscyamine. The accuracy of the validation assay for L-hyoscyamine ranged from -2.7% to 4.5%, and the precision were within 6.3% coefficient of variation. L-hyoscyamine in human plasma were stable at different storage conditions. The method has been successfully applied to plasma samples obtained from a safety study in human.

Keywords:  Atropine; Chiral column; L-hyoscyamine; LC-MS/MS

DOI:  https://doi.org/10.1002/bmc.5485
Drug Test Anal. 2022 Aug 26.

Valproic acid determination by liquid chromatography coupled to mass spectrometry (LC-MS/MS) in whole blood for forensic purposes.

Jennifer P Pascali, Arianna Giorgetti, Rossella Barone, Guido Pelletti, Paolo Fais.

Valproic acid (VPA) is a well-known drug prescribed as anti-epileptic. It has a narrow therapeutic range and shows great individual differences in pharmacodynamics and pharmacokinetics. Consequently, the therapeutical drug monitoring (TDM) in patient's plasma is of crucial importance. Liquid chromatography coupled to mass spectrometry (LC-MS/MS) has gained importance in TDM applications for its features of sensitivity, selectivity and rapidity. However, in case of VPA, the LC-MS/MS selectivity could be hampered by the lack of a sufficient number of MRM transitions describing the molecule. In fact, the product ion scan of deprotonated molecules of VPA does not produce any ion and thus most LC-MS/MS methods are based on the detection of the unique MRM transition m/z 143➔143. In this way, the advantages of selectivity in LC-MS cannot be effectively exploited. In the present method stable analyte adducts were exploited for the determination of VPA in blood. An Acquity HSS C18 column and mobile phases consisting of 5 mM ammonium formate and acetonitrile both added 0.1% formic acid were used. Source worked in negative acquisition mode and parameters were optimized to increase the adduct (m/z 189) and dimer (m/z 287) stability and their fragmentation were used to increase the selectivity of MRM detection. The method has been validated according to the toxicological forensic guidelines and successfully applied to 10 real blood samples. Finally, the present method showed suitable for the rapid LC-MS/MS detection of VPA in whole blood, demonstrating the possibility to increase specificity by exploiting stable in-source adducts. This should be considered of utmost importance in the case of forensic applications.

DOI: https://doi.org/10.1002/dta.3362
Anal Chem. 2022 Aug 24.

Controlled Formation of Protonated and Radical Cation Precursor Ions by Atmospheric Pressure Photoionization with μLC-MS Enables Electron Ionization and MS/MS Library Search.

Patrick Mueller, Ron Bonner, Gérard Hopfgartner.

Atmospheric pressure photoionization (APPI) was developed as an alternative to electrospray ionization (ESI) for the generation of protonated molecules using liquid chromatography and optimized using dopants such as toluene, which predominantly forms protonated molecules, and chlorobenzene, which favors the formation of radical cations, although the latter has not been fully exploited. Based on 40 diverse low-molecular-weight compounds and micro liquid chromatography (μLC) coupled with APPI tandem mass spectrometry (APPI-MS/MS), the potential of radical cations was investigated. Chromatographic and ionization conditions were decoupled by post-column addition of methanol, allowing separate study and optimization. Due to the mass flow sensitive behavior of APPI, sensitivity is not affected by post-column dilution, and for 8 of 35 analytes, the radical cation response with μLC-APPI is better than for protonated molecules using μLC-ESI. Collision-induced fragmentation (CID) of radical cations produced within a collision energy range from 10-115 eV have, in the median, 65% of the fragments found in electron ionization (EI) spectra. This similarity allowed identification of 86% of the analytes using data-dependent acquisition (DDA) of radical cations and NIST EI library searches. We propose a workflow that uses multimodal DDA of protonated precursor molecules using ESI or APPI with toluene as a dopant, and radical cations produced by chlorobenzene-assisted μLC-APPI with post-column addition of methanol. This increases the confidence of molecular identification by allowing orthogonal library searches using MS/MS libraries for protonated precursor CID spectra and EI libraries for radical cation CID spectra.

DOI: https://doi.org/10.1021/acs.analchem.2c02105
J Am Soc Mass Spectrom. 2022 Aug 26.

Quantitation of Enantiomeric Excess in an Achiral Environment Using Trapped Ion Mobility Mass Spectrometry.

Joseph Czekner, Erik K Schneider, Patrick Weis, Manfred M Kappes.

We present a novel, straightforward method to determine the enantiomeric excess (ee) of tryptophan (Trp) and N-tert-butyloxycarbonyl-O-benzylserine (BBS) solutions without chiral additives. For this, lithium carbonate, sodium carbonate, or silver acetate was added to solutions of Trp or BBS. Singly negatively charged dimer and trimer clusters were then formed by electrospray ionization and analyzed using trapped ion mobility spectrometry (TIMS) and time-of-flight mass spectrometry. When a solution contains both enantiomers, homo- and heterochiral clusters are generated which can be separated in the TIMS-tunnel based on their different mobilities using a nitrogen buffer gas. The ratio of homochiral to heterochiral clusters shows a binomial distribution and can be calibrated with solutions of known ee to yield ee measurements of samples with better than 1% accuracy. Samples can be prepared rapidly, and measurements are completed in less than 5 min. Current instrumental limitations restrict this method to rigid molecules with large functional groups adjacent to the chiral centers. Nevertheless, we expect this method to be applicable to many pharmaceuticals and provide the example of 1-methyltryptophan to demonstrate this.

DOI: https://doi.org/10.1021/jasms.2c00136
NMR Biomed. 2022 Aug 23. e4817

Recent Progress in Analysis of Intermediary Metabolism by ex vivo 13 C NMR.

Craig R Malloy, A Dean Sherry, Jeffry R Alger, Eunsook S Jin.

  Advanced imaging technologies, large-scale metabolomics and the measurement of gene transcripts or enzyme expression all enable investigations of intermediary metabolism in human patients. Complementary information about fluxes in individual metabolic pathways may be obtained by ex vivo 13 C NMR of blood or tissue biopsies. Simple molecules such as 13 C-labeled glucose are readily administered to patients prior to surgical biopsies, and 13 C-labeled glycerol is easily administered orally to outpatients. Here we review recent progress in practical applications of 13 C NMR to study cancer biology, the response to oxidative stress, gluconeogenesis, triglyceride synthesis in patients, as well as new insights into compartmentation of metabolism in the cytosol. The technical aspects of obtaining the sample, preparing material for analysis, and acquiring the spectra are relatively simple. This approach enables convenient, valuable and quantitative insights into intermediary metabolism in patients.

Keywords:  13C; NMR; cancer; glucose; glycerol; metabolic syndrome; stable isotope

DOI:  https://doi.org/10.1002/nbm.4817
J Anal Toxicol. 2022 Aug 27. pii: bkac065. [Epub ahead of print]

β'-Phenylfentanyl Metabolism in Primary Human Hepatocyte Incubations: Identification of Potential Biomarkers of Exposure in Clinical and Forensic Toxicology.

Pietro Brunetti, Alfredo F Lo Faro, Annagiulia Di Trana, Angelo Montana, Giuseppe Basile, Jeremy Carlier, Francesco P Busardò.

  From 2014 onwards, illicit fentanyl and analogues have caused numerous intoxications and fatalities worldwide, impacting the demographics of opioid-related overdoses. The identification of cases involving fentanyl analogues is crucial in clinical and forensic settings to treat patients, elucidate intoxications, address drug use disorders, and tackle drug trends. However, in analytical toxicology, the concentration of fentanyl analogues in biological matrices is low, making their detection challenging. Therefore, the identification of specific metabolite biomarkers is often required to document consumption. β'-Phenylfentanyl (N-phenyl-N-[1-(2-phenylethyl)-4-piperidinyl]-benzenepropanamide) is a fentanyl analogue that was first detected in Sweden in 2017 and has recently reemerged onto the American illicit drug market. There is little data available on β'-phenylfentanyl effects and toxicokinetics, and its metabolism is yet to be studied. We aimed to investigate β'-phenylfentanyl human metabolism to identify potential biomarkers of use. To assist in β'-phenylfentanyl metabolite identification, a list of putative reactions was generated using in silico predictions with GLORYx freeware. β'-phenylfentanyl was incubated with cryopreserved 10-donor-pooled human hepatocytes, analyses were performed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS-MS), and data were processed using a partially automated targeted/untargeted approach with Compound Discoverer. We identified 26 metabolites produced by N-dealkylation, oxidation, hydroxylation, O-glucuronidation, O-methylation, and combinations thereof. We suggest β'-phenylnorfentanyl (N-phenyl-N-4-piperidinyl-benzenepropanamide) and further metabolites 1-oxo-N-phenyl-N-4-piperidinyl-benzenepropanamide and 1-hydroxy-N-phenyl-N-4-piperidinyl-benzenepropanamide as major biomarkers of β'-phenylfentanyl use. In silico predictions were mostly wrong, and β'-phenylfentanyl metabolic fate substantially differed from that of a closely related analogue incubated in the same conditions, highlighting the value of the experimental assessment of NPS human metabolism. In vivo data are necessary to confirm the present results. However, the present results may be necessary to help analytical toxicologists identify β'-phenylfentanyl-positive cases to provide authentic samples.

Keywords:   In silico metabolite prediction; 3-Phenylpropanoylfentanyl; Fentanyl analogue; Human hepatocyte metabolism; Liquid chromatography–high-resolution tandem mass spectrometry; Software-assisted data mining; Synthetic opioid; β’-Phenylfentanyl

DOI:  https://doi.org/10.1093/jat/bkac065
J Agric Food Chem. 2022 Aug 24.

Natural Deep Eutectic Solvent-Based Dispersive Liquid-Liquid Microextraction Coupled with Direct Analysis in Real Time Mass Spectrometry: A Green Temperature-Mediated Analytical Strategy.

Meng Chen, Ming Li, Wenxi Zhang, Hua Bai, Qiang Ma.

  Green analytical chemistry (GAC) represents a rapidly growing research field that aims at developing novel analytical approaches with minimal consumption of hazardous reagents and solvents. The current study reports on a GAC methodology exploiting the unique physicochemical properties of natural deep eutectic solvents (NADESs), a supposedly environmentally friendly class of solvents. Based on a temperature-mediated strategy, the NADESs were manipulated to undergo multiple phase transitions for favorable functionality and performance. As proof-of-concept demonstrations, both hydrophobic and hydrophilic NADESs were prepared for the extraction and analysis of eight phthalate esters in aqueous samples (food simulants) and three aflatoxins in oily samples (edible oils), respectively. NADES-based dispersive liquid-liquid microextraction (DLLME) was employed to achieve high-efficiency sample pretreatment. Afterward, the NADESs were transformed from liquids into solids by tuning the peripheral temperature for a convenient phase separation from the sample matrices. The solidified NADES extracts were melted and vaporized at elevated temperatures by transmission-mode direct analysis in real time (DART) for further quadrupole-Orbitrap high-resolution mass spectrometry (Q-Orbitrap HRMS) analysis. The developed protocol was validated, achieving good repeatability with relative standard deviations (RSDs) of less than 9% and satisfactory sensitivity with limits of detection (LODs) and quantitation (LOQs) ranging from 0.1 to 0.8 and 0.2 to 2.0 μg/kg, respectively. The greenness of the analytical methodology was assessed with the calculated scores of 0.66 and 0.57 for the hydrophobic and hydrophilic NADES-based protocols, respectively. The method was applied to marketed samples, highlighting the great potential for green chemical analysis.

Keywords:  direct analysis in real time mass spectrometry; dispersive liquid−liquid microextraction; edible oil; food contact materials; green analytical chemistry; natural deep eutectic solvent

DOI:  https://doi.org/10.1021/acs.jafc.2c03561