bims-metlip 2024-02-18 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2024–02–18
24 papers selected by
Sofia Costa, Matterworks

An IROA Workflow for correction and normalization of ion suppression in mass spectrometry-based metabolomic profiling data.
Dispersive liquid-liquid microextraction followed by sweeping micellar electrokinetic chromatography-tandem mass spectrometry for determination of six breast cancer drugs in human plasma.
A fast method for the determination of personal care product chemicals in human urine using dispersive liquid-liquid extraction and ultra high-performance liquid chromatography-tandem mass spectrometry.
SIMPEL: using stable isotopes to elucidate dynamics of context specific metabolism.
Therapeutic Monitoring of Palbociclib, Ribociclib, Abemaciclib, M2, M20, and Letrozole in Human Plasma: A Novel LC-MS/MS Method.
Studies of Labware Contamination during Lipid Extraction in Mass Spectrometry-Based Lipidome Analysis.
Simultaneously quantifying hundreds of acylcarnitines in multiple biological matrices within ten minutes using ultrahigh-performance liquid-chromatography and tandem mass spectrometry.
Liquid electron ionization-mass spectrometry as a novel strategy for integrating normal-phase liquid chromatography with low and high-resolution mass spectrometry.
Leveraging Unidentified Metabolic Features for Key Pathway Discovery: Chemical Classification-driven Network Analysis in Untargeted Metabolomics.
Routine application of SFC-MS in doping control: Analysis of 3 × 1000 urine samples using three different SFC-MS instruments.
A rapid and sensitive analytical methodology for the simultaneous biomonitoring of two direct oral anticoagulant drugs and their major metabolites in thromboembolic disordered patients samples for clinical evaluations.
Establishing a framework for best practices for quality assurance and quality control in untargeted metabolomics.
Ethanolysis of nitrogen mustards: A novel strategy for nitrogen mustard identification in environmental matrices by liquid chromatography-electrospray ionization-tandem mass spectrometry.
Quantitation of MUC5AC and MUC5B by Stable Isotope Labeling Mass Spectrometry.
Evaluation of normalization strategies for GC-based metabolomics.
Separation and Characterization of Synthetic Cannabinoid Metabolite Isomers Using SLIM High-Resolution Ion Mobility-Tandem Mass Spectrometry (HRIM-MS/MS).
Simultaneous quantification of endogenous biomarkers and detection of testosterone esters in human serum using gas chromatography-high-resolution mass spectrometry.
A validated liquid chromatography-tandem mass spectrometry assay for the analysis of isoniazid and its metabolite acetyl-isoniazid in urine.
Discovery of Glycation Products: Unraveling the Unknown Glycation Space Using a Mass Spectral Library from In Vitro Model Systems.
Chemical analysis of additives included in fully formulated oils using high-performance liquid chromatography-tandem mass spectrometry.
A biomarker panel of secondary hypertension is simultaneously quantified by coupling of magnetic solid-phase extraction and liquid chromatography-tandem mass spectrometry.
Multiscale biochemical mapping of the brain through deep-learning-enhanced high-throughput mass spectrometry.
Comparison of the applicability of electromembrane extraction and liquid-phase microextraction for extraction of non-polar basic drugs from different biological samples: Using clozapine as the model analyte.
Development of a LC-MS/MS Quantification Assay for Human Relaxin H2 and Extraction of Human Relaxin H1 and Relaxin H3.

Res Sq. 2024 Feb 01. pii: rs.3.rs-3914827. [Epub ahead of print]

An IROA Workflow for correction and normalization of ion suppression in mass spectrometry-based metabolomic profiling data.

Iqbal Mahmud, Bo Wei, Lucas Veillon, Lin Tan, Sara Martinez, Bao Tran, Alexander Raskind, Felice de Jong, Rehan Akbani, John N Weinstein, Chris Beecher, Philip L Lorenzi.

Ion suppression is a major problem in mass spectrometry (MS)-based metabolomics; it can dramatically decrease measurement accuracy, precision, and signal-to-noise sensitivity. Here we report a new method, the IROA TruQuant Workflow, that uses a stable isotope-labeled internal standard (IROA-IS) plus novel companion algorithms to 1) measure and correct for ion suppression, and 2) perform Dual MSTUS normalization of MS metabolomic data. We have evaluated the method across ion chromatography (IC), hydrophilic interaction liquid chromatography (HILIC), and reverse phase liquid chromatography (RPLC)-MS systems in both positive and negative ionization modes, with clean and unclean ion sources, and across different biological matrices. Across the broad range of conditions tested, all detected metabolites exhibited ion suppression ranging from 1% to 90+% and coefficient of variations ranging from 1% to 20%, but the Workflow and companion algorithms were highly effective at nulling out that suppression and error. Overall, the Workflow corrects ion suppression across diverse analytical conditions and produces robust normalization of non-targeted metabolomic data.

DOI: https://doi.org/10.21203/rs.3.rs-3914827/v1
J Chromatogr A. 2024 Feb 03. pii: S0021-9673(24)00071-2. [Epub ahead of print]1718 464698

Dispersive liquid-liquid microextraction followed by sweeping micellar electrokinetic chromatography-tandem mass spectrometry for determination of six breast cancer drugs in human plasma.

Zvonimir Mlinarić, Lu Turković, Miranda Sertić.

  Herein, we have developed a novel method of aqueous-sample dispersive liquid-liquid microextraction (AqS-DLLME) followed by sweeping micellar electrokinetic chromatography-tandem mass spectrometry (MEKC-MS/MS) for simultaneous determination of breast cancer drugs letrozole, anastrozole, palbociclib, ribociclib, abemaciclib, and fulvestrant in human plasma. Coupling of MEKC to MS was possible due to the use of ammonium perfluorooctanoate (APFO) as a volatile surfactant. The MEKC and MS conditions were optimized to achieve a fast, sensitive, selective, and green analysis enabling full separation of the analytes within 16 min. Electrophoretic buffer was 125 mM APFO at apparent pH 10.5 in 32 % MeOH, while sheath liquid was 70 % MeOH with 0.2 % formic acid, delivered at 10 µL/min. Excellent extraction recoveries from plasma ranging from 89.4 to 104.9 % were obtained with a combination of protein precipitation and DLLME. The developed method was validated according to the ICH guidelines. Remarkable selectivity, accuracy (bias < 6.7 %), precision (RSD < 15.8 %), and stability (bias < 10.4 %) with insignificant matrix effect (RSD < 14.0 %) and no carry-over were obtained over a wide range of concentrations. Linearity with inter-day slope RSD lower than 8.7 % was demonstrated. With this method, very low concentrations could be detected after the injection of only 68.7 nL of the sample. The method was applied to plasma samples from six women currently receiving breast cancer treatment. Determined concentrations of the drugs of interest agreed with concentrations found in clinical studies, thus proving the suitability of the developed method for therapeutic drug monitoring as a superior alternative to published LC-MS methods.

Keywords:  Breast cancer; CDK4/6 inhibitors; Dispersive liquid-liquid microextraction; Mass spectrometry; Micellar electrokinetic chromatography; Therapeutic drug monitoring

DOI:  https://doi.org/10.1016/j.chroma.2024.464698
Rapid Commun Mass Spectrom. 2024 Mar 15. 38(5): e9684

A fast method for the determination of personal care product chemicals in human urine using dispersive liquid-liquid extraction and ultra high-performance liquid chromatography-tandem mass spectrometry.

Yawen Zheng, Yarui Liu, Zining Zhang, Liting Hua, Jing Fang, Lei Wang, Hongzhi Zhao.

RATIONALE: Personal care product chemicals (PCPCs) are the chemicals used in personal care products. Many of them are endocrine disruptors and have potential adverse effects on humans. The concentrations of PCPCs in urine are the main biomarker for assessing human exposure.
METHODS: A method was developed for the simultaneous determination of 14 PCPCs in human urine using dispersive liquid-liquid extraction combined with ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS).
RESULTS: Compared with liquid-liquid extraction, this method had the advantages of time efficiency, sensitivity, and limited organic solvent consumption. It produced good linearity (0.9965-0.9996), limits of detection (2.82-36.36 pg mL-1 ), limits of quantitation (9.39-121.08 pg mL-1 ), matrix effect (-0.90%-2.55%), intra-day precision (relative standard deviations [RSDs] <15%), and inter-day precision (RSDs <19.9%). The method had satisfactory relative recovery at three concentration levels.
CONCLUSIONS: A rapid method was developed for the simultaneous quantification of 14 PCPCs in human urine. The practicability of the method was verified with 21 urine from university students. It is expected that this method will provide a powerful reference for the assessment of exposure to PCPCs in large populations.

DOI: https://doi.org/10.1002/rcm.9684
Commun Biol. 2024 Feb 12. 7(1): 172

SIMPEL: using stable isotopes to elucidate dynamics of context specific metabolism.

Shrikaar Kambhampati, Allen H Hubbard, Somnath Koley, Javier D Gomez, Frédéric Marsolais, Bradley S Evans, Jamey D Young, Doug K Allen.

The capacity to leverage high resolution mass spectrometry (HRMS) with transient isotope labeling experiments is an untapped opportunity to derive insights on context-specific metabolism, that is difficult to assess quantitatively. Tools are needed to comprehensively mine isotopologue information in an automated, high-throughput way without errors. We describe a tool, Stable Isotope-assisted Metabolomics for Pathway Elucidation (SIMPEL), to simplify analysis and interpretation of isotope-enriched HRMS datasets. The efficacy of SIMPEL is demonstrated through examples of central carbon and lipid metabolism. In the first description, a dual-isotope labeling experiment is paired with SIMPEL and isotopically nonstationary metabolic flux analysis (INST-MFA) to resolve fluxes in central metabolism that would be otherwise challenging to quantify. In the second example, SIMPEL was paired with HRMS-based lipidomics data to describe lipid metabolism based on a single labeling experiment. Available as an R package, SIMPEL extends metabolomics analyses to include isotopologue signatures necessary to quantify metabolic flux.

DOI: https://doi.org/10.1038/s42003-024-05844-z
Ther Drug Monit. 2024 Feb 06.

Therapeutic Monitoring of Palbociclib, Ribociclib, Abemaciclib, M2, M20, and Letrozole in Human Plasma: A Novel LC-MS/MS Method.

Bianca Posocco, Martina Zanchetta, Marco Orleni, Sara Gagno, Marcella Montico, Elena Peruzzi, Rossana Roncato, Lorenzo Gerratana, Serena Corsetti, Fabio Puglisi, Giuseppe Toffoli.

BACKGROUND: Therapeutic drug monitoring (TDM) using cyclin-dependent kinase inhibitors (CDK4/6is) is a novel approach for optimizing treatment outcomes. Currently, palbociclib, ribociclib, and abemaciclib are the available CDK4/6is and are primarily coadministered with letrozole. This study aimed to develop and validate an LC-MS/MS method for the simultaneous analysis of CDK4/6is, 2 active metabolites of abemaciclib (M2 and M20), and letrozole in human plasma for use in TDM studies.
METHODS: Sample pretreatment comprised protein precipitation with methanol and dilution of the supernatant with an aqueous mobile phase. Chromatographic separation was achieved using a reversed-phase XBridge BEH C18 column (2.5 μm, 3.0 × 75 mm XP), with methanol serving as the organic mobile phase and pyrrolidine-pyrrolidinium formate (0.005:0.005 mol/L) buffer (pH 11.3) as the aqueous mobile phase. A triple quadrupole mass spectrometer was used for the detection, with the ESI source switched from negative to positive ionization mode and the acquisition performed in multiple reaction monitoring mode.
RESULTS: The complete validation procedure was successfully performed in accordance with the latest regulatory guidelines. The following analytical ranges (ng/mL) were established for the tested compounds: 6-300, palbociclib and letrozole; 120-6000, ribociclib; 40-800, abemaciclib; and 20-400, M2 and M20. All results met the acceptance criteria for linearity, accuracy, precision, selectivity, sensitivity, matrix effects, and carryover. A total of 85 patient samples were analyzed, and all measured concentrations were within the validated ranges. The percent difference for the reanalyzed samples ranged from -11.2% to 7.0%.
CONCLUSIONS: A simple and robust LC-MS/MS method was successfully validated for the simultaneous quantification of CDK4/6is, M2, M20, and letrozole in human plasma. The assay was found to be suitable for measuring steady-state trough concentrations of the analytes in patient samples.

DOI: https://doi.org/10.1097/FTD.0000000000001174
Anal Chem. 2024 Feb 15.

Studies of Labware Contamination during Lipid Extraction in Mass Spectrometry-Based Lipidome Analysis.

Carlos R Canez, Liang Li.

In lipidomic analysis, plasticware is increasingly being used for lipid extraction and other sample processing procedures over glassware. However, a systematic investigation of the consequences of plasticware use on mass spectrometry (MS)-based lipidome analysis is lacking. In this work, we present an analytical approach for detecting and comparing solvent and labware contaminants encountered in lipidomic workflows. It is shown that the contaminant profiles varied widely between microcentrifuge tubes from different manufacturers. The most suitable polypropylene tubes tested introduced 847 labware-originating contaminant m/z's when three different manufacturing batches were tested for Folch lipid extractions. Of particular concern is that 21 primary amide and fatty acid surfactants were introduced that were identical to biological endogenous lipids, 16 of which had not been previously reported as leachables from polypropylene materials. Alternatively, the use of borosilicate glassware and PTFE-lined screw caps introduced 98 different contaminant m/z's across three manufacturing batches tested for Folch extractions. Despite the overwhelming number of labware contaminants introduced, current databases and literature only facilitated the identification of 32 contaminants. To address the dearth of publicly available contaminant information, we provide a comprehensive labware contamination repository containing high-resolution m/z values, adductation information, retention times, and MS/MS spectra. This resource should prove to be valuable for researchers in detecting and distinguishing contaminants from analytes of interest. A companion paper presents a detailed study of how labware contamination can lead to ion-suppression effects on coeluting lipids and interference in the analysis of endogenous lipids, such as those from human sera.

DOI: https://doi.org/10.1021/acs.analchem.3c05431
J Pharm Anal. 2024 Jan;14(1): 140-148

Simultaneously quantifying hundreds of acylcarnitines in multiple biological matrices within ten minutes using ultrahigh-performance liquid-chromatography and tandem mass spectrometry.

Jingxian Zhang, Qinsheng Chen, Lianglong Zhang, Biru Shi, Men Yu, Qingxia Huang, Huiru Tang.

  Acylcarnitines are metabolic intermediates of fatty acids and branched-chain amino acids having vital biofunctions and pathophysiological significances. Here, we developed a high-throughput method for quantifying hundreds of acylcarnitines in one run using ultrahigh performance liquid chromatography and tandem mass spectrometry (UPLC-MS/MS). This enabled simultaneous quantification of 1136 acylcarnitines (C0-C26) within 10-min with good sensitivity (limit of detection < 0.7 fmol), linearity (correlation coefficient > 0.992), accuracy (relative error < 20%), precision (coefficient of variation (CV), CV < 15%), stability (CV < 15%), and inter-technician consistency (CV < 20%, n = 6). We also established a quantitative structure-retention relationship (goodness of fit > 0.998) for predicting retention time (tR) of acylcarnitines with no standards and built a database of their multiple reaction monitoring parameters (tR, ion-pairs, and collision energy). Furthermore, we quantified 514 acylcarnitines in human plasma and urine, mouse kidney, liver, heart, lung, and muscle. This provides a rapid method for quantifying acylcarnitines in multiple biological matrices.

Keywords:  Acylcarnitine; Molecular phenotype; Quantitative structure-retention relationship; UPLC-MS/MS

DOI:  https://doi.org/10.1016/j.jpha.2023.10.004
Analyst. 2024 Feb 16.

Liquid electron ionization-mass spectrometry as a novel strategy for integrating normal-phase liquid chromatography with low and high-resolution mass spectrometry.

Nicole Marittimo, Genny Grasselli, Adriana Arigò, Giorgio Famiglini, Marco Agostini, Caterina Renzoni, Pierangela Palma, Achille Cappiello.

Normal-phase liquid chromatography (NPLC) plays a pivotal role in the rapid separation of non-polar compounds, facilitating isomer separation and finding applications in various crucial areas where aprotic solvents are necessary. Similar to reversed-phase liquid chromatography (RPLC), NPLC requires a robust and sensitive detector to unequivocally identify the analytes, such as a mass spectrometer. However, coupling NPLC with mass spectrometry (MS) poses challenges due to the incompatibility between the non-polar solvents used as the mobile phase and the primary ionization techniques employed in MS. Several analytical methods have been developed to combine NPLC with electrospray ionization (ESI), but these methods are restricted to the analysis of polar compounds. In most cases, atmospheric pressure chemical ionization (APCI) becomes necessary to expand the range of analysis applications. To overcome these limitations and fully realize the potential of NPLC-MS coupling, a technique termed liquid electron ionization-mass spectrometry (LEI-MS) can be used. LEI-MS offers a straightforward solution by enabling the effective coupling of NPLC with both low and high-resolution MS. LEI allows for the comprehensive analysis of non-polar compounds and provides a powerful tool for isomer separation and precise identification of analytes. Optimal separations, mass spectral qualities, and matches with the NIST library were obtained in both configurations, demonstrating the potential of the proposed approach.

DOI: https://doi.org/10.1039/d3an02109b
Anal Chem. 2024 Feb 14.

Leveraging Unidentified Metabolic Features for Key Pathway Discovery: Chemical Classification-driven Network Analysis in Untargeted Metabolomics.

Xiuqiong Zhang, Zaifang Li, Chunxia Zhao, Tiantian Chen, Xinxin Wang, Xiaoshan Sun, Xinjie Zhao, Xin Lu, Guowang Xu.

Untargeted metabolomics using liquid chromatography-electrospray ionization-high-resolution tandem mass spectrometry (UPLC-ESI-MS/MS) provides comprehensive insights into the dynamic changes of metabolites in biological systems. However, numerous unidentified metabolic features limit its utilization. In this study, a novel approach, the Chemical Classification-driven Molecular Network (CCMN), was proposed to unveil key metabolic pathways by leveraging hidden information within unidentified metabolic features. The method was demonstrated by using the herbivore-induced metabolic response in corn silk as a case study. Untargeted metabolomics analysis using UPLC-MS/MS was performed on wild corn silk and two genetically modified lines (pre- and postinsect treatment). Global annotation initially identified 256 (ESI-) and 327 (ESI+) metabolites. MS/MS-based classifications predicted 1939 (ESI-) and 1985 (ESI+) metabolic features into the chemical classes. CCMNs were then constructed using metabolic features shared classes, which facilitated the structure- or class annotation for completely unknown metabolic features. Next, 844/713 significantly decreased and 1593/1378 increased metabolites in ESI-/ESI+ modes were defined in response to insect herbivory, respectively. Method validation on a spiked maize sample demonstrated an overall class prediction accuracy rate of 95.7%. Potential key pathways were prescreened by a hypergeometric test using both structure- and class-annotated differential metabolites. Subsequently, CCMN was used to deeply amend and uncover the pathway metabolites deeply. Finally, 8 key pathways were defined, including phenylpropanoid (C6-C3), flavonoid, octadecanoid, diterpenoid, lignan, steroid, amino acid/small peptide, and monoterpenoid. This study highlights the effectiveness of leveraging unidentified metabolic features. CCMN-based key pathway analysis reduced the bias in conventional pathway enrichment analysis. It provides valuable insights into complex biological processes.

DOI: https://doi.org/10.1021/acs.analchem.3c04591
Drug Test Anal. 2024 Feb 15.

Routine application of SFC-MS in doping control: Analysis of 3 × 1000 urine samples using three different SFC-MS instruments.

Ivana Gavrilović, Bernhard Wüst, Jonathan Danaceau, Emmanuelle Braidman, Xavier de la Torre, Francesco Botrè, Maria Kristina Parr, David Cowan.

  Supercritical fluid chromatography-mass spectrometry (SFC-MS) has proved to be a beneficial tool for sample analysis for a wide variety of compounds and, as such, has recently gained the attention of the anti-doping community. We have tested the applicability of SFC-MS for routine doping control analysing approximately 3 × 1000 identical anti-doping samples utilising SFC-MS instruments from three different vendors: Agilent Technologies, Waters Corporation and Shimadzu Corporation. A 'dilute and inject' approach either without or after hydrolysis of glucuronide metabolites was applied. Most of the compounds included in our study demonstrated excellent chromatography, whereas some showed co-elution with endogenous interferences requiring MS discrimination. Retention times typically were very stable within batches (%CV ≤ 0.5%), although this appeared to be analyte and column dependent. Chromatographic peak shape was good (symmetrical) and stable over the period of the testing without any change of column. Our results suggest that SFC-MS is a sensitive, reproducible and robust analytical tool ready to be used in anti-doping laboratories alongside the currently applied techniques such as gas and liquid chromatography coupled to mass spectrometry. Even if instruments are designed slightly differently, all three setups demonstrated their fitness for the purpose in anti-doping testing.

Keywords:  SFC-MS; doping control; robustness

DOI:  https://doi.org/10.1002/dta.3652
J Chromatogr A. 2024 Jan 26. pii: S0021-9673(24)00062-1. [Epub ahead of print]1717 464689

A rapid and sensitive analytical methodology for the simultaneous biomonitoring of two direct oral anticoagulant drugs and their major metabolites in thromboembolic disordered patients samples for clinical evaluations.

Karthikeyan Prakasham, Tzu-Yu Pan, Chun-Hsiang Tan, Chia-Fang Wu, Pranjal Chandra, Ching-Mei Cheng, Wei Chen, Wei-Chung Tsai, Vinoth Kumar Ponnusamy, Ming-Tsang Wu.

  Apixaban and dabigatran are the two major direct oral anticoagulant drugs to treat thromboembolic disordered patients. Increasing the clinical application for the thromboembolic disorder and monitoring the concentrations of apixaban, dabigatran, and their metabolites are essential in most clinical circumstances. In this work, we developed a rapid analytical methodology comprising of vortex-assisted salt-enhanced liquid-liquid microextraction technique coupled with UHPLC-MS/MS for the extraction and simultaneous determination of two major direct oral anticoagulant drugs (apixaban, dabigatran), and their two major metabolites from plasma, serum, and urine samples of patients. The developed method was optimized with various procedural steps and validated to study the analytical merits. The developed method yielded a good detection limit of 0.01 ∼ 0.37 ng/mL, 0.01 ∼ 0.32 ng/ml, and 0.01 ∼ 0.27 ng/mL for four target analytes in the plasma, serum, and urine matrices. Moreover, extraction recoveries ranged from 85.11 - 113.57% (for plasma), 89.63 - 110.47% (for serum), and 87.44 -106.79% (for urine samples) with 8.78% RSD. In addition, the method exhibited good R2 values of 0.999 for all four target analytes, and the specificity and carryover study revealed no carryover effect from the UHPLC-MS/MS system for determining the apixaban, dabigatran, and their metabolites. Due to the above advantages, the developed analytical technique was applied to examine 11 real-time clinical patients' samples, and the observed results were satisfactory for all three different sample matrices. Therefore, this analytical method can be applied for biomonitoring apixaban, dabigatran, and their two major metabolites with high sensitivity in a short time for various clinical applications.

Keywords:  Metabolites of apixaban and dabigatran; Targeted metabolomics; UHPLC-MS/MS; Vortex assisted salt enhanced liquid-liquid microextraction (VASE-LLME)

DOI:  https://doi.org/10.1016/j.chroma.2024.464689
Metabolomics. 2024 Feb 12. 20(2): 20

Establishing a framework for best practices for quality assurance and quality control in untargeted metabolomics.

Jonathan D Mosley, Tracey B Schock, Chris W Beecher, Warwick B Dunn, Julia Kuligowski, Matthew R Lewis, Georgios Theodoridis, Candice Z Ulmer Holland, Dajana Vuckovic, Ian D Wilson, Krista A Zanetti.

   BACKGROUND: Quality assurance (QA) and quality control (QC) practices are key tenets that facilitate study and data quality across all applications of untargeted metabolomics. These important practices will strengthen this field and accelerate its success. The Best Practices Working Group (WG) within the Metabolomics Quality Assurance and Quality Control Consortium (mQACC) focuses on community use of QA/QC practices and protocols and aims to identify, catalogue, harmonize, and disseminate current best practices in untargeted metabolomics through community-driven activities.
AIM OF REVIEW: A present goal of the Best Practices WG is to develop a working strategy, or roadmap, that guides the actions of practitioners and progress in the field. The framework in which mQACC operates promotes the harmonization and dissemination of current best QA/QC practice guidance and encourages widespread adoption of these essential QA/QC activities for liquid chromatography-mass spectrometry.
KEY SCIENTIFIC CONCEPTS OF REVIEW: Community engagement and QA/QC information gathering activities have been occurring through conference workshops, virtual and in-person interactive forum discussions, and community surveys. Seven principal QC stages prioritized by internal discussions of the Best Practices WG have received participant input, feedback and discussion. We outline these stages, each involving a multitude of activities, as the framework for identifying QA/QC best practices. The ultimate planned product of these endeavors is a "living guidance" document of current QA/QC best practices for untargeted metabolomics that will grow and change with the evolution of the field.

Keywords:  Guidance; Liquid chromatography–mass spectrometry (LC-MS); Quality assurance (QA); Quality control (QC); Reproducibility; Untargeted metabolomics

DOI:  https://doi.org/10.1007/s11306-023-02080-0
Rapid Commun Mass Spectrom. 2024 Mar 15. 38(5): e9701

Ethanolysis of nitrogen mustards: A novel strategy for nitrogen mustard identification in environmental matrices by liquid chromatography-electrospray ionization-tandem mass spectrometry.

Chen Belay, Avital Shifrovitch, Hagit Prihed, Moran Madmon, Merav Blanca, Tamar Shamai Yamin, Avi Weissberg.

RATIONALE: Nitrogen mustards (NMs) are blistering chemical warfare agents. The ability to detect NMs in environmental samples is very important for obtaining forensic evidence. The most common analytical techniques for NM detection are gas chromatography-mass spectrometry, which detects NMs in their intact form but is disadvantaged by high limits of detection (LODs), and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) of their hydrolysis products, which do not provide robust evidence to support NM use.
METHODS: We developed a novel approach to detect and identify NMs using LC/ESI-MS/MS after chemical derivatization. The method is based on ethoxide-promoted ethanolysis prior to analysis. The effects of reaction time, temperature, ethoxide concentration and chromatography behavior were studied and optimized. In the developed procedure, 0.1% (v/v) sodium ethoxide solution is added to the NMs in ethanol and agitated for 2 h at 50°C, followed by LC/ESI-MS/MS, without any other pretreatment.
RESULTS: The ethanolysis reaction efficiencies were evaluated in ethanolic extracts from soil, asphalt, and ethanol contaminated with 0.5% (v/v) diesel fortified with NMs at a five-point calibration curve. The calibration curves showed good linearity in the range of 0.05-1 ng/mL, with an R2 value of 0.99, and were similar to those of LC/MS-grade ethanol, with almost no observable matrix effects. The derivatization products were stable at room temperature, with LODs of 10 pg/mL, in all investigated extracts.
CONCLUSIONS: Through this newly developed strategy, the derivatization of active NMs by ethanolysis was achieved for the first time, and these derivatization products can serve as specific indicators for the use and presence of NMs. The methodology can also verify trace levels of NM chemical warfare agents collected in war or terror scenarios in forensic investigations.

DOI: https://doi.org/10.1002/rcm.9701
Methods Mol Biol. 2024 ;2763 125-136

Quantitation of MUC5AC and MUC5B by Stable Isotope Labeling Mass Spectrometry.

Giorgia Radicioni, Mehmet Kesimer.

  Mucins MUC5AC and MUC5B are large glycoproteins that play an essential role in the innate defense of epithelial surfaces and their quantitation in biological samples would be informative about the health status of the tissue/samples they are derived from. However, they are difficult to study and quantify with traditional methods such as ELISA and western blot, due to their size, heterogeneity, and high degree of glycosylation. We successfully implemented a stable isotope labeling mass spectrometry approach for absolute quantification of mucin macromolecules. Here, in detail, we describe this accurate and sensitive liquid chromatography and mass spectrometry (LC-MS) method applied for both MUC5AC and MUC5B quantification in diverse and complex biological samples.

Keywords:  MUC5AC; MUC5B; Proteomics; Stable isotope labeling mass spectrometry

DOI:  https://doi.org/10.1007/978-1-0716-3670-1_11
Metabolomics. 2024 Feb 12. 20(2): 22

Evaluation of normalization strategies for GC-based metabolomics.

Seo Lin Nam, Ryland T Giebelhaus, Kieran S Tarazona Carrillo, A Paulina de la Mata, James J Harynuk.

   INTRODUCTION: For many samples studied by GC-based metabolomics applications, extensive sample preparation involving extraction followed by a two-step derivatization procedure of methoximation and trimethylsilylation (TMS) is typically required to expand the metabolome coverage. Performing normalization is critical to correct for variations present in samples and any biases added during the sample preparation steps and analytical runs. Addressing the totality of variations with an adequate normalization method increases the reliability of the downstream data analysis and interpretation of the results.
OBJECTIVES: Normalizing to sample mass is one of the most commonly employed strategies, while the total peak area (TPA) as a normalization factor is also frequently used as a post-acquisition technique. Here, we present a new normalization approach, total derivatized peak area (TDPA), where data are normalized to the intensity of all derivatized compounds. TDPA relies on the benefits of silylation as a universal derivatization method for GC-based metabolomics studies.
METHODS: Two sample classes consisting of systematically incremented sample mass were simulated, with the only difference between the groups being the added amino acid concentrations. The samples were TMS derivatized and analyzed using comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC × GC-TOFMS). The performance of five normalization strategies (no normalization, normalized to sample mass, TPA, total useful peak area (TUPA), and TDPA) were evaluated on the acquired data.
RESULTS: Of the five normalization techniques compared, TUPA and TDPA were the most effective. On PCA score space, they offered a clear separation between the two classes.
CONCLUSION: TUPA and TDPA carry different strengths: TUPA requires peak alignment across all samples, which depends upon the completion of the study, while TDPA is free from the requirement of alignment. The findings of the study would enhance the convenient and effective use of data normalization strategies and contribute to overcoming the data normalization challenges that currently exist in the metabolomics community.

Keywords:  Derivatization; GC × GC-TOFMS; Gas chromatography; Metabolomics; Normalization; Trimethylsilylation

DOI:  https://doi.org/10.1007/s11306-023-02086-8
J Am Soc Mass Spectrom. 2024 Feb 15.

Separation and Characterization of Synthetic Cannabinoid Metabolite Isomers Using SLIM High-Resolution Ion Mobility-Tandem Mass Spectrometry (HRIM-MS/MS).

Ralph Aderorho, Shadrack Wilson Lucas, Christopher D Chouinard.

  Synthetic cannabinoids, a subclass of new psychoactive substances (NPS), are laboratory-made substances that are chemically similar to those found naturally in the cannabis plant. Many of these substances are illicitly manufactured and have been associated with severe health problems, prompting a need to develop analytical methods capable of characterizing both known and previously undetected compounds. This work focuses on a novel Structures for Lossless Ion Manipulations (SLIM) IM-MS approach to the differentiation and structural characterization of synthetic cannabinoid metabolites, specifically MDA-19/BUTINACA, JWH-018, and JWH-250 isomer groups. These different compound classes are structurally very similar, differing only in the position of one or a few functional groups; this yielded similarity in measured collision cross section (CCS) values. However, the high resolution of SLIM IM provided adequate separation of many of these isomers, such as sodiated JWH-250 metabolites N-4-OH, N-5-OH, and 5-OH, which displayed CCS of 187.5, 182.5, and 202.3 Å2, respectively. In challenging cases where baseline separation was precluded due to nearly identical CCS, such as for JWH-018 isomers, simple derivatization by dansyl chloride selectively reacted with the 6-OH compound to provide differentiation of all isomers using a combination of CCS and m/z. Finally, the opportunity to use this method for structural elucidation of unknowns was demonstrated by using SLIM IM mobility-aligned MS/MS fragmentation. Different MDA-19/BUTINACA isomers were first mobility separated and could then be individually activated, yielding unique fragments for both targeted identification and structural determination. Overall, the described SLIM IM-MS/MS workflow provides significant potential as a rapid screening tool for the characterization of emerging NPS such as synthetic cannabinoids and their metabolites.

Keywords:  SLIM; ion mobility-mass spectrometry (IM-MS); isomers; synthetic cannabinoids

DOI:  https://doi.org/10.1021/jasms.3c00419
Rapid Commun Mass Spectrom. 2024 Mar 30. 38(6): e9685

Simultaneous quantification of endogenous biomarkers and detection of testosterone esters in human serum using gas chromatography-high-resolution mass spectrometry.

Dayamín Martínez Brito, Patrizia Leogrande, Daniel Jardines, Xavier de la Torre, Francesco Botrè.

RATIONALE: High-resolution mass spectrometry (HRMS) has been demonstrated to be an alternative platform for quantitative analyses, identifying unknown compounds and gathering information for the elucidation of chemical structures. This work describes a method to detect 13 esters of testosterone (T) and 5 biomarkers in 0.1 mL of human serum using gas chromatography (GC) coupled to HRMS.
METHODS: Analytes were extracted from serum after deproteinization and liquid-liquid extraction. The trimethylsilyl derivatives were analyzed using a gas chromatograph coupled to HRMS at low electron energy to minimize molecule fragmentation. The acquisition in profiling full-scan mode was applied with a resolving power of 30 000 at m/z 400. Linearity, lower limit of quantitation, and measurement uncertainty were assessed. Precision and accuracy were assessed at 0.5 and 2 ng/mL, respectively. Mass accuracy (MA) and mass extraction window (MEW) were also evaluated.
RESULTS: T esters showed a linear response between 0.25 and 10 ng/mL (except for undecanoate, enanthate, and propionate that showed lineal responses between 0.5 and 10 ng/mL and isocaproate between 2 and 10 ng/mL); detection limits remained between 0.1 and 0.5 ng/mL and accuracy between 81% and 119%. The MA (MEW = 10 ppm) was maintained between -2.4 and 4.8 ppm. The biomarkers (T, androstenedione, dehydroepiandrosterone [DHEA], estradiol, and 17-OH-progesterone) showed a linear response within the evaluated range; quantification limits remained between 0.1 and 0.5 ng/mL (except for DHEA), the accuracy between 88% and 99%, and precision between 3.5% and 10.8%. Measurement uncertainties were found between 5.6% and 17.2%. MA (MEW = 3 ppm) was maintained between -0.47 and 0.12 ppm.
CONCLUSIONS: The method to detect T esters and five endogenous biomarkers in serum using GC coupled to HRMS showed linear responses up to 10 ng/mL with adequate precision, accuracy, and uncertainties. It was possible to distinguish cholesterol from T-isocaproate based on the MEW of 10 ppm, preventing false positives. In addition, this method allows searching for other biomarkers and/or unknown metabolites and other ester forms not included here but at a later stage if necessary.

DOI: https://doi.org/10.1002/rcm.9685
J Mass Spectrom Adv Clin Lab. 2024 Apr;32 11-17

A validated liquid chromatography-tandem mass spectrometry assay for the analysis of isoniazid and its metabolite acetyl-isoniazid in urine.

Sydwell Poulo Maputla, Willem Van Dalen, Anton Joubert, Jennifer Norman, Sandra Castel, Marthinus van der Merwe, Lubbe Wiesner.

   Introduction: Isoniazid (INH) is one of the most effective and potent first-line anti-tubercular drug. INH is also effectively administered as a preventative monotherapy and has been shown to significantly reduce TB incidence. INH is primarily metabolised to acetyl-isoniazid (AcINH) in the liver. AcINH is mainly excreted in urine presenting as a target for monitoring adherence to INH therapy.
Objective: The study aimed to develop and fully validate a bioanalytical method using liquid chromatography-tandem mass spectrometry for the quantification of INH and AcINH in human urine.
Methods: The samples were prepared using solid phase extraction, with the internal standards isoniazid-d4 and acetyl-isoniazid-d4 being used. The extracts were chromatographed on an Atlantis T3 analytical column with an isocratic mobile phase. For detection, a AB Sciex™ API 5500 triple quadrupole mass spectrometer was used at unit resolution in the multiple reaction monitoring mode, following positive electrospray ionization.
Results: The analytical method demonstrated sufficient sensitivity, as indicated by average signal-to-noise ratios of 7.07 and 6.23 at the lower limit of quantification for INH and AcINH, respectively. Validation was performed over three consecutive batches, demonstrating accuracy, precision, and overall robustness based on peak area ratios within the analytical range of 0.234-30.0 µg/mL for both INH and AcINH. All required validation experiments were assessed and met the acceptance criteria guidelines of the US Food and Drug Administration and European Medicines Agency. The validated method was utilized to measure concentrations of AcINH in urine as a means of assessing adherence to the intake of isoniazid in order to prevent TB infection during a phase III open-label multicenter trial.
Conclusion: A bioanalytical method was developed and fully validated for quantifying isoniazid (INH) and acetyl-isoniazid (AcINH) in 100 µL of human urine.

Keywords:  Acetyl-isoniazid; Isoniazid; LC-MS/MS; Solid phase extraction; Urine; Validation

DOI:  https://doi.org/10.1016/j.jmsacl.2024.02.001
Anal Chem. 2024 Feb 12.

Discovery of Glycation Products: Unraveling the Unknown Glycation Space Using a Mass Spectral Library from In Vitro Model Systems.

Yingfei Yan, Daniel Hemmler, Philippe Schmitt-Kopplin.

The nonenzymatic reaction between amino acids (AAs) and reducing sugars, also known as the Maillard reaction, is the primary source of free glycation products (GPs) in vivo and in vitro. The limited number of MS/MS records for GPs in public libraries hinders the annotation and investigation of nonenzymatic glycation. To address this issue, we present a mass spectral library containing the experimental MS/MS spectra of diverse GPs from model systems. Based on the conceptional reaction processes and structural characteristics of products, we classified GPs into common GPs (CGPs) and modified AAs (MAAs). A workflow for annotating GPs was established based on the structural and fragmentation patterns of each GP type. The final spectral library contains 157 CGPs, 499 MAAs, and 2426 GP spectra with synthetic model system information, retention time, precursor m/z, MS/MS, and annotations. As a proof-of-concept, we demonstrated the use of the library for screening GPs in unidentified spectra of human plasma and urine. The AAs with the C6H10O5 modification, fructosylation from Amadori rearrangement, were the most found GPs. With the help of the model system, we confirmed the existence of C6H10O5-modified Valine in human plasma by matching both retention time, MS1, and MS/MS without reference standards. In summary, our GP library can serve as an online resource to quickly screen possible GPs in an untargeted metabolomics workflow, furthermore with the model system as a practical synthesis method to confirm their identity.

DOI: https://doi.org/10.1021/acs.analchem.3c05540
Rapid Commun Mass Spectrom. 2024 Mar 15. 38(5): e9682

Chemical analysis of additives included in fully formulated oils using high-performance liquid chromatography-tandem mass spectrometry.

Vincent Basham, Tom Hancock, John McKendrick, Nathalia Tessarolo, Chrissie Wicking.

RATIONALE: Fully formulated oils (FFOs) are chemically complex petrochemical products composed of base oil and additive mixtures that are employed in automotive engines to provide lubrication. In particular, the additive portion of FFOs is often precisely controlled to tailor the resultant formulation to a specific role. Analysis of the additive composition of both used and unused FFOs is therefore of great importance within the petroleum, automotive, and wider engineering industries.
METHODS: A simple and rapid reversed-phase high-performance liquid chromatography-tandem mass spectrometry method is reported herein for the analysis of a range of additives commonly encountered in FFO samples. Mass spectrometry was performed using an LTQ Orbitrap XL instrument using both positive- and negative-ion electrospray ionization. Tandem mass spectra were acquired in the data-dependent mode. FFO samples were analysed with minimal sample preparation, limited in this case to simple dilution steps.
RESULTS: The reported method permits analysis of a range of antioxidant, detergent, and antiwear chemistries from various FFO samples in under 10 min. Additionally, it is demonstrated that additive confirmation can be performed and some structural information obtained using the data-dependent tandem mass spectrometry method. Furthermore, analysis of additives and corresponding degradation products within a used FFO is reported.
CONCLUSIONS: The results obtained using the reported methodology are of demonstrable use in numerous industries and applications, and readily return an abundance of information on the additive composition of a range of FFO samples. Anticipated applications of the methodology include but are not limited to quality control, suspected counterfeit analysis, and FFO degradation analysis.

DOI: https://doi.org/10.1002/rcm.9682
Rapid Commun Mass Spectrom. 2024 Mar 30. 38(6): e9703

A biomarker panel of secondary hypertension is simultaneously quantified by coupling of magnetic solid-phase extraction and liquid chromatography-tandem mass spectrometry.

Xi Gao, Xiaoyong Li, Lingyun Chen, Shuyan Chen, Guixue Hou, Liang Lin, Qunjie Wang, Jiuxin Qu, Siqi Liu.

RATIONALE: Secondary hypertension is often caused by activation of complex multi-organ endocrine systems, while renin activity indicated by angiotensins (Angs), aldosterone (ALD) and cortisol (COR) in such systems are generally accepted as its diagnostic markers. As antibody-based methods cannot offer comparable quantification for these biomarkers, a liquid chromatography (LC)-tandem mass spectrometry (MS/MS)-based approach was developed to quantify them simultaneously and accurately.
METHODS: Five different beads for magnetic solid-phase extraction (MSPE) were evaluated towards their enrichment efficiency for these biomarkers. An LC system with optimized elution gradient and a triple-quadrupole MS with tuned parameters were coupled to quantitatively monitor the extracted analytes. The method performance was further examined such as linearity, precision, stability, recovery rate and matrix effect. Based on the developed method, the abundance of Ang II, ALD and COR in plasma was measured and the quantification was compared with that derived from commercial ELISA kits.
RESULTS: As compared with other MSPEs, Angs, ALD and COR were highly enriched by the HLB magnetic beads with satisfactory recoveries. These analytes were simultaneously quantified by LC/MS/MS and all the method parameters for quantification were well matched with the requirements of clinical testing. Comparison of the quantitative results derived from ELISA and LC/MS/MS exhibited that the two methods offered basically comparable values with Pearson r values at 0.896, 0.895 and 0.835, respectively. The stability test for plasma Angs at room temperature indicated that the abundance of Ang II was relatively stable within 3 h, whereas that of Ang I and Ang 1-7 was time-dependently changed.
CONCLUSIONS: Coupling of HLB beads and LC/MS/MS thus enables simultaneous quantification of a set of biomarkers related to secondary hypertension.

DOI: https://doi.org/10.1002/rcm.9703
Nat Methods. 2024 Feb 16.

Multiscale biochemical mapping of the brain through deep-learning-enhanced high-throughput mass spectrometry.

Yuxuan Richard Xie, Daniel C Castro, Stanislav S Rubakhin, Timothy J Trinklein, Jonathan V Sweedler, Fan Lam.

Spatial omics technologies can reveal the molecular intricacy of the brain. While mass spectrometry imaging (MSI) provides spatial localization of compounds, comprehensive biochemical profiling at a brain-wide scale in three dimensions by MSI with single-cell resolution has not been achieved. We demonstrate complementary brain-wide and single-cell biochemical mapping using MEISTER, an integrative experimental and computational mass spectrometry (MS) framework. Our framework integrates a deep-learning-based reconstruction that accelerates high-mass-resolving MS by 15-fold, multimodal registration creating three-dimensional (3D) molecular distributions and a data integration method fitting cell-specific mass spectra to 3D datasets. We imaged detailed lipid profiles in tissues with millions of pixels and in large single-cell populations acquired from the rat brain. We identified region-specific lipid contents and cell-specific localizations of lipids depending on both cell subpopulations and anatomical origins of the cells. Our workflow establishes a blueprint for future development of multiscale technologies for biochemical characterization of the brain.

DOI: https://doi.org/10.1038/s41592-024-02171-3
J Sep Sci. 2024 Feb;47(3): e2300745

Comparison of the applicability of electromembrane extraction and liquid-phase microextraction for extraction of non-polar basic drugs from different biological samples: Using clozapine as the model analyte.

Jianhua Xiong, Linxin Tian, Xiantao Shen, Chuixiu Huang.

  Understanding and comparing the applicability of electromembrane extraction (EME) and liquid-phase microextraction (LPME) is crucial for selecting an appropriate microextraction approach. In this work, EME and LPME based on supported liquid membranes were compared using biological samples, including whole blood, urine, saliva, and liver tissue. After optimization, efficient EME and LPME of clozapine from four biological samples were achieved. EME provided higher recovery and faster mass transfer for blood and liver tissue than LPME. These advantages were attributed to the electric field disrupting clozapine binding to interfering substances. For urine and saliva, EME demonstrated similar recoveries while achieving faster mass transfer rates. Finally, efficient EME and LPME were validated and evaluated combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The coefficient of determination of all methods was greater than 0.999, and all methods showed acceptable reproducibility (≤14%), accuracy (90%-110%), and matrix effect (85%-112%). For liver and blood with high viscosity and complex matrices, EME-LC-MS/MS provided better sensitivity than LPME-LC-MS/MS. The above results indicated that both EME and LPME could be used to isolate non-polar basic drugs from different biological samples, although EME demonstrated higher recovery rates for liver tissue and blood.

Keywords:  biological samples; electromembrane extraction; equilibrium; liquid-phase microextraction; mass transfer

DOI:  https://doi.org/10.1002/jssc.202300745
Clin Lab. 2024 Feb 01. 70(2):

Development of a LC-MS/MS Quantification Assay for Human Relaxin H2 and Extraction of Human Relaxin H1 and Relaxin H3.

Annabelle Fülöp, Franz P Armbruster, Hans J Grön, Thomas B Dschietzig, Max Diesner.

DOI: https://doi.org/10.7754/Clin.Lab.2024.240131