bims-metlip 2024-01-21 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2024–01–21
twenty-two papers selected by
Sofia Costa, Matterworks

Unraveling the potential of segment scan mass spectral acquisition for chemical isotope labeling LC-MS-based metabolome analysis: Performance assessment across different types of biological samples.
MARS: A Multipurpose Software for Untargeted LC-MS-Based Metabolomics and Exposomics.
HILIC/MS quantitation of low-abundant phospholipids and sphingolipids in human plasma and serum: Dysregulation in pancreatic cancer.
Liquid chromatography-tandem mass spectrometry determination of bumetanide in human plasma and application to a clinical pharmacokinetic study.
IDSL_MINT: a deep learning framework to predict molecular fingerprints from mass spectra.
Enhancing Metabolome Annotation by Electron Impact Excitation of Ions from Organics-Molecular Networking.
Correction of creatine-creatinine conversion during serum creatinine quantification by two-dimensional liquid chromatography and double-spike isotope dilution tandem mass spectrometry.
Simultaneous determination of colistin sulfate and tigecycline in human plasma by liquid chromatography-tandem mass spectrometry method.
Rapid and Bifunctional Chemoselective Metabolome Analysis of Liver Patient Samples Using the Reagent 4-Nitrophenyl-2H-azirine.
Advances in multimodal mass spectrometry for single-cell analysis and imaging enhancement.
Analysis of 14 β-agonists in pork using an automated wooden tip-based solid-phase microextraction device and ultra-high-performance liquid chromatography-tandem mass spectrometry.
Probing Liquid Chromatography-Tandem Mass Spectrometry Response Dynamics and Nonlinear Effects for Response Level Defined Calibration Strategies with Simple Methods To Expand Linear Dynamic Ranges.
Synthesis and quantification of short-chain fatty acid esters of hydroxy fatty acids in rat intestinal contents and fecal samples by LC-MS/MS.
UPLC-MS/MS Method for Detection of Etomidate and Its Metabolite Etomidate Acid Quantity in Blood.
A new adsorptive 3D-printed sampling device for simultaneous determination of 63 urinary organic acids by LC-MS/MS.
Development of an ultra-high-performance liquid chromatography-tandem mass spectrometry method for the simultaneous determination of crassicauline A, fuziline, karacoline, and songorine in rat plasma and application in their pharmacokinetics.
Simultaneous determination of sinomenine and its metabolites desmethyl-sinomenine and sinomenine N-oxide in rat plasma by liquid chromatography tandem mass spectrometry.
A rapid and simple HPLC-MS/MS method for the therapeutic drug monitoring of six special-grade antimicrobials in pediatric patients.
Triplex glycan quantification by metabolic labeling with isotopically labeled glucose in yeast.
Inhibition of anaerobic digestion by various ammonia sources resulted in subtle differences in metabolite dynamics.
Gas chromatography-mass spectrometry-based quantitative method using tert-butyldimethylsilyl derivatization for plasma levels of free amino acids and related metabolites in Japanese Black cattle.
Development and evaluation of a comprehensive workflow for suspect screening of exposome-related xenobiotics and phase II metabolites in diverse human biofluids.

Anal Chim Acta. 2024 Feb 01. pii: S0003-2670(23)01358-2. [Epub ahead of print]1288 342137

Unraveling the potential of segment scan mass spectral acquisition for chemical isotope labeling LC-MS-based metabolome analysis: Performance assessment across different types of biological samples.

Chu-Fan Wang, Liang Li.

   BACKGROUND: Chemical isotope labeling (CIL) LC-MS is a powerful tool for metabolome analysis with high metabolomic coverage and quantification accuracy. In CIL LC-MS, the overall metabolite detection efficiency using Orbitrap MS can be further improved by employing a segment scan method where the full m/z range is divided into multiple segments for spectral acquisition with a significant increase in the in-spectrum dynamic range. Considering the metabolic complexity in different types of biological samples (e.g., feces, urine, serum/plasma, cell/tissue extracts, saliva, etc.), we report the development and evaluation of the segment scan method for metabolome analysis of different sample types.
RESULTS: It was found that sample complexity significantly influenced the performance of the segment scan method. In metabolically complex samples such as feces and urine, the method yielded a substantial increase (up to 94 %) in detected peak pairs or metabolites, compared to conventional full scan. Conversely, less complex samples like saliva exhibited more modest gains (approximately 25 %). Based on the observations, a 120-m/z segment scan method was determined as a routine approach for CIL LC-Orbitrap-MS-based metabolomics with good compatibility with different types of biological samples. For this method, a further investigation on relative quantification accuracy was done. The peak area ratios of 12C-/13-labeled metabolites were slightly reduced with 72%-84 % of peak pairs falling within the ±25 % range of the anticipated peak ratio of 1.0 among different samples, as opposed to 81%-90 % in the full scan, which was attributed to the inclusion of more low-abundance peak pairs within the narrow MS segments. However, the overall peak ratio measurement precision was not significantly affected by the segment scan.
SIGNIFICANCE AND NOVELTY: The segment scan method was found to be useful for CIL LC-Orbitrap-MS-based metabolome analysis of different types of samples with significant improvement in metabolite detectability (25-94 % increase), compared to the conventional full scan method.

Keywords:  Chemical isotope labeling; Metabolic complexity; Metabolomics; Orbitrap MS; Segment scan

DOI:  https://doi.org/10.1016/j.aca.2023.342137
Anal Chem. 2024 Jan 18.

MARS: A Multipurpose Software for Untargeted LC-MS-Based Metabolomics and Exposomics.

Laura Goracci, Paolo Tiberi, Stefano Di Bona, Stefano Bonciarelli, Giovanna Ilaria Passeri, Marta Piroddi, Simone Moretti, Claudia Volpi, Ismael Zamora, Gabriele Cruciani.

Untargeted metabolomics is a growing field, in which recent advances in high-resolution mass spectrometry coupled with liquid chromatography (LC-MS) have facilitated untargeted approaches as a result of improvements in sensitivity, mass accuracy, and resolving power. However, a very large amount of data are generated. Consequently, using computational tools is now mandatory for the in-depth analysis of untargeted metabolomics data. This article describes MetAbolomics ReSearch (MARS), an all-in-one vendor-agnostic graphical user interface-based software applying LC-MS analysis to untargeted metabolomics. All of the analytical steps are described (from instrument data conversion and processing to statistical analysis, annotation/identification, quantification, and preliminary biological interpretation), and tools developed to improve annotation accuracy (e.g., multiple adducts and in-source fragmentation detection, trends across samples, and the MS/MS validator) are highlighted. In addition, MARS allows in-house building of reference databases, to bypass the limits of freely available MS/MS spectra collections. Focusing on the flexibility of the software and its user-friendliness, which are two important features in multipurpose software, MARS could provide new perspectives in untargeted metabolomics data analysis.

DOI: https://doi.org/10.1021/acs.analchem.3c03620
Anal Chim Acta. 2024 Feb 01. pii: S0003-2670(23)01365-X. [Epub ahead of print]1288 342144

HILIC/MS quantitation of low-abundant phospholipids and sphingolipids in human plasma and serum: Dysregulation in pancreatic cancer.

Ondřej Peterka, Alessandro Maccelli, Robert Jirásko, Zuzana Vaňková, Jakub Idkowiak, Roman Hrstka, Denise Wolrab, Michal Holčapek.

  A new hydrophilic interaction liquid chromatography - mass spectrometry method is developed for low-abundant phospholipids and sphingolipids in human plasma and serum. The optimized method involves the Cogent Silica type C hydride column, the simple sample preparation by protein precipitation, and the removal of highly abundant lipid classes using the postcolumn valve directed to waste during two elution windows. The method allows a highly confident and sensitive identification of low-abundant lipid classes in human plasma (246 lipid species from 24 lipid subclasses) based on mass accuracy and retention dependencies in both polarity modes. The method is validated for quantitation using two internal standards (if available) for each lipid class and applied to human plasma and serum samples obtained from patients with pancreatic ductal adenocarcinoma (PDAC), healthy controls, and NIST SRM 1950. Multivariate data analysis followed by various statistical projection methods is used to determine the most dysregulated lipids. Significant downregulation is observed for lysophospholipids with fatty acyl composition 16:0, 18:0, 18:1, and 18:2. Distinct trends are observed for phosphatidylethanolamines (PE) in relation to the bonding type of fatty acyls, where most PE with acyl bonds are upregulated, while ether/plasmenyl PE are downregulated. For the sphingolipid category, sphingolipids with very long N-acyl chains are downregulated, while sphingolipids with shorter N-acyl chains were upregulated in PDAC. These changes are consistently observed for various classes of sphingolipids, ranging from ceramides to glycosphingolipids, indicating a possible metabolic disorder in ceramide biosynthesis caused by PDAC.

Keywords:  Human plasma; Human serum; Hydrophilic interaction liquid chromatography; Lipidomics; Liquid chromatography; Mass spectrometry; Pancreatic cancer

DOI:  https://doi.org/10.1016/j.aca.2023.342144
Biomed Chromatogr. 2024 Jan 17. e5825

Liquid chromatography-tandem mass spectrometry determination of bumetanide in human plasma and application to a clinical pharmacokinetic study.

Ganesan Padmini Tamilarasi, Krishnan Manikandan, Viswas Raja Solomon.

  Determining a drug's bioavailability and bioequivalence is important for developing and approving a drug product. The procedure supports applications for generic drug products and novel therapeutic substances, makes important decisions regarding safety and efficacy, and measures a drug's concentration in biological matrices. This study aimed to develop and validate a specific, simple, sensitive, and accurate method using liquid chromatography-tandem mass spectrometry (LC-MS) for measuring bumetanide (BUM) in human plasma. Chromatographic separation was achieved using a Hypurity C18 column (4.6 × 50 mm, 5 μm) under isocratic conditions, and LC-MS detected positive ionization acquisition modes. Protonated precursor to product ion transitions were observed at m/z 365.08 → 240.10 and 370.04 → 244.52 for BUM and internal standard, respectively. The linear range of BUM in plasma samples was 3.490-401.192 ng/mL. The inter-precision value ranged from 1.76% to 4.75%. The inter-accuracy value ranged from 96.46% to 99.95%. The method was adequately validated per the U.S. Food and Drug Administration guidelines, and the results were within permissible bounds. The Cmax and Tmax values were ~53.097 ± 13.537 ng/mL and 1.25 (0.67-5.00) h, respectively. The new approach showed satisfactory results for studying BUM in human plasma with potential use in pharmacokinetic and bioequivalence investigations.

Keywords:  LC-MS/MS; bumetanide; liquid-liquid extraction; method development; validation

DOI:  https://doi.org/10.1002/bmc.5825
J Cheminform. 2024 Jan 18. 16(1): 8

IDSL_MINT: a deep learning framework to predict molecular fingerprints from mass spectra.

Sadjad Fakouri Baygi, Dinesh Kumar Barupal.

  The majority of tandem mass spectrometry (MS/MS) spectra in untargeted metabolomics and exposomics studies lack any annotation. Our deep learning framework, Integrated Data Science Laboratory for Metabolomics and Exposomics-Mass INTerpreter (IDSL_MINT) can translate MS/MS spectra into molecular fingerprint descriptors. IDSL_MINT allows users to leverage the power of the transformer model for mass spectrometry data, similar to the large language models. Models are trained on user-provided reference MS/MS libraries via any customizable molecular fingerprint descriptors. IDSL_MINT was benchmarked using the LipidMaps database and improved the annotation rate of a test study for MS/MS spectra that were not originally annotated using existing mass spectral libraries. IDSL_MINT may improve the overall annotation rates in untargeted metabolomics and exposomics studies. The IDSL_MINT framework and tutorials are available in the GitHub repository at https://github.com/idslme/IDSL_MINT .Scientific contribution statement.Structural annotation of MS/MS spectra from untargeted metabolomics and exposomics datasets is a major bottleneck in gaining new biological insights. Machine learning models to convert spectra into molecular fingerprints can help in the annotation process. Here, we present IDSL_MINT, a new, easy-to-use and customizable deep-learning framework to train and utilize new models to predict molecular fingerprints from spectra for the compound annotation workflows.

Keywords:  Deep learning; LipidMaps; Lipidomics; Mass spectrometry; Metabolomics; Molecular fingerprint descriptor; PyTorch; Transformer

DOI:  https://doi.org/10.1186/s13321-024-00804-5
Anal Chem. 2024 Jan 19.

Enhancing Metabolome Annotation by Electron Impact Excitation of Ions from Organics-Molecular Networking.

Xinxin Wang, Xiaoshan Sun, Fubo Wang, Chunmeng Wei, Fujian Zheng, Xiuqiong Zhang, Xinjie Zhao, Chunxia Zhao, Xin Lu, Guowang Xu.

Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) is widely used in untargeted metabolomics, but large-scale and high-accuracy metabolite annotation remains a challenge due to the complex nature of biological samples. Recently introduced electron impact excitation of ions from organics (EIEIO) fragmentation can generate information-rich fragment ions. However, effective utilization of EIEIO tandem mass spectrometry (MS/MS) is hindered by the lack of reference spectral databases. Molecular networking (MN) shows great promise in large-scale metabolome annotation, but enhancing the correlation between spectral and structural similarity is essential to fully exploring the benefits of MN annotation. In this study, a novel approach was proposed to enhance metabolite annotation in untargeted metabolomics using EIEIO and MN. MS/MS spectra were acquired in EIEIO and collision-induced dissociation (CID) modes for over 400 reference metabolites. The study revealed a stronger correlation between the EIEIO spectra and metabolite structure. Moreover, the EIEIO spectral network outperformed the CID spectral network in capturing structural analogues. The annotation performance of the structural similarity network for untargeted LC-MS/MS was evaluated. For the spiked NIST SRM 1950 human plasma, the annotation coverage and accuracy were 72.94 and 74.19%, respectively. A total of 2337 metabolite features were successfully annotated in NIST SRM 1950 human plasma, which was twice that of LC-CID MS/MS. Finally, the developed method was applied to investigate prostate cancer. A total of 87 significantly differential metabolites were annotated. This study combining EIEIO and MN makes a valuable contribution to improving metabolome annotation.

DOI: https://doi.org/10.1021/acs.analchem.3c03443
Clin Chim Acta. 2024 Jan 14. pii: S0009-8981(24)00019-6. [Epub ahead of print]554 117778

Correction of creatine-creatinine conversion during serum creatinine quantification by two-dimensional liquid chromatography and double-spike isotope dilution tandem mass spectrometry.

Daniela Pineda-Cevallos, María Funes Menéndez, Adriana González-Gago, Pablo Rodríguez-González, J Ignacio García Alonso.

   BACKGROUND AND AIMS: Development of a candidate reference method based on bidimensional liquid chromatography coupled to ESI-MS/MS and double spike isotope dilution for serum creatinine quantification capable of correcting for creatinine-creatine interconversion during sample pretreatment. Study of the impact of the creatine-creatinine interconversion during the analysis of human serum samples.
MATERIALS AND METHODS: 13C1-creatinine and 13C2-creatine are added to the serum sample. Separation carried out by bidimensional liquid chromatography combining reversed phase and a strong cation exchange chromatography. The heart cut, containing creatine and creatinine, is automatically transferred to the second dimension. Quantification carried out by double spike isotope dilution tandem MS/MS.
RESULTS: Minimization of spectral interferences and ion suppression due to matrix effects while increasing sample throughput compared to the direct coupling of cation exchange chromatography to the ESI source. Trueness of the method studied with the satisfactory analysis of two certified reference materials. Satisfactory intra- and inter-day precisions obtained analysing a serum pool and control sera. Analysis of 93 serum samples revealed negligible interconversions with no correlation with creatine levels.
CONCLUSIONS: The method provides adequate analytical figures of merit for serum creatinine determination according to CSLI guidelines. Negligible creatine-creatinine interconversion is promoted with the applied sample preparation procedure.

Keywords:  Analyte interconversion; Bidimensional chromatography; Double spike isotope dilution; Serum creatinine; Tandem mass spectrometry

DOI:  https://doi.org/10.1016/j.cca.2024.117778
BMC Chem. 2024 Jan 13. 18(1): 12

Simultaneous determination of colistin sulfate and tigecycline in human plasma by liquid chromatography-tandem mass spectrometry method.

Ying-Chao Ma, Xi-Kun Wu, Xiu-Ling Yang, Zhi-Qing Zhang.

   OBJECTIVE: To establish a high-performance liquid chromatography-tandem mass spectrometry method (HPLC-MS/MS) to simultaneously determine colistin sulfate and tigecycline in human plasma.
METHODS: Polymyxin B1 internal standard (20 µL) was added into 200 µL of plasma sample. The samples were treated with methanol-5% trichloroacetic acid (50:50, V/V) solution, and the protein precipitation method was adopted for post-injection analysis. The chromatographic column was a Dikma C18 (4.6 mm × 150 mm, 5 μm). For the mobile phase, 0.1% formic acid in aqueous solution was used for phase A, 0.1% formic acid in acetonitrile solution for phase B, and gradient elution was also applied. The flow rate was 0.8 mL/min, the column temperature was 40 °C, and the injection volume was 10 µL; Electrospray ionization and multiple reaction ion monitoring were adopted and scanned by the HPLC-MS/MS positive ion mode.
RESULTS: The endogenous impurities in the plasma had no interference in the determination of the analytes. There existed a good linear relationship of colistin sulfate within the range of 0.1-10 µg/mL (R2 = 0.9986), with the lower limit of quantification (LLOQ) of 0.1 µg/mL. There existed a good linear relationship of tigecycline within the range of 0.05-5 µg/ mL (R2 = 0.9987), with the LLOQ of 0.05 µg/mL. The intra- and inter-day relative standard deviations of colistin sulfate and tigecycline were both less than 15%, and the accuracy was between 88.21% and 108.24%. The extraction had good stability, the extraction recovery rate was 87.75-91.22%, and the matrix effect was 99.40-105.26%.
CONCLUSION: This study successfully established a method for simultaneously detecting colistin sulfate and tigecycline plasma concentrations. The method was simple, rapid, and highly sensitive and could be applied for therapeutic medication monitoring.

Keywords:  Colistin sulfate; Liquid chromatography–tandem mass spectrometry; Plasma drug concentration; Therapeutic medication monitoring; Tigecycline

DOI:  https://doi.org/10.1186/s13065-023-01109-8
Angew Chem Int Ed Engl. 2024 Jan 18. e202318579

Rapid and Bifunctional Chemoselective Metabolome Analysis of Liver Patient Samples Using the Reagent 4-Nitrophenyl-2H-azirine.

Weifeng Lin, Lars Gerullat, Peder R Braadland, Anaïs Fournier, Johannes R Hov, Daniel Globisch.

  Primary sclerosing cholangitis (PSC) is a chronic inflammatory disease of the bile ducts that has been associated with diverse metabolic carboxylic acids. Mass spectrometric techniques are the method of choice for their analysis. However, the broad investigation of this metabolite class remains challenging. Derivatization of carboxylic acids represents a strategy to overcome these limitations but available methods suffer from diverse analytical challenges. Herein, we have designed a novel strategy introducing 4-nitrophenyl-2H-azirine as a new chemoselective moiety for the first time for carboxylic acid metabolites. This moiety was selected as it rapidly forms a stable amide bond and also generates a new ketone, which can be analyzed by our recently developed quant-SCHEMA method specific for carbonyl metabolites. Optimization of this new method revealed a high reproducibility and robustness, which was utilized to validate 102 metabolic carboxylic acids using authentic synthetic standard conjugates in human plasma samples including nine metabolites that were newly detected. Using this sequential analysis of the carbonyl- and carboxylic acid-metabolomes revealed alterations of the ketogenesis pathway, which demonstrates the vast benefit of our unique methodology. We anticipate that the developed azirine moiety with rapid functional group transformation will find broad application in diverse chemical biology research fields.

Keywords:  2H-Azirine; Bioorganic chemistry; Chemical Biology; Chemical metabolomics; Metabolic carboxylic acids

DOI:  https://doi.org/10.1002/anie.202318579
FEBS Lett. 2024 Jan 19.

Advances in multimodal mass spectrometry for single-cell analysis and imaging enhancement.

Seth W Croslow, Timothy J Trinklein, Jonathan V Sweedler.

  Multimodal mass spectrometry (MMS) incorporates an imaging modality with probe-based mass spectrometry (MS) to enable precise, targeted data acquisition and provide additional biological and chemical data not available by MS alone. Two categories of MMS are covered; in the first, an imaging modality guides the MS probe to target individual cells and to reduce acquisition time by automatically defining regions of interest. In the second category, imaging and MS data are coupled in the data analysis pipeline to increase the effective spatial resolution using a higher resolution imaging method, correct for tissue deformation, and incorporate fine morphological features in an MS imaging dataset. Recent methodological and computational developments are covered along with their application to single-cell and imaging analyses.

Keywords:  cell; imaging; mass spectrometry; microscopy; multimodal; segmentation; subcellular

DOI:  https://doi.org/10.1002/1873-3468.14798
J Sep Sci. 2024 Jan;47(1): e2300716

Analysis of 14 β-agonists in pork using an automated wooden tip-based solid-phase microextraction device and ultra-high-performance liquid chromatography-tandem mass spectrometry.

Kai Li, Xiaoming Gong, Liming Jin, Mengxiao Liu, Mengmeng Hua.

  This study introduces a cost-effective, automated ultra-high-performance liquid chromatography-tandem mass spectrometry method for the detection of 14 β-agonists in pork using a novel solid-phase microextraction probe composed of polyacrylonitrile and molecularly imprinted polymer. Integrated into an automated extraction device, the probe optimizes extraction prior to analysis while reducing expenses and time compared to traditional solid-phase extraction procedures. The method validation followed the Chinese National Standard (GB/T 27404-2008) and examined limits of detection, limits of quantification, matrix effects, linearity, intraday, and interday precision. Average recovery rates ranged from 71.6% to 82.2%, with relative standard deviations less than 15%. Limits of detection and limits of quantification ranged from 0.09 to 0.39 and 0.27 to 0.99 μg/kg, respectively. The new method identified positive samples more accurately than the current National Standard GB/T 31658.22-2022 and demonstrated its potential for routine assessment and regulatory compliance in the detection of β-agonists in pork.

Keywords:  automated solid-phase microextraction; pork; ultra-high-performance liquid chromatography-tandem mass spectrometry; veterinary drug residues; β-agonists

DOI:  https://doi.org/10.1002/jssc.202300716
ACS Omega. 2024 Jan 09. 9(1): 607-617

Probing Liquid Chromatography-Tandem Mass Spectrometry Response Dynamics and Nonlinear Effects for Response Level Defined Calibration Strategies with Simple Methods To Expand Linear Dynamic Ranges.

Rui Zhang.

The response characteristics of liquid chromatography-tandem mass spectrometry (LC-MS/MS) serve as the basis for selecting calibration methods in quantitative analysis. LC-MS/MS inherently exhibits nonlinear detection behavior, primarily attributed to the disproportionate growth observed between peak area and peak height at elevated response levels, potentially leading to signal saturation. This disproportionate peak growth results in reduced unit response (UR), which quantifies the instrument's detection sensitivity. LC-MS/MS typically operates within a narrow near-linear response range (NLRR) due to approximately proportional peak growth, yet the NLRR width varies across different analytes or platforms. Although the inclusion of stable isotope-labeled (SIL) internal standards (IS) in LC-MS/MS analysis can mitigate certain instrument response variations, it does not eliminate the fundamental cause of nonlinearity. Moreover, the concentration range accommodated by the NLRR can significantly fluctuate at different sensitivity levels. LC-MS/MS also encounters various other nonlinear effects, including ion suppression during ionization, signal cross-contribution between the analyte/IS, and matrix effects (ME). Consequently, quadratic regression emerges as a more adaptable approach to LC-MS/MS nonlinear response dynamics, offering a broader calibration range. The application of linear regression, on the other hand, requires strict conditions. Although the signal saturation zone typically remains inaccessible to calibration methods, reducing responses by employing less-optimal selected reaction monitoring (SRM) transitions and/or lower detection gain can facilitate fitting a wide concentration range into the NLRR, thereby enabling accurate linear regression calibration. This report delves into examining the LC-MS/MS response profile, its dynamics, and major nonlinear effects through instrument response mapping to elucidate their influence on the selection of calibration methods.

DOI: https://doi.org/10.1021/acsomega.3c06190
Anal Chim Acta. 2024 Feb 01. pii: S0003-2670(23)01366-1. [Epub ahead of print]1288 342145

Synthesis and quantification of short-chain fatty acid esters of hydroxy fatty acids in rat intestinal contents and fecal samples by LC-MS/MS.

Siddabasave Gowda B Gowda, Fengjue Hou, Divyavani Gowda, Hitoshi Chiba, Kentaro Kawakami, Satoru Fukiya, Atsushi Yokota, Shu-Ping Hui.

  Short-chain fatty acid esters of hydroxy fatty acids (SFAHFAs) are a new class of endogenous lipids belonging to the fatty acid esters of the hydroxy fatty acid family. We previously uncovered their chemical structure and discussed their potential biological significance. We anticipate an increased need for SFAHFA measurements as markers of metabolic and inflammatory health. In this study, we synthesized sixty isomeric SFAHFAs by combining 12 hydroxy fatty acids (C16-C24) and five short-chain fatty acids (C2-C6) including a labelled internal standard. SFAHFA enrichment was achieved by solid-phase extraction and established a sensitive method for their quantitation by targeted LC-MS/MS. The method was applied to profile SFAHFAs in intestinal contents and fecal samples collected from rats fed a high-fat diet (HFD). The results demonstrated a significant decrease in SFAHFAs in the intestinal contents of the HFD group compared with the control group. The fecal time course (0-8 weeks) profile of SFAHFAs showed significant downregulation of acetic and propanoic acid esters in just 2 weeks after HFD administration. This study offers the first synthesis and quantitation method for SFAHFAs, demonstrating their potential use in elucidating SFAHFA sources, their role in various diseases, and potential biochemical signalling pathways.

Keywords:  Chemical synthesis; Gut microbial lipids; Intestinal contents; Liquid chromatography; Mass spectrometry; SFAHFAs

DOI:  https://doi.org/10.1016/j.aca.2023.342145
Fa Yi Xue Za Zhi. 2023 Dec 25. pii: 1004-5619(2023)06-0564-07. [Epub ahead of print]39(6): 564-570

UPLC-MS/MS Method for Detection of Etomidate and Its Metabolite Etomidate Acid Quantity in Blood.

Xing Han, Xin Liu, Ming-Luo DU, Ruo-Lun Xu, Jia-Rong Li, Chao Liu, Wei-Guo Liu.

   OBJECTIVES: To establish a method for the simultaneous quantitative analysis of etomidate and its metabolite etomidate acid in blood, and to discuss its application value in actual cases.
METHODS: Acetonitrile precipitate protein method was used, and C18 column was selected. Gradient elution was performed with acetonitrile and 5 mmol/L ammonium acetate within 6 min. Electrospray ionization source in positive ion mode was used. The internal standard etomidate acid-d5 was obtained by etomidate-d5 alkaline hydrolysis reaction. Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used for quantitative analysis. The methodological verification was conducted.
RESULTS: Etomidate and etomidate acid in blood showed good linear relationship in the quantitative linear range (r>0.999), with the lower limit of quantification was 2.5 ng/mL and 7.5 ng/mL, respectively. The accuracy, precision, recovery rate, and matrix effect of the method met the professional verification standards. The practical application results showed that etomidate and etomidate acid could be detected in the blood of the abusers, and their mass concentrations ranged from 17.24 to 379.93 ng/mL.
CONCLUSIONS: The method established in this study can simultaneously quantify etomidate and etomidate acid in blood, which is simple and convenient to operate with accuracy. It can meet the detection needs of actual cases and provide technical support for law enforcement to crack down on etomidate abuse.

Keywords:  blood; etomidate; etomidate acid; forensic medicine; toxicological analysis; ultra-high performance liquid chromatography-tandem mass spectrometry

DOI:  https://doi.org/10.12116/j.issn.1004-5619.2023.330901
Anal Chim Acta. 2024 Feb 01. pii: S0003-2670(23)01406-X. [Epub ahead of print]1288 342185

A new adsorptive 3D-printed sampling device for simultaneous determination of 63 urinary organic acids by LC-MS/MS.

Mariana Zuccherato Bocato, Reverson Fernandes Quero, Anthony Alexandre Weil, Cibele Aparecida Cesila, Joseph Adewuyi Adeyemi, Fernando Barbosa.

   BACKGROUND: The detection and quantification of urinary metabolites play an important role in disease diagnosis. In most cases, urinary analyses are done with liquid urine samples, which must be quickly transported to the laboratory to avoid metabolites degradation that is associated with temperature fluctuations. Consequently, dried sampling devices have emerged to minimize analyte degradation. However, most commercial dried sampling devices are expensive, aggregate low volumes, and need better analytical sensitivity. Therefore, a new dry urine sampling device that is inexpensive, suitable for domestic sampling operation, and efficient for quantifying metabolites without requiring high-resolution instruments is proposed in the present study.
RESULTS: The newly designed dry urine sampling device was produced by 3D printing that efficiently determines 63 urinary organic acids using liquid chromatography coupled with mass spectrometry (LC-MS/MS). The system's efficiency was demonstrated with analytical figures of merit, such as precision, accuracy, and stability of analytes after the sampling and storing of ordinary urine samples. The limits of quantification ranged from 0.01 to 0.42 ng mL-1. Precision and accuracy tests showed relative standard deviations of less than 15 %. The urine stability in the sampling device was high within seven days without any significant degradation of the metabolites. The method was applied to the analysis of 10 human urine samples and compared to a conventional method without the use of the sampling device. The results showed no statistically significant differences, demonstrating the method's efficiency.
SIGNIFICANCE: The proposed 3-D printing device was developed with fast, low-cost manufacturing features and can be manufactured with different volumetric capacities, adaptable to the needs of each user. Furthermore, it is innovative because this is the first sampling device that is effective for the simultaneous storage and preservation of several important urinary metabolites. Thus, it is anticipated that its application would contribute significantly to the identification of metabolic disorders.

Keywords:  3D–printed; Dried urine; LC–MS/MS; Sampling; Urinary metabolites

DOI:  https://doi.org/10.1016/j.aca.2023.342185
Biomed Chromatogr. 2024 Jan 13. e5821

Development of an ultra-high-performance liquid chromatography-tandem mass spectrometry method for the simultaneous determination of crassicauline A, fuziline, karacoline, and songorine in rat plasma and application in their pharmacokinetics.

Fan Chen, Ziyue Wang, Lvqi Luo, Yifan He, Yizhe Ma, Congcong Wen, Xianqin Wang, Xiuwei Shen.

  In this paper, an ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for quantifying the levels of crassicauline A, fuziline, karacoline, and songorine in rat plasma. After processing the rat plasma, the proteins in the plasma were separated by extracting the analytes with acetonitrile-methanol (9:1, v/v). The chromatographic column used was the UPLC HSS T3 column, and the mobile phase (methanol-water with 0.1% formic acid) under a gradient elution profile was used to separate the four compounds, with elution times for each analyte being less than 5 min. Electrospray ionization in positive-ion mode and operating in multiple reaction monitoring mode was used for quantitative analysis. Crassicauline A, fuziline, karacoline, and songorine were administered to 48 rats (n = 6 per group) orally (5 mg/kg) and intravenously (0.5 mg/kg). The standard curves demonstrated excellent linearity in the range of 1-2500 ng/mL, wherein all r values were greater than 0.99. The UPLC-MS/MS method for the determination of crassicauline A, fuziline, karacoline, and songorine in rat plasma was successfully applied in determining their pharmacokinetics parameters, from which their oral bioavailabilities were calculated to be 18.7%, 4.3%, 6.0%, and 8.4%, respectively.

Keywords:  UPLC-MS/MS; crassicauline A; fuziline; karacoline; pharmacokinetics; songorine

DOI:  https://doi.org/10.1002/bmc.5821
J Sep Sci. 2024 Jan;47(1): e2300790

Simultaneous determination of sinomenine and its metabolites desmethyl-sinomenine and sinomenine N-oxide in rat plasma by liquid chromatography tandem mass spectrometry.

Ning He, Ali Fan, Dongmei Wu, Hongyan Yu, Kun Wang, Weijie Jiao, Xu Zhao.

  Sinomenine is an active ingredient extracted from herb medicine, which has been prescribed to treat rheumatoid arthritis in clinics. The present work was to develop a simple method to simultaneously determine sinomenine and its metabolites desmethyl sinomenine and sinomenine N-oxide in rat plasma by liquid chromatography tandem mass spectrometry. Precursor-to-product transitions for detection were m/z 330.2 > 239.1 for sinomenine, m/z 316.2 > 239.1 for desmethyl-sinomenine, m/z 346.2 > 314.1 for sinomenine N-oxide and m/z 286.2 > 153.2 for morphine (internal standard), respectively. During the validation and sample quantification, an excellent linear calibration range was observed for all the analytes with correlation coefficients more than 0.999 (r > 0.99). The extraction recovery was more than 85%. No significant matrix effect and carryover were observed. The precision was less than 6.45%, whereas accuracy ranged from -4.10% to 7.23%. The validated method has been successfully applied to the pharmacokinetic study of sinomenine, desmethyl sinomenine, and sinomenine N-oxide in rat plasma after oral administration of sinomenine at a single dose of 5 mg/kg. The results suggested that sinomenine was rapidly metabolized into its metabolite desmethyl sinomenine and sinomenine N-oxide.

Keywords:  desmethyl sinomenine; pharmacokinetics; sinomenine; sinomenine N-oxide

DOI:  https://doi.org/10.1002/jssc.202300790
Heliyon. 2024 Jan 15. 10(1): e24198

A rapid and simple HPLC-MS/MS method for the therapeutic drug monitoring of six special-grade antimicrobials in pediatric patients.

Xijuan Jiang, Yabin Qin, Rong Lei, Yu Han, Jing Yang, Guying Zhang, Jianfang Liu.

  Meropenem, linezolid, fluconazole, voriconazole, posaconazole, and vancomycin are six important antimicrobials used for severe infections in critically ill patients listed in special-grade antimicrobials in China. The six antimicrobials' highly variable pharmacodynamics and pharmacokinetics in critically ill pediatric patients present significant challenges to clinicians in ensuring optimal therapeutic targets. Therefore, therapeutic drug monitoring of these antimicrobials in human plasma is necessary to obtain their plasma concentration. A rapid, simple, and sample-saving high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed, which could simultaneously determine all six antimicrobials. It required only 10 μL of plasma and a one-step protein precipitation process. Chromatographic separation was achieved on a reversed-phase column (C18, 30 × 2.1 mm, 2.6 μm) via gradient elution using water and acetonitrile containing 0.1 % formic acid as mobile phase. The injection volume was 2 μL, and the total run time was only 2.5 min. Detection was done using a Triple Quad™ 4500MD tandem mass spectrometer coupled with an electrospray ionization (ESI) source in positive mode. The calibration curves ranged from 0.5 to 64 μg/mL for meropenem and fluconazole, 0.2-25.6 μg/mL for linezolid and voriconazole, 0.1-12.8 μg/mL for posaconazole and 1-128 μg/mL for vancomycin, with the coefficients of correlation all greater than 0.996. Furthermore, the method was validated rigorously according to the European Medicines Agency (EMA) guidelines, demonstrating excellent accuracy (from 93.0 % to 110.6 %) and precision (from 2.0 % to 12.8 %). Moreover, its applicability to various matrices (including serum, hemolytic plasma, and hyperlipidemic plasma) was evaluated. Thus, this method was successfully applied to routine therapeutic drug monitoring for critically ill pediatric patients and other patients in need.

Keywords:  HPLC-MS/MS; Pediatric patients; Special-grade antimicrobials; Therapeutic drug monitoring (TDM)

DOI:  https://doi.org/10.1016/j.heliyon.2024.e24198
Anal Chim Acta. 2024 Feb 01. pii: S0003-2670(23)01335-1. [Epub ahead of print]1288 342114

Triplex glycan quantification by metabolic labeling with isotopically labeled glucose in yeast.

Thao Thi Pham, Ji-Yeon Kim, Sami T Tuomivaara, Yong-Ill Lee, Seonghun Kim, Lance Wells, Jae-Min Lim.

  Mass spectrometry-based approaches encompass a powerful collection of tools for the analysis biological molecules, including glycans and glycoconjugates. Unlike most traditional bioanalytical methods focusing on these molecules, mass spectrometry is especially suited for multiplexing, by utilizing stable-isotope labeling. Indeed, stable isotope-based multiplexing can be regarded as the gold-standard approach in reducing noise and uncertainty in quantitative mass spectrometry and quantitative analyses generally. The increasing sophistication and depth of biological questions being asked continue to challenge the practitioners of mass spectrometry method development. To understand the biological relevance of glycans, many stable isotope labeling-based mass spectrometry methods have been developed. Based on the duplex MILPIG (metabolic isotope labeling of polysaccharides with isotopic glucose), we establish here a novel triplex isotope labeling method using baker's yeast as the model system. Two differentially isotope-labeled glucoses (medium: 1-13C1 and heavy: 1,2-13C2), in addition to natural abundance glucose (light), were successfully used to label each monosaccharide ring in N-linked glycans in three different cell culture conditions, that, after sample mixing, resulted in a predictable triplet spectrum amenable for relative quantitation. We demonstrate excellent accuracy and precision of relative quantitation for a 1:1:1 mixture of glycans labeled in such a fashion. In addition, we applied triplex MILPIG to interrogate differential N-glycan profiles in tunicamycin-treated and control yeast cells and show that different N-glycans respond differently to tunicamycin.

Keywords:  Glycans; Glycomics; MILPIG; Mass spectrometry; Triplex quantification

DOI:  https://doi.org/10.1016/j.aca.2023.342114
Chemosphere. 2024 Jan 11. pii: S0045-6535(24)00050-X. [Epub ahead of print]351 141157

Inhibition of anaerobic digestion by various ammonia sources resulted in subtle differences in metabolite dynamics.

Xiaoqing Wang, Stephany Campuzano, Angéline Guenne, Laurent Mazéas, Olivier Chapleur.

  The impact of ammonia on anaerobic digestion performance and microbial dynamics has been extensively studied, but the concurrent effect of anions brought by ammonium salt should not be neglected. This paper studied this effect using metabolomics and a time-course statistical framework. Metabolomics provides novel perspectives to study microbial processes and facilitates a more profound understanding at the metabolic level. The advanced statistical framework enables deciphering the complexity of large metabolomics data sets. More specifically, a series of lab-scale batch reactors were set up with different ammonia sources added. Samples of nine time points over the degradation were analyzed with liquid chromatography-mass spectrometry. A filtering procedure was applied to select the promising metabolomic peaks from 1262 peaks, followed by modeling their intensities across time. The metabolomic peaks with similar time profiles were clustered, evidencing the correlation of different biological processes. Differential analysis was performed to seek the differences in metabolite dynamics caused by different anions. Finally, tandem mass spectrometry and metabolite annotation provided further information on the molecular structure and possible metabolic pathways. For example, the consumption of 5-aminovaleric acid, a short-chain fatty acid obtained from l-lysine degradation, was slowed down by phosphates. Overall, by investigating the effect of anions on anaerobic digestion, our study demonstrated the effectiveness of metabolomics in providing detailed information in a set of samples from different experimental conditions. With the statistical framework, the approach enables capturing subtle differences in metabolite dynamics between samples while accounting for the differences caused by time variations.

Keywords:  Ammonia inhibition; Anaerobic digestion; Anion; Non-targeted metabolomics; Statistics; Time-course analysis

DOI:  https://doi.org/10.1016/j.chemosphere.2024.141157
Anim Sci J. 2024 Jan-Dec;95(1):95(1): e13896

Gas chromatography-mass spectrometry-based quantitative method using tert-butyldimethylsilyl derivatization for plasma levels of free amino acids and related metabolites in Japanese Black cattle.

Hanwool Do, Emi Yoshida, Tatsunori Masaki, Shigeru Oosaki, Mitsuhiro Sakase, Eiji Iwamoto, Shozo Tomonaga.

  The quantification of amino acid and related metabolite levels is important for evaluating amino acid metabolism and function in animals. However, a useful quantitative method is not enough. In this study, we developed and validated tert-butyldimethylsilyl derivatization method using gas chromatography-mass spectrometry to quantify plasma levels of free amino acids and related metabolites in Japanese Black cattle. Of the 51 metabolites examined, 24, including 20 amino acids, one amine, and three keto acids, could be quantified. Compared with the trimethylsilyl derivatization method using gas chromatography-mass spectrometry, which has been used for untargeted metabolomic analysis, the present method had higher analytical reliability. This method is advantageous for assessing branched-chain amino acid (BCAA) metabolism because it enables the quantification of not only BCAA levels (valine, leucine, and isoleucine) but also their bioactive metabolite keto acid levels (2-ketoisovaleric acid, 2-ketoisocaproic acid, and 2-keto-3-methylvaleric acid) in the plasma. In addition, this method can quantify the plasma levels of not only tryptophan but also its bioactive metabolites kynurenine and serotonin. These results suggest that this quantitative method has the potential to further our understanding of amino acid metabolic processes and their functions in Japanese Black cattle.

Keywords:  Japanese Black cattle; amino acid metabolism; plasma; quantitative analysis

DOI:  https://doi.org/10.1111/asj.13896
Chemosphere. 2024 Jan 13. pii: S0045-6535(24)00114-0. [Epub ahead of print]351 141221

Development and evaluation of a comprehensive workflow for suspect screening of exposome-related xenobiotics and phase II metabolites in diverse human biofluids.

Mikel Musatadi, Inés Baciero-Hernández, Ailette Prieto, Maitane Olivares, Nestor Etxebarria, Olatz Zuloaga.

  Suspect and non-target screening (SNTS) methods are being promoted in order to decode the human exposome since a wide chemical space can be analysed in a diversity of human biofluids. However, SNTS approaches in the exposomics field are infra-studied in comparison to environmental or food monitoring studies. In this work, a comprehensive suspect screening workflow was developed to annotate exposome-related xenobiotics and phase II metabolites in diverse human biofluids. Precisely, human urine, breast milk, saliva and ovarian follicular fluid were employed as samples and analysed by means of ultra-high performance liquid chromatography coupled with high resolution tandem mass spectrometry (UHPLC-HRMS/MS). To automate the workflow, the "peak rating" parameter implemented in Compound Discoverer 3.3.2 was optimized to avoid time-consuming manual revision of chromatographic peaks. In addition, the presence of endogenous molecules that might interfere with the annotation of xenobiotics was carefully studied as the employment of inclusion and exclusion suspect lists. To evaluate the workflow, limits of identification (LOIs) and type I and II errors (i.e., false positives and negatives, respectively) were calculated in both standard solutions and spiked biofluids using 161 xenobiotics and 22 metabolites. For 80.3 % of the suspects, LOIs below 15 ng/mL were achieved. In terms of type I errors, only two cases were identified in standards and spiked samples. Regarding type II errors, the 7.7 % errors accounted in standards increased to 17.4 % in real samples. Lastly, the use of an inclusion list for endogens was favoured since it avoided 18.7 % of potential type I errors, while the exclusion list caused 7.2 % of type II errors despite making the annotation workflow less time-consuming.

Keywords:  Endogenous molecules; Human biofluids; Inclusion and exclusion suspect lists; Suspect and non-target screening (SNTS); Type I and II errors; “Peak rating”

DOI:  https://doi.org/10.1016/j.chemosphere.2024.141221