bims-metlip 2022-11-20 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2022–11–20
twenty-one papers selected by
Sofia Costa, Matterworks

Differential Kendrick's Plots as an Innovative Tool for Lipidomics in Complex Samples: Comparison of Liquid Chromatography and Infusion-Based Methods to Sample Differential Study.
UHPLC-MS/MS method for chiral separation of 3-hydroxy fatty acids on amylose-based chiral stationary phase and its application for the enantioselective analysis in plasma and platelets.
Single sample pathway analysis in metabolomics: performance evaluation and application.
Quantitation of cystic fibrosis triple combination therapy, elexacaftor/tezacaftor/ivacaftor, in human plasma and cellular lysate.
A candidate reference method and multiple commutable control materials for serum 25-hydroxyvitamin D measurement.
Towards inferring absolute concentrations from relative abundance in time-course GC-MS metabolomics data.
Teaching a New Dog Old Tricks: Ultrafast Liquid Chromatography/Tandem-Mass Spectrometry Analysis of Nine ß-Lactam Antibiotics for Improved Therapeutic Drug Monitoring.
High performance liquid chromatography-tandem mass spectrometry quantification of tryptophan metabolites in human serum and stool - Application to clinical cohorts in Inflammatory Bowel Diseases.
[Application progress of liquid chromatography tandem mass spectrometry in precision medicine].
Simultaneous determination of sex hormones and bile acids in rat plasma using a liquid chromatography-tandem mass spectrometry method.
Unsupervised Pharmaceutical Polymorph Identification and Multicomponent Particle Mapping of ToF-SIMS Data by Non-Negative Matrix Factorization.
A sensitive and robust method for the simultaneous determination of thirty-three legacy and emerging per- and polyfluoroalkyl substances in human plasma and serum.
Metabolomic and Lipidomic Profiling of Bacillus Using Two-Dimensional Tandem Mass Spectrometry.
Development of an LC-MS/MS method for simultaneous detection of 11 polyphenols in rat plasma and its pharmacokinetic application after oral administration of Coreopsis tinctoria extract.
Mass Spectrometry for the Advancement of Lipid Analysis in Alzheimer's Research.
Visualizing metabolomics data with R.
A distributable LC-MS/MS method for the measurement of serum thyroglobulin.
Validated LC-MS/MS method for the determination of copanlisib in mouse dried blood spots.
Development and validation of an LC-MS/MS method for determination of 8-iso-prostaglandin f2 Alpha in human saliva.
Leveraging the HMBC to Facilitate Metabolite Identification.
Direct adsorption sampling and ambient mass spectrometry analysis of tobacco smoke with porous paper strips.

J Am Soc Mass Spectrom. 2022 Nov 15.

Differential Kendrick's Plots as an Innovative Tool for Lipidomics in Complex Samples: Comparison of Liquid Chromatography and Infusion-Based Methods to Sample Differential Study.

Justine Hustin, Christopher Kune, Johann Far, Gauthier Eppe, Delphine Debois, Loïc Quinton, Edwin De Pauw.

Lipidomics has developed rapidly over the past decade. Nontargeted lipidomics from biological samples remains a challenge due to the high structural diversity, the concentration range of lipids, and the complexity of biological samples. We introduce here the use of differential Kendrick's plots as a rapid visualization tool for a qualitative nontargeted analysis of lipids categories and classes from data generated by either liquid chromatography-mass spectrometry (LC-MS) or direct infusion (nESI-MS). Each lipid class is easily identified by comparison with the theoretical Kendrick plot pattern constructed from exact mass measurements and by using MSKendrickFilter, an in-house Python software. The lipids are identified with the LIPID MAPS database. In addition, in LC-MS, the software based on the Kendrick plots returns the retention time from all the lipids belonging to the same series. Lipid extracts from a yeast (Saccharomyces cerevisiae) are used as a model. An on/off case comparing Kendrick plots from two cell lines (prostate cancer cell lines treated or not with a DGAT2 inhibition) clearly shows the effect of the inhibition. Our study demonstrates the good performance of direct infusion as a fast qualitative screening method as well as for the analysis of chromatograms. A fast screening semiquantitative approach is also possible, while the targeted mode remains the golden standard for precise quantitative analysis.

DOI: https://doi.org/10.1021/jasms.2c00232
J Pharm Biomed Anal. 2022 Nov 09. pii: S0731-7085(22)00572-6. [Epub ahead of print]223 115151

UHPLC-MS/MS method for chiral separation of 3-hydroxy fatty acids on amylose-based chiral stationary phase and its application for the enantioselective analysis in plasma and platelets.

Xiaoqing Fu, Zhanjian Xu, Meinrad Gawaz, Michael Lämmerhofer.

  3-Hydroxyfatty acids (3-OH-FAs) are formed in the hydration step during mitochondrial β-oxidation of saturated straight-chain fatty acids, which is a catabolic pathway that involves several enzymes. For an unbiased biological interpretation, an enantioselective analysis of 3-OH-FAs including their stereoisomers is necessary, which may contribute to the elucidation of enzymatic mechanisms in the biological pathways. In this work, an enantioselective gradient UHPLC-MS/MS method based on 1.6 µm particle polysaccharide column (Chiralpak IA-U) for chiral separation of 3-hydroxyfatty acids was developed which covers carbon chain length from C8 to C18 with a good resolution of R and S enantiomers. The method is fast and sensitive for detecting enantiomers of 3-OH-FAs by using a triple quadrupole instrument as a detector in a targeted, selected reaction monitoring (SRM) mode. A matrix matched-calibration strategy was applied for quantification of individual 3-OH-FA enantiomers. The method allows the simultaneous quantification of each enantiomer of 3-OH-FAs from C8-C18. One-phase liquid extraction with 2-propanol showed good extraction recoveries with over 90% on average. Further, the validated method was applied to investigate the alteration of 3-OH-FA enantiomers in platelets and plasma samples from human donors with different diagnoses of cardiovascular disease (acute coronary syndrome ACS, chronic coronary syndrome CCS). Both R and S enantiomers were detected in platelets and plasma samples with different predominance for R or S in dependence on carbon chain length, which might be associated with different functional enzymes of mitochondrial and peroxisomal β-oxidation. Finally, our study provides a new strategy for chiral separation and enantioselective analysis, showing great potential for targeted metabolomics in clinical biomarker discovery.

Keywords:  Coronary artery disease; Enantioselective metabolomics; Enzyme steroselectivity; Mitochondrial fatty acid oxidation; Peroxysomal fatty acid oxidation; Targeted lipidomics

DOI:  https://doi.org/10.1016/j.jpba.2022.115151
BMC Bioinformatics. 2022 Nov 14. 23(1): 481

Single sample pathway analysis in metabolomics: performance evaluation and application.

Cecilia Wieder, Rachel P J Lai, Timothy M D Ebbels.

   BACKGROUND: Single sample pathway analysis (ssPA) transforms molecular level omics data to the pathway level, enabling the discovery of patient-specific pathway signatures. Compared to conventional pathway analysis, ssPA overcomes the limitations by enabling multi-group comparisons, alongside facilitating numerous downstream analyses such as pathway-based machine learning. While in transcriptomics ssPA is a widely used technique, there is little literature evaluating its suitability for metabolomics. Here we provide a benchmark of established ssPA methods (ssGSEA, GSVA, SVD (PLAGE), and z-score) alongside the evaluation of two novel methods we propose: ssClustPA and kPCA, using semi-synthetic metabolomics data. We then demonstrate how ssPA can facilitate pathway-based interpretation of metabolomics data by performing a case-study on inflammatory bowel disease mass spectrometry data, using clustering to determine subtype-specific pathway signatures.
RESULTS: While GSEA-based and z-score methods outperformed the others in terms of recall, clustering/dimensionality reduction-based methods provided higher precision at moderate-to-high effect sizes. A case study applying ssPA to inflammatory bowel disease data demonstrates how these methods yield a much richer depth of interpretation than conventional approaches, for example by clustering pathway scores to visualise a pathway-based patient subtype-specific correlation network. We also developed the sspa python package (freely available at https://pypi.org/project/sspa/ ), providing implementations of all the methods benchmarked in this study.
CONCLUSION: This work underscores the value ssPA methods can add to metabolomic studies and provides a useful reference for those wishing to apply ssPA methods to metabolomics data.

Keywords:  Benchmarking; Enrichment analysis; Metabolomics pathway analysis; Pathway visualisation; Simulation; Single-sample pathway analysis

DOI:  https://doi.org/10.1186/s12859-022-05005-1
J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Nov 03. pii: S1570-0232(22)00423-8. [Epub ahead of print]1213 123518

Quantitation of cystic fibrosis triple combination therapy, elexacaftor/tezacaftor/ivacaftor, in human plasma and cellular lysate.

Kevin J Ryan, Jennifer S Guimbellot, Alexander E Dowell, Kedria D Reed-Walker, Corenna D Kerstner-Wood, Justin D Anderson, Zhongyu Liu, Edward P Acosta.

  The triple combination modulator therapy (ETI, elexacaftor (ELX), tezacaftor (TEZ), and ivacaftor (IVA)) is a recent advancement for the care of patients with cystic fibrosis. To aid in the development of clinical pharmacokinetics studies of this treatment, we developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for quantifying the component compounds in human plasma and cell lysate. This assay was optimized for small volumes (10 µL), uses stably labeled isotopes of the ETI compounds as internal standards, and employs a simple methanol protein precipitation method. Chromatography was performed on an ACE Excel C18, 2.1 × 50 mm, reversed phase analytical column, using a step or bump isocratic method, with mobile phases consisting of 0.1% formic acid in water for A, and 0.1% formic acid in acetonitrile for B. Analyte and internal standard detection was conducted with ESI positive ionization tandem mass spectrometry. The precursor/product transitions (m/z) monitored were 598.0/422.0 for ELX, 521.0/449.0 for TEZ, 393.0/172.0 for IVA, 601.0/422.0 for IS-ELX, 525.0/453.0 for IS-TEZ, and 399.0/178.0 for IS-IVA, respectively. The assay has a dynamic range of 10 to 10,000 ng/mL, with a mean coefficient of determination (r2, mean ± SD) of 0.9970 ± 0.0027 (ELX), 0.9989 ± 0.0004 (TEZ), 0.9981 ± 0.0003 (IVA), regardless of specimen matrix. The mean precision values for all calibration standards ranged from 0.0 to 10.8% (ELX), 0.0 to 6.7% (TEZ), and 0.2 to 5.6% (IVA), while the accuracy for calibration standards was within the range of -5.7 to 3.5% (ELX), -3.2 to 6.0% (TEZ), and -3.8 to 5.2% (IVA). Validation results demonstrated high accuracy (≤7.3, ≤9.8, ≤10.6% deviation) and high precision (≤11.5, ≤6.3, ≤11.0% CV) for the respective ETI quality control samples. This method provides a fully validated assay for ETI quantitation for use in clinical research.

Keywords:  CFTR; Cystic Fibrosis; Elexacaftor; Ivacaftor; LC-MS/MS; Tezacaftor

DOI:  https://doi.org/10.1016/j.jchromb.2022.123518
J Clin Lab Anal. 2022 Nov 13. e24756

A candidate reference method and multiple commutable control materials for serum 25-hydroxyvitamin D measurement.

Li Zhang, Qichen Long, Jiangtao Zhang, Qingzhang Zeng, Haijian Zhao, Wenxiang Chen, Tianjiao Zhang, Chuanbao Zhang.

   OBJECTIVES: The aim of the current study was to establish a reliable candidate reference method for serum 25-hydroxyvitamin D [25(OH)D] measurement and to assess the commutability of multiple control materials among liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods.
METHODS: Serum 25-hydroxyvitamin D2 [25(OH)D2] and 25-hydroxyvitamin D3 [25(OH)D3] together with spiked internal standards were extracted with a one-step approach and then analyzed by LC-MS/MS. The commutability assessment for 25(OH)D was conducted according to the Clinical and Laboratory Standards Institute (CLSI) EP14-A3 protocol. 25(OH)D concentrations in 5 levels of unprocessed serum pools, 7 levels of serum pools spiked with 25(OH)D3 or 25(OH)D2, 3 levels of commercial control materials, 2 levels of spiked bovine serum, and 4 levels of external quality assessment (EQA) materials were measured along with 30 single-donor samples using the candidate reference method and two routine LC-MS/MS methods.
RESULTS: The candidate reference method could separate 25(OH)D2 and 25(OH)D3 from 14 potential interfering compounds completely within a 9-min analysis time. Good method precision was obtained, and measurement results on certified reference material NIST SRM 972a were within the uncertainty of the certified values. All candidate materials were assessed commutable for LC-MS/MS methods.
CONCLUSIONS: The candidate reference method for serum 25(OH)D measurement is precise, accurate, and robust against interferences and can provide an accuracy base for routine methods. The multiple alternative control materials with commutability among LC-MS/MS methods will facilitate the further standardization for serum 25(OH)D measurement.

Keywords:  25-hydroxyvitamin D; accuracy-based external quality assessment; commutability; control materials; liquid chromatography-tandem mass spectrometry; reference method

DOI:  https://doi.org/10.1002/jcla.24756
Mol Omics. 2022 Nov 14.

Towards inferring absolute concentrations from relative abundance in time-course GC-MS metabolomics data.

Justin Y Lee, Yue Han, Mark P Styczynski.

Metabolomics, the large-scale study of metabolites, has significant appeal as a source of information for metabolic modeling and other scientific applications. One common approach for measuring metabolomics data is gas chromatography-mass spectrometry (GC-MS). However, GC-MS metabolomics data are typically reported as relative abundances, precluding their use with approaches and tools where absolute concentrations are necessary. While chemical standards can be used to help provide quantification, their use is time-consuming, expensive, or even impossible due to their limited availability. The ability to infer absolute concentrations from GC-MS metabolomics data without chemical standards would have significant value. We hypothesized that when analyzing time-course metabolomics datasets, the mass balances of metabolism and other biological information could provide sufficient information towards inference of absolute concentrations. To demonstrate this, we developed and characterized MetaboPAC, a computational framework that uses two approaches-one based on kinetic equations and another using biological heuristics-to predict the most likely response factors that allow translation between relative abundances and absolute concentrations. When used to analyze noiseless synthetic data generated from multiple types of kinetic rate laws, MetaboPAC performs significantly better than negative control approaches when 20% of kinetic terms are known a priori. Under conditions of lower sampling frequency and high noise, MetaboPAC is still able to provide significant inference of concentrations in 3 of 4 models studied. This provides a starting point for leveraging biological knowledge to extract concentration information from time-course intracellular GC-MS metabolomics datasets, particularly for systems that are well-studied and have partially known kinetic structures.

DOI: https://doi.org/10.1039/d2mo00168c
Ther Drug Monit. 2022 Dec 01. 44(6): 777-783

Teaching a New Dog Old Tricks: Ultrafast Liquid Chromatography/Tandem-Mass Spectrometry Analysis of Nine ß-Lactam Antibiotics for Improved Therapeutic Drug Monitoring.

Christopher Hodgkins, Stuart J Cordwell, Danijela Kocic.

BACKGROUND: Therapeutic drug monitoring (TDM) of β-lactam antibiotics provides critical knowledge in hospital intensive care unit environments to support dosing within the narrow window between therapeutic failure and toxicity. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the most suitable analytical technique for these drugs; however, clinicians, patients, and laboratories would benefit from shortening the timeframe between the collection of samples and reporting of results.
METHODS: The authors developed a very rapid LC-MS/MS method for 9 β-lactam antimicrobial drugs on a commercial core-shell reverse-phase LC column by exploiting the performance of such stationary phase materials at a high mobile-phase linear velocity and using a simple flow split to optimize ionization conditions in the mass spectrometer ion source. The method's performance was assessed using a currently validated routine LC-MS/MS assay performed on the same instrument.
RESULTS: Routine ß-lactam assays were reduced from >6 minutes per sample to less than 2 minutes with improved chromatographic resolution, while still maintaining acceptable analytical performance (average correlation coefficient: 0.99670, interday imprecision: 2.0%-10.8%, and bias: -1.68%), hence generating results in agreement with an existing validated method for patient and quality assurance program samples.
CONCLUSIONS: Time-critical results, such as those for β-lactam antimicrobials, may be reported by the TDM laboratory several hours earlier than current methods allow, providing improved patient care and generating capacity on LC-MS/MS instruments for larger batch sizes and/or additional assays. The simple-to-implement technique demonstrated in this study may be applicable to other TDM assays or any LC-MS/MS method where faster turnaround times are desirable.

DOI: https://doi.org/10.1097/FTD.0000000000001007
J Chromatogr A. 2022 Nov 03. pii: S0021-9673(22)00793-2. [Epub ahead of print]1685 463602

High performance liquid chromatography-tandem mass spectrometry quantification of tryptophan metabolites in human serum and stool - Application to clinical cohorts in Inflammatory Bowel Diseases.

Aurore Desmons, Lydie Humbert, Thibaut Eguether, Pranvera Krasniqi, Dominique Rainteau, Tarek Mahdi, Nathalie Kapel, Antonin Lamazière.

  Tryptophan, an essential amino acid, and its metabolites are involved in many physiological processes including neuronal functions, immune system, and gut homeostasis. Alterations to tryptophan metabolism are associated with various pathologies such as neurologic, psychiatric disorders, inflammatory bowel diseases (IBD), metabolic disorders, and cancer. It is consequently critical to develop a reliable, quantitative method for the analysis of tryptophan and its downstream metabolites from the kynurenine, serotonin, and indoles pathways. An LC-MS/MS method was designed for the analysis of tryptophan and 20 of its metabolites, without derivatization and performed in a single run. This method was validated for both serum and stool. The comparisons between serum and plasma, collected with several differing anticoagulants, showed significant differences only for serotonin. References values were established in sera and stools from healthy donors. For stool samples, as a proof of concept, the developed method was applied to a healthy control group and an IBD patient group. Results showed significant differences in the concentrations of tryptophan, xanthurenic acid, kynurenic acid, indole-3-lactic acid, and picolinic acid. This method allowed an extensive analysis of the three tryptophan metabolic pathways in two compartments. Beyond the application to IBD patients, the clinical use of this method is wide-ranging and may be applied to other pathological conditions involving tryptophan metabolism, such as neurological, psychiatric, or auto-inflammatory pathologies.

Keywords:  Inflammatory bowel diseases; LC-MS/MS; tryptophan metabolites profile

DOI:  https://doi.org/10.1016/j.chroma.2022.463602
Zhonghua Yu Fang Yi Xue Za Zhi. 2022 Nov 06. 56(11): 1675-1684

[Application progress of liquid chromatography tandem mass spectrometry in precision medicine].

J Y Jin, F R Qiu, J Zhang.

Liquid chromatography tandem mass spectrometry (LC-MS/MS) is an analytical method that combines high separation of liquid chromatography with high selectivity and sensitivity of mass spectrometry. In recent years, LC-MS/MS has been widely used in clinical practice, including screening of inherited disorders, determination of endogenous compounds and analysis of biomarkers. LC-MS/MS is of great value to the precision prevention, diagnosis and treatment of some diseases due to its accurate data. This article not only illustrates the advantages of LC-MS/MS in precision medicine, but also prospects the future trend of LC-MS/MS in clinical practice, which expects to promote the development of clinical LC-MS/MS in the prevention, diagnosis and treatment of diseases.

DOI: https://doi.org/10.3760/cma.j.cn112150-20211207-01128
J Pharm Biomed Anal. 2022 Nov 01. pii: S0731-7085(22)00560-X. [Epub ahead of print]223 115139

Simultaneous determination of sex hormones and bile acids in rat plasma using a liquid chromatography-tandem mass spectrometry method.

Yun Li, Dan Zhang, Yan Mo, Teng Zeng, Tongzhi Wu, Lingqiao Liu, Hua Zhang, Chang Chen.

  Endogenous steroids, including sex hormones and bile acids, are a group of essential compounds with various biological functions. In this study, we developed an LC-MS method that simultaneously measures 14 sex hormones and metabolites (SH) and 32 bile acids (BA) in rat plasma. Multiple innovative approaches were applied to increase the sensitivity and specificity, including optimization of the mobile phases, gradients, and dynamic multiple reaction monitoring (DMRM) transitions. The method was validated and applied on plasma samples from pregnant rats before and 0.5 h after oral glucose tolerance test (OGTT) at gestational days 0.5 and 18.5. Results showed that the method was applicable, and 9 SH and 30 BA were measurable in the samples. In summary, this method is applicable in studies on SH and BA in rat plasma, and may also be used on other matrix and species.

Keywords:  Bile acid; Mass spectrometry; Pregnant; Rat; Sex hormone

DOI:  https://doi.org/10.1016/j.jpba.2022.115139
Anal Chem. 2022 Nov 15.

Unsupervised Pharmaceutical Polymorph Identification and Multicomponent Particle Mapping of ToF-SIMS Data by Non-Negative Matrix Factorization.

Thomas P Forbes, John Greg Gillen, Amanda J Souna, Jeffrey Lawrence.

Crystal polymorphism of pharmaceutical compounds directly impacts resulting physicochemical characteristics, a critical aspect in active pharmaceutical ingredient (API) production. Tools to characterize and chemically map these polymorphs at the single particle scale remain important to advancing directed manufacture of targeted polymorphs. Here, time-of-flight secondary ion mass spectrometry (ToF-SIMS) was employed for chemically imaging inkjet printed acetaminophen samples. ToF-SIMS generates large data sets of high spatial resolution images. Extracting relevant data and peaks of interest can be laborious for, and biased by, users. Advances in machine learning approaches have introduced many supervised and unsupervised methods for data analysis. In this study, we apply non-negative matrix factorization (NMF) for the unsupervised analysis of ToF-SIMS chemical image data. More specifically, an expanded variant of NMF, NMFk, was employed to determine the data set's latent dimensionality. NMFk combines the spectral unmixing of traditional NMF with k-means clustering of the resulting factors and an optimization of the reconstruction and clustering. The method was used to identify the number of polymorph phases-and their representative mass spectra-generated from inkjet printed acetaminophen samples. Amorphous, crystalline form I, and crystalline form II polymorphs were observed. The learned polymorph mass spectra were then used to map the learned polymorphs onto subsequent particle samples of acetaminophen. Finally, NMFk also enabled the decomposition of mixed particle samples (i.e., migraine medicine), learning the number of compounds and their composition. The extracted constituent phase mass spectra-representing single compounds-were searched against mass spectral libraries for identification.

DOI: https://doi.org/10.1021/acs.analchem.2c03913
Anal Bioanal Chem. 2022 Nov 16.

A sensitive and robust method for the simultaneous determination of thirty-three legacy and emerging per- and polyfluoroalkyl substances in human plasma and serum.

Yan Ao, Min Nian, Weifeng Tang, Jun Zhang, Qianlong Zhang, Junjie Ao.

  Legacy and emerging per- and polyfluoroalkyl substances (PFAS) have attracted growing attention due to their potential adverse effects on humans. We developed a method to simultaneously determine thirty-three PFAS (legacy PFAS, precursors, and alternatives) in human plasma and serum using solid phase extraction coupled to ultra-performance liquid chromatography-tandem mass spectrometry (SPE-UPLC-MS/MS). The method yielded good linearity (>0.995) and excellent limits of detection (LODs) (0.0005~0.012 ng mL-1 in plasma and 0.002~0.016 ng mL-1 in serum). The relative recoveries ranged from 80.1 to 116%, with intra- and inter-day precision less than 14.3%. The robustness of this method has been tested continuously for 10 months (coefficients of variation <14.9%). Our method was successfully applied to the PFAS analysis of 42 real human plasma and serum samples collected from women. The proposed method is attractive for the biomonitoring of multi-class PFAS in human health risk assessment and epidemiological studies.

Keywords:  Biomonitoring; Emerging PFAS; Per- and polyfluoroalkyl substances; Plasma; Serum

DOI:  https://doi.org/10.1007/s00216-022-04426-4
Anal Chem. 2022 Nov 17.

Metabolomic and Lipidomic Profiling of Bacillus Using Two-Dimensional Tandem Mass Spectrometry.

L Edwin Gonzalez, Lucas J Szalwinski, Thomas C Sams, Eric T Dziekonski, R Graham Cooks.

Lipidomic and metabolomic profiles of sporulated and vegetative Bacillus subtilis and Bacillus thuringiensis from irradiated lysates were recorded using a quadrupole ion trap mass spectrometer modified to perform two-dimensional tandem mass spectrometry (2D MS/MS). The 2D MS/MS data domains, acquired using a 1.2 s scan of negative ions generated by nanoelectrospray ionization of microwave irradiated spores, showed the presence of dipicolinic acid (DPA) as well as various lipids. Aside from microwave radiation to extract DPA and lipids from spores, sample preparation was minimal. Characteristic lipid and metabolic profiles were observed using 107─108 cells of the two Bacillus species. Major features of the lipid profiles observed for the vegetative states included sets of phosphatidylglycerol (PG) lipids. Product ion spectra were extracted from the 2D MS/MS data, and they provided structural information on the fatty acid components of the PG lipids. The study demonstrates the flexibility, speed, and informative power of metabolomic and lipidomic fingerprinting for identifying the presence of spore-forming biological agents using 2D MS/MS as a rapid profiling screening method.

DOI: https://doi.org/10.1021/acs.analchem.2c03961
Chem Biodivers. 2022 Nov 16.

Development of an LC-MS/MS method for simultaneous detection of 11 polyphenols in rat plasma and its pharmacokinetic application after oral administration of Coreopsis tinctoria extract.

Haixia Han, Rui Ma, Aidi Xie, Juanjuan Gao, Zhen Wang, Yi Zhao, Huanming Pang, Wei Zhang.

  Eleven polyphenols, classified as flavonoid glycosides, flavonoid aglycones, and phenolic acids, are important bioactive components in the capitula of Coreopsis tinctoria (CCT). Nevertheless, their full pharmacokinetic profiles have not been demonstrated simultaneously. Therefore, a liquid chromatography - tandem mass spectrometry (LC-MS/MS) method was developed in the present work and used it to study the pharmacokinetics of these 11 compounds. We performed LC-MS/MS with a gradient mobile phase composed of water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid on a Proshell 120 SB C18 column (2.1 mm × 100 mm, 2.7 μm). We achieved a good chromatographic peak shape, resolution, and mass signal response, and multiple reaction monitoring facilitated the simultaneous detection of 11 analytes. In addition, we validated the selectivity, correlation coefficient, precision, extraction recovery, matrix effects, and stability of the LC-MS/MS method to be acceptable for 11 analytes in rat plasma. Subsequently, rats were orally administered with 50% ethanol eluent of CCT (ECCT). Nine of 11 polyphenols were absorbed quickly (except for QCD and TCA), and their plasma levels peaked within 40 min. The exposure and Cmax values of flavonoid glycosides and phenolic acids were lower than those of flavonoid aglycones. This is the first report to demonstrate the pharmacokinetics of 11 polyphenols in ECCT, which may play an important role in future studies of the bioactive components of ECCT and their bioactive mechanisms.

Keywords:  Coreopsis tinctoria; LC-MS/MS; biological sample analysis; multi-component pharmacokinetics; polyphenols

DOI:  https://doi.org/10.1002/cbdv.202200574
Methods Mol Biol. 2023 ;2561 245-259

Mass Spectrometry for the Advancement of Lipid Analysis in Alzheimer's Research.

Jonatan Martínez-Gardeazabal, Marta Moreno-Rodríguez, Estíbaliz González de San Román, Beatriz Abad, Iván Manuel, Rafael Rodríguez-Puertas.

  Recent technical advances in mass spectrometry, as applied to the analytical chemistry of lipid molecules, enable the simultaneous detection of the multiplicity of lipid complex species present in the human brain. This, in combination with quantitative studies carried out in plasma samples, helps to identify disease biomarkers including for Alzheimer's disease (AD). Mass spectrometry imaging (MSI) is particularly powerful for the anatomical localization of lipids in brain slices, identifying lipid modifications in postmortem frozen samples from AD patients.Human brain tissues are sectioned in a cryostat and then covered with a chemical matrix, such as mercaptobenzothiazole (MBT) or α-cyano-4-hydroxycinnamic acid (CHCA), to ionize the lipid molecules either by sublimation or by spraying. We describe the use of matrix-assisted laser desorption ionization (MALDI) in an LTQ-Orbitrap-XL mass spectrometer to scan brain tissue slices with high spatial resolution, analyzing 50 μm cell layers. The lipid spectra obtained for each pixel are transformed to color-coded intensity maps of hundreds of lipid species included those within a single tissue slice.

Keywords:  Alzheimer’s disease; Biomarkers; Lipid; MALDI-MSI; Mass spectroscopy; Neurolipid; UHPLC-MS

DOI:  https://doi.org/10.1007/978-1-0716-2655-9_13
NMR Biomed. 2022 Nov 13. e4865

Visualizing metabolomics data with R.

Yannick Berker, Isabella H Muti, Leo L Cheng.

  In communicating scientific results, convincing data visualization is of utmost importance. Especially in metabolomics, results based on large numbers of dimensions and variables necessitate particular attention in order to convey their message unambiguously to the reader; and in the era of open science, traceability and reproducibility are becoming increasingly important. This paper describes the use of the R programming language to visualize published metabolomics data resulting from ex-vivo NMR spectroscopy and mass spectrometry experiments with a special focus on reproducibility, including example figures as well as associated R code for ease of reuse. Examples include various types of plots (bar plots, swarm plots, and violin plots; volcano plots, heatmaps, Euler diagrams, Kaplan-Meier survival plots) and annotations (groupings, intra-group line connections, significance brackets, text annotations). Advantages of code-generated plots as well as advanced techniques and best practices are discussed.

Keywords:  Metabolomics; R (Word count: ≈ 2750); data visualization

DOI:  https://doi.org/10.1002/nbm.4865
J Mass Spectrom Adv Clin Lab. 2022 Nov;26 28-33

A distributable LC-MS/MS method for the measurement of serum thyroglobulin.

Junyan Shi, William S Phipps, Benjamin Y Owusu, Clark M Henderson, Thomas J Laha, Jessica O Becker, Morteza Razavi, Michelle A Emrick, Andrew N Hoofnagle.

   Background: Despite its clear advantages over immunoassay-based testing, the measurement of serum thyroglobulin by mass spectrometry remains limited to a handful of institutions. Slow adoption by clinical laboratories could reflect limited accessibility to existing methods that have sensitivity comparable to modern immunoassays, as well as a lack of tools for calibration and assay harmonization.
Methods: We developed and validated a liquid chromatography-tandem mass spectrometry-based assay for the quantification of serum thyroglobulin. The protocol combined peptide immunoaffinity purification using a commercially available, well-characterized monoclonal antibody and mobile phase modification with dimethylsulfoxide (DMSO) for enhanced sensitivity. To facilitate harmonization with other laboratories, we developed a novel, serum-based 5-point distributable reference material (Husky Ref).
Results: The assay demonstrated a lower limit of quantification of 0.15 ng/mL (<20 %CV). Mobile phase DMSO increased signal intensity of the target peptide at least 3-fold, improving quantification at low concentrations. Calibration traceable to Husky Ref enabled harmonization between laboratories in an interlaboratory study.
Conclusions: Sensitive mass spectrometry-based thyroglobulin measurement can be achieved using a monoclonal antibody during peptide immunoaffinity purification and the addition of mobile phase DMSO. Laboratories interested in deploying this assay can utilize the provided standard operating procedure and freely-available Husky Ref reference material.

Keywords:  Autoantibody; CPTAC, Clinical Proteomic Tumor Analysis Consortium; DMSO, dimethylsulfoxide; DTC, differentiated thyroid cancer; Differentiated thyroid cancer; IAE, immunoaffinity enrichment; LC-MS/MS, liquid chromatography-tandem mass spectrometry; Mass spectrometry; TLCK, tosyllysine chloromethyl ketone hydrochloride; Tg, thyroglobulin; Thyroglobulin

DOI:  https://doi.org/10.1016/j.jmsacl.2022.09.005
Biomed Chromatogr. 2022 Nov 17. e5548

Validated LC-MS/MS method for the determination of copanlisib in mouse dried blood spots.

Harsha K Tripathy, Vinay Kiran, Ashok Zakkula, A B Vinod, Rama Murthy Bestha, Sreekanth Dittakavi, Ramesh Mullangi.

  Copanlisib is a dual PI3K-δ inhibitor, being used in follicular lymphoma treatment. In this research, we report a validated LC-MS/MS method for quantifying copanlisib from mouse dried blood spot (DBS). We validated the method in-line with the FDA. Liquid-liquid extraction technique was used to extract copanlisib from the DBS discs. We used an Atlantis dC18 column and isocratic mobile phase for the chromatographic separation of copanlisib and the internal standard (idelalisib). The flow was 0.90 mL/min. In the optimized chromatographic conditions, the retention of copanlisib and the internal standard was 0.98 and 0.93 min, respectively. Each injection total run time was 2.50 min. The MS/MS ion transitions monitored were m/z 481.31→128.00 and 416.10→176.10 for copanlisib and the internal standard (IS) idelalisib, respectively. We have used a broad calibration range (1.01-4797 ng/mL) with a determination coefficient (r2 ) of 0.997. All the evaluated parameters met the acceptance criteria. Haematocrit did not influence DBS copanlisib concentrations. We have used the validated method to derive the intravenous pharmacokinetic parameters by quantifying copanlisib in mouse plasma.

Keywords:  Copanlisib; DBS; LC-MS/MS; method validation; mouse blood; pharmacokinetics

DOI:  https://doi.org/10.1002/bmc.5548
J Med Biochem. 2022 Oct 15. 41(4): 466-473

Development and validation of an LC-MS/MS method for determination of 8-iso-prostaglandin f2 Alpha in human saliva.

Zlatina Tomova, Desislav Tomov, Angelina Vlahova, Veneta Chaova-Gizdakova, Lyubka Yoanidu, Dobrin Svinarov.

   Background: Increased formation of reactive oxygen species may be caused by the ion release of the metal alloys used in prosthetic dental restorations due to the corrosion process. As products of lipid peroxidation, isoprostanes can be used as a marker for oxidative stress in the body. There are two significant advantages of using isoprostanes as an oxidative stress marker - presence in all fluids in the body and low reactivity. Saliva provides noninvasive, painless, and cost-effective sample collection and can be used as an alternative testing medium of blood and urine.
Methods: This study presents the development and validation of a sample LC-MS/MS method to quantify 8-isoprostaglandin F2-a in human saliva using salt-out assisted liquid-liquid extraction (SALLE).
Results: The selected sample preparation procedure optimized chromatographic separation and mass detection provided high recovery and sensitivity of the analysis. The calibration curve was obtained in the predefined range 25-329 ng/L with R2 larger than 0.995. Normalized matrix varied between 89.7 % and 113.5%. The method showed sufficient accuracy and precision - accuracy in the range 89.7 %-113.9 %, and precision between 2.3% and 5.4%.
Conclusions: The proposed method is validated according to current EMA/FDA industrial guidance for bioanalysis and offers an appropriate level of sensitivity and sufficient accuracy and precision.

Keywords:  8-isoPGF2a; LC-MS/MS; SALLE; metal alloy; saliva

DOI:  https://doi.org/10.5937/jomb0-33556
Anal Chem. 2022 Nov 14.

Leveraging the HMBC to Facilitate Metabolite Identification.

Fatema Bhinderwala, Thao Vu, Thomas G Smith, Julian Kosacki, Darrell D Marshall, Yuhang Xu, Martha Morton, Robert Powers.

The accuracy and ease of metabolite assignments from a complex mixture are expected to be facilitated by employing a multispectral approach. The two-dimensional (2D) 1H-13C heteronuclear single quantum coherence (HSQC) and 2D 1H-1H-total correlation spectroscopy (TOCSY) are the experiments commonly used for metabolite assignments. The 2D 1H-13C HSQC-TOCSY and 2D 1H-13C heteronuclear multiple-bond correlation (HMBC) are routinely used by natural products chemists but have seen minimal usage in metabolomics despite the unique information, the nearly complete 1H-1H and 1H-13C and spin systems provided by these experiments that may improve the accuracy and reliability of metabolite assignments. The use of a 13C-labeled feedstock such as glucose is a routine practice in metabolomics to improve sensitivity and to emphasize the detection of specific metabolites but causes severe artifacts and an increase in spectral complexity in the HMBC experiment. To address this issue, the standard HMBC pulse sequence was modified to include carbon decoupling. Nonuniform sampling was also employed for rapid data collection. A dataset of reference 2D 1H-13C HMBC spectra was collected for 94 common metabolites. 13C-13C spin connectivity was then obtained by generating a covariance pseudo-spectrum from the carbon-decoupled HMBC and the 1H-13C HSQC-TOCSY spectra. The resulting 13C-13C pseudo-spectrum provides a connectivity map of the entire carbon backbone that uniquely describes each metabolite and would enable automated metabolite identification.

DOI: https://doi.org/10.1021/acs.analchem.2c02902
Front Chem. 2022 ;10 1037542

Direct adsorption sampling and ambient mass spectrometry analysis of tobacco smoke with porous paper strips.

Ji Yang, Wen Xiong, Chunbo Liu, Juan Li, Ruizhi Zhu, Jianjun Xia, Zhijiang Yin, Ran Tian, Shiyun Tang, Zhenjie Li, Hui Li, Ying Han, Xiaoxi Si, Wei Jiang, Pei He, Fengmei Zhang, Yanqin Xu, Zhihua Liu.

  Chemical analysis of atmospheric aerosols by conventional analytical methods is usually required to perform complicated and time-consuming sample preparation processes. In recent decades, ambient ionization mass spectrometry (AI-MS) methods have been proven to be simple, rapid, and effective analytical tools for direct analysis of various complex samples. In this work, we applied porous paper filters for direct adsorptive sampling of tobacco smoke, and then the sampled paper filters were performed the emitters of the paper spray ionization (PSI) device. An auto-sampling device was made to control the generation and collection of tobacco smoke. Nicotine, the typical compound of tobacco smoke, was used to optimize the key conditions of auto-sampling. Moreover, different types of tobacco smoke were also compared with multivariate variable analysis, and the makers of tobacco smoke from different sources of tobacco smoke were investigated. By using this method, direct sampling and analysis of a single tobacco sample can be completed within minutes. Overall, our results show that PSI-MS is a powerful tool that integrates collection, extraction, ionization, and identification analytes in smoke.

Keywords:  ambient mass spectrometry; paper spray; paper strip; sampling; tobacco smoke

DOI:  https://doi.org/10.3389/fchem.2022.1037542