bims-metlip 2024-02-11 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2024–02–11
23 papers selected by
Sofia Costa, Matterworks

Development and validation of a simple, fast and sensitive liquid chromatography-tandem mass spectrometry method to establish reference intervals for 24-h urinary free normetanephrine, metanephrine and methoxytyramine.
Sample preparation for fatty acid analysis in biological samples with mass spectrometry-based strategies.
Validation of a LC-MS/MS method for establishing reference intervals and assessing diagnostic performance of urinary free cortisol and cortisone.
Lipid Isobar and Isomer Imaging Using Nanospray Desorption Electrospray Ionization Combined with Triple Quadrupole Mass Spectrometry.
Lipidome isotope labelling of gut microbes (LILGM): A method of discovering endogenous microbial lipids.
AutonoMS: Automated Ion Mobility Metabolomic Fingerprinting.
A liquid chromatographic-mass spectrometric procedure for analysis of pentaerythrityl tetranitrate metabolites - Development, validation and application to ovine serum and human plasma samples.
[Determination of triclocarban and triclosan in urine by QuEChERS extraction and ultra-performance liquid chromatography-tandem mass spectrometry].
Separation of Lipoproteins for Quantitative Analysis of 14C-Labeled Lipid-Soluble Compounds by Accelerator Mass Spectrometry.
Resolution and quantification of carbohydrates by enantioselective comprehensive two-dimensional gas chromatography.
MRMPro: a web-based tool to improve the speed of manual calibration for multiple reaction monitoring data analysis by mass spectrometry.
Integrative open workflow for confident annotation and molecular networking of metabolomics MSE/DIA data.
Bioassay-Driven, Fractionation-Empowered, Focused Metabolomics for Discovering Bacterial Activators of Aryl Hydrocarbon Receptor.
Comparing the Extraction Performance in Mouse Plasma of Different Biphasic Methods for Polar and Nonpolar Compounds.
Computer-assisted multifactorial method development for the streamlined separation and analysis of multicomponent mixtures in (Bio)pharmaceutical settings.
Quantification of derivatized phenylalanine and tyrosine in dried blood spots using liquid chromatography with tandem spectrometry for newborn screening of phenylketonuria.
Total serum pentosidine quantification using liquid chromatography-tandem mass spectrometry.
Pharmacokinetics of Anti-Rheumatic drugs Methotrexate and Tofacitinib with its metabolite M9 in Rats by UPLC-MS/MS.
microbeMASST: a taxonomically informed mass spectrometry search tool for microbial metabolomics data.
Identification of oxygenated triacylglycerols in pistachio nuts' lipids by reverse-phase high-resolution-liquid chromatography-ESI mass spectrometry.
Mass spectrometry imaging methods for visualizing tumor heterogeneity.
Development of a Two-Dimensional Liquid Chromatographic Method for Analysis of Urea Cycle Amino Acids.
Liquid Chromatography Tandem Mass Spectrometric Method for Quantification of Margetuximab in Rat Plasma and Application to a Pharmacokinetic Study.

Pract Lab Med. 2024 Mar;39 e00358

Development and validation of a simple, fast and sensitive liquid chromatography-tandem mass spectrometry method to establish reference intervals for 24-h urinary free normetanephrine, metanephrine and methoxytyramine.

Yan Song, Runhao Xu, Dan Liu, Jie Zhang.

   Objective: To develop and validate a rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to detect urinary free metanephrines and methoxytyramine, establishing reference intervals.
Methods: Urine samples were diluted with isotope internal standard solution, then analyzed directly using tandem mass spectrometry with multiple reaction monitoring measurement and electrospray ionization source in positive ion mode. Analytical parameters including linearity, lower limit of quantitation, imprecision and accuracy of the method were evaluated. The reference intervals for urinary catecholamine metabolites were established by analyzing 24-h urine samples collected from 81 apparently healthy volunteers.
Results: The analytical times for MN, NMN, and 3-MT were at 2.79, 2.80, and 2.74 min, respectively. The method displayed excellent linearity (r > 0.99) in the range of 1-1000 ng/mL, with lower limits of quantification (LLOQ) at 0.50 ng/mL for MN and NMN, and 0.25 ng/mL for 3-MT. The method's intra-day and inter-day imprecisions were less than 8 %. The method recovery ranged from 96.8% to 105.8 % for MN, 89.7%-106.4 % for NMN, and 93.5%-106.2 % for 3-MT. No carry-over was observed during the analysis of all analytes. The LC-MS/MS method was used to establish reference intervals in 24-h urine samples from 81 apparently healthy volunteers. There was no association of sex with urinary free metabolites.
Conclusion: This study established a novel, fast and sensitive LC-MS/MS method for determining urinary free catecholamine metabolites, which could facilitate screening and diagnosis for catecholamine-related tumors more conveniently and quickly.

Keywords:  Catecholamine metabolites; Liquid chromatography-tandem mass spectrometry; Urine; Validation

DOI:  https://doi.org/10.1016/j.plabm.2024.e00358
Anal Bioanal Chem. 2024 Feb 06.

Sample preparation for fatty acid analysis in biological samples with mass spectrometry-based strategies.

Li Yang, Jie Yuan, Bolin Yu, Shuang Hu, Yu Bai.

  Fatty acids (FAs) have attracted many interests for their pivotal roles in many biological processes. Imbalance of FAs is related to a variety of diseases, which makes the measurement of them important in biological samples. Over the past two decades, mass spectrometry (MS) has become an indispensable technique for the analysis of FAs owing to its high sensitivity and precision. Due to complex matrix effect of biological samples and inherent poor ionization efficiency of FAs in MS, sample preparation including extraction and chemical derivatization prior to analysis are often employed. Here, we describe an updated overview of FA extraction techniques, as well as representative derivatization methods utilized in different MS platforms including gas chromatography-MS, liquid chromatography-MS, and mass spectrometry imaging based on different chain lengths of FAs. Derivatization strategies for the identification of double bond location in unsaturated FAs are also summarized and highlighted. The advantages, disadvantages, and prospects of these methods are compared and discussed. This review provides the development and valuable information for sample pretreatment approaches and qualitative and quantitative analysis of interested FAs using different MS-based platforms in complex biological matrices. Finally, the challenges of FA analysis are summarized and the future perspectives are prospected.

Keywords:  Derivatization; Extraction; Fatty acids; Mass spectrometry

DOI:  https://doi.org/10.1007/s00216-024-05185-0
Steroids. 2024 Feb 01. pii: S0039-128X(24)00016-3. [Epub ahead of print] 109378

Validation of a LC-MS/MS method for establishing reference intervals and assessing diagnostic performance of urinary free cortisol and cortisone.

Yan Song, Juan Zhao, Dan Liu, Jie Zhang.

   BACKGROUND: 24-h urinary free cortisol (UFF) is recommended for screening of Cushing's syndrome (CS), a rare disease characterized by apparent cortisol and cortisone excess. We aimed to validate a simple LC-MS/MS method for accurate measurement of UFF and urinary free cortisone (UFE), establishment of reference ranges, and evaluation of performance for CS diagnosis.
METHODS: Urine samples were processed using solid-phase extraction cartridges, followed by elution with methanol and acetonitrile. Analysis was performed via tandem mass spectrometry, utilizing multiple reaction monitoring and electrospray ionization source in positive ion mode.
RESULTS: The assay displayed excellent linearity (r > 0.99) in the range of 0.05-100 ng/mL for cortisol and 0.25-500 ng/mL for cortisone, with lower limits of quantification (LLOQ) at 0.05 ng/mL for cortisol and 0.25 ng/mL for cortisone. The obtained results for intra-day and inter-day imprecision for both analytes were within the acceptable range of less than 10%. The trueness values for both compounds were also within the acceptable limit of 15%. No significant matrix effects or carry over observed in our method. The reference intervals of UFF, UFE and UFF:UFE ratio were 7.01-45.66 µg/24-h, 27.97-139.21 µg/24-h and 0.17-0.56, respectively. UFF > 56.75 µg/24-h showed 100% specificity and 100% sensitivity for CS diagnosis, which was superior to UFF:UFE ratio.
CONCLUSIONS: We developed and validated a sensitive LC-MS/MS method to detect UFF and UFE. Our data indicate that UFF measured by the current LC-MS/MS assay exhibited high diagnostic performance for CS.

Keywords:  Cortisol; Cortisone; Cushing syndrome; Liquid chromatography-tandem mass spectrometry; Ratio

DOI:  https://doi.org/10.1016/j.steroids.2024.109378
Anal Chem. 2024 Feb 06.

Lipid Isobar and Isomer Imaging Using Nanospray Desorption Electrospray Ionization Combined with Triple Quadrupole Mass Spectrometry.

Miranda R Weigand, Daisy M Unsihuay Vila, Manxi Yang, Hang Hu, Emerson Hernly, Matthew Muhoberac, Shane Tichy, Julia Laskin.

Mass spectrometry imaging (MSI) is widely used for examining the spatial distributions of molecules in biological samples. Conventional MSI approaches, in which molecules extracted from the sample are distinguished based on their mass-to-charge ratio, cannot distinguish between isomeric species and some closely spaced isobars. To facilitate isobar separation, MSI is typically performed using high-resolution mass spectrometers. Nevertheless, the complexity of the mixture of biomolecules observed in each pixel of the image presents a challenge, even for modern mass spectrometers with the highest resolving power. Herein, we implement nanospray desorption electrospray ionization (nano-DESI) MSI on a triple quadrupole (QqQ) mass spectrometer for the spatial mapping of isobaric and isomeric species in biological tissues. We use multiple reaction monitoring acquisition mode (MRM) with unit mass resolution to demonstrate the performance of this new platform by imaging lipids in mouse brain and rat kidney tissues. We demonstrate that imaging in MRM mode may be used to distinguish between isobaric phospholipids requiring a mass resolving power of 3,800,000. Additionally, we have been able to image eicosanoid isomers, a largely unexplored class of signaling molecules present in tissues at low concentrations, in rat kidney tissue. This new capability substantially enhances the specificity and selectivity of MSI, enabling spatial localization of species that remain unresolved in conventional MSI experiments.

DOI: https://doi.org/10.1021/acs.analchem.3c04705
Talanta. 2024 Feb 01. pii: S0039-9140(24)00109-7. [Epub ahead of print]271 125730

Lipidome isotope labelling of gut microbes (LILGM): A method of discovering endogenous microbial lipids.

Li Chen, Rui Xu, Jiangjiang Zhu.

  Lipidomics analysis of gut microbiome has become critical in recent surge of extensive human disease studies that investigate microbiome contributions. However, challenges remain in comprehending the origins of thousands of lipid species produced by the diverse microbes. Here, we proposed the development and utilization of a liquid chromatography-mass spectrometry-based approach, named lipidome isotope labelling of gut microbes (LILGM), which enables confident detection and identification of endogenous gut microbial lipidome via 13C/15N labeling strategy and high-resolution mass spectrometry. Our method leveraged in vitro microbial cultures and stable isotope-labeled 13C and 15N, allowing a reasonable degree of isotope incorporation into microbial lipids over short-term of inoculation. We then systematically detected the mass spectral patterns of 182 labeled lipid species by our in-house data analysis pipeline. Further bioinformatics analyses confidently identified biologically relevant microbial lipids from lipid classes such as diacylglycerols (DGs), fatty acids (FAs), phosphatidylglycerols (PGs), and phosphatidylethanolamines (PEs) that may have profound impacts to human physiology. Our study also demonstrated the application of LILGM by showcasing the confident detection of dysregulated microbial lipids post antibiotic perturbation. The debiased sparse partial correlation analysis provides insights into lipid metabolism intricacies. Overall, our method can provide unambiguous analyses to the endogenous microbial lipids in given biological context, and can also instantly reflect the lipidomic changes of gut microbes in response to environmental factors. We believe our LILGM approach has the potential to provide new body of knowledge by combining promising analytical approaches for sensitive and specific lipid detection to support functional microbiome studies.

Keywords:  Anaerobic microbes; Endogenous metabolites; Isotope labeling; Lipidomics; Metabolism

DOI:  https://doi.org/10.1016/j.talanta.2024.125730
J Am Soc Mass Spectrom. 2024 Feb 04.

AutonoMS: Automated Ion Mobility Metabolomic Fingerprinting.

Gabriel K Reder, Erik Y Bjurström, Daniel Brunnsåker, Filip Kronström, Praphapan Lasin, Ievgeniia Tiukova, Otto I Savolainen, James N Dodds, Jody C May, John P Wikswo, John A McLean, Ross D King.

Automation is dramatically changing the nature of laboratory life science. Robotic lab hardware that can perform manual operations with greater speed, endurance, and reproducibility opens an avenue for faster scientific discovery with less time spent on laborious repetitive tasks. A major bottleneck remains in integrating cutting-edge laboratory equipment into automated workflows, notably specialized analytical equipment, which is designed for human usage. Here we present AutonoMS, a platform for automatically running, processing, and analyzing high-throughput mass spectrometry experiments. AutonoMS is currently written around an ion mobility mass spectrometry (IM-MS) platform and can be adapted to additional analytical instruments and data processing flows. AutonoMS enables automated software agent-controlled end-to-end measurement and analysis runs from experimental specification files that can be produced by human users or upstream software processes. We demonstrate the use and abilities of AutonoMS in a high-throughput flow-injection ion mobility configuration with 5 s sample analysis time, processing robotically prepared chemical standards and cultured yeast samples in targeted and untargeted metabolomics applications. The platform exhibited consistency, reliability, and ease of use while eliminating the need for human intervention in the process of sample injection, data processing, and analysis. The platform paves the way toward a more fully automated mass spectrometry analysis and ultimately closed-loop laboratory workflows involving automated experimentation and analysis coupled to AI-driven experimentation utilizing cutting-edge analytical instrumentation. AutonoMS documentation is available at https://autonoms.readthedocs.io.

DOI: https://doi.org/10.1021/jasms.3c00396
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Jan 24. pii: S1570-0232(24)00036-9. [Epub ahead of print]1234 124028

A liquid chromatographic-mass spectrometric procedure for analysis of pentaerythrityl tetranitrate metabolites - Development, validation and application to ovine serum and human plasma samples.

Mariann Städtler, Daniela Wissenbach, Dirk K Wissenbach, Laura Franke, Jana Pastuschek, Ekkehard Schleussner, Beth Allison, Frank T Peters, Tanja Groten.

  Pentaerythrityl tetranitrate (PETN) is an established drug in the treatment of coronary heart disease and heart failure. It is assumed, that the vasodilative and vasoprotective effects of PETN also have a positive impact on pregnant patients with impaired placental perfusion and studies evaluating the effect of PETN in risk pregnancies have been carried out. In the context of these clinical trials, measuring of serum levels of PETN and its metabolites pentaerythrityl trinitrate (PETriN), pentaerythrityl dinitrate (PEDN), pentaerythrityl mononitrate (PEMN) and pentaerythritol (PE) were required. To evaluate the transfer of PETN and its metabolites (PEXN) from the mother to the fetus using samples from a human clinical trial and animal study, the present work aimed to develop a rapid and simple method to simultaneously analyze PEXN in human and ovine samples. A method employing a rapid and simple liquid-liquid extraction followed by reversed-phase (C18) liquid chromatography coupled to high-resolution mass spectrometry with negative electrospray ionization was developed and validated for the detection of PETN and PEXN in human and ovine samples. PE could only be qualitatively detected at higher concenrations. Method validation requirements, including accuracy, repeatability and intermediate precision were fulfilled in ovine and human samples for all other PEXN with exception PETriN in human samples. The recovery (RE) in ovine samples was 76.7 % ± 12 % for PEMN, 98 % ± 23 % for PEDN, 94 % ± 22 % for PETriN, in human samples RE was 59 % ± 16 % for PEMN, 67 % ± 19 % for PEDN, 71 % ± 17 %. The matrix effects (ME) in ovine samples were 90 % ± 11 % for PEMN, 70 % ± 30 % for PEDN, 107 % ± 17 % for PETriN, in human samples the ME were 93 % ± 13 % for PEMN, 84 % ± 17 % for PEDN, 98 % ± 16 % for PETriN. The limits of quantification (LOQ) in ovine samples were 1.0 ng/mL for PETriN and 0.1 ng/mL for PEMN and PEDN. The LOQs in human samples were 5.0 ng/mL for PETriN and 0.3 ng/mL for PEMN und PEDN. The newly developed method was used to analyze 184 ovine serum samples and 18 human plasma samples. In ovine maternal samples, the highest observed PEDN concentration was 3.5 ng/mL and the highest PEMN concentration was 10 ng/mL, the respective concentrations in fetal serum samples were 4.9 ng/mL for PEDN and 5.4 ng/mL for PEMN. PETriN was only detected in traces in maternal and fetal samples, whereas PETN could not be detected at all. In human maternal samples, the highest concentration for PEDN was 27 ng/mL and for PEMN 150 ng/mL. In umbilical cord plasma, concentrations of 2.3 ng/mL for PEDN and 73 ng/mL for PEMN were detected. Although the PEMN and PEDN concentrations in the human samples were several times higher than in ovine samples, neither PETN nor PETriN signals could be detected. These results demonstrated that the metabolites were transferred from mother to fetus with a slight time delay.

Keywords:  Fetal growth retardation; Human; Liquid chromatography- high resolution mass spectrometry; Ovine; Pentaerythrityl tetranitrate; Pregnant

DOI:  https://doi.org/10.1016/j.jchromb.2024.124028
Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi. 2024 Jan 20. 42(1): 46-49

[Determination of triclocarban and triclosan in urine by QuEChERS extraction and ultra-performance liquid chromatography-tandem mass spectrometry].

Q L Qiu, X H Chen, S S Yao, X P Yao, L Y Fang, M C Jin.

  Objective: To establish a method for the determination of triclocarban (TCC) and triclosan (TCS) in urine by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) after purification by QuEChERS. Methods: In May 2022, urine samples were extracted by acetonitrile, purified by QuEChERS, separated by Waters Acquity UPLC BEH C18 column (100 mm×2.1 mm, 1.7 μm), and eluated with water-acetonitrile as mobile phase gradient at a flow rate of 0.3 ml/min. The detection was conducted in negative ion mode (ESI(-)) and multiple reaction monitoring (MRM) scanning, it was quantified with a internal standard method, and the methodology was verified. Results: The linear ranges of TCC and TCS were 0.5-100.0 μg/L and 1.0-100.0 μg/L, and the correlation coefficients were 0.9997 and 0.9991, respectively. The limits of detection and quantitation of TCC and TCS were 0.17 and 0.33 μg/L, and 0.5 and 1.0 μg/L, respectively. The recoveries of TCC and TCS were 100.1%-102.8% and 96.7%-108.6%, and the relative standard deviations were 4.9%-6.7% and 4.1%-8.3%, respectively, at 2.0, 10.0 and 80.0 μg/L. Conclusion: QuEChERS-UPLC-MS/MS method is simple, rapid, sensitive and reproducible, and can be used for rapid and accurate simultaneous detection of TCC and TCS exposure levels in occupational population.

Keywords:  QuEChERS; Tandem mass spectrometry; Triclocarban; Triclosan; Ultra-performance liquid chromatography; Urine

DOI:  https://doi.org/10.3760/cma.j.cn121094-20220913-00452
Int J Mol Sci. 2024 Feb 03. pii: 1856. [Epub ahead of print]25(3):

Separation of Lipoproteins for Quantitative Analysis of 14C-Labeled Lipid-Soluble Compounds by Accelerator Mass Spectrometry.

Jennifer C Chuang, Andrew J Clifford, Seung-Hyun Kim, Janet A Novotny, Peter B Kelly, Dirk M Holstege, Rosemary L Walzem.

  To date, 14C tracer studies using accelerator mass spectrometry (AMS) have not yet resolved lipid-soluble analytes into individual lipoprotein density subclasses. The objective of this work was to develop a reliable method for lipoprotein separation and quantitative recovery for biokinetic modeling purposes. The novel method developed provides the means for use of small volumes (10-200 µL) of frozen plasma as a starting material for continuous isopycnic lipoprotein separation within a carbon- and pH-stable analyte matrix, which, following post-separation fraction clean up, created samples suitable for highly accurate 14C/12C isotope ratio determinations by AMS. Manual aspiration achieved 99.2 ± 0.41% recovery of [5-14CH3]-(2R, 4'R, 8'R)-α-tocopherol contained within 25 µL plasma recovered in triacylglycerol rich lipoproteins (TRL = Chylomicrons + VLDL), LDL, HDL, and infranatant (INF) from each of 10 different sampling times for one male and one female subject, n = 20 total samples. Small sample volumes of previously frozen plasma and high analyte recoveries make this an attractive method for AMS studies using newer, smaller footprint AMS equipment to develop genuine tracer analyses of lipophilic nutrients or compounds in all human age ranges.

Keywords:  accelerator mass spectrometry; fluorescent imaging; isopycnic centrifugation; lipoproteins; α-tocopherol tracer analysis

DOI:  https://doi.org/10.3390/ijms25031856
Talanta. 2024 Feb 01. pii: S0039-9140(24)00107-3. [Epub ahead of print]271 125728

Resolution and quantification of carbohydrates by enantioselective comprehensive two-dimensional gas chromatography.

Adrien D Garcia, Vanessa Leyva, Jana Bocková, Raphaël L Pepino, Cornelia Meinert.

  Carbohydrates, in particular the d-enantiomers of ribose, 2-deoxyribose, and glucose, are essential to life's informational biopolymers (RNA/DNA) and for supplying energy to living cells through glycolysis. Considered to be potential biosignatures in the search of past or present life, our capacity to detect and quantify these essential sugars is crucial for future space missions to the Moon, Mars or Titan as well as for sample-return missions. However, the enantioselective analysis of carbohydrates is challenging and both research and routine applications, are lacking efficient methods that combine highly sensitive and reproducible detection with baseline enantioselective resolution and reliable enantiomeric excess (ee) measurements. Here, we present four different derivatization strategies in combination with multidimensional gas chromatography coupled to a reflectron time-of-flight mass spectrometer (GC×GC-TOF-MS) for the enantioselective resolution of C3 to C6 carbohydrates potentially suitable for sample-return analyses. Full mass spectral interpretation and calibration curves for one single-step (cyclic boronate derivatives) and three two-step derivatization protocols (aldononitrile-acetate, hemiacetalization-trifluoroacetylation, and hemiacetalization-permethylation) are presented for concentrations ranging from 1 to 50 pmol μL⁻1 with correlation coefficients R2 > 0.94. We compared several analytical parameters including reproducibility, sensitivity (LOD and LOQ), overall separation, chiral resolution (RS), mass spectrum selectivity, stability during long term storage, and reliability of ee measurements to guide the application-dependent selection of optimal separation and quantification performance.

Keywords:  Biosignature; Chirality; Enantioseparation; Sugars; Two-dimensional gas chromatography

DOI:  https://doi.org/10.1016/j.talanta.2024.125728
BMC Bioinformatics. 2024 Feb 06. 25(1): 60

MRMPro: a web-based tool to improve the speed of manual calibration for multiple reaction monitoring data analysis by mass spectrometry.

Ruimin Wang, Hengxuan Jiang, Miaoshan Lu, Junjie Tong, Shaowei An, Jinyin Wang, Changbin Yu.

   BACKGROUND: As a gold-standard quantitative technique based on mass spectrometry, multiple reaction monitoring (MRM) has been widely used in proteomics and metabolomics. In the analysis of MRM data, as no peak picking algorithm can achieve perfect accuracy, manual inspection is necessary to correct the errors. In large cohort analysis scenarios, the time required for manual inspection is often considerable. Apart from the commercial software that comes with mass spectrometers, the open-source and free software Skyline is the most popular software for quantitative omics. However, this software is not optimized for manual inspection of hundreds of samples, the interactive experience also needs to be improved.
RESULTS: Here we introduce MRMPro, a web-based MRM data analysis platform for efficient manual inspection. MRMPro supports data analysis of MRM and schedule MRM data acquired by mass spectrometers of mainstream vendors. With the goal of improving the speed of manual inspection, we implemented a collaborative review system based on cloud architecture, allowing multiple users to review through browsers. To reduce bandwidth usage and improve data retrieval speed, we proposed a MRM data compression algorithm, which reduced data volume by more than 60% and 80% respectively compared to vendor and mzML format. To improve the efficiency of manual inspection, we proposed a retention time drift estimation algorithm based on similarity of chromatograms. The estimated retention time drifts were then used for peak alignment and automatic EIC grouping. Compared with Skyline, MRMPro has higher quantification accuracy and better manual inspection support.
CONCLUSIONS: In this study, we proposed MRMPro to improve the usability of manual calibration for MRM data analysis. MRMPro is free for non-commercial use. Researchers can access MRMPro through http://mrmpro.csibio.com/ . All major mass spectrometry formats (wiff, raw, mzML, etc.) can be analyzed on the platform. The final identification results can be exported to a common.xlsx format for subsequent analysis.

Keywords:  Batch inspection; MRM; MRMPro; Mass spectrometry; Web service

DOI:  https://doi.org/10.1186/s12859-024-05685-x
Brief Bioinform. 2024 Jan 22. pii: bbae013. [Epub ahead of print]25(2):

Integrative open workflow for confident annotation and molecular networking of metabolomics MSE/DIA data.

Albert Katchborian-Neto, Matheus F Alves, Paula C P Bueno, Karen de Jesus Nicácio, Miller S Ferreira, Tiago B Oliveira, Henrique Barbosa, Michael Murgu, Ana C C de Paula Ladvocat, Danielle F Dias, Marisi G Soares, João H G Lago, Daniela A Chagas-Paula.

  Liquid chromatography coupled with high-resolution mass spectrometry data-independent acquisition (LC-HRMS/DIA), including MSE, enable comprehensive metabolomics analyses though they pose challenges for data processing with automatic annotation and molecular networking (MN) implementation. This motivated the present proposal, in which we introduce DIA-IntOpenStream, a new integrated workflow combining open-source software to streamline MSE data handling. It provides 'in-house' custom database construction, allows the conversion of raw MSE data to a universal format (.mzML) and leverages open software (MZmine 3 and MS-DIAL) all advantages for confident annotation and effective MN data interpretation. This pipeline significantly enhances the accessibility, reliability and reproducibility of complex MSE/DIA studies, overcoming previous limitations of proprietary software and non-universal MS data formats that restricted integrative analysis. We demonstrate the utility of DIA-IntOpenStream with two independent datasets: dataset 1 consists of new data from 60 plant extracts from the Ocotea genus; dataset 2 is a publicly available actinobacterial extract spiked with authentic standard for detailed comparative analysis with existing methods. This user-friendly pipeline enables broader adoption of cutting-edge MS tools and provides value to the scientific community. Overall, it holds promise for speeding up metabolite discoveries toward a more collaborative and open environment for research.

Keywords:  Ocotea; chemical annotation; data-independent acquisition; mass spectrometry; open software

DOI:  https://doi.org/10.1093/bib/bbae013
J Am Soc Mass Spectrom. 2024 Feb 03.

Bioassay-Driven, Fractionation-Empowered, Focused Metabolomics for Discovering Bacterial Activators of Aryl Hydrocarbon Receptor.

Huidi Tian, Lei Wang, Rachel Hardy, Lina Kozhaya, Lindsey Placek, Elizabeth Fleming, Julia Oh, Derya Unutmaz, Xudong Yao.

Aryl hydrocarbon receptor (AhR) is a transcription factor that regulates gene expression upon ligand activation, enabling microbiota-dependent induction, training, and function of the host immune system. A spectrum of metabolites, encompassing indole and tryptophan derivatives, have been recognized as activators. This work introduces an integrated, mass spectrometry-centric workflow that employs a bioassay-guided, fractionation-based methodology for the identification of AhR activators derived from human bacterial isolates. By leveraging the workflow efficiency, the complexities inherent in metabolomics profiling are significantly reduced, paving the way for an in-depth and focused mass spectrometry analysis of bioactive fractions isolated from bacterial culture supernatants. Validation of AhR activator candidates used multiple criteria─MS/MS of the synthetic reference compound, bioassay of AhR activity, and elution time confirmation using a C-13 isotopic reference─and was demonstrated for N-formylkynurenine (NFK). The workflow reported provides a roadmap update for improved efficiency of identifying bioactive metabolites using mass spectrometry-based metabolomics. Mass spectrometry datasets are accessible at the National Metabolomics Data Repository (PR001479, Project DOI: 10.21228/M8JM7Q).

DOI: https://doi.org/10.1021/jasms.3c00386
J Proteome Res. 2024 Feb 06.

Comparing the Extraction Performance in Mouse Plasma of Different Biphasic Methods for Polar and Nonpolar Compounds.

Friederike Gutmann, Raphaela Fritsche-Guenther, Daniela B Dias, Jennifer A Kirwan.

  Many metabolomic studies are interested in both polar and nonpolar analyses. However, the available sample volume often precludes multiple separate extractions. Therefore, there are major advantages in performing a biphasic extraction and retaining both phases for subsequent separate analyses. To be successful, such approaches require the method to be robust and repeatable for both phases. Hence, we determined the performance of three extraction protocols, plus two variant versions, using 25 μL of commercially available mouse plasma. The preferred option for nonpolar lipids was a modified diluted version of a method employing methyl tert-butyl ether (MTBE) suggested by Matyash and colleagues due to its high repeatability for nonpolar compounds. For polar compounds, the Bligh-Dyer method performs best for sensitivity but with consequentially poorer lipid performance. Overall, the scaled-down version of the MTBE method gave the best overall performance, with high sensitivity for both polar and nonpolar compounds and good repeatability for polar compounds in particular.

Keywords:  biphasic extraction; central carbon metabolism; lipidomics; targeted methods; validation

DOI:  https://doi.org/10.1021/acs.jproteome.3c00596
Anal Chim Acta. 2024 Mar 08. pii: S0003-2670(23)01399-5. [Epub ahead of print]1293 342178

Computer-assisted multifactorial method development for the streamlined separation and analysis of multicomponent mixtures in (Bio)pharmaceutical settings.

Mohamed Hemida, Imad A Haidar Ahmad, Rodell C Barrientos, Erik L Regalado.

  The (bio)pharmaceutical industry is rapidly moving towards complex drug modalities that require a commensurate level of analytical enabling technologies that can be deployed at a fast pace. Unsystematic method development and unnecessary manual intervention remain a major barrier towards a more efficient deployment of meaningful analytical assay across emerging modalities. Digitalization and automation are key to streamline method development and enable rapid assay deployment. This review discusses the use of computer-assisted multifactorial chromatographic method development strategies for fast-paced downstream characterization and purification of biopharmaceuticals. Various chromatographic techniques such as reversed-phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC), ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), and supercritical fluid chromatography (SFC) are addressed and critically reviewed. The most significant parameters for retention mechanism modelling, as well as mapping the separation landscape for optimal chromatographic selectivity and resolution are also discussed. Furthermore, several computer-assisted approaches for optimization and development of chromatographic methods of therapeutics, including linear, nonlinear, and multifactorial modelling are outlined. Finally, the potential of the chromatographic modelling and computer-assisted optimization strategies are also illustrated, highlighting substantial productivity improvements, and cost savings while accelerating method development, deployment and transfer processes for therapeutic analysis in industrial settings.

Keywords:  (Bio)pharmaceuticals; Complex mixtures; Computer-assisted; Liquid chromatography; Method development; Optimization

DOI:  https://doi.org/10.1016/j.aca.2023.342178
Eur J Mass Spectrom (Chichester). 2024 Feb 06. 14690667241229626

Quantification of derivatized phenylalanine and tyrosine in dried blood spots using liquid chromatography with tandem spectrometry for newborn screening of phenylketonuria.

Tsai-Hui Duh, Yu-Ching Liang, Po Tsun Shen, Yi-Wen Ke, Yan-Tian Nian, Shih-Shin Liang.

  Phenylketonuria (PKU) is an autosomal genetic disorder caused by a deficiency of the phenylalanine hydroxylase (PAH) enzyme. The lack of PAH results in the inability of phenylalanine (PHE) to transform into tyrosine (TYR). Consequently, this leads to the accumulation of PHE in the blood samples of newborns causing metabolic diseases such as irreversible neurological problems. An analysis was required for determining the values of PHE and TYR in blood samples from newborn babies. In this study, therefore, we developed a derivatized method to monitor PHE and TYR in plasma samples using liquid phase chromatography linked with quadrupole mass spectrometry. Accessible formaldehyde isotopes and cyanoborohydride were used to react with PHE and TYR amino groups to generate h2-formaldehyde-modified PHE and TYR (as standards) and d2-formaldehyde-modified PHE and TYR (as internal standards). We used tandem mass spectrometry for multiple reaction monitoring. We demonstrated a derivatized method suitable for the PKU screening of newborns. The recoveries for PHE and TYR were 85% and 90%, respectively. Furthermore, we compared the values of PHE and TYR in different human plasma sample storage methods, including direct plasma and dried blood spots, and the results showed no significant difference.

Keywords:  dried blood spots; newborn screening; phenylalanine; phenylketonuria; tyrosine

DOI:  https://doi.org/10.1177/14690667241229626
Bone Rep. 2024 Mar;20 101737

Total serum pentosidine quantification using liquid chromatography-tandem mass spectrometry.

Lindsie A Blencowe, Andrea Božović, Evelyn Wong, Vathany Kulasingam, Angela M Cheung.

  Pentosidine (PEN) is an Advanced Glycation End-product (AGE) that is known to accumulate in bone collagen with aging and contribute to fracture risk. The PEN content in bone is correlated with serum PEN, making it an attractive, potential osteoporosis biomarker. We sought to develop a method for quantifying PEN in stored serum. After conducting a systematic narrative review of PEN quantification methodologies, we developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantifying total serum PEN. Our method is both sensitive and precise (LOD 2 nM, LOQ 5 nM, %CV < 6.5 % and recovery 91.2-100.7 %). Our method is also equivalent or better than other methods identified in our review. Additionally, LC-MS/MS avoids the pitfalls and limitations of using fluorescence as a means of detection and could be adapted to investigate a broad range of AGE compounds.

Keywords:  Advanced glycation end product; Liquid chromatography-tandem mass spectrometry; Osteoporosis; Pentosidine

DOI:  https://doi.org/10.1016/j.bonr.2024.101737
Curr Med Chem. 2024 Jan 17.

Pharmacokinetics of Anti-Rheumatic drugs Methotrexate and Tofacitinib with its metabolite M9 in Rats by UPLC-MS/MS.

Er-Min Gu, Lingjie Xue, Chenjian Zhou, Yang Xia, Ge-Xin Dai.

   BACKGROUND: Tofacitinib is an oral JAK inhibitor for the treatment of rheumatoid arthritis (RA). The clinical efficacy and safety of an administered tofacitinib, either monotherapy or in combination with conventional synthetic disease-modifying anti-rheumatic drugs, mainly methotrexate (MTX), have been evaluated. The high plasma concentration with delayed medicine clearance may affect the liver and/or kidney functions. In this study, an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC- MS/MS) method for the quantitative analysis of methotrexate, tofacitinib, and metabolite M9 in plasma of Sprague Dawley (SD) rats was developed, and its effectiveness was validated as well.
METHODS: Methotrexate, tofacitinib, M9 and fedratinib (internal standard, IS) were separated by gradient elution. The chromatography was performed on an Acquity BEH C18 (2.1 mm × 50 mm, 1.7 μm) column with the mobile phases of acetonitrile and 0.1% formic acid aqueous solution with different proportions at the flow rate of 0.30 mL/min. In the positive ionization mode, the analyzes were detected using a Xevo TQ-S triple quadrupole tandem mass spectrometer, with the following mass transition pairs: m/z 313.12 → 148.97 for tofacitinib, m/z 329.10 → 165.00 for M9 and m/z 455.12 → 308.05 for methotrexate.
RESULTS: The obtained results manifested good calibration linearity over the ranges of tofacitinib at 0.1-100 ng/mL, M9 at 0.05-100 ng/mL, and methotrexate at 0.05-100 ng/mL. The lower limit of quantifications (LLOQs) of methotrexate, tofacitinib and M9 were 0.05 ng/mL, 0.1 ng/mL and 0.05 ng/mL, respectively. Intra-day and inter-day accuracy values were confirmed with a range of -6.3% to 12.7%, while intra-day and inter-- day precision values were ≤14.4%. Additionally, recoveries were greater than 86.5% for each compound without significant matrix effects.
CONCLUSION: The currently established analytical method exhibited great potential for the evaluation of plasma concentrations of methotrexate, tofacitinib and M9 simultaneously, greatly reducing the detection time, which would serve as a supplementary role in formulating dose decisions to achieve personalized treatment, identify drugs that cause adverse reactions and finally, to assess drug-drug interactions on clinical studies.

Keywords:  SD rat.; UPLC-MS/MS; methotrexate; pharmacokinetics; rheumatoid arthritis; tofacitinib

DOI:  https://doi.org/10.2174/0109298673256258231219060950
Nat Microbiol. 2024 Feb;9(2): 336-345

microbeMASST: a taxonomically informed mass spectrometry search tool for microbial metabolomics data.

Simone Zuffa, Robin Schmid, Anelize Bauermeister, Paulo Wender P Gomes, Andres M Caraballo-Rodriguez, Yasin El Abiead, Allegra T Aron, Emily C Gentry, Jasmine Zemlin, Michael J Meehan, Nicole E Avalon, Robert H Cichewicz, Ekaterina Buzun, Marvic Carrillo Terrazas, Chia-Yun Hsu, Renee Oles, Adriana Vasquez Ayala, Jiaqi Zhao, Hiutung Chu, Mirte C M Kuijpers, Sara L Jackrel, Fidele Tugizimana, Lerato Pertunia Nephali, Ian A Dubery, Ntakadzeni Edwin Madala, Eduarda Antunes Moreira, Leticia Veras Costa-Lotufo, Norberto Peporine Lopes, Paula Rezende-Teixeira, Paula C Jimenez, Bipin Rimal, Andrew D Patterson, Matthew F Traxler, Rita de Cassia Pessotti, Daniel Alvarado-Villalobos, Giselle Tamayo-Castillo, Priscila Chaverri, Efrain Escudero-Leyva, Luis-Manuel Quiros-Guerrero, Alexandre Jean Bory, Juliette Joubert, Adriano Rutz, Jean-Luc Wolfender, Pierre-Marie Allard, Andreas Sichert, Sammy Pontrelli, Benjamin S Pullman, Nuno Bandeira, William H Gerwick, Katia Gindro, Josep Massana-Codina, Berenike C Wagner, Karl Forchhammer, Daniel Petras, Nicole Aiosa, Neha Garg, Manuel Liebeke, Patric Bourceau, Kyo Bin Kang, Henna Gadhavi, Luiz Pedro Sorio de Carvalho, Mariana Silva Dos Santos, Alicia Isabel Pérez-Lorente, Carlos Molina-Santiago, Diego Romero, Raimo Franke, Mark Brönstrup, Arturo Vera Ponce de León, Phillip Byron Pope, Sabina Leanti La Rosa, Giorgia La Barbera, Henrik M Roager, Martin Frederik Laursen, Fabian Hammerle, Bianka Siewert, Ursula Peintner, Cuauhtemoc Licona-Cassani, Lorena Rodriguez-Orduña, Evelyn Rampler, Felina Hildebrand, Gunda Koellensperger, Harald Schoeny, Katharina Hohenwallner, Lisa Panzenboeck, Rachel Gregor, Ellis Charles O'Neill, Eve Tallulah Roxborough, Jane Odoi, Nicole J Bale, Su Ding, Jaap S Sinninghe Damsté, Xue Li Guan, Jerry J Cui, Kou-San Ju, Denise Brentan Silva, Fernanda Motta Ribeiro Silva, Gilvan Ferreira da Silva, Hector H F Koolen, Carlismari Grundmann, Jason A Clement, Hosein Mohimani, Kirk Broders, Kerry L McPhail, Sidnee E Ober-Singleton, Christopher M Rath, Daniel McDonald, Rob Knight, Mingxun Wang, Pieter C Dorrestein.

microbeMASST, a taxonomically informed mass spectrometry (MS) search tool, tackles limited microbial metabolite annotation in untargeted metabolomics experiments. Leveraging a curated database of >60,000 microbial monocultures, users can search known and unknown MS/MS spectra and link them to their respective microbial producers via MS/MS fragmentation patterns. Identification of microbe-derived metabolites and relative producers without a priori knowledge will vastly enhance the understanding of microorganisms' role in ecology and human health.

DOI: https://doi.org/10.1038/s41564-023-01575-9
Food Res Int. 2024 Feb;pii: S0963-9969(23)01298-X. [Epub ahead of print]178 113750

Identification of oxygenated triacylglycerols in pistachio nuts' lipids by reverse-phase high-resolution-liquid chromatography-ESI mass spectrometry.

Fariba Sabzi, Nikolai Kuhnert.

  Hydroxy- and peroxy-triacylglycerols are common products of lipid peroxidation formed during oil storage or heating or as enzymatic oxidation product of arachidonic acid as signaling molecules in mammals. In this study, oxygenated triacylglycerides (TAG) were identified in pistachio oil based on reverse phase(RP), high-performance liquid chromatography coupled with electrospray ionization and mass spectrometry (HPLC- ESI -MS). 20 novel lipid plant metabolites, classified based on their fragment spectra into a hydroxy (TAG-OH), an epoxy (TAG-O), and hydroperoxide (TAG-OOH) groups. We believe that this class of compounds has been for the first time observed as genuine secondary plant metabolites in a natural source in this case pistachio lipids of dietary relevance.

Keywords:  Electrospray ionization; Lipid oxidation; Liquid chromatography; Mass spectrometry; Pistachio; Triacylglycerol

DOI:  https://doi.org/10.1016/j.foodres.2023.113750
Curr Opin Biotechnol. 2024 Feb 03. pii: S0958-1669(24)00004-1. [Epub ahead of print]86 103068

Mass spectrometry imaging methods for visualizing tumor heterogeneity.

Kyle D Duncan, Helena Pětrošová, Julian J Lum, David R Goodlett.

Profiling spatial distributions of lipids, metabolites, and proteins in tumors can reveal unique cellular microenvironments and provide molecular evidence for cancer cell dysfunction and proliferation. Mass spectrometry imaging (MSI) is a label-free technique that can be used to map biomolecules in tumors in situ. Here, we discuss current progress in applying MSI to uncover molecular heterogeneity in tumors. First, the analytical strategies to profile small molecules and proteins are outlined, and current methods for multimodal imaging to maximize biological information are highlighted. Second, we present and summarize biological insights obtained by MSI of tumor tissue. Finally, we discuss important considerations for designing MSI experiments and several current analytical challenges.

DOI: https://doi.org/10.1016/j.copbio.2024.103068
Molecules. 2024 Feb 02. pii: 700. [Epub ahead of print]29(3):

Development of a Two-Dimensional Liquid Chromatographic Method for Analysis of Urea Cycle Amino Acids.

Yuko Sumida, Makoto Tsunoda.

  The urea cycle has been found to be closely associated with certain types of cancers and other diseases such as cardiovascular disease and chronic kidney disease. An analytical method for the precise quantification of urea cycle amino acids (arginine, ornithine, citrulline, and argininosuccinate) by off-line two-dimensional liquid chromatography (2D-LC) combined with fluorescence-based detection was developed. Before analysis, the amino acids were derivatised with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) to obtain NBD-amino acids. The first dimension involved the reversed-phase separation, in which NBD derivatives of urea cycle amino acids were completely separated from each other and mostly separated from the 18 NBD-proteinogenic amino acids. The samples were eluted with stepwise gradient using 0.02% trifluoroacetic acid in water-acetonitrile as the mobile phase. In the second dimension, an amino column was used for the separation of NBD-ornithine, -citrulline, and -argininosuccinate, while a sulfonic acid column was used to separate NBD-arginine. The developed 2D-LC system was used to analyse human plasma samples. The fractions of NBD-urea cycle amino acids obtained in the first dimension were collected manually and introduced into the second dimension. By choosing appropriate mobile phases for the second dimension, each NBD-urea cycle amino acid eluted in the first dimension was well separated from the other proteinogenic amino acids and interference from endogenous substance. This could not be achieved in the first dimension. The urea cycle amino acids in human plasma sample were quantified, and the method was well validated. The calibration curves for each NBD-urea cycle amino acid showed good linearity from 3 (ASA) or 15 (Orn, Cit, and Arg) to 600 nM, with correlation coefficients higher than 0.9969. The intraday and interday precisions were less than 7.9% and 15%, respectively. The 2D-LC system is expected to be useful for understanding the involvement of the urea cycle in disease progression.

Keywords:  arginine; argininosuccinate; citrulline; fluorescence; ornithine

DOI:  https://doi.org/10.3390/molecules29030700
AAPS PharmSciTech. 2024 Feb 08. 25(2): 33

Liquid Chromatography Tandem Mass Spectrometric Method for Quantification of Margetuximab in Rat Plasma and Application to a Pharmacokinetic Study.

Pridhvi Krishna Gaddey, Raja Sundararajan.

  Margetuximab was approved for the treatment of advanced HER2+ breast cancer. A feasible analytical technique that can measure this drug was obligatory. In light of this, a novel and thoroughly validated liquid chromatographic (LC)-tandem mass spectrometric (MS/MS) approach was developed for the quantification of margetuximab in rat plasma. The liquid-liquid extraction method was used to extract the analyte from rat plasma. The analyte was separated using acetonitrile and formic acid buffer (30:70) as a mobile phase on Waters, alliance e-2695 model HPLC having Symmetry C18 column, 150 mm × 4.6 mm, 3.5-µm column. The overall runtime was 6 min at a flow rate of 1.0 ml/min. The method showed significant sensitivity and acceptable linearity over the concentration range of 6-120 ng/ml. Accuracy was within 98.51-99.92%. The intraday precision ranged between 0.41 and 8.98% CV. Also, the findings of pharmacokinetic parameters such as Cmax, tmax, AUC0-∞, AUC0-t, and half-life results of margetuximab showed that the technique was helpful for accurately measuring drug concentrations in rat plasma. The method that was developed was useful and effective for quantifying margetuximab.

Keywords:  liquid chromatography; margetuximab; rat plasma; tandem mass spectrometry; validation

DOI:  https://doi.org/10.1208/s12249-024-02755-4