bims-metlip 2024-09-15 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2024–09–15
twenty-one papers selected by
Sofia Costa, Matterworks

LC-HRMS analysis of phospholipids bearing oxylipins.
Tracing de novo Lipids using Stable Isotope Labeling LC-TIMS-TOF MS/MS.
Dual column chromatography combined with high-resolution mass spectrometry improves coverage of non-targeted analysis of plant root exudates.
MassLite: An integrated python platform for single cell mass spectrometry metabolomics data pretreatment with graphical user interface and advanced peak alignment method.
MetMiner: A user-friendly pipeline for large-scale plant metabolomics data analysis.
Development of a UPLC-MS/MS Method to Simultaneously Measure 13 Antiepileptic Drugs with Deuterated Internal Standards.
Development and Validation of Methodologies for the Identification of Specialized Pro-Resolving Lipid Mediators and Classic Eicosanoids in Biological Matrices.
LAMAIS: A library-aided approach for efficient 1D 1H NMR qualitative analysis in plant metabolomics.
Quantifying Phospholipids in Organic Samples Using a Hydrophilic Interaction Liquid Chromatography-Inductively Coupled Plasma High-Resolution Mass Spectrometry (HILIC-ICP-HRMS) Method.
A rapid and reliable targeted LC-MS/MS method for quantitative analysis of the Tryptophan-NAD metabolic network disturbances in tissues and blood of sleep deprivation mice.
Complete resolution across the neodymium/samarium isotopic envelope with a liquid sampling-atmospheric pressure glow discharge - Orbitrap mass spectrometer.
SIL-IS LC-ESI-MS/MS method for simultaneous quick detection of amoxicillin and clavulanic acid in human plasma: Development, validation and its application to a pharmacokinetics study.
Advancing the Prediction of MS/MS Spectra Using Machine Learning.
Pharmaceutical metabolite identification in lettuce (Lactuca sativa) and earthworms (Eisenia fetida) using liquid chromatography coupled to high-resolution mass spectrometry and in silico spectral library.
Application of a Novel UPLC-MS/MS Method for Analysis of Rivaroxaban Concentrations in Dried Blood Spot and Plasma Samples Collected from Patients with Venous Thrombosis.
Separation of triacylglycerol (TAG) isomers by cyclic ion mobility mass spectrometry.
Simultaneous quantification of alkanolamines and their dehydrogenation products in aqueous samples using high-performance liquid chromatography with pre-column derivatization with 2,4-dinitrofluorobenzene.
An LC-MS/MS method for the quantification of 3-bromotyrosine in plasma from patients diagnosed with eosinophilic esophagitis.
Simultaneous observation of 2H and 13C enrichment of methyl phosphonic acid via Orbitrap-IRMS with applications to nerve agent forensics.
Advantages of online supercritical fluid extraction and chromatography hyphenated to mass spectrometry to analyse plastic additives in laboratory gloves.
Dynamics of the Aspiration of Charged Droplets into a LC-ESI-MS System.

Anal Chim Acta. 2024 Oct 16. pii: S0003-2670(24)00940-1. [Epub ahead of print]1326 343139

LC-HRMS analysis of phospholipids bearing oxylipins.

Laura Carpanedo, Katharina M Rund, Luca M Wende, Nadja Kampschulte, Nils Helge Schebb.

   BACKGROUND: Several oxylipins including hydroxy- and epoxy-polyunsaturated fatty acids act as lipid mediators. In biological samples they can be present as non-esterified form, but the major part occurs esterified in phospholipids (PL) or other lipids. Esterified oxylipins are quantified indirectly after alkaline hydrolysis as non-esterified oxylipins. However, in this indirect analysis the information in which lipid class oxylipins are bound is lost. In this work, an untargeted liquid chromatography high-resolution mass spectrometry (LC-HRMS) method for the direct analysis of PL bearing oxylipins was developed.
RESULTS: Optimized reversed-phase LC separation achieved a sufficient separation of isobaric and isomeric PL from different lipid classes bearing oxylipin positional isomers. Individual PL species bearing oxylipins were identified based on retention time, precursor ion and characteristic product ions. The bound oxylipin could be characterized based on product ions resulting from the α-cleavage occurring at the hydroxy/epoxy group. PL sn-1/sn-2 isomers were identified based on the neutral loss of the fatty acyl in the sn-2 position. A total of 422 individual oxPL species from 7 different lipid classes i.e., PI, PS, PC, PE, PC-P, PC-O, and PE-P were detected in human serum and cells. This method enabled to determine in which PL class supplemented oxylipins are incorporated in HEK293 cells: 20:4;15OH, 20:4;14Ep, and 20:5;14Ep were mostly bound to PI. 20:4;8Ep and 20:5;8Ep were esterified to PC and PE while other oxylipins were mainly found in PC.
SIGNIFICANCE: The developed LC-HRMS method enables the comprehensive detection as well as the semi-quantification of isobaric and isomeric PL species bearing oxylipins. With this method, we show that the position of the oxidation has a great impact and directs the incorporation of oxylipins into the different PL classes in human cells.

Keywords:  Chromatographic separation; Esterified oxylipins; Fragmentation spectra; High-resolution mass spectrometry; Oxidized phospholipids; Oxylipin-supplementation; Untargeted lipidomics

DOI:  https://doi.org/10.1016/j.aca.2024.343139
J Vis Exp. 2024 Aug 23.

Tracing de novo Lipids using Stable Isotope Labeling LC-TIMS-TOF MS/MS.

Katherine D Castro, Lilian Valadares Tose, Matthew Willetts, Melvin A Park, Marcela Nouzova, Fernando G Noriega, Francisco Fernandez-Lima.

Lipids are highly diverse, and small changes in lipid structures and composition can have profound effects on critical biological functions. Stable isotope labeling (SIL) offers several advantages for the study of lipid distribution, mobilization, and metabolism, as well as de novo lipid synthesis. The successful implementation of the SIL technique requires the removal of interferences from endogenous molecules. In the present work, we describe a high-throughput analytical protocol for the screening of SIL lipids from biological samples; examples will be shown of lipid de novo identification during mosquito ovary development. The use of complementary liquid chromatography trapped ion mobility spectrometry and mass spectrometry allows for the separation and lipids assignment from a single sample in a single scan (<1 h). The described approach takes advantage of recent developments in data-dependent acquisition and data-independent acquisition, using parallel accumulation in the mobility trap followed by sequential fragmentation and collision-induced dissociation. The measurement of SIL at the fatty acid chain level reveals changes in lipid dynamics during the ovary development of mosquitoes. The lipids de novo structures are confidently assigned based on their retention time, mobility, and fragmentation pattern.

DOI: https://doi.org/10.3791/65590
Anal Chim Acta. 2024 Oct 23. pii: S0003-2670(24)00927-9. [Epub ahead of print]1327 343126

Dual column chromatography combined with high-resolution mass spectrometry improves coverage of non-targeted analysis of plant root exudates.

Alexandra A Bennett, Teresa Steininger-Mairinger, Çağla Görkem Eroğlu, Aurélie Gfeller, Judith Wirth, Markus Puschenreiter, Stephan Hann.

   BACKGROUND: Within the plant kingdom, there is an exceptional amount of chemical diversity that has yet to be annotated. It is for this reason that non-targeted analysis is of interest for those working in novel natural products. To increase the number and diversity of compounds observable in root exudate extracts, several workflows which differ at three key stages were compared: 1) sample extraction, 2) chromatography, and 3) data preprocessing.
RESULTS: Plants were grown in Hoagland's solution for two weeks, and exudates were initially extracted with water, followed by a 24-h regeneration period with subsequent extraction using methanol. Utilizing the second extraction showed improved results with less ion suppression and reduced retention time shifting compared to the first extraction. A single column method, utilizing a pentafluorophenyl column, paired with high-resolution mass spectrometry ionized and correctly identified 34 mock root exudate compounds, while the dual column method, incorporating a pentafluorophenyl column and a porous graphitic carbon column, retained and identified 43 compounds. In a pooled quality control sample of exudate extracts, the single column method detected 1,444 compounds. While the dual method detected fewer compounds overall (1,050), it revealed a larger number of small polar compounds. Three preprocessing methods (targeted, proprietary, and open source) successfully identified 43, 31, and 38 mock root exudate compounds to confidence level 1, respectively.
SIGNIFICANCE: Enhancing signal strength and analytical method stability involves removing the high ionic strength nutrient solution before sampling root exudate extracts. Despite signal intensity loss, a dual column method enhances compound coverage, particularly for small polar metabolites. Open-source software proves a viable alternative for non-targeted analysis, even surpassing proprietary software in peak picking.

Keywords:  Data dependent acquisition; Dual column chromatography; High-resolution mass spectrometry; Non-targeted analysis; Rhizosphere

DOI:  https://doi.org/10.1016/j.aca.2024.343126
Anal Chim Acta. 2024 Oct 09. pii: S0003-2670(24)00925-5. [Epub ahead of print]1325 343124

MassLite: An integrated python platform for single cell mass spectrometry metabolomics data pretreatment with graphical user interface and advanced peak alignment method.

Zhu Zou, Zongkai Peng, Deepti Bhusal, Shakya Wije Munige, Zhibo Yang.

Mass spectrometry (MS) has been one of the most widely used tools for bioanalytical analysis due to its high sensitivity, capability of quantitative analysis, and compatibility with biomolecules. Among various MS techniques, single cell mass spectrometry (SCMS) is an advanced approach to molecular analysis of cellular contents in individual cells. In tandem with the creation of novel experimental techniques, the development of new SCMS data analysis tools is equally important. As most published software packages are not specifically designed for pretreatment of SCMS data, including peak alignment and background removal, their applicability on processing SCMS data is generally limited. Hereby we introduce a Python platform, MassLite, specifically designed for rapid SCMS metabolomics data pretreatment. This platform is made user-friendly with graphical user interface (GUI) and exports data in the forms of each individual cell for further analysis. A core function of this tool is to use a novel peak alignment method that avoids the intrinsic drawbacks of traditional binning method, allowing for more effective handling of MS data obtained from high resolution mass spectrometers. Other functions, such as void scan filtering, dynamic grouping, and advanced background removal, are also implemented in this tool to improve pretreatment efficiency.

DOI: https://doi.org/10.1016/j.aca.2024.343124
J Integr Plant Biol. 2024 Sep 10.

MetMiner: A user-friendly pipeline for large-scale plant metabolomics data analysis.

Xiao Wang, Shuang Liang, Wenqi Yang, Ke Yu, Fei Liang, Bing Zhao, Xiang Zhu, Chao Zhou, Luis A J Mur, Jeremy A Roberts, Junli Zhang, Xuebin Zhang.

  The utilization of metabolomics approaches to explore the metabolic mechanisms underlying plant fitness and adaptation to dynamic environments is growing, highlighting the need for an efficient and user-friendly toolkit tailored for analyzing the extensive datasets generated by metabolomics studies. Current protocols for metabolome data analysis often struggle with handling large-scale datasets or require programming skills. To address this, we present MetMiner (https://github.com/ShawnWx2019/MetMiner), a user-friendly, full-functionality pipeline specifically designed for plant metabolomics data analysis. Built on R shiny, MetMiner can be deployed on servers to utilize additional computational resources for processing large-scale datasets. MetMiner ensures transparency, traceability, and reproducibility throughout the analytical process. Its intuitive interface provides robust data interaction and graphical capabilities, enabling users without prior programming skills to engage deeply in data analysis. Additionally, we constructed and integrated a plant-specific mass spectrometry database into the MetMiner pipeline to optimize metabolite annotation. We have also developed MDAtoolkits, which include a complete set of tools for statistical analysis, metabolite classification, and enrichment analysis, to facilitate the mining of biological meaning from the datasets. Moreover, we propose an iterative weighted gene co-expression network analysis strategy for efficient biomarker metabolite screening in large-scale metabolomics data mining. In two case studies, we validated MetMiner's efficiency in data mining and robustness in metabolite annotation. Together, the MetMiner pipeline represents a promising solution for plant metabolomics analysis, providing a valuable tool for the scientific community to use with ease.

Keywords:  data mining; iterative WGCNA; metabolomics; pipeline; shinyapp

DOI:  https://doi.org/10.1111/jipb.13774
Clin Lab. 2024 Sep 01. 70(9):

Development of a UPLC-MS/MS Method to Simultaneously Measure 13 Antiepileptic Drugs with Deuterated Internal Standards.

Jae Hyun Cha, Hyebin Choi, Jisook Yim, Keun Ju Kim, Minjeong Nam, Myung Hyun Nam, Chang Kyu Lee, Dae Won Kim, Yunjung Cho, Seung Gyu Yun.

BACKGROUND: The goal of this study was to develop and validate a UPLC-MS/MS method for simultaneous mea-surement of 13 AEDs, including carbamazepine, oxcarbazepine, lamotrigine, levetiracetam, topiramate, primidone, zonisamide, gabapentin, lacosamide, perampanel, pregabalin, rufinamide, and vigabatrin, in whole blood samples.
METHODS: A UPLC-MS/MS method for simultaneous determination of 13 AEDs in whole blood was developed, and validation was conducted for accuracy, precision, limit of quantification (LOQ), matrix effect, and stability. Our method was compared to two different hospitals using UPLC-MS/MS.
RESULTS: All AEDs exhibited linearity across the AMR (analytical measurement range), with R2 values ranging from 0.994 to 1.000. The imprecision and inaccuracy for low and high quality control (QC) levels were within an acceptable range, with the coefficient of variation (CV) < 15%. The LOQ was 0.62 µg/mL for carbamazepine, 1.61 µg/mL for oxcarbazepine, 1.30 µg/mL for lamotrigine, 13.20 µg/mL for levetiracetam, 1.26 µg/mL for topira-mate, 1.01 µg/mL for primidone, 1.59 µg/mL for zonisamide, 1.09 µg/mL for lacosamide, 1.61 µg/mL for gabapentin, 0.50 µg/mL for pregabalin, 0.07 ng/mL for perampanel, 3.00 µg/mL for rufinamide, and 2.06 µg/mL for vigabatrin. All AEDs demonstrated acceptable assay parameters for carryover, stability, and matrix effects. Moreover, the assay showed satisfactory results compared to two different hospitals with a bias of less than 15%.
CONCLUSIONS: We successfully developed and validated a fast and robust UPLC-MS/MS method for routine therapeutic drug monitoring of thirteen antiepileptic drugs simultaneously.

DOI: https://doi.org/10.7754/Clin.Lab.2024.240206
J Am Soc Mass Spectrom. 2024 Sep 09.

Development and Validation of Methodologies for the Identification of Specialized Pro-Resolving Lipid Mediators and Classic Eicosanoids in Biological Matrices.

Matthew Dooley, Amitis Saliani, Jesmond Dalli.

  Lipid mediators, which include specialized pro-resolving mediators and classic eicosanoids, are pivotal in both initiating and resolving inflammation. The regulation of these molecules determines whether inflammation resolves naturally or persists. However, our understanding of how these mediators are regulated over time in various inflammatory contexts is limited. This gap hinders our grasp of the mechanisms underlying the disease onset and progression. Due to their localized action and low endogenous levels in many tissues, developing robust and highly sensitive methodologies is imperative for assessing their endogenous regulation in diverse inflammatory settings. These methodologies will help us gain insight into their physiological roles. Here, we establish methodologies for extracting, identifying, and quantifying these mediators. Using our methods, we identified a total of 37 lipid mediators. Additionally, by employing a reverse-phase HPLC method, we successfully separated both double-bond and chiral isomers of select lipid mediators, including Lipoxin (LX) A4, 15-epi-LXA4, Protectin (PD) D1, PDX, and 17R-PD1. Validation of the method was performed in both solvent and surrogate matrix for linearity of the standard curves, lower limits of quantitation (LLOQ), accuracy, and precision. Results from these studies demonstrated that linearity was good with r2 values > 0.98, and LLOQ for the mediators ranged from 0.01 to 0.9 pg in phase and from 0.1 to 8.5 pg in surrogate matrix. The relative standard deviation (RSD) for inter- and intraday precision in solvent ranged from 5% to 12% at low, intermediate, and high concentrations, whereas the RSD for the inter- and intraday variability in the accuracy ranged from 95% to 87% at low to high concentrations. The recovery in biological matrices (plasma and serum) for the internal standards used ranged from 60% to 118%. We observed a marked ion suppression for molecules evaluated in negative ionization mode, while there was an ion enhancement effect by the matrix for molecules evaluated in positive ionization mode. Comparison of the integration algorithms, namely, AutoPeak and MQ4, and approaches for calculating signal-to-noise ratios (i.e., US Pharmacopeia, relative noise, peak to peak, and standard deviation) demonstrated that different integration algorithms tested had little influence on signal-to-noise ratio calculations. In contrast, the method used to calculate the signal-to-noise ratio had a more significant effect on the results, with the relative noise approach proving to be the most robust. The methods described herein provide a platform to study the SPM and classic eicosanoids in biological tissues that will help further our understanding of disease mechanisms.

Keywords:  Eicosanoids; lipid mediators; liquid chromatography-tandem mass spectrometry; specialized pro-resolving mediators

DOI:  https://doi.org/10.1021/jasms.4c00211
Anal Chim Acta. 2024 Oct 16. pii: S0003-2670(24)00901-2. [Epub ahead of print]1326 343100

LAMAIS: A library-aided approach for efficient 1D 1H NMR qualitative analysis in plant metabolomics.

Xinyuan Xie, Jiayu Yang, Yuting Lu, Yingting Shi, Jianyang Pan, Haibin Qu.

   BACKGROUND: One-dimensional proton nuclear magnetic resonance (1D 1H NMR) spectroscopy is a non-destructive, non-targeted analytical technique providing both qualitative and quantitative insights, particularly beneficial for mixture analysis. However, the qualitative analysis of 1D 1H NMR spectra for mixture samples is laborious and time-consuming, involving extensive database searches and verification experiments like spiking. This process heavily relies on the analyst's expertise, leading to efficiency discrepancies. There is a pressing need for a reliable method to streamline operations and enhance the efficiency of qualitative analysis in complex mixtures.
RESULTS: We introduce a library-aided method for spectral profiling, named LAMAIS. This method achieves compound identification through similarity assessment between samples and template data, allowing rapid, automatic compound identification and full-spectrum peak assignment without the need for fitting. LAMAIS correctly identifies over 90 % of components in synthetic mixtures and more than 75 % in experimental mixtures, surpassing other representative methods with a higher F2 score. Our reference library, which currently includes 71 compounds, is tailored to capture the commonality of primary metabolites across diverse plant species. The analysis of real-world samples yielded encouraging results, underscoring LAMAIS's versatility as an auxiliary tool suitable for a variety of botanical sources. For analyst convenience, interactive graphics are utilized as the output format.
SIGNIFICANCE: LAMAIS excels, demonstrating competitiveness and reliability. The approach minimizes repetitive tasks and sample wastage, improving the efficiency of 1D 1H NMR qualitative analysis. Constructing a reference library effectively preserves knowledge, mitigates reliance on human experience, and addresses gaps in the analysis of plant source samples.

Keywords:  (1)H NMR spectroscopy; Library-aided; Mixture analysis; Plant primary metabolites; Spectral profiling

DOI:  https://doi.org/10.1016/j.aca.2024.343100
Anal Chem. 2024 Sep 12.

Quantifying Phospholipids in Organic Samples Using a Hydrophilic Interaction Liquid Chromatography-Inductively Coupled Plasma High-Resolution Mass Spectrometry (HILIC-ICP-HRMS) Method.

Wladimir Ruiz, Stéphanie Fontagné-Dicharry, Sylvain Verdier, David C Dayton, Julie Guillemant, Rémi Moulian, Pierre Giusti, Caroline Barrère-Mangote, Brice Bouyssiere.

In this study, a novel method using hydrophilic interaction liquid chromatography (HILIC) coupled with inductively coupled plasma high-resolution mass spectrometry (ICP-HRMS) was introduced for the quantification of phospholipids in oil samples. The method employed a bridged ethyl hybrid (BEH) stationary phase HILIC column with a tetrahydrofuran (THF)/water mobile phase, enhancing the solubility and detection of phospholipids. During the study, a gradient/matrix effect on ICP-HRMS sensitivity was observed and successfully compensated for experimentally, ensuring reliable quantification results. This approach has proven effective for a wide range of different oil samples including vegetable oils, animal fats, and phospholipid supplements. Notably, this method allowed the direct quantification of phospholipids in oil samples, bypassing the need for prior sample preparation methods, such as solid phase extraction (SPE), thereby streamlining the analytical process. The precision, accuracy, and reduced need for extensive sample preparation offered by this method mark a significant advancement in lipids analysis. Its robustness and broad applicability have substantial implications for industries such as food and renewable energy production, where both efficient and accurate lipid identification and quantification are crucial.

DOI: https://doi.org/10.1021/acs.analchem.4c01883
Anal Chim Acta. 2024 Nov 01. pii: S0003-2670(24)00926-7. [Epub ahead of print]1328 343125

A rapid and reliable targeted LC-MS/MS method for quantitative analysis of the Tryptophan-NAD metabolic network disturbances in tissues and blood of sleep deprivation mice.

Peiling Wu, Wenjie Wang, Chuan Huang, Le Sun, Xiaoli Wu, Lijia Xu, Peigen Xiao.

   BACKGROUND: TRY-NAD metabolic network includes TRY (tryptophan), 5-HT (5-hydroxytryptamine), KYN (kynurenine), and NAD (nicotinamide adenine dinucleotide) pathway, which plays a significant role in neurological diseases and ageing. It is important to monitor these metabolites for studying the pathological anatomy of disease and treatment of responses evaluation. Although previous studies have reported quantitative methods for several metabolites in the network, the bottlenecks of simultaneously quantifying the whole metabolic network are their similar structures, diverse physico-chemical properties, and instability. Standardized protocols for the whole metabolic network are still missing, which hinders the in-depth study of TRY-NAD metabolic network in laboratory research and clinical screening.
RESULTS: We developed a LC-MS/MS method for quantifying 28 metabolites in the TRY-NAD network simultaneously. Optimization was done for the mass spectral parameters, chromatographic conditions and sample pretreatment process. The developed method was fully validated in terms of standard curves, sensitivity, carryover, recovery, matrix effect, accuracy, precision, and stability. The pretreatment of 30 samples only takes 90 min, and the LC-MS/MS running time of one sample is only 13 min. With this method, we bring to light the chaos of global TRY-NAD metabolic network in sleep deprivation mice for the first time, including serum, clotted blood cells, hippocampus, cerebral cortex, and liver. NAD pathway levels in brain and blood decreased, whereas the opposite happened in the liver. The 5-HT pathway decreased and the concentration of KYN increased significantly in the brain. The concentration of many metabolites in KYN pathway (NAD+ de novo synthesis pathway) increased in the liver.
SIGNIFICANCE: This method is the first time to determine the metabolites of KYN, 5-HT and NAD pathway at the same time, and it is found that TRY-NAD metabolic network will be disordered after sleep deprivation. This work clarifies the importance of the pH of the extraction solution, the time and temperature control in pretreatment in standardized protocols building, and overcoming the problems of inconsistent sample pretreatment, separation, matrix effect interference and potential metabolite degradation. This method exhibits great prospects in providing more information on metabolic disturbances caused by sleep deprivation as well as neurological diseases and ageing.

Keywords:  LC-MS/MS; Metabolomic; NAD; Sleep deprivation; Tryptophan

DOI:  https://doi.org/10.1016/j.aca.2024.343125
Rapid Commun Mass Spectrom. 2024 Dec 15. 38(23): e9912

Complete resolution across the neodymium/samarium isotopic envelope with a liquid sampling-atmospheric pressure glow discharge - Orbitrap mass spectrometer.

Joseph V Goodwin, Suraj Shrestha, Benjamin T Manard, R Kenneth Marcus.

RATIONALE: Nd and Sm isotope ratios play an important role in geological dating and as nuclear forensic signatures; however, the overlap of the respective 144, 148, 150 Nd/Sm isobars requires prior separations to be performed before analysis on typical MS platforms. The work presented here overcomes these isobaric interferences using ultrahigh-mass resolution to alleviate interference without prior chemical separations.
METHODS: A liquid sampling-atmospheric pressure glow discharge ion source was coupled to a standard, QExactive Focus Orbitrap mass spectrometer, providing a mass resolution of ~80 k. A Spectroswiss FTMS booster X2 data acquisition package was used to collect extended transients, providing much higher mass resolution; ~230 k and ~600 k are employed here for Nd and Sm isotopes.
RESULTS: While the standard Orbitrap resolution is far greater than typical "atomic" MS platforms, it was insufficient to alleviate all isobars. The use of a resolution of ~230 k resulted in baseline separation across the entire isotopic envelope for both Nd and Sm. Isotope ratios obtained from Nd:Sm mixtures using high-resolution were equivalent to those found for individual-element solutions, while isotope ratios obtained at a resolution of ~80 k (standard for the OEM data system) showed large deviations.
CONCLUSIONS: Use of ultrahigh-resolution is an attractive alternative to extensive chemical separations to alleviate severe isobaric interferences. Sufficient mass resolution greatly reduces/eliminates the need for sample manipulations (separations) before analysis while reducing costs and total analysis times.

DOI: https://doi.org/10.1002/rcm.9912
Biomed Chromatogr. 2024 Sep 10. e5964

SIL-IS LC-ESI-MS/MS method for simultaneous quick detection of amoxicillin and clavulanic acid in human plasma: Development, validation and its application to a pharmacokinetics study.

Jianbang Wu, Changmao Wang, Rong Zhang, Pengfei Du, Yaqin Wang, Ping Wu, Xinyan Chen, Yunzhe Huang, Yuanwei Jia, Jie Shen.

  A liquid chromatography electrospray ionization tandem mass spectrometry method with amoxicillin-d4 as the stable isotope-labeled internal standard for simultaneous quick detection of amoxicillin and clavulanic acid in human plasma was developed and validated. Chromatographic separations were performed on a Hedera ODS-2 column (2.1 × 150 mm, 5 μm). The mobile phases for gradient elution were aqueous solution containing 0.2% acetic acid (AA) (mobile phase A) together with organic phase solution (acetonitrile and methanol mixed solution, mobile phase B). Mass spectrometry was performed using negative electrospray ionization in multiple reaction monitoring mode. The target fragment ion pairs of amoxicillin, clavulanic acid and amoxicillin-d4 were m/z 364.1 → 223.1, 198.1 → 135.9 and 368.1 → 227.1, respectively. The linear ranges of this method were 40-5,000 ng/ml for amoxicillin and 30-2,500 ng/ml for clavulanic acid, with coefficient of determination > 0.9900. This method validation included selectivity, standard curve, lower limit of quantitation, accuracy, precision, recovery, matrix effect (hemolytic matrix and hyperlipidemic matrix), carryover, stability, dilution reliability and incurred sample reanalysis study. A successful application of this method was realized in a pharmacokinetic study after administration of amoxicillin-clavulanic acid potassium granules.

Keywords:  LC–MS/MS; amoxicillin; clavulanic acid; human plasma; pharmacokinetics

DOI:  https://doi.org/10.1002/bmc.5964
J Am Soc Mass Spectrom. 2024 Sep 11.

Advancing the Prediction of MS/MS Spectra Using Machine Learning.

Julia Nguyen, Richard Overstreet, Ethan King, Danielle Ciesielski.

Tandem mass spectrometry (MS/MS) is an important tool for the identification of small molecules and metabolites where resultant spectra are most commonly identified by matching them with spectra in MS/MS reference libraries. While popular, this strategy is limited by the contents of existing reference libraries. In response to this limitation, various methods are being developed for the in silico generation of spectra to augment existing libraries. Recently, machine learning and deep learning techniques have been applied to predict spectra with greater speed and accuracy. Here, we investigate the challenges these algorithms face in achieving fast and accurate predictions on a wide range of small molecules. The challenges are often amplified by the use of generic machine learning benchmarking tactics, which lead to misleading accuracy scores. Curating data sets, only predicting spectra for sufficiently high collision energies, and working more closely with experimental mass spectrometrists are recommended strategies to improve overall prediction accuracy in this nuanced field.

DOI: https://doi.org/10.1021/jasms.4c00154
Anal Bioanal Chem. 2024 Sep 10.

Pharmaceutical metabolite identification in lettuce (Lactuca sativa) and earthworms (Eisenia fetida) using liquid chromatography coupled to high-resolution mass spectrometry and in silico spectral library.

Jan Fučík, Stanislav Fučík, Sascha Rexroth, Marian Sedlář, Helena Zlámalová Gargošová, Ludmila Mravcová.

  Pharmaceuticals released into the aquatic and soil environments can be absorbed by plants and soil organisms, potentially leading to the formation of unknown metabolites that may negatively affect these organisms or contaminate the food chain. The aim of this study was to identify pharmaceutical metabolites through a triplet approach for metabolite structure prediction (software-based predictions, literature review, and known common metabolic pathways), followed by generating in silico mass spectral libraries and applying various mass spectrometry modes for untargeted LC-qTOF analysis. Therefore, Eisenia fetida and Lactuca sativa were exposed to a pharmaceutical mixture (atenolol, enrofloxacin, erythromycin, ketoprofen, sulfametoxazole, tetracycline) under hydroponic and soil conditions at environmentally relevant concentrations. Samples collected at different time points were extracted using QuEChERS and analyzed with LC-qTOF in data-dependent (DDA) and data-independent (DIA) acquisition modes, applying both positive and negative electrospray ionization. The triplet approach for metabolite structure prediction yielded a total of 3762 pharmaceutical metabolites, and an in silico mass spectral library was created based on these predicted metabolites. This approach resulted in the identification of 26 statistically significant metabolites (p < 0.05), with DDA + and DDA - outperforming DIA modes by successfully detecting 56/67 sample type:metabolite combinations. Lettuce roots had the highest metabolite count (26), followed by leaves (6) and earthworms (2). Despite the lower metabolite count, earthworms showed the highest peak intensities, closely followed by roots, with leaves displaying the lowest intensities. Common metabolic reactions observed included hydroxylation, decarboxylation, acetylation, and glucosidation, with ketoprofen-related metabolites being the most prevalent, totaling 12 distinct metabolites. In conclusion, we developed a high-throughput workflow combining open-source software with LC-HRMS for identifying unknown metabolites across various sample types.

Keywords:  High-resolution mass spectrometry; In silico spectral library; Liquid chromatography; Metabolite identification in Eisenia fetida and Lactuca sativa ; Pharmaceuticals; Software prediction

DOI:  https://doi.org/10.1007/s00216-024-05515-2
Molecules. 2024 Aug 31. pii: 4140. [Epub ahead of print]29(17):

Application of a Novel UPLC-MS/MS Method for Analysis of Rivaroxaban Concentrations in Dried Blood Spot and Plasma Samples Collected from Patients with Venous Thrombosis.

Kornel Pawlak, Łukasz Kruszyna, Marta Miecznikowska, Marta Karaźniewicz-Łada.

  Despite a higher safety profile compared to vitamin K antagonists, rivaroxaban therapy is still connected with multiple adverse effects, such as a high risk of bleeding. Thus, therapeutic drug monitoring (TDM) of rivaroxaban concentrations is suggested. An alternative to plasma samples can be dried blood spots (DBS), which minimize the cost of sample storage and transport. In this study, we developed a UPLC-MS/MS method for the analysis of rivaroxaban in DBS and plasma samples. Chromatographic separation was achieved on a Zorbax Eclipse Plus C18 column (2.1 × 100 mm; 3.5 µm, Agilent Technologies Inc., Santa Clara, CA, USA) with a mobile phase consisting of water and acetonitrile, both containing 0.1% formic acid. The analytes were detected using a positive ionization mode by multiple reaction monitoring. We validated the method according to ICH guidelines. The precision and accuracy were satisfactory. Extraction recovery was approximately 57% and 66% for DBS and plasma samples, respectively. A high correlation between rivaroxaban concentrations in plasma and DBS samples collected from patients was confirmed with Deming regression. The suitability of both sampling techniques for the rivaroxaban TDM was also verified by Bland-Altman plots based on DBS-predicted and observed plasma concentrations. In addition, we found a significant relationship between rivaroxaban concentrations and coagulation parameters, including prothrombin time (PT) and international normalized ratio (INR).

Keywords:  DBS; UPLC-MS/MS; plasma; rivaroxaban; venous thrombosis

DOI:  https://doi.org/10.3390/molecules29174140
Talanta. 2024 Sep 03. pii: S0039-9140(24)01183-4. [Epub ahead of print]281 126804

Separation of triacylglycerol (TAG) isomers by cyclic ion mobility mass spectrometry.

Carlo R de Bruin, Wouter J C de Bruijn, Mirjam A Hemelaar, Jean-Paul Vincken, Marie Hennebelle.

  Triacylglycerols (TAGs), a major lipid class in foods and the human body, consist of three fatty acids esterified to a glycerol backbone. They can occur in various isomeric forms, including sn-positional, cis/trans configurational, acyl chain length, double bond positional, and mixed type isomers. Separating isomeric mixtures is of great interest as different isomers can have distinct influence on mechanisms, such as digestibility, oxidative stability, or lipid metabolism. However, TAG isomer separation remains challenging with established analytical methodologies such as liquid-chromatography coupled to mass spectrometry (LC-MS). In this study, we developed a method with cyclic ion mobility mass spectrometry (cIMS-MS) for the separation and identification of all types of TAG isomers. First, the influence of different adducts (Li+, NH4+, Na+, and K+) on the separation was studied. Overall, it was concluded that the sodium adduct is the best choice to efficiently separate all types of TAG isomers. In addition, trends were found in the influence of specific structural features on the drift time order. An order of relative influence (from high to low) was established; (1) degree of unsaturation of the fatty acid(s) on an exterior position (if the total degree of unsaturation(s) is equal in both TAGs), (2) acyl chain length on the exterior positions, (3) cis/trans configuration, and (4) double bond (DB)-position. Finally, various cIMS-MS strategies were developed for the separation of mixtures containing four, five, and six isomers. To conclude, the developed methods can be used for separation of complex mixtures of TAG isomers and have great potential to be expanded to isomers of similar types of lipids such as di- and monoacylglycerols. This study also shows the potential of cIMS-MS to be used for the application on real TAG samples.

Keywords:  Adduct formation; Isomeric lipids; Space charge effects; Triglycerides; cIMS-MS analysis

DOI:  https://doi.org/10.1016/j.talanta.2024.126804
Anal Chim Acta. 2024 Oct 23. pii: S0003-2670(24)00957-7. [Epub ahead of print]1327 343156

Simultaneous quantification of alkanolamines and their dehydrogenation products in aqueous samples using high-performance liquid chromatography with pre-column derivatization with 2,4-dinitrofluorobenzene.

Yonglong Huang, Zhengkang Duan, Chuan Li, Yingying Wu.

   BACKGROUND: Diethanolamine, monoethanolamine, iminodiacetic acid, and glycine are important fine chemical intermediates, often requiring simultaneous quantitative analysis in various applications. This presents the challenge of accurately quantifying multiple substances within a single sample. The catalytic dehydrogenation of diethanolamine and monoethanolamine has garnered significant research interest, yet no analytical method has been reported for the simultaneous quantification of reactants and products in the dehydrogenation reaction mixtures of different alkanolamines.
RESULTS: A high-performance liquid chromatography (HPLC) method has been developed for the simultaneous quantification of diethanolamine (DEA), iminodiacetic acid (IDA), glycine (Gly), and monoethanolamine (MEA) in aqueous solutions using 2,4-dinitrofluorobenzene (DNFB) for pre-column derivatization. The method demonstrated excellent linearity, with correlation coefficients (R2) of 0.9999, 0.9997, and 0.9998 for IDA, DEA, and Gly, respectively. The detection limits (LODs) were 0.02, 0.08, and 0.01 mg L-1, respectively. The quantification limits (LOQs) were 0.06, 0.24, and 0.03 mg L-1, respectively. Spiked recovery rates ranged from 96.99 % to 104.32 %, with relative standard deviations (RSDs) between 0.70 % and 3.03 %. For MEA and Gly, the R2 values were 0.9970 and 0.9990, the LODs were 0.12 and 0.01 mg L-1, the LOQs were 0.36 and 0.03 mg L-1, and spiked recoveries ranged from 96.24 % to 104.82 %, with RSDs between 1.22 % and 3.68 %. Compared to other methods, this HPLC approach offers superior sensitivity, accuracy, and precision.
SIGNIFICANCE: This method provides a robust reference for the individual or simultaneous quantification of alkanolamines, glycine, and iminodiacetic acid in aqueous matrices. It offers new insights into the simultaneous analysis of alkanolamines with multiple organic acids in complex matrices. Additionally, the method can guide the optimization of catalytic dehydrogenation processes for alkanolamines, potentially extending the advantages of dehydrogenation catalysts to other reactions.

Keywords:  Diethanolamine; Glycine; HPLC; Iminodiacetic acid; Monoethanolamine; Pre-column derivatization

DOI:  https://doi.org/10.1016/j.aca.2024.343156
Anal Methods. 2024 Sep 09.

An LC-MS/MS method for the quantification of 3-bromotyrosine in plasma from patients diagnosed with eosinophilic esophagitis.

Morgan E Thomas, Jonathan E Markowitz, Ada C Arwood, Joshua M Germany, William M Gilliland.

Eosinophilic esophagitis (EoE) is a disease marked by a surplus of eosinophils, a type of white blood cell that causes inflammation and irritation. The current diagnostic and monitoring procedure for EoE is endoscopy with biopsy, which is invasive, expensive, and leads to tissue tearing in patients. A biomarker in plasma would offer a much less invasive form of disease monitoring for patients with EoE. Eosinophils have been shown to make eosinophil peroxidase, an enzyme that produces hypobromous acid, reacts with primary amines, and forms bromoamides. One product of this biochemical reaction is 3-bromotyrosine. We have optimized a selective, sensitive, and reproducible method to detect and quantify L-tyrosine and 3-bromotyrosine in human plasma using high-pressure liquid chromatography and tandem mass spectrometry (HPLC-MS/MS). Our sample preparation and analysis method requires fewer steps and provides a faster analysis than previous methods. Method validation yielded limits of quantification of 50 ng mL-1 for L-tyrosine and 10 ng mL-1 for 3-bromotyrosine. Calibration curves for quantification were linear from 50 to 500 ng mL-1 with an R2 value of 0.9995 for L-tyrosine and 10 to 300 ng mL-1 with an R2 value of 0.9998 for 3-bromotyrosine. Method variability was assessed resulting in relative standard deviations of 0.98-4.6% for 3-bromotyrosine (n = 18) and 0.20-0.58% for L-tyrosine (n = 18). Method applicability was tested with patients with a confirmed diagnosis of EoE, initially suggesting little to no correlation between eosinophil count and 3-bromotyrosine concentration in plasma. However, we do observe a relationship between eosinophil count and esophageal deformities. More research must be conducted to determine a more definitive correlation.

DOI: https://doi.org/10.1039/d4ay00697f
Talanta. 2024 Sep 02. pii: S0039-9140(24)01181-0. [Epub ahead of print]281 126802

Simultaneous observation of 2H and 13C enrichment of methyl phosphonic acid via Orbitrap-IRMS with applications to nerve agent forensics.

Timothy Csernica, James J Moran, Carlos G Fraga, John M Eiler.

  Quantification of the stable isotopes within a compound aids forensic investigations as it provides a fingerprint which can determine that compound's source substrates, synthetic route, and possible mechanisms of degradation. Previous stable isotope studies have explored 13C and 2H measurements of the sarin precursors methylphosphonic dichloride (DC) and methylphosphonic difluoride (DF) as forensic signatures. However, these measurements required different sample preparations and measurement techniques. Orbitrap isotope ratio mass spectrometry (Orbitrap-IRMS) is a developing technique which can characterize multiple stable isotopes simultaneously. Here, we apply Orbitrap-IRMS to simultaneously observe the 13C and 2H content of methylphosphonic acid (MPA), the hydrolysis product of DC and DF, which can be used as a proxy for the isotopic content of DC and DF. Our method requires 20 min analyses and consumes ≈60 nmol of sample, with precisions of ≈0.9 ‰ (13C) and ≈3.6 ‰ (2H). We apply our method to both commercially acquired MPA and MPA obtained from the hydrolysis of commercially acquired DC. We validate our methods via comparison to elemental-analyzer isotope ratio mass spectrometry (EA-IRMS). The combined 13C and 2H measurement creates a more robust forensic tool than either isotope individually. Our results demonstrate the viability of Orbitrap-IRMS for chemical forensic measurements.

Keywords:  Chemical forensics; Orbitrap isotope ratio mass spectrometry; Sarin; Stable isotopes

DOI:  https://doi.org/10.1016/j.talanta.2024.126802
J Chromatogr A. 2024 Aug 30. pii: S0021-9673(24)00697-6. [Epub ahead of print]1735 465323

Advantages of online supercritical fluid extraction and chromatography hyphenated to mass spectrometry to analyse plastic additives in laboratory gloves.

Benjamin Caux, Clément De Saint Jores, Ramy Abou-Naccoul, Shinnosuke Horie, Caroline West.

  Plastic additives are introduced in plastic material formulations, along with organic polymers, to offer different properties such as stability, plasticity or color. However, plastic additives may migrate from the plastic material to the content (in case of plastic containers) or to the material in contact with the plastic, like human skin. In the case of plastic medical devices, this migration is of particular interest, as plastic additives may be deleterious to health. In the present paper, we examined the interest of combining supercritical fluid extraction (SFE) to supercritical fluid chromatography (SFC) hyphenated to mass spectrometry (MS) in an online system to characterize plastic additives in laboratory gloves, taken as samples of medical devices. A set of target compounds comprising 18 plasticizers, 4 antioxidants and 2 lubricants was defined and their detectability with MS was examined, where it appeared that electrospray ionization (ESI) provided better detectability than atmospheric pressure chemical ionization (APCI). After examining possible stationary phases with the help of Derringer desirability function, an isocratic chromatographic method (CO2:methanol 95:5) was developed on Shim-pack UC Phenyl column. The extraction method was examined with a 3-level full factorial design of experiments to optimize the extraction temperature (40 °C) and pressure (200 bar). The online SFE-SFC-MS method was compared to offline methods where the samples were extracted with liquid solvents at atmospheric pressure or high pressure then analysed with SFC-MS. In all cases, offline methods showed significant contaminants (like the oleamide lubricant) issuing from laboratory plastic materials as nitrogen drying station, syringes and filters, while the online method allowed a complete elimination of laboratory contaminations. Furthermore, the online method saved time, solvents and laboratory consumables. It will also show that transferring a compressible fluid from a loading loop is favourable to high efficiency, as the resulting chromatographic peaks are much thinner than when transferring a liquid. Compared to injecting liquid heptane, the efficiency increase was 3.4-fold, while compared to injecting liquid methanol (a common practice in SFC), the efficiency increase was 13-fold. Finally, the additive composition of different laboratory gloves was compared.

Keywords:  Mass spectrometry; Medical devices; Plastic additives; Supercritical fluid chromatography; Supercritical fluid extraction

DOI:  https://doi.org/10.1016/j.chroma.2024.465323
J Am Soc Mass Spectrom. 2024 Sep 10.

Dynamics of the Aspiration of Charged Droplets into a LC-ESI-MS System.

Patricia Itzenhäuser, Ferdinand Max Wachter, Laura Lehmann, Michelle Rajkovic, Thorsten Benter, Walter Wißdorf.

Electrospray ionization (ESI) enables coupling between liquid chromatography (LC) and mass spectrometry (MS). Since it is a gentle ionization method, it is frequently used for the analysis of large biomolecules. In recent years, several experimental setups have demonstrated that the use of ESI results in the formation of charged droplets that are aspirated into the vacuum systems of mass spectrometers. This results in a variety of consequences, such as instrument contamination, which can impede the analytical performance. We investigate the signatures of aspirated charged droplets with a commercial LC-ESI-MS system at analytical conditions. Previous observations without LC coupling are reproduced and show that significant droplet aspiration is probably taking place at analytical LC-ESI-MS conditions. This common phenomenon likely decreases the instrument sensitivity. Analyte can be released by isolation and fragmentation of droplet fragments; thus, aspirated droplets can mask analyte even in the mass analyzer region. The complex morphology of droplet MS/MS mass spectra is highly reproducible at the same experimental conditions. This implies the existence of distinct molecular reaction pathways of the droplet fragments. To assess the effect of droplet aspiration on analytical applications, relevant method and ion source parameters, which are commonly varied during method optimization, were investigated. Further variations of the solvent composition revealed that the aspirated droplets and their fragmentation are particularly sensitive to the solvent composition and thus also to the LC solvent gradient in an analytical experiment.

DOI: https://doi.org/10.1021/jasms.4c00238