bims-metlip 2023-12-17 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2023–12–17
twenty-six papers selected by
Sofia Costa, Matterworks

Database-Driven Spatially Resolved Lipidomics Highlights Heterogeneous Metabolic Alterations in Type 2 Diabetic Mice.
Separation of bile acid isomer plays a pivotal role in bioequivalence evaluation of ursodeoxycholic acid.
A simple liquid chromatography method running in dual modes for quantification of short and medium chain fatty acids.
Deep Learning Based Metabolite Annotation.
A simple reliable quantification of glyphosate in human urine using MonoSpin TiO extraction and isotope dilution mass spectrometry.
[Determination of 14 β-agonists in animal meat by ultra high performance liquid chromatography-tandem mass spectrometry].
Mass spectrometry imaging of natural carbonyl products directly from agar-based microbial interactions using 4-APEBA derivatization.
[Simultaneous determination of 83 glucocorticoids in cosmetics by ultra performance liquid chromatography-tandem mass spectrometry].
An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure for the quantification of carbamazepine in human serum and plasma.
Data Processing of Product Ion Spectra: Redundancy of Product Ion Spectra of Small Molecules in Data-Dependent Acquisition Dataset.
Toward Omics-Scale Quantitative Mass Spectrometry Imaging of Lipids in Brain Tissue Using a Multiclass Internal Standard Mixture.
Sample preparation for suspect and non-target screening of xenobiotics in human biofluids by liquid chromatography-High resolution tandem mass spectrometry.
Development and validation of a sensitive UPLC-MS/MS analytical method for GFH009 in rat plasma and its application to toxicokinetics studies.
Simultaneous quantitation of urinary albumin and creatinine for rapid clinical albuminuria diagnostics using high-throughput paper spray mass spectrometry.
PMart Web Application: Marketplace for Interactive Analysis of Panomics Data.
Challenges in the Analysis of Gangliosides by LC-MS.
DeepRTAlign: toward accurate retention time alignment for large cohort mass spectrometry data analysis.
Application of a capillary electrophoresis-mass spectrometry metabolomics workflow in zebrafish larvae reveals new effects of cortisol.
Bioanalytical Method Development and Validation of Doxorubicin and Lapatinib in Rat Plasma Using UHPLC-HESI-LTQ-MS.
Combination of in silico prediction and convolutional neural network framework for targeted screening of metabolites from LC-HRMS fingerprints: A case study of "Pericarpium Citri Reticulatae - FructusAurantii".
Extraction and analysis by liquid chromatography - tandem mass spectrometry of intra- and extracellular microcystins and nodularin to study the fate of cyanobacteria and cyanotoxins across the freshwater-marine continuum.
Study of sample preparation influence on bacterial lipids profile in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
Development of a column switching for direct online enrichment and separation of polar and nonpolar analytes from aqueous matrices.
A Novel and Fast Online-SPE-LC-MS/MS Method to Quantify Thyroid Hormone Metabolites in Rat Plasma.
Challenges in ESI-MS-based Untargeted Metabolomics.
Sub-5-min RP-UHPLC-TIMS for high-throughput untargeted lipidomics and its application to multiple matrices.

Anal Chem. 2023 Dec 13.

Database-Driven Spatially Resolved Lipidomics Highlights Heterogeneous Metabolic Alterations in Type 2 Diabetic Mice.

Xinzhu Li, Qingce Zang, Ying Zhu, Xinyi Tu, Jialin Liu, Ting Li, Shiyu Zhu, Lingzhi Wang, Zeper Abliz, Ruiping Zhang.

Spatially resolved lipidomics is pivotal for detecting and interpreting lipidomes within spatial contexts using the mass spectrometry imaging (MSI) technique. However, comprehensive and efficient lipid identification in MSI remains challenging. Herein, we introduce a high-coverage, database-driven approach combined with air-flow-assisted desorption electrospray ionization (AFADESI)-MSI to generate spatial lipid profiles across whole-body mice. Using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), we identified 2868 unique lipids in the serum and various organs of mice. Subsequently, we systematically evaluated the distinct ionization properties of the lipids between LC-MS and MSI and created a detailed MSI database containing 14 123 ions. This method enabled the visualization of aberrant fatty acid and phospholipid metabolism across organs in a diabetic mouse model. As a powerful extension incorporated into the MSIannotator tool, our strategy facilitates the rapid and accurate annotation of lipids, providing new research avenues for probing spatially resolved heterogeneous metabolic changes in response to diseases.

DOI: https://doi.org/10.1021/acs.analchem.3c03765
J Pharm Biomed Anal. 2023 Nov 28. pii: S0731-7085(23)00651-9. [Epub ahead of print]239 115882

Separation of bile acid isomer plays a pivotal role in bioequivalence evaluation of ursodeoxycholic acid.

Yanbo Chang, Xuejing Li, Jinping Jiang, Lanlan Gui, Linfei Wan, Xiangxiang Zhou, Linchuan Liao, Kexin Li, Ke Lan.

  Based on our experiences in bile acid profiling, this work developed and validated a liquid chromatography electrospray ionization tandem mass spectrometry method to separate endogenous bile acid isomers and quantitatively determine ursodeoxycholic acid (UDCA), glycoursodeoxycholic acid (GUDCA) and tauroursodeoxycholic acid (TUDCA) in human plasma. The separation was performed on a CORTECS C18 column with the mobile phase consisting of 1.0 mM ammonium acetate and acetonitrile-methanol (80:20, v/v). UDCA, GUDCA and TUDCA were detected in the negative mode on a triple-quadrupole mass spectrometer at the ion transitions of m/z 391 > 391, m/z 448 > 74, m/z 498 > 80, respectively. Phosphate buffer was employed as the surrogate matrix to establish the isotope internal standard corrected calibration curves of analytes. The background-method with a linearity range of 10-200 ng/mL was partially validated to determine the endogenous levels of analytes in blank human plasma, which was incorporated into the validation of bioequivalence-method with a linearity range of 50-10000 ng/mL. The bioequivalence (BE)-method was fully validated with special focus on matrix effects, which have been critically evaluated using the precision and accuracy of quality control samples prepared from the blank human plasma of 12 individuals. It is disclosed for the first time that the BE results of UDCA formulation may yield false results when the method is insufficient to separate UDCA from isoursodeoxycholic acid, a microbial metabolite of both endogenous and exogenous UDCA. The present method has established a milestone for the evaluation of UDCA formulations and is expected to provide a valuable reference for the bioanalytical development of endogenous medicinal products.

Keywords:  Bioequivalence; Glycoursodeoxycholic acid; Isoursodeoxycholic acid; LC-MS; Pharmacokinetics; Tauroursodeoxycholic acid; Ursodeoxycholic acid

DOI:  https://doi.org/10.1016/j.jpba.2023.115882
J Chromatogr A. 2023 Dec 10. pii: S0021-9673(23)00791-4. [Epub ahead of print]1714 464566

A simple liquid chromatography method running in dual modes for quantification of short and medium chain fatty acids.

Dean Lee, Michael S Kerry.

  Sample preparation is often a rate-limiting step in quantification of short chain and medium chain fatty acids (SFAs and MFAs) in biological samples. A novel liquid chromatography (LC) method with simple sample preparation is introduced in this report. The method is performed on a column packed with polyvinyl alcohol sorbents. In separation of a mixture of bovine serum albumin (BSA) with SFAs and MFAs, the protein is eluted in size-exclusion mode in the first part of the chromatogram. SFAs and MFAs are then eluted in the second part of the chromatogram in reverse-phase mode where fatty acids with longer carbon lengths are eluted at longer retention times. SFAs and MFAs are well resolved from each other without pre-column derivatization. The size-exclusion mode affords good tolerance to macromolecules in sample matrixes. The dual mode separation allows sample injections without complicated sample preparation steps, such as derivatization, extraction, evaporation, and reconstitution.

Keywords:  Medium chain fatty acid; Reverse phase liquid chromatography; Short chain fatty acid; Size exclusion chromatography

DOI:  https://doi.org/10.1016/j.chroma.2023.464566
Annu Int Conf IEEE Eng Med Biol Soc. 2023 Jul;2023 1-4

Deep Learning Based Metabolite Annotation.

Hoi Yan Katharine Chau, Hongyu Ao, Xinran Zhang, Shijinqiu Gao, Rency S Varghese, Habtom W Ressom.

Metabolite annotation is a major bottleneck in untargeted metabolomics studies by liquid chromatography coupled with mass spectrometry (LC-MS). This is in part due to the limited publicly available spectral libraries, which consist of tandem mass spectrometry (MS/MS) data acquired from just a fraction of known compounds. Machine learning and deep learning methods provide the opportunity to predict molecular fingerprints based on MS/MS data. The predicted molecular fingerprints can then be used to help rank candidate metabolite IDs obtained based on predicted formula or measured precursor m/z of the unknown metabolite. This approach is particularly useful to help annotate metabolites whose corresponding MS/MS spectra cannot be matched with those in spectral libraries. We previously reported application of a convolutional neural network (CNN) for molecular fingerprint prediction using MS/MS spectra obtained from the MoNA repository and NIST 20. In this paper, we investigate high-dimensional representation of the spectral data and molecular fingerprints to improve accuracy in molecular fingerprint prediction.

DOI: https://doi.org/10.1109/EMBC40787.2023.10341007
J Pestic Sci. 2023 Nov 20. 48(4): 137-148

A simple reliable quantification of glyphosate in human urine using MonoSpin TiO extraction and isotope dilution mass spectrometry.

Takamitsu Otake, Keisuke Nakamura, Nobuyasu Hanari.

  A method of quantifying glyphosate (Gly) in human urine by means of MonoSpin TiO extraction and 9-fluorenylmethoxycarbonyl chloride (FMOC-Cl) derivatization with isotope dilution mass spectrometry (IDMS) was investigated and optimized. The method's quantification limit under optimized conditions was 0.3 µg/kg for FMOC-Gly, which was comparable to or lower than those described in previous studies. When a spike test using human urine samples was carried out with optimized analytical conditions, the trueness for FMOC-Gly was as follows: 101.6-104.9% for a spike level of 0.5 µg/kg and 99.2-101.0% for a spike level of 30 µg/kg. The intra-day repeatability and inter-day reproducibility were <6.5%. The spike test results for validation between the "with" and "without" derivatization methods were comparable at 1 µg/kg. Our results indicate that using MonoSpin TiO extraction and FMOC-Cl derivatization with IDMS is an accurate method for analyzing Gly in human urine.

Keywords:  FMOC-Cl derivatization; MonoSpin TiO extraction; glyphosate; human urine; isotope dilution mass spectrometry (IDMS)

DOI:  https://doi.org/10.1584/jpestics.D23-030
Se Pu. 2023 Dec;41(12): 1106-1114

[Determination of 14 β-agonists in animal meat by ultra high performance liquid chromatography-tandem mass spectrometry].

Jieqiong Dong, Jin Xiao, Xin Zhou, Ning Li, Xuesong Wang, Junjie Kang.

  The addition of β-agonists to animal feed can significantly improve the lean-meat rate of pigs, cattle, sheep, and other animals. However, the food residues of β-agonists are harmful to human health. When meat with β-agonist residues is consumed, poisoning symptoms such as palpitation, dizziness, and muscle tremors may develop, and damage to the cardiovascular system, liver, and kidney may occur. In this study, a method based on ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was established for the rapid detection of 14 β-agonists (clenbuterol, salbutamol, ractopamine, clorprenaline, terbutaline, tulobuterol, bromobuterol, bambuterol, zilpaterol, mabuterol, fenoterol, arformoterol, cimaterol, and cimbuterol) in animal food sources. The sample pretreatment method and chromatographic conditions were optimized. The samples were hydrolyzed with β-glucuronidase hydrochloride/aryl sulfate esterase in ammonium acetate buffer (pH 5.2). Enzymatic hydrolysis was performed in a constant-temperature water bath ((36±2) ℃) oscillator for 16 h. The samples were cooled to room temperature and extracted with 0.5% formic acid acetonitrile. NaCl was added to separate the organic and aqueous phases, and 5 mL of the upper organic layer was purified using a one-step purification solid-phase extraction column. After drying with nitrogen at 50 ℃, the residue was dissolved in 0.4 mL of 0.2% formic acid aqueous solution. The samples were passed through a 0.22 μm filter and detected by UHPLC-MS/MS with gradient elution using acetonitrile and 0.1% formic acid aqueous solution as the mobile phases. The analytes were separated on a Phenomenex Kinetex F5 column and detected by positive-ion scanning in multiple-reaction monitoring (MRM) mode. Internal and external standard methods were used for quantitative analysis. The effects of the extract pH, solid-phase extraction column, purification method, and dissolved solution on the extraction efficiency were optimized during pretreatment. UHPLC-quadrupole time-of-flight MS was used to verify the purification effect of the one-step purification solid-phase extraction column, and the results indicated that this type of column could remove most of the phospholipids, sphingolipids, and glycerides in the sample extract. The factors influencing the different chromatographic columns and mobile phases were investigated. MS scanning was conducted in positive-ion mode with needle pump injection in mass-only mode, and the two daughter ions with the highest responses for each target were selected as the quantitative and qualitative ions. The declustering potential (DP) and collision energy (CE) of each ion were separately optimized in MRM mode. The switching mode of the mass spectrum and waste liquid was used, and the mobile phase was switched to waste liquid after all the target peaks were removed. These steps ensured that impurities in the sample flowed out of the column in a timely manner and that the effects of excessive impurities on the mass spectra were avoided. The 14 β-agonists showed good linear relationships in the range of 1.0-50 μg/L, with correlation coefficients of >0.99. The limits of detection (LODs) and quantification (LOQs) were in the range of 0.1-0.2 and 0.3-0.6 μg/kg, respectively. The average recoveries of the 14 β-agonists ranged from 70.25% to 117.48%, with relative standard deviations (RSDs) in the range of 0.63%-14.29% at low, medium, and high spiked levels. Pork, beef, and mutton samples were selected and analyzed using the developed method. The results were close to those of the national standard method, indicating that the method is accurate and reliable. Moreover, the proposed method has good stability and high accuracy; thus, it is suitable for the qualitative and quantitative determination of β-agonists in animal meat.

Keywords:  animal meat; one-step purification solid-phase column; ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS); β-agonist

DOI:  https://doi.org/10.3724/SP.J.1123.2023.03008
mSystems. 2023 Dec 08. e0080323

Mass spectrometry imaging of natural carbonyl products directly from agar-based microbial interactions using 4-APEBA derivatization.

Dušan Veličković, Kevin J Zemaitis, Arunima Bhattacharjee, Christopher R Anderton.

   IMPORTANCE: The metabolic profiles within microbial biofilms and interkingdom interactions are extremely complex and serve a variety of functions, which include promoting colonization, growth, and survival within competitive and symbiotic environments. However, measuring and differentiating many of these molecules, especially in an in situ fashion, remains a significant analytical challenge. We demonstrate a chemical derivatization strategy that enabled highly sensitive, multiplexed mass spectrometry imaging of over 300 metabolites from a model microbial co-culture. Notably, this approach afforded us to visualize over two dozen classes of ketone-, aldehyde-, and carboxyl-containing molecules, which were previously undetectable from colonies grown on agar. We also demonstrate that this chemical derivatization strategy can enable the discrimination of isobaric and isomeric metabolites without the need for orthogonal separation (e.g., online chromatography or ion mobility). We anticipate that this approach will further enhance our knowledge of metabolic regulation within microbiomes and microbial systems used in bioengineering applications.

Keywords:  Bacillus subtilis; Fusarium; MALDI; aldehydes; carboxylic acids; ketones; metabolomics

DOI:  https://doi.org/10.1128/msystems.00803-23
Se Pu. 2023 Dec;41(12): 1084-1094

[Simultaneous determination of 83 glucocorticoids in cosmetics by ultra performance liquid chromatography-tandem mass spectrometry].

Qianru Zhao, Hua Liu, Yaping Meng, Xiang Li, Ruifang Gao, Xiangsheng Li.

  Glucocorticoids, which are a class of steroidal hormones secreted by the adrenal cortex, have significant anti-inflammatory, immunosuppressive, and anti-allergic effects. Thus, these compounds are widely used in clinical practice. However, the long-term use of cosmetics containing glucocorticoids can lead to serious consequences, such as hormone-dependent dermatitis, hypertension, and other serious injuries. The Safety and Technical Specification for Cosmetics (2015 edition) and Regulation (EC) No. 1223/2009 of the European Parliament and Council on cosmetic products list glucocorticoids as prohibited raw materials. According to the National Medical Products Administration, reports on the illegal addition of glucocorticoids to cosmetics by manufacturers have increased in recent years. Therefore, establishing high-throughput screening methods to ensure the quality and safety of cosmetics is imperative. In this study, a comprehensive analytical method based on ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for the rapid screening of 83 glucocorticoids in cosmetics. A series of conditions were optimized using three matrices that are commonly used in cosmetics: water, lotion, and cream (o/w-type). Four mobile-phase systems and three chromatographic columns were then optimized to achieve the best separation effects. Various MS parameters, such as the capillary voltages, cone voltages, desolvation gas flow rates, and collision energies of the ion pairs of the target compounds, were also optimized. Furthermore, pretreatment was essential for glucocorticoid determination owing to the complex matrix effects of cosmetics. The analytes were divided into two groups, with lg Kow=4 as the limit, to compare the effects of the extraction solvent on recoveries. The extraction recoveries of target analytes with six extraction methods, namely, extraction with acetonitrile, extraction with acetone, extraction with ethyl acetate, dispersion in saturated sodium chloride solution followed by extraction with acetonitrile, dispersion in saturated sodium chloride solution followed by extraction with acetone, and dispersion in saturated sodium chloride solution followed by extraction with ethyl acetate, were compared. The recoveries from QuEChERS and solid-phase extraction (SPE) purification were also compared. Based on the experimental results, the final sample pretreatment method included acetonitrile vortex dispersion, ultrasonic extraction, and sample loading after filtration. The 83 target compounds were separated on a Thermo Accucore PFP column (100 mm×2.1 mm, 2.6 μm) with 0.1% (v/v) acetic acid in acetonitrile and 0.1% (v/v) acetic acid in water as the mobile phases. The analytes were determined by dynamic multiple-reaction monitoring (MRM) in electrospray positive ionization mode (ESI+) and quantified using the external standard method. Matrix standard curves were used to reduce matrix effects. The calibration curves of the 83 target compounds were linear in the mass concentration range of 2-200 μg/L (r>0.995). At three levels of addition, the recoveries were 74.5%-112.4%, and the relative standard deviations (RSDs, n=6) were 0.8%-9.9%. The limits of detection (LODs, S/N≥3) were 0.001-0.023 μg/g, and the limits of quantification (LOQs, S/N≥10) were 0.002-0.076 μg/g. The developed method was used to detect glucocorticoids in 41 cosmetic samples. Fluocinolone acetonide, beclomethasone dipropionate, desonide 21-acetate, and desonide were detected in four samples. The content range of glucocorticoids in the positive samples was 0.53-634.27 μg/g. Notably, desonide 21-acetate, which is not included in the scope of the statutory detection method, was detected in two batches of samples. In conclusion, the proposed method is simple, sensitive, reliable, and suitable for the high-throughput analysis of the 83 glucocorticoids in cosmetics with different matrices. This method could provide reliable technical support for the daily supervision of cosmetics and serve as a supplement to current glucocorticoid standards.

Keywords:  cosmetics; glucocorticoids; illegal addition; ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)

DOI:  https://doi.org/10.3724/SP.J.1123.2023.04009
Clin Chem Lab Med. 2023 Dec 13.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure for the quantification of carbamazepine in human serum and plasma.

Tobias Schierscher, Linda Salzmann, Neeraj Singh, Martina Bachmann, Friederike Bauland, Andrea Geistanger, Lorenz Risch, Christian Geletneky, Christoph Seger, Judith Taibon.

   OBJECTIVES: An isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) was developed and validated to accurately measure serum and plasma concentrations of carbamazepine.
METHODS: Quantitative nuclear magnetic resonance (qNMR) spectroscopy was used to determine the absolute content of the reference material, ensuring its traceability to SI units. The separation of carbamazepine from potential interferences, whether known or unknown, was achieved using a C18 column. A protein precipitation protocol followed by a high dilution step was established for sample preparation. Assay validation and determination of measurement uncertainty were performed in accordance with the guidelines of the Clinical and Laboratory Standards Institute, the International Conference on Harmonization (ICH), and the Guide to the Expression of Uncertainty in Measurement (GUM). In order to demonstrate equivalence to the already existing RMP a method comparison study was performed.
RESULTS: The RMP was proven to be highly selective and specific with no evidence of a matrix effect, allowing for quantification of carbamazepine within the range of 0.800-18.0 μg/mL. Intermediate precision and repeatability (n=60 measurements) was found to be <1.6 % and <1.3 % over all concentration levels and independent from the matrix. The relative mean bias ranged from -0.1 to 0.6 % for native serum and from -0.3 to -0.1 % for Li-heparin plasma levels. The measurement uncertainties for single measurements and target value assignment were found to be <1.8 % and <1.3 %, respectively. Method comparison showed a good agreement between the Joint Committee of Traceability in Laboratory Medicine (JCTLM) listed RMP and the candidate RMP resulting in a Passing-Bablok regression equation with a slope of 1.01 and an intercept of -0.01. The bias in the patient cohort was found to be 0.9 %.
CONCLUSIONS: We present a novel LC-MS/MS-based candidate RMP for carbamazepine in human serum and plasma which provides a traceable and reliable platform for the standardization of routine assays and evaluation of clinically relevant samples.

Keywords:  ID-LC-MS/MS; carbamazepine; qNMR; reference measurement procedure; standardization; traceability

DOI:  https://doi.org/10.1515/cclm-2023-0943
Mass Spectrom (Tokyo). 2023 ;12(1): A0138

Data Processing of Product Ion Spectra: Redundancy of Product Ion Spectra of Small Molecules in Data-Dependent Acquisition Dataset.

Fumio Matsuda.

  Non-targeted metabolome analysis studies comprehensively acquire product ion spectra from the observed ions by the data-dependent acquisition (DDA) mode of tandem mass spectrometry (MS). A DDA dataset redundantly contains closely similar product ion spectra of metabolites commonly existing among the biological samples analyzed in a metabolome study. Moreover, a single DDA data file often includes two or more closely similar raw spectra obtained from an identical precursor ion. The redundancy of product ion spectra has been used to generate an averaged product ion spectrum from a set of similar product ion spectra recorded in a DDA dataset. The spectral averaging improved the accuracy of m/z values and signal-to-noise levels of product ion spectra. However, the origins of redundancy, variations among datasets, and these effects on the spectral averaging procedure needed to be better characterized. This study investigated the nature of the redundancy by comparing the averaging results of eight DDA datasets of non-targeted metabolomics studies. The comparison revealed a significant variation in redundancy among datasets. The DDA datasets obtained by the quadrupole (Q)-Orbitrap-MS datasets had more significant intrafile redundancy than that of the Q-time-of-flight-MS. For evaluating the similarity score between two production spectra, the optimal threshold level of the cosine-product method was approximately 0.8-0.9. Moreover, contamination of biological samples such as plasticizers was another origin of spectral redundancy. The results will be the basis for further development of methods for processing of product ion spectra data. Copyright © 2023 Fumio Matsuda. This is an open-access article distributed under the terms of Creative Commons Attribution Non-Commercial 4.0 International License, which permits use, distribution, and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. Please cite this article as: Mass Spectrom (Tokyo) 2023; 12(1): A0138.

Keywords:  data-dependent acquisition mode; metabolomics; redundancy; spectra averaging; spectra similarity

DOI:  https://doi.org/10.5702/massspectrometry.A0138
Anal Chem. 2023 Dec 11.

Toward Omics-Scale Quantitative Mass Spectrometry Imaging of Lipids in Brain Tissue Using a Multiclass Internal Standard Mixture.

Michiel Vandenbosch, Shadrack M Mutuku, Maria José Q Mantas, Nathan H Patterson, Tucker Hallmark, Marc Claesen, Ron M A Heeren, Nathan G Hatcher, Nico Verbeeck, Kim Ekroos, Shane R Ellis.

Mass spectrometry imaging (MSI) has accelerated our understanding of lipid metabolism and spatial distribution in tissues and cells. However, few MSI studies have approached lipid imaging quantitatively and those that have focused on a single lipid class. We overcome this limitation by using a multiclass internal standard (IS) mixture sprayed homogeneously over the tissue surface with concentrations that reflect those of endogenous lipids. This enabled quantitative MSI (Q-MSI) of 13 lipid classes and subclasses representing almost 200 sum-composition lipid species using both MALDI (negative ion mode) and MALDI-2 (positive ion mode) and pixel-wise normalization of each lipid species in a manner analogous to that widely used in shotgun lipidomics. The Q-MSI approach covered 3 orders of magnitude in dynamic range (lipid concentrations reported in pmol/mm2) and revealed subtle changes in distribution compared to data without normalization. The robustness of the method was evaluated by repeating experiments in two laboratories using both timsTOF and Orbitrap mass spectrometers with an ∼4-fold difference in mass resolution power. There was a strong overall correlation in the Q-MSI results obtained by using the two approaches. Outliers were mostly rationalized by isobaric interferences or the higher sensitivity of one instrument for a particular lipid species. These data provide insight into how the mass resolving power can affect Q-MSI data. This approach opens up the possibility of performing large-scale Q-MSI studies across numerous lipid classes and subclasses and revealing how absolute lipid concentrations vary throughout and between biological tissues.

DOI: https://doi.org/10.1021/acs.analchem.3c02724
MethodsX. 2024 Jun;12 102501

Sample preparation for suspect and non-target screening of xenobiotics in human biofluids by liquid chromatography-High resolution tandem mass spectrometry.

I Baciero-Hernández, M Musatadi, M Olivares, A Prieto, N Etxebarria, O Zuloaga.

  The concept of exposome covers all the exposures an individual suffers from conception to death, which can be partially assessed through the monitoring of human biofluids. In there, target analytical approaches tend to focus on a limited set of xenobiotics, whereas exposomic studies need broad scopes in search of a full understanding. Given the issue, suspect and non-target screening are feasible alternatives. However, adequate sample preparation procedures should minimize interferences without significantly reducing the number of xenobiotics. Within this context, the present article aims to describe comprehensive sample preparation procedures for suspect or non-target screening of organic xenobiotics in several human biofluids, all coupled to unified separation and detection conditions based on ultra-high performance liquid chromatography-high resolution tandem mass spectrometry (UHPLC-HRMS/MS). The referred biofluids consist of human urine, breast milk, saliva and ovarian follicular fluid.•Analytical methods for untargeted analysis of a wide range of xenobiotics in human biofluids are fully described in order to ensure reproducibility.•The sample preparation procedures balance selectivity and sensitivity.•Unified analysis conditions allow simultaneous analysis of diverse biofluids.

Keywords:  Biomonitoring; Breast milk; Extraction & clean-up; Follicular fluid; Human exposome; LC-HRMS/MS analysis; Saliva; Suspect and non-target screening of xenobiotics in human biofluids; Urine

DOI:  https://doi.org/10.1016/j.mex.2023.102501
Biomed Chromatogr. 2023 Dec 11. e5788

Development and validation of a sensitive UPLC-MS/MS analytical method for GFH009 in rat plasma and its application to toxicokinetics studies.

Qiang Liu, Jin-Zhu Zhao, Fu-Sheng Zhou, Jiong Lan, Qiang Lu, Hong-Can Ren.

  GFH009 is a potent, highly selective, small molecule that targets and inhibits the activity of the CDK9/cyclin T1 regulatory complex of P-TEFb. This study aimed to develop and validate a highly selective and sensitive ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for precise quantification of GFH009 in rat plasma. This method was subsequently employed for conducting toxicokinetic studies of GFH009 in rats. Plasma was prepared using a simple protein precipitation method by acetonitrile. Chromatographic separation of the analytes was achieved on a BEH C18 analytical column with a rapid 3.0 min run time and a flow rate of 0.5 ml/min. The calibration curves for plasma samples exhibited excellent linearity over a wide concentration range of 1.0-1,000 ng/ml for GFH009. Intra- and inter-day accuracies were within 92.7-105.7%, and precisions were no more than 6.7%. Furthermore, the analyte demonstrated stability under four different storage conditions, with variations of <15.0%. This study pioneers a methodological innovation by introducing a highly reliable, specific and sensitive analytical method for GFH009 in rat plasma. The successful application of this method in toxicokinetic studies further underscores its significance, offering valuable insights for the methodology of clinical pharmacokinetic research.

Keywords:  CDK9 inhibitor; UHPLC-MS/MS; toxicokinetic

DOI:  https://doi.org/10.1002/bmc.5788
Analyst. 2023 Dec 14.

Simultaneous quantitation of urinary albumin and creatinine for rapid clinical albuminuria diagnostics using high-throughput paper spray mass spectrometry.

Igor Pereira, Jason L Robinson, Chris G Gill.

Albuminuria is a clinical condition associated with poor kidney function, diagnosed by determining the ratio of albumin to creatinine concentrations in patient urine samples. We present a high-throughput paper spray mass spectrometry (PS-MS) method for simultaneous quantitation of urinary albumin and creatinine for potential diagnosis of albuminuria. Minimal (urine dilution) or no sample preparation is required. The analytical performance of the method was evaluated, achieving linear calibration curves (R2 > 0.99) with little inter-day variability in the slope (N = 5 days), exhibiting coefficient of variation (CV) of 8% and 3% for albumin and creatinine, respectively. LOD and LOQ for albumin were 2.1 and 7.0 mg L-1, and for creatinine were 0.01 and 0.03 mmol L-1, respectively. Intra- and inter-day (N = 5) precisions (%CV) and accuracies (%bias) were <10% and ±11%, respectively, for both analytes. The method was applied to determine albumin-to-creatinine ratios in anonymous human patient urine samples (N = 56), and a correlation of R2 = 0.9744 was achieved between the PS-MS results and validated clinical method results. This work demonstrates the utility of PS-MS to simultaneously quantify a large (albumin) and a small (creatinine) molecule directly in patient urine samples, and its potential as a tool for clinical albuminuria diagnostics.

DOI: https://doi.org/10.1039/d3an01855e
J Proteome Res. 2023 Dec 12.

PMart Web Application: Marketplace for Interactive Analysis of Panomics Data.

Kelly G Stratton, Daniel M Claborne, David J Degnan, Rachel E Richardson, Amanda M White, Lee Ann McCue, Bobbie-Jo M Webb-Robertson, Lisa M Bramer.

  PMart is a web-based tool for reproducible quality control, exploratory data analysis, statistical analysis, and interactive visualization of 'omics data, based on the functionality of the pmartR R package. The newly improved user interface supports more 'omics data types, additional statistical capabilities, and enhanced options for creating downloadable graphics. PMart supports the analysis of label-free and isobaric-labeled (e.g., TMT, iTRAQ) proteomics, nuclear magnetic resonance (NMR) and mass-spectrometry (MS)-based metabolomics, MS-based lipidomics, and ribonucleic acid sequencing (RNA-seq) transcriptomics data. At the end of a PMart session, a report is available that summarizes the processing steps performed and includes the pmartR R package functions used to execute the data processing. In addition, built-in safeguards in the backend code prevent users from utilizing methods that are inappropriate based on omics data type. PMart is a user-friendly interface for conducting exploratory data analysis and statistical comparisons of omics data without programming.

Keywords:  RNA-seq; exploratory data analysis; lipidomics; metabolomics; proteomics; reproducibility; software; statistics; visualization; web service

DOI:  https://doi.org/10.1021/acs.jproteome.3c00512
Chimia (Aarau). 2022 Feb 23. 76(1-2): 109-113

Challenges in the Analysis of Gangliosides by LC-MS.

Thomas Muggli, Claudia Bühr, Stefan Schürch.

  Gangliosides are a family of conjugates consisting of a polar sialoglycan head group and a hydrophobic ceramide tail. Gangliosides are of high abundance in neuronal tissues and are involved in numerous biological processes, such as cell-cell recognition, adhesion, and signal transduction. Alteration of the ganglioside profile is associated with various neurodegenerative diseases and there is indication that gangliosides are involved in the pathogenesis of Parkinson's and Huntington's disease. The development of refined methods for the analysis of gangliosides by high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS) has supported research with qualitative and quantitative data. However, the amphiphilic character of gangliosides renders their separation and mass spectrometric analysis challenging. In this article, the strengths of hydrophilic interaction liquid chromatography (HILIC) for baseline separation of gangliosides, including two structural isomers, and their structural characterization by tandem mass spectrometry are demonstrated. The importance of ion source parameter optimization is highlighted to prevent misleading ganglioside transformation due to in-source dissociation.

Keywords:  Gangliosides; Glycosphingolipids; HILIC; In-source dissociation; Mass spectrometry

DOI:  https://doi.org/10.2533/chimia.2022.109
Nat Commun. 2023 Dec 11. 14(1): 8188

DeepRTAlign: toward accurate retention time alignment for large cohort mass spectrometry data analysis.

Yi Liu, Yun Yang, Wendong Chen, Feng Shen, Linhai Xie, Yingying Zhang, Yuanjun Zhai, Fuchu He, Yunping Zhu, Cheng Chang.

Retention time (RT) alignment is a crucial step in liquid chromatography-mass spectrometry (LC-MS)-based proteomic and metabolomic experiments, especially for large cohort studies. The most popular alignment tools are based on warping function method and direct matching method. However, existing tools can hardly handle monotonic and non-monotonic RT shifts simultaneously. Here, we develop a deep learning-based RT alignment tool, DeepRTAlign, for large cohort LC-MS data analysis. DeepRTAlign has been demonstrated to have improved performances by benchmarking it against current state-of-the-art approaches on multiple real-world and simulated proteomic and metabolomic datasets. The results also show that DeepRTAlign can improve identification sensitivity without compromising quantitative accuracy. Furthermore, using the MS features aligned by DeepRTAlign, we trained and validated a robust classifier to predict the early recurrence of hepatocellular carcinoma. DeepRTAlign provides an advanced solution to RT alignment in large cohort LC-MS studies, which is currently a major bottleneck in proteomics and metabolomics research.

DOI: https://doi.org/10.1038/s41467-023-43909-5
Electrophoresis. 2023 Dec 10.

Application of a capillary electrophoresis-mass spectrometry metabolomics workflow in zebrafish larvae reveals new effects of cortisol.

Marlien van Mever, Maricruz Mamani-Huanca, Erin Faught, Ángeles López-Gonzálvez, Thomas Hankemeier, Coral Barbas, Marcel J M Schaaf, Rawi Ramautar.

  In contemporary biomedical research, the zebrafish (Danio rerio) is increasingly considered a model system, as zebrafish embryos and larvae can (potentially) fill the gap between cultured cells and mammalian animal models, because they can be obtained in large numbers, are small and can easily be manipulated genetically. Given that capillary electrophoresis-mass spectrometry (CE-MS) is a useful analytical separation technique for the analysis of polar ionogenic metabolites in biomass-limited samples, the aim of this study was to develop and assess a CE-MS-based analytical workflow for the profiling of (endogenous) metabolites in extracts from individual zebrafish larvae and pools of small numbers of larvae. The developed CE-MS workflow was used to profile metabolites in extracts from pools of 1, 2, 4, 8, 12, 16, 20, and 40 zebrafish larvae. For six selected endogenous metabolites, a linear response (R2 > 0.98) for peak areas was obtained in extracts from these pools. The repeatability was satisfactory, with inter-day relative standard deviation values for peak area of 9.4%-17.7% for biological replicates (n = 3 over 3 days). Furthermore, the method allowed the analysis of over 70 endogenous metabolites in a pool of 12 zebrafish larvae, and 29 endogenous metabolites in an extract from only 1 zebrafish larva. Finally, we applied the optimized CE-MS workflow to identify potential novel targets of the mineralocorticoid receptor in mediating the effects of cortisol.

Keywords:  capillary electrophoresis-mass spectrometry; glucocorticoid receptor; metabolomics; mineralocorticoid receptor; zebrafish larvae

DOI:  https://doi.org/10.1002/elps.202300186
J Chromatogr Sci. 2023 Dec 12. pii: bmad090. [Epub ahead of print]

Bioanalytical Method Development and Validation of Doxorubicin and Lapatinib in Rat Plasma Using UHPLC-HESI-LTQ-MS.

Shaik Khaja Moinuddin, Pirangi Srikanth, Parul Sharma, Sukhendu Nandi.

Cancer is considered a silent killer. The complexity of cancer makes it earn that title. So far there are only a few approaches to treat cancer. Among them, chemotherapy is considered the best approach. Many chemotherapeutical compounds are commercially available in the market. Among them, doxorubicin (DOX) and lapatinib (LAP) are considered blockbuster molecules. However, DOX suffers from poor bioavailability and exhibits cardiotoxicity. Interestingly, a fixed dose combination of DOX and LAP significantly decreases the cardiotoxic effect of DOX. To enhance the oral bioavailability of DOX and to avail the synergistic effect of LAP, many formulations have been made. To quantify both compounds in any formulation or biological matrix, an Liquid chromatography-Mass Spectrometry (LC-MS) method is required. In this present study, a simple and rapid Ultra High-Performance Liquid Chromatography - Heated Electron Spray Ionization - Mass Spectrometry (UHPLC-HESI-MS) bioanalytical method was developed. The developed method was validated as per the regulatory guidelines. The validated bioanalytical method had a lower limit of quantification of 0.75 ng. A simple protein precipitation technique was optimized to extract the compounds from the rat plasma. All the validation parameters were found to be within the limits as per the regulatory guidelines. A novel and rapid analytical method was successfully developed and validated. This developed method can be used to quantify the DOX and LAP in any formulation and biological matrix.

DOI: https://doi.org/10.1093/chromsci/bmad090
Talanta. 2023 Dec 05. pii: S0039-9140(23)01265-1. [Epub ahead of print]269 125514

Combination of in silico prediction and convolutional neural network framework for targeted screening of metabolites from LC-HRMS fingerprints: A case study of "Pericarpium Citri Reticulatae - FructusAurantii".

Jun Zeng, Yaping Li, Chuanlin Wang, Sheng Fu, Min He.

  In this study, a novel approach is introduced, merging in silico prediction with a Convolutional Neural Network (CNN) framework for the targeted screening of in vivo metabolites in Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) fingerprints. Initially, three predictive tools, supplemented by literature, identify potential metabolites for target prototypes derived from Traditional Chinese Medicines (TCMs) or functional foods. Subsequently, a CNN is developed to minimize false positives from CWT-based peak detection. The Extracted Ion Chromatogram (EIC) peaks are then annotated using MS-FINDER across three levels of confidence. This methodology focuses on analyzing the metabolic fingerprints of rats administered with "Pericarpium Citri Reticulatae - Fructus Aurantii" (PCR-FA). Consequently, 384 peaks in positive mode and 282 in negative mode were identified as true peaks of probable metabolites. By contrasting these with "blank serum" data, EIC peaks of adequate intensity were chosen for MS/MS fragment analysis. Ultimately, 14 prototypes (including flavonoids and lactones) and 40 metabolites were precisely linked to their corresponding EIC peaks, thereby providing deeper insight into the pharmacological mechanism. This innovative strategy markedly enhances the chemical coverage in the targeted screening of LC-HRMS metabolic fingerprints.

Keywords:  Convolutional neural network; Fructus Aurantii; In silico mass spectra; LC-HRMS; Metabolite prediction; Pericarpium citri Reticulatae; Targeted screening

DOI:  https://doi.org/10.1016/j.talanta.2023.125514
Toxicon. 2023 Dec 07. pii: S0041-0101(23)00362-8. [Epub ahead of print]237 107551

Extraction and analysis by liquid chromatography - tandem mass spectrometry of intra- and extracellular microcystins and nodularin to study the fate of cyanobacteria and cyanotoxins across the freshwater-marine continuum.

Damien Réveillon, Maxime Georges des Aulnois, Véronique Savar, Elise Robert, Amandine M N Caruana, Enora Briand, Myriam Bormans.

  The presence of microcystins (MCs) is increasingly being reported in coastal areas worldwide. To provide reliable data regarding this emerging concern, reproducible and accurate methods are required to quantify MCs in salt-containing samples. Herein, we characterized methods of extraction and analysis by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) for nine MCs and one nodularin (NOD) variants in both cyanobacteria (intracellular) and dissolved forms (extracellular). Different approaches have been used to cope with salinity for the extraction of dissolved MCs but none assessed solid phase extraction (SPE) so far. It was found that salt had negligible effect on the SPE recovery of dissolved MCs using the C18 cartridge while an overestimation up to 67% was noted for some variants with a polymeric sorbent. The limits of detection (LOD) and quantification (LOQ) were 1.0-22 and 5.5-124 pg on column for the intracellular toxins, while 0.05-0.81 and 0.13-2.4 ng/mL were obtained for dissolved toxins. Extraction recoveries were excellent for intracellular (89-121%) and good to excellent for extracellular cyanotoxins (73-102%) while matrix effects were considered neglectable (<12% for 16/20 toxin-matrix combinations), except for the two MC-RR variants. The strategy based on the application of a corrective factor to compensate for losses proved useful as the accuracy was satisfactory (73-117% for intra- and 81-139% for extracellular cyanotoxins, bias <10% for 46/60 conditions, with a few exceptions), with acceptable precisions (intra- and inter-days variabilities <11%). We then applied this method on natural colonies of Microcystis spp. subjected to a salt shock, mimicking their estuarine transfer, in order to assess their survival and to quantify their toxins. The colonies of Microcystis spp. had both their growth and photosynthetic activity impaired at salinities from 10, while toxins remained mainly intracellular (>76%) even at salinity 20, suggesting a potential health risk and contamination of estuarine organisms.

Keywords:  Coastal water; Colonial Microcystis; LC-MS/MS; Microcystins; Salinity; Solid phase extraction

DOI:  https://doi.org/10.1016/j.toxicon.2023.107551
Lipids. 2023 Dec 07.

Study of sample preparation influence on bacterial lipids profile in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Adrian Arendowski, Ewelina Sibińska, Wioletta Miśta, Piotr Fijałkowski, Michał Złoch, Dorota Gabryś, Paweł Pomastowski.

  Lipids are one of the cell components therefore it is important to be able to accurately assess them. One of the analytical techniques used to study lipid profiles is matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS). The present study attempted to select optimal conditions for sample preparation and MALDI MS analysis of bacterial lipidome in both positive and negative ion modes using different extraction protocols-Folch, Matyash, and Bligh & Dyer, solvents used to apply samples, and matrices such as 9-aminoacridine (9-AA), α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), 2-mercaptobenzothiazole (MBT), and 2,4,6-trihydroxyacetophenone (THAP). The obtained results allowed concluding that DHB or CHCA matrices are suitable for lipid analysis in the positive mode, and in the negative mode THAP or 9-AA. The most appropriate protocol for extracting lipids from bacterial cells was the Bligh & Dyer method in both ionization modes. The use of the solvent TA30, which was a mixture of acetonitrile and 0.1% trifluoroacetic acid in water, provided on the spectra a significant number of signals from lipids in all groups analyzed, such as fatty acyls, glycerolipids, and glycerophospholipids.

Keywords:  MALDI; extracts; lipids; mass spectrometry; matrix; microorganisms

DOI:  https://doi.org/10.1002/lipd.12383
J Chromatogr A. 2023 Dec 02. pii: S0021-9673(23)00779-3. [Epub ahead of print]1714 464554

Development of a column switching for direct online enrichment and separation of polar and nonpolar analytes from aqueous matrices.

Kjell Kochale, Ricardo Cunha, Thorsten Teutenberg, Torsten C Schmidt.

  Trace substances in surface waters may threaten health and pose a risk for the aquatic environment. Moreover, separation and detection by instrumental analysis is challenging due to the low concentration and the wide range of polarities. Separation of polar and nonpolar analytes can be achieved by using stationary phases with different selectivity. Lower limits of detection of trace substances can be obtained by offline enrichment on solid phase materials. However, these practices require substantial effort and are time consuming and costly. Therefore, in this study, a column switching was developed to enrich and separate both polar and nonpolar analytes by an on-column large volume injection of aqueous samples. The column switching can significantly reduce the effort and time for analyzing trace substances without compromising on separation and detection. A reversed phase (RP) column is used to trap the nonpolar analytes. The polar analytes are enriched on a porous graphitized carbon column (PGC) coupled serially behind the RP column. A novel valve switching system is implemented to enable elution of the nonpolar analytes from the RP column and, subsequently, elution of polar analytes from the PGC column and separation on a hydrophilic interaction liquid chromatography (HILIC) column. To enable separation of polar analytes dissolved in an aqueous matrix by HILIC, the water plug that is flushed from the PGC column is diluted by dosing organic solvent directly upstream of the HILIC column. The developed method was tested by applying target analysis and non-target screening, highlighting the advantage to effectively separate and detect both polar and nonpolar compounds in a single chromatographic run. In the target analysis, the analytes, with a logD at pH 3 ranging from -2.8 to + 4.5, could be enriched and separated. Besides the 965 features in the RP phase, 572 features from real wastewater were observed in the HILIC phase which would otherwise elute in the void time in conventional one-dimensional RP methods.

Keywords:  Column switching; Large volume injection; Non-target screening; Porous graphitic carbon; Water analysis

DOI:  https://doi.org/10.1016/j.chroma.2023.464554
Chem Res Toxicol. 2023 Dec 11.

A Novel and Fast Online-SPE-LC-MS/MS Method to Quantify Thyroid Hormone Metabolites in Rat Plasma.

Christiane Hindrichs, Tilmann Walk, Stephanie Melching-Kollmuss, Robert Landsiedel, Hennicke Kamp, Dorothee Funk-Weyer.

Since the focus in regulatory toxicology has drifted toward the identification of endocrine disruptors, the improvement in determination of alterations in the thyroid hormone system has become more important. THs are involved in several molecular processes important for a proper pre- and postnatal development so that disturbances can inter alia lead to incorrect brain maturation and/or disturbed metabolic processes (thermogenesis or lipolysis). In this publication, a new automated online solid-phase extraction (SPE)-liquid chromatography (LC)-tandem mass spectrometry (MS/MS, xLC-MS/MS) is introduced which simultaneously analyzes total T4, T3, rT3, T2, and T1. Method validation parameters are presented, and the method was positively verified by analyzing control and PTU-treated rat plasma samples (time points day 7, 14, and 28) for their total TH content. The obtained results were compared to published results by using a radioimmunoassay method. The automated SPE system ensures a consistent unified sample preparation, and this method overall showed sufficient specificity and accuracy to detect the given analytes in rat plasma. For the preparation of 50 μL of rat plasma, the following LOQs were established: 0.020 nM for T1, 0.029 nM for T2, 0.023 nM for rT3 and T3, and 3.22 nM for T4. This method is suitable to assess the identification of mechanisms leading to adverse effects, such as disturbed TH metabolism and regulation.

DOI: https://doi.org/10.1021/acs.chemrestox.3c00152
Chimia (Aarau). 2022 Feb 23. 76(1-2): 90-100

Challenges in ESI-MS-based Untargeted Metabolomics.

Elena Tobolkina, Víctor González-Ruiz, Isabel Meister, Miguel De Figueiredo, Davy Guillarme, Julien Boccard, Serge Rudaz.

  Untargeted metabolomics is now widely recognized as a useful tool for exploring metabolic changes taking place in biological systems under different conditions. In this article, we aim to provide a short overview of the liquid-phase separation methods hyphenated to MS to perform untargeted metabolomics of biological samples. Each approach is complemented by up-to-date literature to guide readers, as well as practical information for avoiding or fixing some of the most frequently encountered pitfalls. This article covers mainly data acquisition, but a short discussion is provided regarding signal processing and data treatment, as well as data analysis and its biological interpretation in the context of metabolomic studies.

Keywords:  Annotation; Capillary electrophoresis; Liquid chromatography; Mass spectrometry; Metabolomics; Supercritical fluid chromatography; Toxicological and doping analysis

DOI:  https://doi.org/10.2533/chimia.2022.90
Anal Bioanal Chem. 2023 Dec 11.

Sub-5-min RP-UHPLC-TIMS for high-throughput untargeted lipidomics and its application to multiple matrices.

Fabrizio Merciai, Manuela Giovanna Basilicata, Danila La Gioia, Emanuela Salviati, Vicky Caponigro, Tania Ciaglia, Simona Musella, Carlo Crescenzi, Eduardo Sommella, Pietro Campiglia.

  Untargeted lipidomics, with its ability to take a snapshot of the lipidome landscape, is an important tool to highlight lipid changes in pathology or drug treatment models. One of the shortcomings of most untargeted lipidomics based on UHPLC-HRMS is the low throughput, which is not compatible with large-scale screening. In this contribution, we evaluate the application of a sub-5-min high-throughput four-dimensional trapped ion mobility mass spectrometry (HT-4D-TIMS) platform for the fast profiling of multiple complex biological matrices. Human AC-16 cells and mouse brain, liver, sclera, and feces were used as samples. By using a fast 4-min RP gradient, the implementation of TIMS allows us to differentiate coeluting isomeric and isobaric lipids, with correct precursor ion isolation, avoiding co-fragmentation and chimeric MS/MS spectra. Globally, the HT-4D-TIMS allowed us to annotate 1910 different lipid species, 1308 at the molecular level and 602 at the sum composition level, covering 58 lipid subclasses, together with quantitation capability covering more than three orders of magnitude. Notably, TIMS values were highly comparable with respect to longer LC gradients (CV% = 0.39%). These results highlight how HT-4D-TIMS-based untargeted lipidomics possess high coverage and accuracy, halving the analysis time with respect to conventional UHPLC methods, and can be used for fast and accurate untargeted analysis of complex matrices to rapidly evaluate changes of lipid metabolism in disease models or drug discovery campaigns.

Keywords:  High-throughput; PASEF; Trapped ion mobility; Untargeted lipidomics

DOI:  https://doi.org/10.1007/s00216-023-05084-w