bims-metlip 2021-05-09 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2021–05–09
seventeen papers selected by
Sofia Costa, Cold Spring Harbor Laboratory

High Confidence Shotgun Lipidomics Using Structurally Selective Ion Mobility-Mass Spectrometry.
Global Lipidomics Profiling by a High Resolution LC-MS Platform.
Quo Vadis Caenorhabditis elegans Metabolomics-A Review of Current Methods and Applications to Explore Metabolism in the Nematode.
Quantification of Plasma Oxylipins Using Solid-Phase Extraction and Reversed-Phase Liquid Chromatography-Triple Quadrupole Mass Spectrometry.
LipidSuite: interactive web server for lipidomics differential and enrichment analysis.
Non-target bioanalytical eight-dimensional hyphenation including bioassay, heart-cut trapping, online desalting, orthogonal separations and mass spectrometry.
Validation of a Gas Chromatography-Mass Spectrometry Method for the Measurement of the Redox State Metabolic Ratios Lactate/Pyruvate and β-Hydroxybutyrate/Acetoacetate in Biological Samples.
Data Processing Optimization in Untargeted Metabolomics of Urine Using Voigt Lineshape Model Non-Linear Regression Analysis.
Determination of tetrabromobisphenol-A/S and their eight derivatives in abiotic (soil/dust) samples using ultra-high performance liquid chromatography-tandem mass spectrometry.
Mass Spectrometry-Based Shotgun Lipidomics Using Charge-Switch Derivatization for Analysis of Complex Long-Chain Fatty Acids.
Strategy for the identification of flavor compounds in e-liquids by correlating the analysis of GCxIMS and GC-MS.
Comparison of analyte identification criteria and other aspects in triple quadrupole tandem mass spectrometry: Case study using UHPLC-MS/MS for regulatory analysis of veterinary drug residues in liquid and powdered eggs.
Quantitative Analysis of Polyphosphoinositide, Bis(monoacylglycero)phosphate, and Phosphatidylglycerol Species by Shotgun Lipidomics After Methylation.
Mass Spectrometry-Based Lipidomics: An Overview.
Three-in-One Simultaneous Extraction of Proteins, Metabolites and Lipids for Multi-Omics.
Rapid and sensitive LC-MS/MS method for the enantioanalysis of verapamil in rat plasma using superficially porous silica isopropyl-cyclofructan 6 chiral stationary phase after SPE: Application to a stereoselective pharmacokinetic study.
Micro-droplet-based calibration for quantitative elemental bioimaging by LA-ICPMS.

Methods Mol Biol. 2021 ;2306 11-37

High Confidence Shotgun Lipidomics Using Structurally Selective Ion Mobility-Mass Spectrometry.

Bailey S Rose, Katrina L Leaptrot, Rachel A Harris, Stacy D Sherrod, Jody C May, John A McLean.

  Ion mobility (IM) is a gas phase separation strategy that can either supplement or serve as a high-throughput alternative to liquid chromatography (LC) in shotgun lipidomics. Incorporating the IM dimension in untargeted lipidomics workflows can help resolve isomeric lipids, and the collision cross section (CCS) values obtained from the IM measurements can provide an additional molecular descriptor to increase lipid identification confidence. This chapter provides a broad overview of an untargeted ion mobility-mass spectrometry (IM-MS) workflow using a commercial drift tube ion mobility-quadrupole-time-of-flight mass spectrometer (IM-QTOF) for high confidence lipidomics.

Keywords:  Collision cross section; Drift tube ion mobility spectrometry; Ion mobility spectrometry; Lipidomics; Tandem MS/MS

DOI:  https://doi.org/10.1007/978-1-0716-1410-5_2
Methods Mol Biol. 2021 ;2306 39-51

Global Lipidomics Profiling by a High Resolution LC-MS Platform.

Thomas Züllig, Martin Trötzmüller, Harald C Köfeler.

  Lipidomics is the determination of big lipid assemblies by mass spectrometry. When using chromatography coupled high resolution mass spectrometry, lipids can be identified by exact mass, fragment spectra, and retention time. This protocol covers lipid extraction, LC-MS data acquisition by Orbitrap mass spectrometry and data processing by Lipid Data Analyzer, a custom developed open source software.

Keywords:  Chromatography; LC-MS; Lipid data analyzer; Lipidomics; Lipids; Mass spectrometry

DOI:  https://doi.org/10.1007/978-1-0716-1410-5_3
Metabolites. 2021 Apr 29. pii: 284. [Epub ahead of print]11(5):

Quo Vadis Caenorhabditis elegans Metabolomics-A Review of Current Methods and Applications to Explore Metabolism in the Nematode.

Liesa Salzer, Michael Witting.

  Metabolomics and lipidomics recently gained interest in the model organism Caenorhabditis elegans (C. elegans). The fast development, easy cultivation and existing forward and reverse genetic tools make the small nematode an ideal organism for metabolic investigations in development, aging, different disease models, infection, or toxicology research. The conducted type of analysis is strongly depending on the biological question and requires different analytical approaches. Metabolomic analyses in C. elegans have been performed using nuclear magnetic resonance (NMR) spectroscopy, direct infusion mass spectrometry (DI-MS), gas-chromatography mass spectrometry (GC-MS) and liquid chromatography mass spectrometry (LC-MS) or combinations of them. In this review we provide general information on the employed techniques and their advantages and disadvantages in regard to C. elegans metabolomics. Additionally, we reviewed different fields of application, e.g., longevity, starvation, aging, development or metabolism of secondary metabolites such as ascarosides or maradolipids. We also summarised applied bioinformatic tools that recently have been used for the evaluation of metabolomics or lipidomics data from C. elegans. Lastly, we curated metabolites and lipids from the reviewed literature, enabling a prototypic collection which serves as basis for a future C. elegans specific metabolome database.

Keywords:  Caenorhabditis elegans; NMR; lipidomics; mass-spectrometry; metabolomics

DOI:  https://doi.org/10.3390/metabo11050284
Methods Mol Biol. 2021 ;2306 171-186

Quantification of Plasma Oxylipins Using Solid-Phase Extraction and Reversed-Phase Liquid Chromatography-Triple Quadrupole Mass Spectrometry.

Guan-Yuan Chen, Qibin Zhang.

  Oxylipins are an important class of bioactive lipids derived from polyunsaturated fatty acids. They can be both pro- and anti-inflammatory and function as important mediators in various pathological conditions. However, comprehensive analysis of oxylipins still remains a challenge because of their low abundance in plasma and the dominance of structurally similar isomeric species. Herein, we describe a simple and rapid method to comprehensively analyze oxylipins in blood plasma, which utilizes solid-phase extraction in 96-well format for efficient sample cleanup. Separation and detection of more than 130 oxylipins is accomplished by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring in negative-ion mode. The absolute concentrations of oxylipins in human plasma are determined using the calibration curves constructed from internal standards. Detailed methods and precautions are presented for a successful adoption of this method in analytical laboratory.

Keywords:  Human plasma; LC-MS/MS; Lipid mediators; MRM; Oxylipins; SPE

DOI:  https://doi.org/10.1007/978-1-0716-1410-5_12
Nucleic Acids Res. 2021 May 05. pii: gkab327. [Epub ahead of print]

LipidSuite: interactive web server for lipidomics differential and enrichment analysis.

Ahmed Mohamed, Michelle M Hill.

Advances in mass spectrometry enabled high throughput profiling of lipids but differential analysis and biological interpretation of lipidomics datasets remains challenging. To overcome this barrier, we present LipidSuite, an end-to-end differential lipidomics data analysis server. LipidSuite offers a step-by-step workflow for preprocessing, exploration, differential analysis and enrichment analysis of untargeted and targeted lipidomics. Three lipidomics data formats are accepted for upload: mwTab file from Metabolomics Workbench, Skyline CSV Export, and a numerical matrix. Experimental variables to be used in analysis are uploaded in a separate file. Conventional lipid names are automatically parsed to enable lipid class and chain length analyses. Users can interactively explore data, choose subsets based on sample types or lipid classes or characteristics, and conduct univariate, multivariate and unsupervised analyses. For complex experimental designs and clinical cohorts, LipidSuite offers confounding variables adjustment. Finally, data tables and plots can be both interactively viewed or downloaded for publication or reports. Overall, we anticipate this free, user-friendly webserver to facilitate differential lipidomics data analysis and re-analysis, and fully harness biological interpretation from lipidomics datasets. LipidSuite is freely available at http://suite.lipidr.org.

DOI: https://doi.org/10.1093/nar/gkab327
J Chromatogr A. 2021 Apr 20. pii: S0021-9673(21)00278-8. [Epub ahead of print]1647 462154

Non-target bioanalytical eight-dimensional hyphenation including bioassay, heart-cut trapping, online desalting, orthogonal separations and mass spectrometry.

Tamara Schreiner, Gertrud E Morlock.

  It is still a challenge to discover and identify individual bioactive compounds directly in multicomponent mixtures. Current workflows are too tedious for routine use. Hence, the hyphenation of separation and detection techniques is a powerful tool to maximize the information obtained by a single sample run. A robust eight-dimensional (8D) hyphenation was developed. Orthogonal separations, biological assay detection, analyte trapping, desalting, and physico-chemical detections were arranged in the following order, i.e. 1) normal phase high-performance thin-layer chromatography (NP-HPTLC) separation, 2) Vis detection, 3) UV detection, 4) fluorescence detection (FLD), 5) bioassay for effect-directed analysis (EDA), 6) heart-cut trapping/desalting/elution to reversed phase high-performance liquid chromatography (RP-HPLC) separation, 7) photodiode array (PDA) and 8) mass spectrometry (MS) detection. For the first time, the hyphenation exploited online analyte trapping to desalt the eluted bioactive zone from the plate containing highly salted bioassay media. Subsequent valve switching guided the trapped analyte(s) to the main column, followed by multiple detection. As proof-of-principle, cinnamon samples were analyzed by NP-HPTLC-UV/Vis/FLD-EDA-RP-HPLC-PDA-MS, whereby a bioactive zone was separated into two distinct peaks detected by PDA and MS to be 2-methoxy cinnamaldehyde and cinnamaldehyde. The developed 8D hyphenation is applicable for routine, allowing the non-target high-throughput screening of complex samples for individual bioactive compounds.

Keywords:  Aliivibrio fischeri bioassay; Effect-directed analysis of cinnamon; Eight-dimensional hyphenation; High-performance thin-layer chromatography; Non-target screening; Online desalting

DOI:  https://doi.org/10.1016/j.chroma.2021.462154
Int J Mol Sci. 2021 Apr 30. pii: 4752. [Epub ahead of print]22(9):

Validation of a Gas Chromatography-Mass Spectrometry Method for the Measurement of the Redox State Metabolic Ratios Lactate/Pyruvate and β-Hydroxybutyrate/Acetoacetate in Biological Samples.

Robin Wijngaard, Meritxell Perramón, Marina Parra-Robert, Susana Hidalgo, Gina Butrico, Manuel Morales-Ruiz, Muling Zeng, Eudald Casals, Wladimiro Jiménez, Guillermo Fernández-Varo, Gerald I Shulman, Gary W Cline, Gregori Casals.

  The metabolic ratios lactate/pyruvate and β-hydroxybutyrate/acetoacetate are considered valuable tools to evaluate the in vivo redox cellular state by estimating the free NAD+/NADH in cytoplasm and mitochondria, respectively. The aim of the current study was to validate a gas-chromatography mass spectrometry method for simultaneous determination of the four metabolites in plasma and liver tissue. The procedure included an o-phenylenediamine microwave-assisted derivatization, followed by liquid-liquid extraction with ethyl acetate and silylation with bis(trimethylsilyl)trifluoroacetamide:trimethylchlorosilane 99:1. The calibration curves presented acceptable linearity, with a limit of quantification of 0.001 mM for pyruvate, β-hydroxybutyrate and acetoacetate and of 0.01 mM for lactate. The intra-day and inter-day accuracy and precision were within the European Medicines Agency's Guideline specifications. No significant differences were observed in the slope coefficient of three-point standard metabolite-spiked curves in plasma or liver and water, and acceptable recoveries were obtained in the metabolite-spiked samples. Applicability of the method was tested in precision-cut liver rat slices and also in HepG2 cells incubated under different experimental conditions challenging the redox state. In conclusion, the validated method presented good sensitivity, specificity and reproducibility in the quantification of lactate/pyruvate and β-hydroxybutyrate/acetate metabolites and may be useful in the evaluation of in vivo redox states.

Keywords:  GC-MS; ketone bodies; microwave-assisted derivatization; nicotinamide adenine dinucleotide; redox state

DOI:  https://doi.org/10.3390/ijms22094752
Metabolites. 2021 Apr 29. pii: 285. [Epub ahead of print]11(5):

Data Processing Optimization in Untargeted Metabolomics of Urine Using Voigt Lineshape Model Non-Linear Regression Analysis.

Kristina E Haslauer, Philippe Schmitt-Kopplin, Silke S Heinzmann.

  Nuclear magnetic resonance (NMR) spectroscopy is well-established to address questions in large-scale untargeted metabolomics. Although several approaches in data processing and analysis are available, significant issues remain. NMR spectroscopy of urine generates information-rich but complex spectra in which signals often overlap. Furthermore, slight changes in pH and salt concentrations cause peak shifting, which introduces, in combination with baseline irregularities, un-informative noise in statistical analysis. Within this work, a straight-forward data processing tool addresses these problems by applying a non-linear curve fitting model based on Voigt function line shape and integration of the underlying peak areas. This method allows a rapid untargeted analysis of urine metabolomics datasets without relying on time-consuming 2D-spectra based deconvolution or information from spectral libraries. The approach is validated with spiking experiments and tested on a human urine 1H dataset compared to conventionally used methods and aims to facilitate metabolomics data analysis.

Keywords:  NMR; data processing; metabolomics; voigt-fitting

DOI:  https://doi.org/10.3390/metabo11050285
J Chromatogr A. 2021 Apr 20. pii: S0021-9673(21)00276-4. [Epub ahead of print]1647 462152

Determination of tetrabromobisphenol-A/S and their eight derivatives in abiotic (soil/dust) samples using ultra-high performance liquid chromatography-tandem mass spectrometry.

Hui Yang, Xiao-Jun Luo, Yan-Hong Zeng, Bi-Xian Mai.

  Tetrabromobisphenol-A and Tetrabromobisphenol-S (TBBPA/S) and their derivatives have attracted widespread attention owing to their environmental risks and adverse effects on human health. In this study, we developed an analytical method based on ultra-high performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UPLC-MS/MS) for the simultaneous determination of TBBPA/S and their eight derivatives in soil samples. After ultrasonic extraction, TBBPA/S and their derivatives were purified using an LC-Si cartridge with 1 mL acetone and 30 mL dichloromethane/n-hexane (1/1, v/v) as the eluent. In the multiple reaction monitoring (MRM) mode, TBBPA and TBBPS were quantified with electrospray ionization (ESI), whereas their derivatives were quantified with atmospheric pressure chemical ionization (APCI). The proposed method was verified with blank spiking and matrix spiking experiments. All target compounds were recovered at the range of 78-124% and the average recoveries of surrogate standard 13C12-labeled TBBPA were 103% and 99% in spiked blank and soil samples, respectively. The method quantitative limits (MQLs) of TBBPA/S and their derivatives in soil samples ranged from 0.22 to 8.8 pg/g dw. These results indicated that an effective method was provided for the analysis of TBBPA/S and their derivatives in abiotic matrices.

Keywords:  Abiotic samples; Analytical method; Derivatives; Tetrabromobisphenol A; Tetrabromobisphenol S

DOI:  https://doi.org/10.1016/j.chroma.2021.462152
Methods Mol Biol. 2021 ;2306 93-103

Mass Spectrometry-Based Shotgun Lipidomics Using Charge-Switch Derivatization for Analysis of Complex Long-Chain Fatty Acids.

Cheryl Frankfater, Fong-Fu Hsu.

  Charge-switch derivatization to convert long-chain fatty acids (LCFAs) to their N-(4-aminomethylphenyl) pyridinium (AMPP) derivatives (FA-AMPP derivative) drastically increases their sensitivity (>102) detected by electrospray ionization (ESI) or matrix assisted laser desorption ionization (MALDI). Lipidomic analyses of the FA-AMPP derivatives by ESI combined with CID tandem mass spectrometry (MS2), or by MALDI-TOF/TOF affords unambiguous structural characterization of LCFAs, including many unusual microbial LCFAs that contain various functional groups such as methyl, hydroxyl, cyclopropyl, and double bond(s). The ease of preparation of the FA-AMPP derivatives, the tremendous gain in sensitivity after derivatization, and more importantly, the readily recognizable product ion spectra that contain rich structurally informative fragment ions for locating functional groups make this method one of the most powerful techniques for LCFA identification and quantification.

Keywords:  Charge conversion; Long-chain fatty acids; MALDI-TOF/TOF; Mass spectrometry; Microbial lipids; Tandem mass spectrometry

DOI:  https://doi.org/10.1007/978-1-0716-1410-5_7
Talanta. 2021 Aug 01. pii: S0039-9140(21)00239-3. [Epub ahead of print]230 122318

Strategy for the identification of flavor compounds in e-liquids by correlating the analysis of GCxIMS and GC-MS.

Alexander L R M Augustini, Stefanie Sielemann, Ursula Telgheder.

  This work presents a strategy to correlate the results from gas chromatography coupled ion mobility spectrometry (GCxIMS) and mass spectrometry (GC-MS) to enable a simpler and cheaper analysis of flavor compounds in e-liquids. The use of the retention index for GCxIMS measurements was validated for its application to correlate results with GC-MS data. The easy detection of the GCxIMS for substances at concentrations as low as 1 μg/L can therefore be combined with the identification power of the MS. The use of the MS' mass signals and wide-spread availability of mass spectra libraries reduces the effort necessary to choose the correct reference standards for the identification of unknown substances. Between both detectors, correlating of the retention time indices was achieved for ± 1%. 2-Alkanones were used as an alternative reference point for the IMS and the well-established alkanes for the MS. The application on flavor compounds in e-liquids shows equal or better results than those presented for more complex, hardware-based correlations like line splitting. Additionally, the inverted reduced mobility combined with the retention index of a non-polar column enables simple extrapolation for the confirmation of expected substances as well as the use in a transferable database. For the first time, this comprehensive application allows an extensive, simplified, and cheap identification of flavor compounds in e-liquids by GCxIMS and GC-MS.

Keywords:  Electronic cigarettes; GC-MS; GCxIMS; Retention time index; e-liquids

DOI:  https://doi.org/10.1016/j.talanta.2021.122318
Anal Bioanal Chem. 2021 May 07.

Comparison of analyte identification criteria and other aspects in triple quadrupole tandem mass spectrometry: Case study using UHPLC-MS/MS for regulatory analysis of veterinary drug residues in liquid and powdered eggs.

Steven J Lehotay.

  Ultrahigh-performance liquid chromatography (UHPLC) coupled with triple quadrupole tandem mass spectrometry (MS/MS) is one of the most powerful tools for the multiclass, multiresidue analysis of veterinary drugs, pesticides, mycotoxins, and other chemical contaminants in foods and other sample types. Until approximately 2010, commercial MS/MS instruments using multiple reaction monitoring (MRM) were generally limited to minimum dwell (and inter-dwell) times of 10 ms per ion transition. To achieve the needed accuracy and detection limits for hundreds of targeted analytes, older UHPLC-MS/MS methods typically acquired only two ion transitions per analyte (yielding only one ion ratio for qualitative identification purposes), which is still the norm despite technological advancements. Newer instruments permit as little as 1 ms (inter-)dwell times to afford monitoring of more MRMs/analyte with minimal sacrifices in accuracy and sensitivity. In this study, quantification and identification were assessed in the validation of 169 veterinary drugs in liquid and powdered eggs. Quantitatively, an "extract-and-inject" sample preparation method yielded acceptable 70-120% recoveries and < 25% RSD for 139-141 (82-83%) of the 169 diverse drug analytes spiked into powdered and liquid eggs, respectively, at three levels of regulatory interest. Qualitatively, rates of false positives and negatives were compared when applying three different regulatory identification criteria in which two or three MRMs/drug were used in each case. Independent of the identification criteria, rates of false positives remained <10% for 95-99% of the drugs whether 2 or 3 ions were monitored, but the percent of drugs with >10% false negatives decreased from 25-45 to 10-12% when using 2 vs. 3 MRMs/analyte, respectively. Use of a concentration threshold at 10% of the regulatory level as an identification criterion was also very useful to reduce rates of false positives independent of ion ratios. Based on these results, monitoring >2 ion transitions per analyte is advised when using MS/MS for analysis, independent of SANTE/12682/2019, FDA/USDA, or 2002/657/EC identification criteria. (Quant)identification results using all three criteria were similar, but the SANTE criteria were advantageous in their greater simplicity and practical ease of use.

Keywords:  Eggs; Liquid chromatography–tandem mass spectrometry (LC-MS/MS); Qualitative identification; Quantification; Veterinary drug residue analysis

DOI:  https://doi.org/10.1007/s00216-021-03380-x
Methods Mol Biol. 2021 ;2306 77-91

Quantitative Analysis of Polyphosphoinositide, Bis(monoacylglycero)phosphate, and Phosphatidylglycerol Species by Shotgun Lipidomics After Methylation.

Meixia Pan, Chao Qin, Xianlin Han.

  Phospholipids play important roles in biological process even at a very low level. For example, bis(monoacylglycerol)phosphate (BMP) is involved in the pathogenesis of lysosomal storage diseases, and polyphosphoinositides (PPI) play critical roles in cellular signaling and functions. Phosphatidylglycerol (PG), a structural isomer of BMP, mediates lipid-protein and lipid-lipid interactions, and inhibits platelet activating factor and phosphatidylcholine transferring. However, due to their low abundance, the analysis of these phospholipids from biological samples is technically challenging. Therefore, the cellular function and metabolism of these phospholipids are still elusive. This chapter overviews a novel method of shotgun lipidomics after methylation with trimethylsilyl-diazomethane (TMS-D) for accurate and comprehensive analysis of these phospholipid species in biological samples. Firstly, a modified Bligh and Dyer procedure is performed to extract tissue lipids for PPI analysis, whereas modified methyl-tert-butylether (MTBE) extraction and modified Folch extraction methods are described to extract tissue lipids for PPI analysis. Secondly, TMS-D methylation is performed to derivatize PG/BMP and PPI, respectively. Then, we described the shotgun lipidomics strategies that can be used as cost-effective and relatively high-throughput methods to determine BMP, PG, and PPI species and isomers with different phosphate position(s) and fatty acyl chains. The described method of shotgun lipidomics after methylation achieves feasible and reliable quantitative analysis of low-abundance lipid classes. The application of this novel method should enable us to reveal the metabolism and functions of these phospholipids in healthy and disease states.

Keywords:  Bis(monoacylglycerol)phosphate; Mass spectrometry; Methylation; Phosphatidylglycerol; Polyphosphoinositide; Shotgun lipidomics

DOI:  https://doi.org/10.1007/978-1-0716-1410-5_6
Methods Mol Biol. 2021 ;2306 1-10

Mass Spectrometry-Based Lipidomics: An Overview.

Fong-Fu Hsu.

  Over the last few decades, MS-based lipidomics has emerged as a powerful tool to study lipids in biological systems. This success is driven by the constant demand for complete and reliable data. The improvement of MS-based lipidomics will continue to be dependent on the advances in the technology of mass spectrometry and related techniques including separation and bioinformatics, and more importantly, on gaining insight into the knowledge of lipid chemistry essential to develop methodology for lipid analysis. It is hoped that the protocols in this book, collected from experts in their fields, can offer the beginner and the advanced user alike, useful tips toward successful lipidomic analysis.

Keywords:  Overview; Shotgun lipidomics; Tandem mass spectrometry

DOI:  https://doi.org/10.1007/978-1-0716-1410-5_1
Front Genet. 2021 ;12 635971

Three-in-One Simultaneous Extraction of Proteins, Metabolites and Lipids for Multi-Omics.

Jianing Kang, Lisa David, Yangyang Li, Jing Cang, Sixue Chen.

  Elucidation of complex molecular networks requires integrative analysis of molecular features and changes at different levels of information flow and regulation. Accordingly, high throughput functional genomics tools such as transcriptomics, proteomics, metabolomics, and lipidomics have emerged to provide system-wide investigations. Unfortunately, analysis of different types of biomolecules requires specific sample extraction procedures in combination with specific analytical instrumentation. The most efficient extraction protocols often only cover a restricted type of biomolecules due to their different physicochemical properties. Therefore, several sets/aliquots of samples are needed for extracting different molecules. Here we adapted a biphasic fractionation method to extract proteins, metabolites, and lipids from the same sample (3-in-1) for liquid chromatography-tandem mass spectrometry (LC-MS/MS) multi-omics. To demonstrate utility of the improved method, we used bacteria-primed Arabidopsis leaves to generate multi-omics datasets from the same sample. In total, we were able to analyze 1849 proteins, 1967 metabolites, and 424 lipid species in single samples. The molecules cover a wide range of biological and molecular processes, and allow quantitative analyses of different molecules and pathways. Our results have shown the clear advantages of the multi-omics method, including sample conservation, high reproducibility, and tight correlation between different types of biomolecules.

Keywords:  3-in-1 method; Arabidopsis; disease; lipidomics; metabolomics; multi-omics; proteomics

DOI:  https://doi.org/10.3389/fgene.2021.635971
J Pharm Biomed Anal. 2021 Apr 29. pii: S0731-7085(21)00219-3. [Epub ahead of print]201 114108

Rapid and sensitive LC-MS/MS method for the enantioanalysis of verapamil in rat plasma using superficially porous silica isopropyl-cyclofructan 6 chiral stationary phase after SPE: Application to a stereoselective pharmacokinetic study.

Mohamed Hefnawy, Abdulrhman Al-Majed, Haitham Alrabiah, Nasser Algrain, Mostafa Mohammed, Yousef Bin Jardan.

  A novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the rapid and sensitive enantioselective analysis of verapamil (VER) in rat plasma was developed and validated using new superficially porous silica isopropyl-cyclofructan 6 chiral column (LarihcShell-P, LSP). The isocratic mobile phase composed of acetonitrile: trifluoroacetic acid: 10 mM ammonium formate (100 : 0.1 : 0.1, v/v/v) at a flow rate of 0.3 mL/min was applied. Sulpride was utilized as the internal standard (IS). Positive multiple reaction monitoring (MRM) mode was used for mass spectrometry analysis, and the process of analysis was run for 5.2 min. The (S)-(-)- and (R)-(+)-VER enantiomers with the IS were extracted from plasma by using solid-phase extraction (SPE) procedure before the analysis. The C18 cartridge gave good recovery rates for both enantiomers without interference from plasma endogenous. The developed assay was successfully validated following the US-FDA guidelines. The method was linear over concentration ranges of 0.5-500 ng/mL (r2 ≥ 0.997) for each enantiomer (plasma). The lower limits of quantification (LLOQ) for both isomers were 0.5 ng/mL. The intra- and inter-day relative standard deviations (RSD) were less than 8.7 % and the recoveries of (S)-(-)- and (R)-(+)-VER at three spiked levels of 1.5, 250.0 and 450.0 ranged from 92.0%-98.6%. The developed assay was effectively applied in monitoring the stereoselective pharmacokinetic study of VER enantiomers in rat plasma following oral administration of racemic VER. The pharmacokinetic parameters revealed that (S)-(-)-VER demonstrated prominently higher Cmax and AUC values than (R)-(+)-enantiomer. The newly developed approach is the first chiral LC-MS/MS for the quantification of (S)-(-)- and (R)-(+)-VER utilizing superficially porous silica isopropyl-cyclofructan 6 chiral column in rat plasma after SPE.

Keywords:  LC–MS/MS; Pharmacokinetics; Rat plasma; Solid-phase extraction; Superficially porous silica isopropyl-cyclofructan chiral column; Verapamil enantiomers

DOI:  https://doi.org/10.1016/j.jpba.2021.114108
Anal Bioanal Chem. 2021 May 05.

Micro-droplet-based calibration for quantitative elemental bioimaging by LA-ICPMS.

Andreas Schweikert, Sarah Theiner, Debora Wernitznig, Anna Schoeberl, Martin Schaier, Sophie Neumayer, Bernhard K Keppler, Gunda Koellensperger.

  In this work, a novel standardization strategy for quantitative elemental bioimaging is evaluated. More specifically, multi-element quantification by laser ablation-inductively coupled plasma-time-of-flight mass spectrometry (LA-ICP-TOFMS) is performed by multi-point calibration using gelatin-based micro-droplet standards and validated using in-house produced reference materials. Fully automated deposition of micro-droplets by micro-spotting ensured precise standard volumes of 400 ± 5 pL resulting in droplet sizes of around 200 μm in diameter. The small dimensions of the micro-droplet standards and the use of a low-dispersion laser ablation setup reduced the analysis time required for calibration by LA-ICPMS significantly. Therefore, as a key advance, high-throughput analysis (pixel acquisition rates of more than 200 Hz) enabled to establish imaging measurement sequences with quality control- and standardization samples comparable to solution-based quantification exercises by ICP-MS. Analytical figures of merit such as limit of detection, precision, and accuracy of the calibration approach were assessed for platinum and for elements with biological key functions from the lower mass range (phosphorus, copper, and zinc). As a proof-of-concept application, the tool-set was employed to investigate the accumulation of metal-based anticancer drugs in multicellular tumor spheroid models at clinically relevant concentrations. Graphical abstract.

Keywords:  Bioimaging; ICP-MS; Laser ablation; Mass spectrometry; Quantification strategy

DOI:  https://doi.org/10.1007/s00216-021-03357-w