bims-metlip 2021-01-03 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2021–01–03
23 papers selected by
Sofia Costa, Cold Spring Harbor Laboratory

An exploratory approach for an oriented development of an untargeted hydrophilic interaction liquid chromatography-mass spectrometry platform for polar metabolites in biological matrices.
Simultaneous determination of methionine cycle metabolites, urea cycle intermediates and polyamines in serum, urine and intestinal tissue by using UHPLC-MS/MS.
Exploring the Use of Gas Chromatography Coupled to Chemical Ionization Mass Spectrometry (GC-CI-MS) for Stable Isotope Labeling in Metabolomics.
A fast and simple method for the simultaneous analysis of midazolam, 1-hydroxymidazolam, 4-hydroxymidazolam and 1-hydroxymidazolam glucuronide in human serum, plasma and urine.
Assessment of Tissue Distribution and Metabolism of MP1, a Novel Pyrrolomycin, in Mice Using a Validated LC-MS/MS Method.
Broad-range RNA modification analysis of complex biological samples using rapid C18-UPLC-MS.
Combination of UHPLC-MS/MS-molecular networking approach and FTICR-MS for the metabolic profiling of Saccharomyces cerevisiae.
A preliminary study procedure for detection of polyamines in plasma samples as a potential diagnostic tool in prostate cancer.
Data-Independent Acquisition for the Quantification and Identification of Metabolites in Plasma.
High Coverage Profiling of Carboxylated Metabolites in HepG2 Cells Using Secondary Amine-Assisted Ultrahigh-Performance Liquid Chromatography Coupled to High-Resolution Mass Spectrometry.
Post-Chromatographic Dicationic Ionic Liquid-Based Charge Complexation for Highly Sensitive Analysis of Anionic Compounds by Ultra-High-Performance Supercritical Fluid Chromatography Coupled with Electrospray Ionization Mass Spectrometry.
Structure-Preserving and Perceptually Consistent Approach for Visualization of Mass Spectrometry Imaging Datasets.
Determination of Antidepressants in Human Plasma by Modified Cloud-Point Extraction Coupled with Mass Spectrometry.
Simultaneous determination of 10 new psychoactive piperazine derivatives in urine using ultrasound-assisted low-density solvent dispersive liquid-liquid microextraction combined with gas chromatography-tandem mass spectrometry.
Quantitative Assessment of Dietary (Poly)phenol Intake: A High-Throughput Targeted Metabolomics Method for Blood and Urine Samples.
Quantitative MALDI-MS assay of steroid hormones in plasma based on hydroxylamine derivatization.
Positive/negative ion-switching-based LC-MS/MS method for quantification of cytosine derivatives produced by the TET-family 5-methylcytosine dioxygenases.
Single cell metabolomics using mass spectrometry: Techniques and data analysis.
Anion exchange chromatography - Mass spectrometry for monitoring multiple quality attributes of erythropoietin biopharmaceuticals.
Optimization of QuEChERS extraction for detection and quantification of 20 antidepressants in postmortem blood samples by LC-MS/MS.
Plasma and serum oxylipin, endocannabinoid, bile acid, steroid, fatty acid and nonsteroidal anti-inflammatory drug quantification in a 96-well plate format.
The software for interactive evaluation of mass spectra stability and reproducibility.
Fully automated method for quantitative determination of steroids in serum: An approach to evaluate steroidogenesis.

J Chromatogr A. 2020 Dec 15. pii: S0021-9673(20)31081-5. [Epub ahead of print]1637 461807

An exploratory approach for an oriented development of an untargeted hydrophilic interaction liquid chromatography-mass spectrometry platform for polar metabolites in biological matrices.

Elias Iturrospe, Katyeny Manuela Da Silva, Begoña Talavera Andújar, Matthias Cuykx, Joost Boeckmans, Tamara Vanhaecke, Adrian Covaci, Alexander L N van Nuijs.

  The analysis of polar metabolites based on liquid chromatography-mass spectrometry (LC-MS) methods should take into consideration the complexity of interactions in LC columns to be able to cover a broad range of metabolites of key biological pathways. Therefore, in this study, different chromatographic columns were tested for polar metabolites including reversed-phase and hydrophilic interaction liquid chromatography (HILIC) columns. Based on a column screening, two new generations of zwitterionic HILIC columns were selected for further evaluation. A tree-based method optimization was applied to investigate the chromatographic factors affecting the retention mechanisms of polar metabolites with zwitterionic stationary phases. The results were evaluated based on a scoring system which was applied for more than 80 polar metabolites with a high coverage of key human metabolic pathways. The final optimized methods showed high complementarity to analyze a wide range of metabolic classes including amino acids, small peptides, sugars, amino sugars, phosphorylated sugars, organic acids, nucleobases, nucleosides, nucleotides and acylcarnitines. Optimized methods were applied to analyze different biological matrices, including human urine, plasma and liver cell extracts using an untargeted approach. The number of high-quality features (< 30% median relative standard deviation) ranged from 3,755 for urine to 5,402 for the intracellular metabolome of liver cells, showing the potential of the methods for untargeted purposes.

Keywords:  Hydrophilic interaction chromatography; Liquid chromatography-high resolution mass spectrometry; Metabolomics; Method optimization; Polar metabolites

DOI:  https://doi.org/10.1016/j.chroma.2020.461807
Talanta. 2021 Mar 01. pii: S0039-9140(20)31159-0. [Epub ahead of print]224 121868

Simultaneous determination of methionine cycle metabolites, urea cycle intermediates and polyamines in serum, urine and intestinal tissue by using UHPLC-MS/MS.

Xiuli Su, Xiaona Li, Haojiang Wang, Zongwei Cai.

  Metabolites of methionine cycle, urea cycle and polyamine metabolism play important roles in regulating the metabolic processes and the development of diseases. It is rewarding and interesting to monitor the levels of the above metabolites in biological matrices to investigate pathological mechanisms. However, their quantitation is still unsatisfactory due to the poor retention behavior of the analytes on the traditional reversed-phase column. And never a single analytical method simultaneously quantify these three classes of metabolites. Besides, the concentrations of some metabolites are too low to be detected in the biological samples. In this study, we developed a UHPLC-ESI-MS/MS method to simultaneously determine the levels of 14 metabolites, including 4 methionine metabolism metabolites (methionine, homocysteine, S-adenosylmethionine and S-adenosylhomocysteine), 3 urea cycle intermediates (arginine, citrulline and ornithine) and 7 polyamines (putrescine, spermidine, spermine, N1-acetylputrescine, N1-acetylspermidine, N1-acetylspermine and N1,N12-diacetylspermine). The chromatographic separation was performed on the BEH amide column within 14 min using water and acetonitrile (both with 0.1% formic acid) as the mobile phases. The results of method validation showed good selectivity, linearity (r2 > 0.99), recovery (93.1%-112.1%), inter-day and intra-day precision (RSD < 13.6% and RSD < 11.0%, respectively), stability (RSD < 15.1%) and matrix effect (76.0%-113.2%). The method is simple, quick and sensitive without derivatization processes and the use of ion-pairing reagents. This approach was successfully applied in urine, serum and tissue matrices, as well as in identifying potential biomarkers for hyperthyroidism and hypothyroidism. The method is promising to provide more information on pathophysiological mechanisms in metabolomics study.

Keywords:  Biological matrices; Methionine cycle metabolites; Polyamines; Thyroid disorder; UHPLC-MS/MS; Urea cycle intermediates

DOI:  https://doi.org/10.1016/j.talanta.2020.121868
Anal Chem. 2020 Dec 28.

Exploring the Use of Gas Chromatography Coupled to Chemical Ionization Mass Spectrometry (GC-CI-MS) for Stable Isotope Labeling in Metabolomics.

Jordi Capellades, Alexandra Junza, Sara Samino, Julia S Brunner, Gernot Schabbauer, Maria Vinaixa, Oscar Yanes.

Isotopic-labeling experiments have been valuable to monitor the flux of metabolic reactions in biological systems, which is crucial to understand homeostatic alterations with disease. Experimental determination of metabolic fluxes can be inferred from a characteristic rearrangement of stable isotope tracers (e.g., 13C or 15N) that can be detected by mass spectrometry (MS). Metabolites measured are generally members of well-known metabolic pathways, and most of them can be detected using both gas chromatography (GC)-MS and liquid chromatography (LC)-MS. In here, we show that GC methods coupled to chemical ionization (CI) MS have a clear advantage over alternative methodologies due to GC's superior chromatography separation efficiency and the fact that CI is a soft ionization technique that yields identifiable protonated molecular ion peaks. We tested diverse GC-CI-MS setups, including methane and isobutane reagent gases, triple quadrupole (QqQ) MS in SIM mode, or selected ion clusters using optimized narrow windows (∼10 Da) in scan mode, and standard full scan methods using high resolution GC-(q)TOF and GC-Orbitrap systems. Isobutane as a reagent gas in combination with both low-resolution (LR) and high-resolution (HR) MS showed the best performance, enabling precise detection of isotopologues in most metabolic intermediates of central carbon metabolism. Finally, with the aim of overcoming manual operations, we developed an R-based tool called isoSCAN that automatically quantifies all isotopologues of intermediate metabolites of glycolysis, TCA cycle, amino acids, pentose phosphate pathway, and urea cycle, from LRMS and HRMS data.

DOI: https://doi.org/10.1021/acs.analchem.0c02998
J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Dec 03. pii: S1570-0232(20)31352-0. [Epub ahead of print]1162 122476

A fast and simple method for the simultaneous analysis of midazolam, 1-hydroxymidazolam, 4-hydroxymidazolam and 1-hydroxymidazolam glucuronide in human serum, plasma and urine.

A Mireille A Wessels, Mathieu S Bolhuis, Wouter Bult, Maarten W N Nijsten, Martin C J Kneyber, Daan J Touw.

  For the quantification of the sedative and anesthetic drug midazolam and its main (active) metabolites 1-hydroxymidazolam, 4-hydroxymidazolam and 1-hydroxymidazolam glucuronide in human serum, human EDTA plasma, human heparin plasma and human urine a single accurate method by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) has been developed. Protein precipitation as sample preparation, without the need of a time-consuming deglucuronidation step for the quantification of 1-hydroxymidazolam glucuronide, resulted in a simple and rapid assay suitable for clinical practice with a total runtime of only 1.1  min. The four components and the isotope-labeled internal standards were separated on a C18 column and detection was performed with a triple-stage quadrupole mass spectrometer operating in positive ionization mode. The method was validated based on the "Guidance for Industry Bioanalytical Method Validation" (Food and Drug Administration, FDA) and the "Guideline on bioanalytical method validation" of the European Medicines Agency (EMA). Linearity was proven over the ranges of 5-1500 μg/L for midazolam, 1-hydroxymidazolam and 4-hydroxymidazolam and 25-5000 μg/L for 1-hydroxymidazolam glucuronide, using a sample volume of 100 μL. Matrix comparison indicated that the assay is also applicable to other human matrices like EDTA and heparin plasma and urine. Stability experiments showed good results for the stability of midazolam, 1-hydroxymidazolam and 1-hydroxymidazolam glucuronide in serum, EDTA and heparin plasma and urine stored for 7 days under different conditions. At room temperature, 4-hydroxymidazo-lam is stable for 7 days in EDTA plasma, but stable for only 3 days in serum and heparin plasma and less than 24 h in urine. All four compounds were found to be stable in serum, EDTA plasma, heparin plasma and urine for 7 days after sample preparation and for 3 freeze-thaw cycles. The assay has been applied in therapeutic drug monitoring of midazolam for (pediatric) intensive care patients.

Keywords:  1-Hydroxymidazolam; 1-Hydroxymidazolam glucuronide; 4-Hydroxymidazolam; Midazolam; UHPLC-MS/MS

DOI:  https://doi.org/10.1016/j.jchromb.2020.122476
Molecules. 2020 Dec 13. pii: E5898. [Epub ahead of print]25(24):

Assessment of Tissue Distribution and Metabolism of MP1, a Novel Pyrrolomycin, in Mice Using a Validated LC-MS/MS Method.

Wafaa N Aldhafiri, Yashpal S Chhonker, Yuning Zhang, Don W Coutler, Timothy R McGuire, Rongshi Li, Daryl J Murry.

  MP1 is a novel marinopyrrole analogue with activity in MYCN amplified neuroblastoma cell lines. A rapid, selective, and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantitation of MP1 in mouse plasma. Analyte separation was achieved using a Waters Acquity UPLC®BEH C18 column (1.7 µm, 100 × 2.1 mm). Mobile phase consisted of 0.1% acetic acid in water (10%) and methanol (90%) at a total flow rate of 0.25 mL/min. The mass spectrometer was operated at unit resolution in the multiple reaction monitoring (MRM) mode, using precursor ion > product ion transitions of 324.10 > 168.30 m/z for MP1 and 411.95 > 224.15 m/z for PL-3. The MS/MS response was linear over the concentration range from 0.2-500 ng/mL for MP1, correlation coefficient (r2) of 0.988. Precision (% RSD) and accuracy (% bias) were within the acceptable limits as per FDA guidelines. MP1 was stable under storage and laboratory handling conditions. The validated method was successfully applied to assess the solubility, in-vitro metabolism, plasma protein binding, and bio-distribution studies of MP1.

Keywords:  LC-MS/MS; biodistribution; in-vitro metabolism; pyrrolomycin

DOI:  https://doi.org/10.3390/molecules25245898
RNA Biol. 2020 Dec 23. 1-8

Broad-range RNA modification analysis of complex biological samples using rapid C18-UPLC-MS.

Pavlina Gregorova, Nina H Sipari, L Peter Sarin.

  Post-transcriptional RNA modifications play an important role in cellular metabolism with homoeostatic disturbances manifesting as a wide repertoire of phenotypes, reduced stress tolerance and translational perturbation, developmental defects, and diseases, such as type II diabetes, leukaemia, and carcinomas. Hence, there has been an intense effort to develop various methods for investigating RNA modifications and their roles in various organisms, including sequencing-based approaches and, more frequently, liquid chromatography-mass spectrometry (LC-MS)-based methods. Although LC-MS offers numerous advantages, such as being highly sensitive and quantitative over a broad detection range, some stationary phase chemistries struggle to resolve positional isomers. Furthermore, the demand for detailed analyses of complex biological samples often necessitates long separation times, hampering sample-to-sample turnover and making multisample analyses time consuming. To overcome this limitation, we have developed an ultra-performance LC-MS (UPLC-MS) method that uses an octadecyl carbon chain (C18)-bonded silica matrix for the efficient separation of 50 modified ribonucleosides, including positional isomers, in a single 9-min sample-to-sample run. To validate the performance and versatility of our method, we analysed tRNA modification patterns of representative microorganisms from each domain of life, namely Archaea (Methanosarcina acetivorans), Bacteria (Pseudomonas syringae), and Eukarya (Saccharomyces cerevisiae). Additionally, our method is flexible and readily applicable for detection and relative quantification using stable isotope labelling and targeted approaches like multiple reaction monitoring (MRM). In conclusion, this method represents a fast and robust tool for broad-range exploration and quantification of ribonucleosides, facilitating future homoeostasis studies of RNA modification in complex biological samples.

Keywords:  C18; UPLC-MS; post-transcriptional nucleoside modification; quantification; transfer RNA; translation

DOI:  https://doi.org/10.1080/15476286.2020.1853385
J Pharm Biomed Anal. 2020 Dec 19. pii: S0731-7085(20)31744-1. [Epub ahead of print]195 113857

Combination of UHPLC-MS/MS-molecular networking approach and FTICR-MS for the metabolic profiling of Saccharomyces cerevisiae.

Olivier Perruchon, Isabelle Schmitz-Afonso, Cécile Grondin, Serge Casaregola, Carlos Afonso, Abdelhakim Elomri.

  Natural products are a reliable source of bioactive molecules and represent an industrial and pharmaceutical stake. Indeed, the model yeast species Saccharomyces cerevisiae is a well-known eukaryotic organism largely used as a biotechnological tool, but still a topical subject of study. In this work, the exploration of Saccharomyces cerevisiae is taken further through an untargeted metabolomics workflow. The aim is to enrich databases and bring new information about the standard S. cerevisiae strain in a given medium. Analytical methods and bioinformatics tools were combined in a high-throughput methodology useable to dereplicate many types of biological extracts and cartography secondary metabolites. Ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analyses were carried out and spectral data were pre-processed to build molecular networks. Annotations were attributed to compounds through comparison with databases and manual investigation of networks. Ultra-high-resolution Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) brought additional information thanks to a higher dynamic range and enhanced UHPLC-MS/MS results by unveiling ambiguities and bringing accurate molecular formulae. Therefore, accurate and reliable annotated features resulted from the UHPLC-MS/MS data while FTICR-MS provided an overall cartography of metabolites thanks to van Krevelen diagrams. Various small molecules such as amino acids derivatives and indole alkaloids have been determined for the first time in this yeast. The complementarity of FTICR-MS and UHPLC-MS/MS for secondary metabolite annotation brought this new mapping of S. cerevisiae.

Keywords:  Dereplication; FTICR-MS; Mass spectrometry; Metabolomics; Molecular networking; Saccharomyces cerevisiae

DOI:  https://doi.org/10.1016/j.jpba.2020.113857
J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Nov 30. pii: S1570-0232(20)31344-1. [Epub ahead of print]1162 122468

A preliminary study procedure for detection of polyamines in plasma samples as a potential diagnostic tool in prostate cancer.

Donatella Coradduzza, Emanuela Azara, Serenella Medici, Caterina Arru, Tatiana Solinas, Massimo Madonia, Angelo Zinellu, Ciriaco Carru.

   BACKGROUND: Many scientific contributions recognize polyamines as important biomarkers for the diagnosis and treatment of cancer. Several authors have suggested the use of LC/MS instruments as an elective method for their measurement, providing good detection limits and specificity; however, many of these procedures suffer from long chromatographic run times, high detection limits and lengthy and expensive sample pre-treatment steps.
METHODS: UHPLC coupled with high-resolution Orbitrap mass spectrometry (UHPLC/Orbitrap) was set up for the identification and separation ofpolyamines, together with some of their metabolites and catabolites, in the plasma of healthy and prostate cancer human patients. Thirteen metabolites were measured in deproteinized plasma samples through a new analytical approach known as the parallel reaction monitoring (PRM) for targeted quantitative analysis.
RESULTS: The calibration curves were linear and R2 ranged from 0.9913 to 0.9995 for all analytes. LOQ values are between 0.382 and 25 ng mL-1 and LOD values are between 0.109 and 7.421 ng mL-1. The method shows an accuracy and precision for intra-day and inter-day < 15% RSD and R.E.% for all the QC samples. The matrix effect calculated at different concentration levels did not exceed 15%.
CONCLUSIONS: The method developed provides rapid, easy and robust identification and measurement of a wide range of polyamines, and some of their metabolites that can be evaluated as biomarkers to predict the clinical features of prostate cancer patients, avoiding invasive diagnostic procedures.

Keywords:  Biomarkers; Orbitrap mass spectrometer; PRM mode; Plasma polyamines; Prostate cancer; UHPLC

DOI:  https://doi.org/10.1016/j.jchromb.2020.122468
Metabolites. 2020 Dec 18. pii: E514. [Epub ahead of print]10(12):

Data-Independent Acquisition for the Quantification and Identification of Metabolites in Plasma.

Tom van der Laan, Isabelle Boom, Joshua Maliepaard, Anne-Charlotte Dubbelman, Amy C Harms, Thomas Hankemeier.

  A popular fragmentation technique for non-targeted analysis is called data-independent acquisition (DIA), because it provides fragmentation data for all analytes in a specific mass range. In this work, we demonstrated the strengths and weaknesses of DIA. Two types of chromatography (fractionation/3 min and hydrophilic interaction liquid chromatography (HILIC)/18 min) and three DIA protocols (variable sequential window acquisition of all theoretical mass spectra (SWATH), fixed SWATH and MSALL) were used to evaluate the performance of DIA. Our results show that fast chromatography and MSALL often results in product ion overlap and complex MS/MS spectra, which reduces the quantitative and qualitative power of these DIA protocols. The combination of SWATH and HILIC allowed for the correct identification of 20 metabolites using the NIST library. After SWATH window customization (i.e., variable SWATH), we were able to quantify ten structural isomers with a mean accuracy of 103% (91-113%). The robustness of the variable SWATH and HILIC method was demonstrated by the accurate quantification of these structural isomers in 10 highly diverse blood samples. Since the combination of variable SWATH and HILIC results in good quantitative and qualitative fragmentation data, it is promising for both targeted and untargeted platforms. This should decrease the number of platforms needed in metabolomics and increase the value of a single analysis.

Keywords:  MSALL; SWATH; chromatography; data-independent acquisition; mass spectrometry; metabolomics

DOI:  https://doi.org/10.3390/metabo10120514
Anal Chem. 2020 Dec 27.

High Coverage Profiling of Carboxylated Metabolites in HepG2 Cells Using Secondary Amine-Assisted Ultrahigh-Performance Liquid Chromatography Coupled to High-Resolution Mass Spectrometry.

Jie Zheng, Ge-Ge Gong, Shu-Jian Zheng, Yu Zhang, Yu-Qi Feng.

Carboxylic metabolites are an important class of metabolites, which widely exist in mammals with various types. Chemical isotope labeling liquid chromatography-mass spectrometry (CIL-LC-MS) has been widely used for the detection of carboxylated metabolites. However, high coverage analysis of carboxylated metabolites in biological samples is still challenging due to improper reactivity and selectivity of labeling reagents to carboxylated metabolites. In this study, we used N-methylphenylethylamine (MPEA) to label various types of carboxylated metabolites including short-chain fatty acids (SCFAs), medium-chain fatty acids (MCFAs), long-chain fatty acids (LCFAs), polycarboxylic acids (polyCAs), amino acids (AAs), and aromatic acids. Additionally, metabolites containing other functional groups, such as phenol, sulfhydryl, and phosphate groups, could not be labeled under the conditions of MPEA labeling. After MPEA labeling, the detection sensitivity of carboxylic acids was increased by 1-2 orders of magnitude, and their chromatographic retention on a reversed-phase (RP) column was enhanced (RT > 3 min). Under optimized labeling conditions, we used MPEA and d3-N-methylphenylethylamine (d3-MPEA) for high coverage screening of carboxylated metabolites in HepG2 cells by ultrahigh-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). As a result, a total of 403 potential carboxylated metabolites were obtained of which 68 were confirmed based on our established in-house chemically labeled metabolite database (CLMD). SCFAs, MCFAs, LCFAs, polyCAs, AAs, and aromatic acids were all detected in HepG2 cell extracts. Due to the successful identification of AAs, the current method increased the coverage of carboxylated metabolites compared with our previous work. Moreover, 133 and 109 carboxylated metabolites with changed contents were obtained in HepG2 cells incubated with curcumin and R-3-hydroxybutyric acid, respectively. In general, our established method realized high coverage analysis of carboxylated metabolites in HepG2 cells.

DOI: https://doi.org/10.1021/acs.analchem.0c04048
Anal Chem. 2020 Dec 31.

Post-Chromatographic Dicationic Ionic Liquid-Based Charge Complexation for Highly Sensitive Analysis of Anionic Compounds by Ultra-High-Performance Supercritical Fluid Chromatography Coupled with Electrospray Ionization Mass Spectrometry.

Guoping Li, Yueguang Lv, Meng Chen, Xiwen Ye, Zengyuan Niu, Hua Bai, Haimin Lei, Qiang Ma.

A green analytical strategy has been developed for the analysis of 10 perfluorinated compounds (PFCs) incorporating supramolecular solvent (SUPRAS)-based extraction and ultra-high-performance supercritical fluid chromatography (UHPSFC)-tandem mass spectrometry. The SUPRAS was prepared through self-assembly of reverse micelles by mixing heptanol, tetrahydrofuran, and water at optimized volume ratios. An imidazolium-based germinal dicationic ionic liquid (DIL), 1,1-bis(3-methylimidazolium-1-yl) butylene difluoride ([C4(MIM)2]F2), was dissolved in the make-up solvent of UHPSFC and introduced post-column but before the electrospray ionization source. After chromatographic separation on a Torus DIOL analytical column (100 mm × 2.1 mm, 1.7 μm), the PFC analytes associated with the DIL reagent through charge complexation. The formation of positively charged complexes resulted in improved ionization efficiency and analytical sensitivity. Enhancement in signal intensity by one to two magnitudes was achieved in the positive ionization mode compared to the negative ionization mode without using the dicationic ion-pairing reagent. The developed protocol was applied to 32 samples of real textiles and 6 samples of real food packaging materials, which exhibited great potential for the analysis of anionic compounds.

DOI: https://doi.org/10.1021/acs.analchem.0c04612
Anal Chem. 2020 Dec 29.

Structure-Preserving and Perceptually Consistent Approach for Visualization of Mass Spectrometry Imaging Datasets.

Anastasia Sarycheva, Anton Grigoryev, Dmitry Sidorchuk, Gleb Vladimirov, Philipp Khaitovich, Olga Efimova, Olga Gavrilenko, Elena Stekolshchikova, Evgeny N Nikolaev, Yury Kostyukevich.

Mass spectrometry imaging (MSI) has become an important tool for 2D profiling of biological tissues, allowing for the visualization of individual compound distributions in the sample. Based on this information, it is possible to investigate the molecular organization within any particular tissue and detect abnormal regions (such as tumor regions) and many other biologically relevant phenomena. However, the large number of compounds present in the spectra hinders the productive analysis of large MSI datasets when utilizing standard tools. The heterogeneity of samples makes exploratory visualization (a presentation of the general idea of the molecular and structural organization of the inspected tissues) challenging. Here, we explore the application of various dimensionality reduction techniques that have been used extensively in the visualization of hyperspectral images and the MSI data specifically, such as principal component analysis, independent component analysis, non-negative matrix factorization, t-distributed stochastic neighbor embedding, and uniform manifold approximation and projection. Further, we propose a new approach based on a combination of structure preserving visualization with nonlinear manifold embedding of normalized spectral data. This way, we aim to preserve as much spatially overlapping signals as possible while augmenting them with information on compositional (spectral) variation. The proposed approach can be used for exploratory visualization of MSI datasets without prior deep chemical or histological knowledge of the sample. Thus, different datasets can be visually compared employing the proposed method. The proposed approach allowed for the clear visualization of the molecular layer, granular layer, and white matter in chimpanzee and macaque cerebellum slices.

DOI: https://doi.org/10.1021/acs.analchem.0c04256
Pharmaceuticals (Basel). 2020 Dec 12. pii: E458. [Epub ahead of print]13(12):

Determination of Antidepressants in Human Plasma by Modified Cloud-Point Extraction Coupled with Mass Spectrometry.

Elżbieta Gniazdowska, Natalia Korytowska, Grzegorz Kłudka, Joanna Giebułtowicz.

  Cloud-point extraction (CPE) is rarely combined with liquid chromatography coupled to mass spectrometry (LC-MS) in drug determination due to the matrix effect (ME). However, we have recently shown that ME is not a limiting factor in CPE. Low extraction efficiency may be improved by salt addition, but none of the salts used in CPE are suitable for LC-MS. It is the first time that the influences of a volatile salt-ammonium acetate (AA)-on the CPE extraction efficiency and ME have been studied. Our modification of CPE included also the use of ethanol instead of acetonitrile to reduce the sample viscosity and make the method more environmentally friendly. We developed and validated CPE-LC-MS for the simultaneous determination of 21 antidepressants in plasma that can be useful for clinical and forensic toxicology. The selected parameters included Triton X-114 concentration (1.5 and 6%, w/v), concentration of AA (0, 10, 20 and 30%, w/v), and pH (3.5, 6.8 and 10.2). The addition of 10% of AA increased recovery twice. For 20 and 30% (w/v) of AA, three phases were formed that prolonged the extraction process. The developed CPE method (6% Triton X-114, 10% AA, pH 10.2) was successfully validated through LC-MS/MS simultaneous determination of 21 antidepressants in human plasma. The linearity was in the range of 10-750 ng/mL (r2 > 0.990).

Keywords:  LC–MS/MS; antidepressant; bioanalytical methods; sample preparation

DOI:  https://doi.org/10.3390/ph13120458
J Forensic Sci. 2020 Dec 28.

Simultaneous determination of 10 new psychoactive piperazine derivatives in urine using ultrasound-assisted low-density solvent dispersive liquid-liquid microextraction combined with gas chromatography-tandem mass spectrometry.

Binling Zhu, Liang Meng, Jie Cao, Wenrong Yang, Xavier A Conlan.

  With the rapid development of synthetic drugs, novel piperazine derivatives, as an increasingly important class of new psychoactive substances (NPS), have attracted global attention owing to their increasing demand in the illicit drug market. In this study, ten piperazine derivatives were analyzed in urine samples after pre-treatment with ultrasound-assisted low-density solvent dispersive liquid-liquid microextraction (UA-LDS-DLLME) combined with gas chromatography-tandem mass spectrometry (GC-MS/MS). This simple approach involved the use of urine samples (1 mL) adjusted to pH 12, which was added to 100 µL of n-hexane and subjected to ultrasonication for 3 min to completely disperse the sample in the n-hexane solution. The resulting turbid suspension was centrifuged at 10,000 rpm for 3 min, and the supernatant was extracted and analyzed using GC-MS/MS. Under the optimized conditions presented in this study, the linear relationship between the analytes was good within 10-1500 ng/mL, and the correlation coefficient (r) was between .9914 and .9983. The limit of detection (LOD) was 0.3-2 ng/mL (S/N = 3), and the lower limit of quantification (LLOQ) was 10 ng/mL (S/N = 10) with the recovery of the analytes of interest from the spiked samples being 76.3%-93.3%. This method has been used to analyze real-world samples; our study shows that the UA-LDS-DLLME approach can be used for rapid analysis while consuming minimal solvent for the simultaneous determination of a range of analytes. This method has the potential for use in clinical analyses and forensic toxicology.

Keywords:  GC-MS/MS; forensic toxicology; piperazines; solvent dispersive liquid-liquid microextraction; urine

DOI:  https://doi.org/10.1111/1556-4029.14624
J Agric Food Chem. 2020 Dec 29.

Quantitative Assessment of Dietary (Poly)phenol Intake: A High-Throughput Targeted Metabolomics Method for Blood and Urine Samples.

Maite Domínguez-Fernández, Yifan Xu, Paul Young Tie Yang, Wafa Alotaibi, Rachel Gibson, Wendy L Hall, Leon Barron, Iziar A Ludwig, Concepción Cid, Ana Rodriguez-Mateos.

  Many studies have associated the consumption of (poly)phenol-rich diets with health benefits. However, accurate high-throughput quantitative methods for estimating exposure covering a broad spectrum of (poly)phenols are lacking. We have developed and validated a high-throughput method for the simultaneous quantification of 119 (poly)phenol metabolites in plasma and urine using ultra high-performance liquid chromatography coupled with triple quadrupole mass spectrometry, with a very fast sample treatment and a single run time of 16 min. This method is highly sensitive, precise, accurate, and shows good linearity for all compounds (R2 > 0.992). This novel method will allow a quantitative assessment of habitual (poly)phenol intake in large epidemiological studies as well as clinical studies investigating the health benefits of dietary (poly)phenols.

Keywords:  (poly)phenols; LC-MS; dietary assessment; flavonoids; food bioactives; metabolomics; nutrition; phenolic acids; plant foods

DOI:  https://doi.org/10.1021/acs.jafc.0c07055
Anal Biochem. 2020 Dec 22. pii: S0003-2697(20)30621-7. [Epub ahead of print] 114089

Quantitative MALDI-MS assay of steroid hormones in plasma based on hydroxylamine derivatization.

Zhijing Song, Huanyu Gao, Wenchun Xie, Qing Sun, Kai Liang, Yan Li.

  Measuring the concentrations of steroid hormones in plasma is critical for understanding their role in various vital physiological processes. The detection of underivatized steroid hormones in biofluids through mass spectrometry (MS) is typically hindered by low ionization efficiency. We described a novel matrix-assisted laser desorption/ionization-MS (MALDI-MS) approach based on hydroxylamine derivatization (HA-D) to analyze low-concentration steroid hormones in plasma. The ketonic carbonyl group containing steroid hormones could be derivatized using HA to form oxime derivatives, which considerably enhanced the MS sensitivity for detecting steroid hormones. By using the optimized conditions, estrone (E1), testosterone (T), and progesterone (Prog), could be simultaneously quantified in plasma with a limit of detection (LOD) from 0.019 to 0.031 nM, recoveries from 86% to 108%, and coefficient of variation (CV%) from 4.59% to 11.90%. HA-D/MALDI-MS exhibited higher sensitivity than those using Girard T (GT). To establish potential utility of our method, we characterized fatter liver patient plasmas to demonstrate that the HA-D/MALDI-MS procedure could generate quantitative results comparable to the current clinical liquid chromatography-electrospray ionization tandem MS (LC-ESI MS/MS) method. This approach facilitates the rapid and accurate characterization of plasma hormones, and renders the MALDI-MS approach for steroid hormones more adaptable for clinical research and use.

Keywords:  Hydroxylamine (HA) derivatization; MALDI-MS; Oxime; Plasma; Steroid hormone

DOI:  https://doi.org/10.1016/j.ab.2020.114089
Biol Methods Protoc. 2020 ;5(1): bpaa019

Positive/negative ion-switching-based LC-MS/MS method for quantification of cytosine derivatives produced by the TET-family 5-methylcytosine dioxygenases.

Aninda Sundar Dey, Navid J Ayon, Chayan Bhattacharya, William G Gutheil, Mridul Mukherji.

  Cytosine methylation at carbon-5 (5mC) in DNA plays crucial roles in epigenetic transcriptional regulation during metazoan development. The iron (II), 2-oxoglutarate-dependent Ten-Eleven Translocation (TET)-family dioxygenases initiate active demethylation of 5mC. TET2 oxidizes 5mC in nucleic acids into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine by iterative oxidation. Mutations in the TET2 gene are frequently detected in myeloid malignancies. Despite the established and emerging roles of TET oxygenases in health and diseases, in vitro characterization of these enzymes and their mutants is still in rudimentary stages. Here, we describe an improved positive/negative ion-switching-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) method that can separate and quantify modified cytosine bases produced by TET-family 5-methylcytosine dioxygenases. This method will help in further elucidate the function of epigenetically important cytosine modifications. To the best of our knowledge, this is the first study reporting ion-switching-based LC-MS/MS method to analyse cytosine variants produced in TET catalysed reactions.

Keywords:  DNA demethylation; LC-MS/MS; TET dioxygenases; epigenetics; positive/negative ion-switching

DOI:  https://doi.org/10.1093/biomethods/bpaa019
Anal Chim Acta. 2021 Jan 25. pii: S0003-2670(20)31144-2. [Epub ahead of print]1143 124-134

Single cell metabolomics using mass spectrometry: Techniques and data analysis.

Renmeng Liu, Zhibo Yang.

  Mass spectrometry (MS) based techniques are gaining popularity for metabolomics research due to their high sensitivity, wide detection range, and capability of molecular identification. Utilizing such powerful technique to explore the cellular metabolism at the single cell level not only appreciates the subtle cell-to-cell difference (i.e., cell heterogeneity), but also gains biological merits corresponding to individual cells or small cell subpopulations. In this review article, we first briefly summarize recent advances in single cell MS experimental techniques, and then emphasize on the single cell metabolomics data analysis approaches. Through implementation of statistical analysis and more advanced data analysis methods, single cell metabolomics is expected to find more potential applications in the translational and clinical fields in the future.

Keywords:  Biological variance vs technical variance; Machine learning; Single cell mass spectrometry; Single cell metabolomics; Univariate and multivariate analysis; Vacuum-based and ambient mass spectrometry

DOI:  https://doi.org/10.1016/j.aca.2020.11.020
Anal Chim Acta. 2021 Jan 25. pii: S0003-2670(20)31151-X. [Epub ahead of print]1143 166-172

Anion exchange chromatography - Mass spectrometry for monitoring multiple quality attributes of erythropoietin biopharmaceuticals.

Guusje van Schaick, Christoph Gstöttner, Alexander Büttner, Dietmar Reusch, Manfred Wuhrer, Elena Domínguez-Vega.

  Assessment of critical quality attributes of the biopharmaceutical erythropoietin (EPO) prior to product release requires the use of several analytical methods. We developed an MS-compatible anion exchange (AEX) method for monitoring multiple quality attributes of EPO biopharmaceuticals. AEX was performed using a stationary phase with quaternary ammonium functional groups and a pH gradient for elution. Baseline separation of charge variants and high-quality MS data were achieved using 30 mM ammonium formate pH 5.5 and 30 mM formic acid pH 2.5 as mobile phases. In a single experiment, assessment of critical quality attributes, such as charge heterogeneity, sialic acid content and number of N-acetyllactosamine units, was possible while providing additional information on other modifications such as O-acetylation and deamidation. In addition, good repeatability and robustness for the relative areas of the individual glycoforms and average number of Neu5Ac per EPO molecule were observed. The results were comparable to common pharmacopeia and standard methods with the advantage of requiring fewer analytical methods and less sample treatment saving time and costs.

Keywords:  Anion exchange chromatography; Biopharmaceuticals; Critical quality attributes; Erythropoietin; High resolution mass spectrometry; Multiple attribute method

DOI:  https://doi.org/10.1016/j.aca.2020.11.027
Forensic Sci Int. 2020 Dec 29. pii: S0379-0738(20)30522-3. [Epub ahead of print]319 110660

Optimization of QuEChERS extraction for detection and quantification of 20 antidepressants in postmortem blood samples by LC-MS/MS.

Jacqueline de M Campêlo, Taís B Rodrigues, Jose L Costa, Jandyson M Santos.

  In this study, a comprehensively optimization of QuEChERS (quick, easy, cheap, effective, rugged and safe) method using design of experiments (DOE) was conducted to evaluate the best conditions to obtain the most effective extraction. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis was performed to identify and quantify the antidepressants, with electrospray ionization acquired in positive mode. The method was validated for all analytes; the calibration curves were linear from 10-1000ng/mL, with R2>0.98, and with LOD and LOQ defined as 10ng/mL. Method imprecision and bias were less than 14.3% and 18.9%, respectively. Neither carryover nor interferences were observed. Overall, the optimized method was applied in postmortem real sample analysis to quantify the antidepressants. This study showed a viable method that can be applied for routine forensic analysis, with a quick and easy sample preparation and a rapid total run time of 8min for each analysis.

Keywords:  Antidepressants; Design of experiments; Forensic toxicology; LC–MS/MS; Postmortem blood; QuEChERS

DOI:  https://doi.org/10.1016/j.forsciint.2020.110660
Anal Chim Acta. 2021 Jan 25. pii: S0003-2670(20)31143-0. [Epub ahead of print]1143 189-200

Plasma and serum oxylipin, endocannabinoid, bile acid, steroid, fatty acid and nonsteroidal anti-inflammatory drug quantification in a 96-well plate format.

Theresa L Pedersen, Ira J Gray, John W Newman.

  The goal of this research was to develop a high-throughput, cost-effective method for metabolic profiling of lipid mediators and hormones involved in the regulation of inflammation and energy metabolism, along with polyunsaturated fatty acids and common over-the-counter non-steroidal anti-inflammatory drugs (NSAIDs). We describe a 96-well plate protein precipitation and filtration procedure for 50 μL of plasma or serum in the presence of 37 deuterated analogs and 2 instrument internal standards. Data is acquired in two back-to-back UPLC-MS/MS analyses using electrospray ionization with positive/negative switching and scheduled multiple reaction monitoring for the determination of 145 compounds, including oxylipins, endocannabinoids and like compounds, bile acids, glucocorticoids, sex steroids, polyunsaturated fatty acids, and 3 NSAIDs. Intra- and inter-batch variability was <25% for >70% of metabolites above the LOQ in both matrices, but higher inter-batch variability was observed for serum oxylipins and some bile acids. Results for NIST Standard Reference Material 1950, compared favorably with the 20 certified metabolite values covered by this assay, and we provide new data for oxylipins, N-acylethanolamides, glucocorticoids, and 17-hydroxy-progesterone in this material. Application to two independent cohorts of elderly men and women showed the routine detection of 86 metabolites, identified fasting state influences on essential fatty acid-derived oxylipins, N-acylethanolamides and conjugated bile acids, identified rare presence of high and low testosterone levels and the presence of NSAIDs in ∼10% of these populations. The described method appears valuable for investigations in large cohort studies to provide insight into metabolic cross-talk between the array of mediators assessed here.

Keywords:  High-throughput; Lipid mediators; Metabolic profiling; NIST SRM 1950

DOI:  https://doi.org/10.1016/j.aca.2020.11.019
Bioinformatics. 2020 Dec 26. pii: btaa1072. [Epub ahead of print]

The software for interactive evaluation of mass spectra stability and reproducibility.

E S Zhvansky, A A Sorokin, D S Bormotov, K V Bocharov, D S Zavorotnyuk, D G Ivanov, E N Nikolaev, I A Popov.

SUMMARY: Mass spectrometry methods are widely used for the analysis of biological and medical samples. Recently developed methods such as DESI, REIMS, NESI allow fast analyses without sample preparation at the cost of higher variability of spectra. In biology and medicine, MS profiles are often used with machine learning (classification, regression, etc.) algorithms and statistical analysis, which are sensitive to outliers and intraclass variability. Here we present SSM Display software, a tool for fast visual outlier detection and variance estimation in mass spectrometric profiles. The tool speeds up the process of manual spectra inspection, improves accuracy and explainability of outlier detection, and decreases the requirements to the operator experience. It was shown that the batch effect could be revealed through SSM analysis and that the SSM calculation can also be used for tuning novel ion sources concerning the quality of obtained mass spectra.
AVAILABILITY: Source code, example datasets, binaries, and other information are available at https://github.com/EvgenyZhvansky/R_matrix.
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

DOI: https://doi.org/10.1093/bioinformatics/btaa1072
Talanta. 2021 Mar 01. pii: S0039-9140(20)31214-5. [Epub ahead of print]224 121923

Fully automated method for quantitative determination of steroids in serum: An approach to evaluate steroidogenesis.

D Luque-Córdoba, F Priego-Capote.

  Steroidogenesis is a set of metabolic reactions where the enzymes play a key role to control the physiological levels of steroids. A deficiency in steroidogenesis induces an accumulation and/or insufficiency of steroids in human blood and can lead to different pathologies. This issue added to the low levels of steroids (pg mL-1 to ng mL-1) in this biofluid make of their determination an analytical challenge. In this research, we present a high-throughtput and fully automated method based on solid-phase extraction on-line coupled to liquid chromatography with tandem mass spectrometry detection (SPE-LC-MS/MS) to quantify estrogens (estrone and estradiol), androgens (testosterone, androstenedione, dihydrotestosterone and dehydroepiandrosterone), progestogens (progesterone, pregnenolone, 17-hydroxyprogesterone and 17-hydroxypregnenolone), glucocorticoids (21-hydroxyprogesterone, 11-deoxycortisol, cortisone, corticosterone and cortisol) and one mineralocorticoid (aldosterone) in human serum. The performance of the SPE step and the multiple reaction monitoring (MRM) mode allowed reaching a high sensitivity and selectivity levels without any derivatization reaction. The fragmentation mechanisms of the steroids were complementary studied by LC-MS/MS in high-resolution mode to confirm the MRM transitions. The method was characterized with two SPE sorbents with similar physico-chemical properties. Thus, limits of quantification were at pg mL-1 levels, the variability was below 25% (except for pregnenolone and cortisone), and the accuracy, expressed as bias, was always within ±25%. The proposed method was tested in human serum from ten volunteers, who reported levels for the sixteen target steroids that were satisfactorily in agreement with the physiological ranges reported in the literature.

Keywords:  Automation; Hormones; SPE–LC–MS/MS; Serum; Steroidogenesis; Steroids

DOI:  https://doi.org/10.1016/j.talanta.2020.121923