bims-metlip 2020-05-10 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2020–05–10
nineteen papers selected by
Sofia Costa, Cold Spring Harbor Laboratory

Direct liquid chromatography tandem mass spectrometry analysis of amino acids in human plasma.
Validation and Optimization of Ultrasound-Assisted Dispersive Liquid-Liquid Microextraction as a Preparation Method for Detection of Methadone in Saliva with Gas Chromatography-Mass Spectrometry Technique.
Metabolic profiling of different wild and cultivated Allium species based on high-resolution mass spectrometry, high-performance liquid chromatography-photodiode array detector, and color analysis.
Development of a thin-film solid-phase microextraction (TF-SPME) method coupled to liquid chromatography and tandem mass spectrometry for high-throughput determination of steroid hormones in white sucker fish plasma.
Network based strategies in metabolomics data analysis and interpretation: from molecular networking to biological interpretation.
Pressurized liquid extraction followed by liquid chromatography coupled to a fluorescence detector and atmospheric pressure chemical ionization mass spectrometry for the determination of benzo(a)pyrene metabolites in liver tissue of an animal model of colon cancer.
Preparation of frozen sections of multicellular tumor spheroids coated with ice for mass spectrometry imaging.
Serum metabolomic profiling in patients with Alzheimer Disease and Amnestic Mild Cognitive Impairment by GC/MS.
Development and Application of a Novel Pluri-Residue Method to Determine Polar Pesticides in Fruits and Vegetables through Liquid Chromatography High Resolution Mass Spectrometry.
Pathway-Activity Likelihood Analysis and Metabolite Annotation for Untargeted Metabolomics Using Probabilistic Modeling.
Liquid chromatography above 20,000 PSI.
PiTMaP: A new analytical platform for high-throughput direct metabolome analysis by probe electrospray ionization/tandem mass spectrometry using an R software-based data pipeline.
Separation of triacylglycerols containing allenic and acetylenic fatty acids by enantiomeric liquid chromatography-mass spectrometry.
A UHPLC-MS-MS Method for the Determination of 84 Drugs of Abuse and Pharmaceuticals in Blood.
Analytical method development and validation of reverse-phase high-performance liquid chromatography (RP-HPLC) method for simultaneous quantifications of quercetin and piperine in dual-drug loaded nanostructured lipid carriers.
In-vitro NMR Studies of Prostate Tumor Cell Metabolism by Means of Hyperpolarized [1-13C]Pyruvate Obtained Using the PHIP-SAH Method.
Development of ion pairing LC-MS/MS method for itaconate and cis-aconitate in cell extract and cell media.
Scientific workflow managers in metabolomics: an overview.
An analytical method for the simultaneous quantification of 30 bioactive compounds in spent coffee ground by HPLC-MS/MS.

J Chromatogr A. 2020 Apr 17. pii: S0021-9673(20)30364-2. [Epub ahead of print] 461135

Direct liquid chromatography tandem mass spectrometry analysis of amino acids in human plasma.

Aurore Desmons, Elizabeth Thioulouse, Jean-Yves Hautem, Anne Saintier, Bruno Baudin, Antonin Lamazière, Claude Netter, Fathi Moussa.

  Here we describe a new HPLC-MS/MS method using a mixed mode stationary phase and a binary gradient of elution for the rapid separation and quantification of AAs in human plasma without derivatization or ion pairing reagent addition. The sample preparation procedure consists in a single dilution step after protein precipitation with sulfosalicylic acid. The proposed method allows for the unambiguous identification and analysis of 52 AAs and related compounds including the separation of isomers and isobars in an 18 min chromatographic run including the conditioning and the equilibration times. AAs were detected by selective reaction monitoring. Internal calibration was used for the quantification of 37 AAs, including 25 using the corresponding isotopically labeled internal standards. External calibration (no internal standard) was used for five additional analytes. Qualitative detection was achieved for the remaining compounds. Validation studies evaluated accuracy, linearity, within- and between-run precision, lower limits of detection and quantification for 37 amino acids present in commonly used quality control samples. For within-run precision CVs averaged 3.8 % (n = 30) for all compounds. For between-run precision, CVs averaged 8.6 % for all compounds (n = 20). Correlation with the common standard ion-exchange chromatography with post-column derivatization method was also performed for 32 plasma samples. While the proposed method is at least 50 times more sensitive, the data showed good correlation with slopes equal or higher than 0.9 and correlation coefficients mostly higher than 0.90. The method was successfully applied for analysis of plasma samples for detection of inherited disorders of amino acid metabolism.

Keywords:  Amino acid profiling; Human plasma; LC-MS/MS; Mixed-mode stationary phases

DOI:  https://doi.org/10.1016/j.chroma.2020.461135
Adv Pharm Bull. 2020 Jun;10(2): 329-333

Validation and Optimization of Ultrasound-Assisted Dispersive Liquid-Liquid Microextraction as a Preparation Method for Detection of Methadone in Saliva with Gas Chromatography-Mass Spectrometry Technique.

Ahmad Shekari, Mehdi Forouzesh, Roohollah Valipour, Fardin Fallah, Pardis Shojaei.

  Purpose: We investigated validation and optimization of ultrasound-assisted dispersive liquidliquid microextraction (UADLLME) as a preparation method for detection of methadone in saliva samples. Methods: We used blank and methadone-containing saliva samples and also standard methadone solution. Sodium hydroxide and chloroform were added to samples and they were held in ultrasonic bath. Then preparations were centrifuged and extracted analyte was analyzed by gas chromatography-mass spectrometry (GC-MS). Accuracy was measured by Intra and between-day mean relative errors (RE). Precision was assessed by coefficient of variation (CV). Recovery, specificity, linearity and limits of detection and quantification were also determined. Optimization was conducted for ultrasound duration, pH and extraction phase volume. Efficiency of dispersive liquid-liquid microextraction (DLLME) and UADLLME were compared. Results: Intra and between-day accuracies (2.3 -7.5%), recovery (89.4-115.5%) and precision (5.2-11.3%) were all acceptable. Calibration curve was linear in the concentration range of 150 ng/mL-10 µL/mL with R2 >0.9995 and equation of y=86.901x-5342.5. Limits of detection and quantification were 50 and 150 ng/mL, respectively. Specificity was measured by comparing retention times of saliva samples (containing methadone metabolites and other commonly used drugs) during UADLLME/GC-MS analysis and no interference was observed. Recovery of UADLLME was 1.4 of DLLME. Solvent and sample volumes required for UADLLME were 1/200 and 1/20 of DLLME. The greatest efficiency obtained at pH of 10, with ultrasound treatment duration of 5 minutes and extraction phase volume of 1000 µL. Conclusion: Study found that UADLLME/GC-MS is a valid and efficient method for detection of methadone in oral fluid.

Keywords:  Dispersive liquid-liquid microextraction; Gas chromatography-mass spectrometry; Methadone; Saliva; Ultrasound; Validation

DOI:  https://doi.org/10.34172/apb.2020.040
J Mass Spectrom. 2020 Apr 14. e4525

Metabolic profiling of different wild and cultivated Allium species based on high-resolution mass spectrometry, high-performance liquid chromatography-photodiode array detector, and color analysis.

Alessandro Maccelli, Stefania Cesa, Francesco Cairone, Daniela Secci, Luigi Menghini, Barbara Chiavarino, Simonetta Fornarini, Maria Elisa Crestoni, Marcello Locatelli.

  Many plants of the genus Allium are widely cultivated and consumed for their nutraceutical and health-enhancing bioactive components effective in many metabolic and infectious diseases. In particular, Allium sativum L. (garlic), the most economically important Allium species, is known to present volatile, comparatively polar sulfur-containing compounds responsible for both the typical garlic aroma and antimicrobial property. More recently, the (moderately) polar portion of garlic metabolome, rich of polyphenols and amino acids, is gaining increasing interest as a source of antioxidants and primary nutrients. In this study, we have explored the chemical diversity of eight different hydroalcoholic extracts obtained by microwave-assisted extraction of white and red crop A. sativum and wild Allium triquetrum, Allium roseum, and Allium ampeloprasum, all originating from the Mediterranean Basin. The aim is to appraise their potential dietetic and healing value through an in-depth chemical characterization and contribute to preserve and exploit natural resources. The multimethodological method applied here is based on an untargeted metabolic profiling by means of high-resolution electrospray ionization Fourier-transform ion cyclotron resonance (ESI FT-ICR) mass spectrometry. More than 850 by ESI(+) and 450 by ESI(-) putative metabolites have been annotated covering all main classes of primary and secondary metabolites, including amino acids, alkaloids, organic and fatty acids, nucleotides, vitamins, organosulfur compounds, and flavonoids. The pigment and polyphenol components have been separated and quantified by a targeted chromatographic high-performance liquid chromatography-photodiode array detector (HPLC-PDA) and CIEL*a*b* colorimetric assay, showing characteristic yellow and red components in each extract, related to a different milieu of anthocyanins and flavonoids as assigned by high-resolution mass spectrometry (MS).

Keywords:  Allium sativum; FT-ICR mass spectrometry; food analysis; metabolomics; wild Allium species

DOI:  https://doi.org/10.1002/jms.4525
Anal Bioanal Chem. 2020 May 02.

Development of a thin-film solid-phase microextraction (TF-SPME) method coupled to liquid chromatography and tandem mass spectrometry for high-throughput determination of steroid hormones in white sucker fish plasma.

Małgorzata Maciążek-Jurczyk, Vincent Bessonneau, Jennifer Ings, Leslie Bragg, Mark McMaster, Mark R Servos, Barbara Bojko, Janusz Pawliszyn.

  Steroid hormones (SH) play a number of important physiological roles in vertebrates including fish. Changes in SH concentration significantly affect reproduction, differentiation, development, or metabolism. The objective of this study was to develop an in vitro high-throughput thin-film solid-phase microextraction (TF-SPME)-liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for targeted analysis of endogenous SH (cortisol, testosterone, progesterone, estrone (E1), 17β-estradiol (E2), and 17α-ethinylestradiol (EE2)) in wild white sucker fish plasma where the concentrations of the analytes are substantially low. A simple TF-SPME method enabled the simultaneous determination of free and total SH concentrations. The use of biocompatible coating allowed direct extraction of these hormones from complex biological samples without prior preparation. The carryover was less than 3%, thereby ensuring reusability of the devices and reproducibility. The results showed that TF-SPME was suitable for the analysis of compounds in the polarity range between 1.28 and 4.31 such as SH at different physicochemical properties. The proposed method was validated according to bioanalytical method validation guidelines. The limit of detection (LOD) and limit of quantification(LOQ) for cortisol, testosterone, progesterone, E1, E2, and EE2 were from 0.006 to 0.150 ng/mL and from 0.020 to 0.500 ng/mL, respectively. The recovery for the method was about 85%, and the accuracy and precision of the method for cortisol, testosterone, and progesterone were ≤ 6.0% and ≤ 11.2%, respectively, whereas those for E1, E2, and EE2 were ≤ 15.0% and ≤ 10.2%, respectively. On the basis of this study, TF-SPME demonstrated several important advantages such as simplicity, sensitivity, and robustness under laboratory conditions. Graphical abstract.

Keywords:  Fish steroid hormones; LC–MS/MS; Thin-film coated blades; Thin-film solid-phase microextraction

DOI:  https://doi.org/10.1007/s00216-020-02657-x
Expert Rev Proteomics. 2020 May 07.

Network based strategies in metabolomics data analysis and interpretation: from molecular networking to biological interpretation.

Leonardo Perez de Souza, Saleh Alseekh, Yariv Brotman, Alisdair R Fernie.

  Introduction: Metabolomics has become a crucial part of systems biology; however data analysis is still often undertaken in a reductionist way focusing on changes in individual metabolites. Whilst such approaches indeed provide relevant insights into the metabolic phenotype of an organism, the intricate nature of metabolic relationships may be better explored when considering the whole system.Areas covered: This review highlights multiple network strategies that can be applied for metabolomics data analysis from different perspectives including: association networks based on quantitative information, mass spectra similarity networks to assist metabolite annotation and biochemical networks for systematic data interpretation. We also highlight some relevant insights into metabolic organization obtained through the exploration of such approaches.Expert opinion: Network based analysis is an established method that allows the identification of non-intuitive metabolic relationships as well as the identification of unknown compounds in mass spectrometry. Additionally, the representation of data from metabolomics within the context of metabolic networks is intuitive and allows for the use of statistical analysis that can better summarize relevant metabolic changes from a systematic perspective.

Keywords:  Correlation; Metabolomics; Network; Pathway enrichment; Spectra similarity

DOI:  https://doi.org/10.1080/14789450.2020.1766975
J Chromatogr A. 2020 Apr 27. pii: S0021-9673(20)30354-X. [Epub ahead of print] 461126

Pressurized liquid extraction followed by liquid chromatography coupled to a fluorescence detector and atmospheric pressure chemical ionization mass spectrometry for the determination of benzo(a)pyrene metabolites in liver tissue of an animal model of colon cancer.

Kenneth J Harris, Seenivasan Subbiah, Mohammad Tabatabai, Anthony E Archibong, Kamaleshwar P Singh, Todd A Anderson, Samuel E Adunyah, Aramandla Ramesh.

  Since metabolism is implicated in the carcinogenesis of toxicants, an efficient extraction method together with an analytical method is warranted to quantify tissue burdens of a carcinogen and/or its metabolites. Therefore, the aim of this study was to validate a pressurized liquid extraction (PLE) method for measuring metabolites of benzo(a)pyrene [B(a)P; a food-borne carcinogen] from tissue samples. The sample extraction was performed separately by PLE and liquid-liquid extraction (LLE). PLE followed by high-performance liquid chromatography coupled to online fluorescence detector (HPLC-FLD) was used to quantify separated analytes; and by ultra-high-performance liquid chromatography (UHPLC) coupled to atmospheric pressure chemical ionization tandem mass spectrometry (UHPLC-APCI-MS/MS) were used for confirmation purposes. The UHPLC-MS/MS was set-up in the atmospheric pressure chemical ionization (APCI) positive interface with selective reaction monitoring (SRM). The analytical performance characteristics of the PLE technique was assessed at different temperatures, pressure, number of cycles and solvent types. A methanol + chloroform + water mixture (30:15:10, v/v/v) yielded greater recoveries at an extraction temperature range of 60-80°C, pressure of 10 MPa and an extraction time of 10 min. The PLE method was validated by the analysis of spiked tissue samples and measuring recoveries and limits of quantitation for the analytes of interest using HPLC-FLD equipment. The optimized PLE-HPLC-FLD method was used to quantify the concentrations of B(a)P metabolites in liver samples obtained from a colon cancer animal model. Overall, PLE performed better in terms of extraction efficiency, recovery of B(a)P metabolites and shortened sample preparation time when compared with the classic LLE method.

Keywords:  Accelerated solvent extraction; Benzo(a)pyrene; Colon cancer; Liquid–liquid extraction; Metabolites; Pressurized liquid extraction

DOI:  https://doi.org/10.1016/j.chroma.2020.461126
Anal Chem. 2020 May 06.

Preparation of frozen sections of multicellular tumor spheroids coated with ice for mass spectrometry imaging.

Peisi Xie, Chao Zhao, Xiaoping Liang, Wei Huang, Yanyan Chen, Zongwei Cai.

Increasing studies have utilized mass spectrometry imaging (MSI) that is a label-free tool to investigate drug penetration and drug biotransformation in multicellular tumor spheroids (MCTS). Currently, the gelatin-assisted sectioning method is widely used to prepare frozen sections of MCTS for MSI. However, owing to the limited transparency of frozen gelatin, MCTS with diameters less than 500 μm that closely mimic solid tumors are difficult to be detected when cryosectioning. In order to identify the presence of MCTS, hematoxylin and eosin staining for frozen sections and dye pretreatment for MCTS were employed in previous works, which either increased the analytical time and cost in sample preparation or caused signal suppression in sample analysis. Herein, a new sectioning method was developed to prepare MCTS frozen sections. MCTS was coated with ice to ensure a good visibility for small-size MCTS. The optimal cutting temperature compound was added around the ice block to assist the formation of frozen sections. A precast frozen mold was prepared to allow the acquisition of complete MCTS frozen sections. The developed method was applied to investigate lipid distribution in MCTS by using matrix-assisted laser desorption/ionization MSI. Compared to the gelatin-assisted sectioning method, our method did not cause signal suppression and analyte delocalization. Thus, this method provides an easy, universal, and innovative strategy to prepare MCTS frozen sections for further MSI analysis. Besides, we applied our method to investigate the penetration of bisphenol A in MCTS.

DOI: https://doi.org/10.1021/acs.analchem.9b05812
Biomed Chromatogr. 2020 May 08. e4875

Serum metabolomic profiling in patients with Alzheimer Disease and Amnestic Mild Cognitive Impairment by GC/MS.

Congcong Sun, Meimei Gao, Feifei Wang, Yan Yun, Qianwen Sun, Ruichen Guo, Chuanzhu Yan, Xiulian Sun, Yi Li.

   OBJECTIVES: The aim of this study is to characterize the serum metabolic profiles of patients with Alzheimer Disease (AD) and Amnestic Mild Cognitive Impairment (AMCI) using metabolomics based on GC/MS.
METHODS: Serum samples were collected from patients with AD (n=30), AMCI (n=32), and normal healthy controls (NOR, n=40). Metabolite profiles were performed with gas chromatography-mass spectrometry (GC/MS) in conjunction with multivariate statistical analysis, and possible biomarker metabolites were identified.
RESULTS: 31 kinds of endogenous metabolites could be identified simultaneously. 11 components were chosen as biomarker metabolites between AD and NOR groups, and these metabolites were closely related to 7 biological pathways: arginine and proline metabolism, phenylalanine metabolism, beta-alanine metabolism, primary bile acid synthesis, glutathione metabolism, starch and sucrose metabolism and steroid hormone biosynthesis. Meanwhile,10 components were chosen as biomarker metabolites between AMCI and NOR groups and 7 biological pathways were closely related: arginine and proline metabolism, phenylalanine metabolism, citrate cycle, alanine, aspartate and glutamate metabolism, taurine and hypotaurine metabolism, starch and sucrose metabolism, and steroid hormone biosynthesis.
CONCLUSIONS: Our study distinguished serum metabotypes between AD, AMCI and NOR patients successfully. The implementation of this metabolomic strategy may help to develop biochemical insight into the metabolic alterations in AD/AMCI and will be helpful for the further understanding of pathogenesis.

Keywords:  Alzheimer; Biomarker; GC/MS; Metabolomics; Mild Cognitive Impairment

DOI:  https://doi.org/10.1002/bmc.4875
Foods. 2020 May 01. pii: E553. [Epub ahead of print]9(5):

Development and Application of a Novel Pluri-Residue Method to Determine Polar Pesticides in Fruits and Vegetables through Liquid Chromatography High Resolution Mass Spectrometry.

Lorena Manzano-Sánchez, José Antonio Martínez-Martínez, Irene Domínguez, José Luis Martínez Vidal, Antonia Garrido Frenich, Roberto Romero-González.

  Nowadays, highly polar pesticides are not included in multiresidue methods due to their physico-chemical characteristics and therefore, specific analytical methodologies are required for their analysis. Laboratories are still looking for a pluri-residue method that encompasses the largest number of polar pesticides. The aim of this work was the simultaneous determination of ethephon, 2-hydroxyethylphosphonic acid (HEPA), fosetyl aluminum, glyphosate, aminomethylphosphonic acid (AMPA), N-acetyl-glyphosate and N-acetyl-AMPA in tomatoes, oranges, aubergines and grapes. For that purpose, an ultra high performance liquid chromatography (UHPLC) coupled to a high resolution single mass spectrometer Orbitrap-MS were used. Different stationary phases were evaluated for chromatographic separation, and among them, the stationary phase Torus DEA provided the best separation of the selected compounds. The QuPPe method was used for the extraction of the analytes, but slight modifications were needed depending on the matrix. The developed method was validated, observing matrix effect in all matrices. Intra- and inter-day precision were estimated, and relative standard deviation were lower than 19%. Recoveries were satisfactory, and mean values ranged from 70% to 110%. Limits of quantification were between 25 and 100 µg kg-1. Finally, the analytical method was applied to different fruits and vegetables (oranges, tomatoes, aubergines and grapes).

Keywords:  QuPPe; UHPLC-Orbitrap-MS; high polar pesticides; pluri-residue analysis

DOI:  https://doi.org/10.3390/foods9050553
Metabolites. 2020 May 03. pii: E183. [Epub ahead of print]10(5):

Pathway-Activity Likelihood Analysis and Metabolite Annotation for Untargeted Metabolomics Using Probabilistic Modeling.

Ramtin Hosseini, Neda Hassanpour, Li-Ping Liu, Soha Hassoun.

  Motivation: Untargeted metabolomics comprehensively characterizes small molecules and elucidates activities of biochemical pathways within a biological sample. Despite computational advances, interpreting collected measurements and determining their biological role remains a challenge. Results: To interpret measurements, we present an inference-based approach, termed Probabilistic modeling for Untargeted Metabolomics Analysis (PUMA). Our approach captures metabolomics measurements and the biological network for the biological sample under study in a generative model and uses stochastic sampling to compute posterior probability distributions. PUMA predicts the likelihood of pathways being active, and then derives probabilistic annotations, which assign chemical identities to measurements. Unlike prior pathway analysis tools that analyze differentially active pathways, PUMA defines a pathway as active if the likelihood that the path generated the observed measurements is above a particular (user-defined) threshold. Due to the lack of "ground truth" metabolomics datasets, where all measurements are annotated and pathway activities are known, PUMA is validated on synthetic datasets that are designed to mimic cellular processes. PUMA, on average, outperforms pathway enrichment analysis by 8%. PUMA is applied to two case studies. PUMA suggests many biological meaningful pathways as active. Annotation results were in agreement to those obtained using other tools that utilize additional information in the form of spectral signatures. Importantly, PUMA annotates many measurements, suggesting 23 chemical identities for metabolites that were previously only identified as isomers, and a significant number of additional putative annotations over spectral database lookups. For an experimentally validated 50-compound dataset, annotations using PUMA yielded 0.833 precision and 0.676 recall.

Keywords:  biological network; inference; machine learning; metabolic model; untargeted metabolomics

DOI:  https://doi.org/10.3390/metabo10050183
Trends Analyt Chem. 2020 Mar;pii: 115810. [Epub ahead of print]124

Liquid chromatography above 20,000 PSI.

Matthew J Sorensen, Brady G Anderson, Robert T Kennedy.

  Continued improvements in HPLC have led to faster and more efficient separations than previously possible. One important aspect of these improvements has been the increase in instrument operating pressure and the advent of ultrahigh pressure LC (UHPLC). Commercial instrumentation is now capable of up to ~20 kpsi, allowing fast and efficient separations with 5-15 cm columns packed with sub-2 μm particles. Home-built instruments have demonstrated the benefits of even further increases in instrument pressure. The focus of this review is on recent advancements and applications in liquid chromatography above 20 kpsi. We outline the theory and advantages of higher pressure and discuss instrument hardware and design capable of withstanding 20 kpsi or greater. We also overview column packing procedures and stationary phase considerations for HPLC above 20 kpsi, and lastly highlight a few recent applicatioob pressure instruments for the analysis of complex mixtures.

Keywords:  Column packing; Long columns; Omics; Small particles; Ultrahigh pressure LC

DOI:  https://doi.org/10.1016/j.trac.2020.115810
Anal Chem. 2020 May 06.

PiTMaP: A new analytical platform for high-throughput direct metabolome analysis by probe electrospray ionization/tandem mass spectrometry using an R software-based data pipeline.

Kei Zaitsu, Seiichiro Eguchi, Tomomi Ohara, Kenta Kondo, Akira Ishii, Hitoshi Tsuchihashi, Takakazu Kawamata, Akira Iguchi.

A new analytical platform called PiTMaP was developed for high-throughput direct metabolome analysis by probe electrospray ionization/tandem mass spectrometry (PESI/MS/MS) using an R software-based data pipeline. PESI/MS/MS was used as the data acquisition technique, applying a scheduled-selected reaction monitoring method to expand the targeted metabolites. Seventy-two metabolites mainly related to the central energy metabolism were selected; data acquisition time was optimized using mouse liver and brain samples, indicating that the 2.4 min data acquisition method had a higher repeatability than the 1.2 min and 4.8 min methods. A data pipeline was constructed using the R software, and it was proven that it can i) automatically generate box-and-whisker plots for all metabolites, ii) perform multivariate analyses such as principal component analysis (PCA) and projections to latent structures-discriminant analysis (PLS-DA), iii) generate score and loading plots of PCA and PLS-DA, iv) calculate variable importance of projection (VIP) values, v) determine a statistical family by VIP value criterion, vi) perform tests of significance with the false discovery rate (FDR) correction method, and vii) draw box-and-whisker plots only for significantly changed metabolites. These tasks could be completed within ca. 1 min. Finally, PiTMaP was applied to two cases: 1) an acetaminophen (APAP)-induced acute liver injury model and control mice and 2) human meningioma samples with different grades (G1-G3), demonstrating the feasibility of PiTMaP. PiTMaP was found to perform data acquisition without tedious sample preparation and a post-hoc data analysis within ca. 1 min. Thus, it would be a universal platform to perform rapid metabolic profiling of biological samples.

DOI: https://doi.org/10.1021/acs.analchem.0c01271
J Chromatogr A. 2020 Apr 28. pii: S0021-9673(20)30401-5. [Epub ahead of print] 461161

Separation of triacylglycerols containing allenic and acetylenic fatty acids by enantiomeric liquid chromatography-mass spectrometry.

Andrea Palyzová, Tomáš Řezanka.

  Triacylglycerols (TAGs) containing less common fatty acids (FAs) were isolated from the seeds of three plants (Santalum album, Crepis foetida, and Leucas aspera). These FAs had allenic (laballenic acid, Lb) and acetylenic (crepenynic, C; ximenynic acids, Xi) bonds. TAGs were analyzed on reversed-phase and chiral columns. High-resolution tandem mass spectrometry identified TAGs by positive electrospray ionization (ESI+). Twenty-two molecular species of TAGs isolated from the seed oil of Santalum album were separated by RP-HPLC and chiral HPLC methods and identified by positive electrospray ionization tandem MS detection (ESI+-MS). Two major enantiomers, i.e., sn-OOLb and sn-LLLb (O represents oleic acid; and L represents linoleic acid), were synthesized from the appropriate phosphatidylcholines. This allowed the identification of enantiomers after separation by chiral chromatography by tandem mass spectrometry. Similarly, TAGs from the seeds of Crepis foetida, and Leucas aspera were analyzed by reversed-phase chromatography and identified by mass spectrometry. Four enantiomers (sn-OOC, sn-LLC, sn-OOXi, and sn-LLXi) were synthesized. A total of six and three enantiomers of TAGs containing crepenynic and ximenynic acids, respectively, were identified by chiral column analysis. The retention times of TAGs containing allenic and acetylenic bonds were always greater on the reversed-phase column than TAGs with the same number of carbon atoms and the same unsaturation (e.g., LLL versus LLLb). From the chiral column, the regioisomers and enantiomers were eluted in the order of symmetric-asymmetric-asymmetric (i.e., sn-OCO, sn-COO, and sn-OOC). Through tandem mass spectrometry, we were able to identify and distinguish regioisomer [DAG]+-type ions, i.e., [MNH4NH3RCOOH]+, that can be considered diagnostic. Unfortunately, enantiomers and TAGs with the same numbers of carbon atoms and the same unsaturation levels have identical mass spectra, such as LLL and LLLb.

Keywords:  Acetylenic fatty acids; Allenic fatty acids; Crepenynic acid; Enantiomeric separation; Triacylglycerols; Ximenynic acid

DOI:  https://doi.org/10.1016/j.chroma.2020.461161
J Anal Toxicol. 2020 May 05. pii: bkaa032. [Epub ahead of print]

A UHPLC-MS-MS Method for the Determination of 84 Drugs of Abuse and Pharmaceuticals in Blood.

Amvrosios Orfanidis, Helen Gika, Georgios Theodoridis, Orthodoxia Mastrogianni, Nikolaos Raikos.

  The analysis of blood samples for forensic or clinical intoxication cases is a daily routine in an analytical laboratory. The list of "suspect" drugs of abuse and pharmaceuticals that should be ideally screened is large, so multi-targeted methods for comprehensive detection and quantification is a useful tool in the hands of a toxicologist. In this study the development of an UHPLC-MS-MS method is described for the detection and quantification of 84 drugs and pharmaceuticals in postmortem blood. The target compounds comprise pharmaceutical drugs (antipsychotics, antidepressants etc.), some of the most important groups of drugs of abuse: opiates, cocaine, cannabinoids, amphetamines, benzodiazepines and new psychoactive substances (NPS). Sample pretreatment was studied applying a modified Mini-QuEChERS single step, and the best results were obtained after adding in 200 μL of sample, a mixture of 20 mg MgSO4, 5 mg K2CO3 and 5 mg NaCl together with 600 μL of cold acetonitrile. After centrifugation, the supernatant was collected for direct injection. LC-MS analysis took place on a C18 column with a gradient elution over 17 minutes. The method was found to be selective, and sensitive offering limits of detection (LOD) ranging from 0.01 to 9.07 ng/mL. Validation included evaluation of limit of quantification (LOQ), recovery, carry-over, matrix effect, accuracy and precision of the method. The method performed satisfactory in relation to established bioanalytical criteria and was therefore applied to the analysis of blood obtained postmortem from chronic drug abusers, offering unambiguous identification and quantitative determination of drugs in postmortem blood.

Keywords:  LC–MS-MS; QuEChERS; Systematic Toxicological Analysis; drugs of abuse; forensic toxicology; pharmaceuticals; postmortem blood

DOI:  https://doi.org/10.1093/jat/bkaa032
J Pharm Biomed Anal. 2020 Apr 29. pii: S0731-7085(20)30012-1. [Epub ahead of print]186 113325

Analytical method development and validation of reverse-phase high-performance liquid chromatography (RP-HPLC) method for simultaneous quantifications of quercetin and piperine in dual-drug loaded nanostructured lipid carriers.

Vishal Sharad Chaudhari, Roshan M Borkar, Upadhyayula Suryanarayana Murty, Subham Banerjee.

  Quercetin and piperine are often used as an add-on therapy for various diseases, however both drug exhibits poor aqueous solubility and photosensitivity issue. Therefore, the aim of the present study is to improve the pharmaceutical challenges by incorporating both the drugs in nanostructured lipid carriers (NLCs) and to develop a sensitive, selective, accurate and precise reverse-phase high performance liquid chromatography (RP-HPLC) method for the simultaneous analysis of both drugs in NLCs. Effective chromatographic separation of quercetin and piperine was achieved on Hypersil gold C-18 column and mobile phase consisting of a mixture of acetonitrile and HPLC grade water (pH 2.6, adjusted with 2%v/v glacial acetic acid) in an isocratic elution mode. The flow rate of the mobile phase was 1 mL/min, column temperature at 35 ± 0.2 °C and the injection volume was 20 μL. The retention time for quercetin and piperine were found to be at 2.80 min and 10.36 min, respectively and detected at an isobestic wavelength of 346 nm using a photodiode array (PDA) detector. The method was found to be specific for the simultaneous analysis of quercetin and piperine in presence of NLCs matrix, accurate (>90%) and precise (%RSD < 2%). The validated RP-HPLC method effectively utilised to determine the percentage drug entrapment efficiency cum percentage drug loading of quercetin and piperine in NLCs enriched formulations along with the secondary estimation of in vitro cumulative percentage drug release study. The results were found to be reliable, hence the validated RP-HPLC method could be further used for the simultaneous detection and quantification of both these drugs in other lipid-based nano-formulations such as solid-lipid nanoparticles, polymer-lipid hybrid nanoparticles, lipid drug conjugates, etc. in in vitro and in vivo.

Keywords:  Analytical method validation; Nanostructured lipid carriers (NLCs); Piperine; Quercetin; RP-HPLC

DOI:  https://doi.org/10.1016/j.jpba.2020.113325
Front Oncol. 2020 ;10 497

In-vitro NMR Studies of Prostate Tumor Cell Metabolism by Means of Hyperpolarized [1-13C]Pyruvate Obtained Using the PHIP-SAH Method.

Eleonora Cavallari, Carla Carrera, Ginevra Di Matteo, Oksana Bondar, Silvio Aime, Francesca Reineri.

  Nuclear Magnetic Resonance allows the non-invasive detection and quantitation of metabolites to be carried out in cells and tissues. This means that that metabolic changes can be revealed without the need for sample processing and the destruction of the biological matrix. The main limitation to the application of this method to biological studies is its intrinsic low sensitivity. The introduction of hyperpolarization techniques and, in particular, of dissolution-Dynamic Nuclear Polarization (d-DNP) and ParaHydrogen Induced Polarization (PHIP) is a significant breakthrough for the field as the MR signals of molecules and, most importantly, metabolites, can be increased by some orders of magnitude. Hyperpolarized pyruvate is the metabolite that has been most widely used for the investigation of metabolic alterations in cancer and other diseases. Although d-DNP is currently the gold-standard hyperpolarization method, its high costs and intrinsically slow hyperpolarization procedure are a hurdle to the application of this tool. However, PHIP is cost effective and fast and hyperpolarized pyruvate can be obtained using the so-called Side Arm Hydrogenation approach (PHIP-SAH). The potential toxicity of a solution of the hyperpolarized metabolite that is obtained in this way is presented herein. HP pyruvate has then been used for metabolic studies on different prostate cancer cells lines (DU145, PC3, and LnCap). The results obtained using the HP metabolite have been compared with those from conventional biochemical assays.

Keywords:  hyperpolarization; metabolism; nuclear magnetic resonance; para-hydrogen; pyruvate

DOI:  https://doi.org/10.3389/fonc.2020.00497
J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Apr 15. pii: S1570-0232(19)31737-4. [Epub ahead of print]1146 122120

Development of ion pairing LC-MS/MS method for itaconate and cis-aconitate in cell extract and cell media.

Bo Tan, Shruti Malu, Kenneth D Roth.

  Accumulation of Immune Responsive Gene 1(IRG1) in macrophage induced by lipopolysaccharide (LPS) and interferon gamma (IFN-γ) leads to production of itaconate by decarboxylation of cis-aconitate. The biology associated with IRG1 and itaconate is not fully understood. A rapid and sensitive method for measurement of itaconate will benefit the study of IRG1 biology. Multiple HPLC and derivatization methods were tested. An ion pairing LC-MS/MS method using tributylamine/formic acid as ion pairing agents and a HypercarbTM guard column we proposed demonstrated better peak shape and better sensitivity for itaconate. The current protocol allows baseline separation of itaconate, citraconate, and cis-aconitate without derivatization and direct analysis of analytes in 80% methanol/water solution to avoid the dry-down step. It provides the limit of quantitation (LOQ) of 30 pg itaconate on column with a 4.5-minute run time. This method is validated for measurement of itaconate and cis-aconitate in RAW264.7 cell extract and cell media in a 96-well plate format. We applied this method to successfully measure the increase of itaconate and the decrease of cis-aconitate in RAW cell extract and cell media after LPS/IFN-γ treatment.

Keywords:  Cis-aconitate; Ion pairing; Itaconate; LC-MS/MS

DOI:  https://doi.org/10.1016/j.jchromb.2020.122120
Analyst. 2020 May 06.

Scientific workflow managers in metabolomics: an overview.

Aswin Verhoeven, Martin Giera, Oleg A Mayboroda.

Providing maximum information on the provenance of scientific results in life sciences is getting considerable attention since the widely publicized reproducibility crisis. Improving the reproducibility of data processing and analysis workflows is part of this movement and may help achieve clinical deployment quicker. Scientific workflow managers can be valuable tools towards achieving this goal. Although these platforms are already well established in the field of genomics and other omics fields, in metabolomics scripts and dedicated software packages are still more popular. However, versatile workflows for metabolomics exist in the KNIME and Galaxy platforms. We will here summarize the available options of scientific workflow managers dedicated to metabolomics analysis.

DOI: https://doi.org/10.1039/d0an00272k
J Mass Spectrom. 2020 Apr 04. e4519

An analytical method for the simultaneous quantification of 30 bioactive compounds in spent coffee ground by HPLC-MS/MS.

Simone Angeloni, Franks Kamgang Nzekoue, Luciano Navarini, Gianni Sagratini, Elisabetta Torregiani, Sauro Vittori, Giovanni Caprioli.

  Spent coffee ground (SCG) is the remaining residue produced after extraction of coffee, and it is considered a source of unextracted bioactive compounds. For this, in the latest years, the attention has been focused to innovative reuses that can exploit the potentiality of SCG. Unfortunately, the content of bioactive compounds has not been thoroughly studied yet, and the major of publication has investigated the caffeine and chlorogenic acids levels, total polyphenol contents, and total flavonoid content. Hence, these approaches have determined only an estimation of flavonoids and polyphenols content and lack on single polyphenols investigation. Therefore, the objective of the current work was to provide a deep characterization of bioactive compounds in SCG. For this purpose, a new analytical method for the quantification of 30 molecules, including caffeine, chlorogenic acids, phenolic acids, flavonoids, and secoiridoids, has been developed using high-performance liquid chromatography tandem mass spectrometry. Moreover, several extraction procedures, that is, liquid-solid extraction assisted and not by ultrasounds, testing diverse solvents, were evaluated. Liquid-solid extraction assisted by sonication, with water/ethanol (30/70, v/v), resulted the best in terms of total bioactive compounds, and, once validated, the new analytical method was applied to five different espresso SCG samples. Data showed that caffeine (means: 1193.886 ± 57.307 mg kg-1 ) and chlorogenic acids (means of total CQAs: 1705.656 ± 88.694 mg kg-1 ) were the most abundant compounds in all SCG samples followed by phenolic acids such as caffeic, ferulic, gallic, p-coumaric, syringic, trans-cinnamic, and vanillic acid. Moreover, some flavonoids, that is, rutin, cyanidin 3-glucoside, and quercetin, occurred in almost all samples. This work provided a deepened characterization of bioactive compounds in SCG and can contribute to develop new strategies of reuses.

Keywords:  HPLC-MS/MS; coffee; coffee by-product; flavonoid; polyphenol; spent coffee ground

DOI:  https://doi.org/10.1002/jms.4519