bims-metlip 2020-02-09 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2020–02–09
twenty-two papers selected by
Sofia Costa, Cold Spring Harbor Laboratory

Mass spectrometric investigation of cardiolipins and their oxidation products after two-dimensional heart-cut liquid chromatography.
Quantitation of Urinary Acylcarnitines by DMS-MS/MS Uncovers the Effects of Total Body Irradiation in Cancer Patients.
The use of a double coaxial electrospray ionization sprayer improves the peak resolutions of anionic metabolites in capillary ion chromatography-mass spectrometry.
Determination of Optimal Electrospray Parameters for Lipidomics in Infrared-Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry Imaging.
Determination of Tryptophan and Kynurenine by LC-MS/MS by Using Amlodipine as an Internal Standard.
Development of a sensitive and quantitative method for the identification of two major furan fatty acids in human plasma.
Advancement in Determination of Phthalate Metabolites by Gas Chromatography Eliminating Derivatization Step.
Molecular Characterization of White Wines Antioxidant Metabolome by Ultra High Performance Liquid Chromatography High-Resolution Mass Spectrometry.
Separation of Structurally Similar Anabolic Steroids as Cation Adducts in FAIMS-MS.
An automatic online solid-phase dehydrate extraction-ultra-high performance supercritical fluid chromatography-tandem mass spectrometry system using a dilution strategy for the screening of doping agents in human urine.
A surrogate analyte-based LC-MS/MS method for the determination of 5-hydroxytryptamine, kynurenine and tryptophan.
Evaluating MISER chromatography as a tool for characterizing HILIC column equilibration.
Evaluation of Lipid In-Source Fragmentation on Different Orbitrap-based Mass Spectrometers.
Comparison of N-ethyl maleimide and N-(1-phenylethyl) maleimide for derivatization of biological thiols using liquid chromatography-mass spectrometry.
A Simplified Method for Simultaneous Determination of α-Lipoic Acid and Low-Molecular-Mass Thiols in Human Plasma.
Metabolomic tools used in marine natural product drug discovery.
Comparison of an Offline SPE-GC-MS and Online HS-SPME-GC-MS Method for the Analysis of Volatile Terpenoids in Wine.
An Interface for Reproducible, Multi-shot Direct Analysis of Solid-phase Microextraction Samples.
High-Temperature Liquid Chromatography and the Hyphenation with Mass Spectrometry Using High-Pressure Electrospray Ionization.
Influence of Solvent Composition and Surface Tension on the Signal Intensity of Amino Acids in Electrospray Ionization Mass Spectrometry.
Disposable Paper-on-CMOS Platform for Real-time Simultaneous Detection of Metabolites.
Fast quantification of free amino acids in food by microfluidic voltage-assisted liquid desorption electrospray ionization-tandem mass spectrometry.

J Chromatogr A. 2020 Jan 25. pii: S0021-9673(20)30095-9. [Epub ahead of print] 460918

Mass spectrometric investigation of cardiolipins and their oxidation products after two-dimensional heart-cut liquid chromatography.

Patrick O Helmer, Carina M Wienken, Ansgar Korf, Heiko Hayen.

  The anionic phospholipid class of cardiolipins (CL) is increasingly attracting scientific attention in the recent years. CL can be found as a functional component of mitochondrial membranes in almost all living organisms. Changes in the CL composition are favored by oxidative stress. Based on this finding, the investigation of CL and their oxidation products in relation to various disease patterns, including neurodegenerative ones, is moving into the focus of current research. The analysis of this diverse lipid class is still challenging and requires sensitive and selective methods. In this work, we demonstrate an online two-dimensional liquid chromatography (2D-LC) approach by means of a heart-cut setup. In the first dimension, a fast hydrophilic interaction liquid chromatography (HILIC) method was developed for the separation of CL and their oxidation products from other phospholipid classes, but more important from nonpolar lipid classes, such as triacylglycerol and cholesterol. Those classes can negatively affect the electrospray ionization and also the chromatography. For the heart-cut approach, the CL fraction was selectively transferred to a loop using a six-port valve followed by the transfer to a reversed phase (RP) column in second dimension. On the RP column, the transferred CL fraction including the oxidation products were separated according to the hydrophobicity of acyl chain moieties. Matrix effects were significantly reduced compared to the one-dimensional LC-MS method. In addition, the total separation time had not to be prolonged by shifting the equilibration step of the RP column parallel to the separation in first dimension. The heart-cut LC-LC approach was applied to artificially oxidized lipid extracts of bovine heart and yeast by means of Fenton reaction. In summary, 42 species have been identified by high resolution mass spectrometry and database matching. 31 species thereof have been further characterized by MS/MS experiments.

Keywords:  2D-LC/MS; Cardiolipin; Cardiolipin oxidation; Fenton reaction; Heart-cut; Phospholipids

DOI:  https://doi.org/10.1016/j.chroma.2020.460918
J Am Soc Mass Spectrom. 2020 Jan 28.

Quantitation of Urinary Acylcarnitines by DMS-MS/MS Uncovers the Effects of Total Body Irradiation in Cancer Patients.

Nicholas B Vera, Stephen L Coy, Evagelia C Laiakis, Albert J Fornace, Michelle Clasquin, Christopher A Barker, Jeffrey A Pfefferkorn, Paul Vouros.

Acylcarnitines have been identified in human and animal metabolomic-profiling studies as urinary markers of radiation exposure, a result which is consistent with their cytoprotective effects and roles in energy metabolism. In the present work, a rapid method for quantitation of the more abundant acylcarnitines in human urine is developed using a valuable set of samples from cancer patients who received total body irradiation (TBI) at Memorial Sloan Kettering Cancer Center. The method uses solid-phase extraction (SPE) processing followed by differential mobility spectrometry (DMS with ethyl acetate modifier) tandem mass spectrometry (ESI-DMS-MS/MS) with deuterated internal standards. The analyzed human urine samples were collected from 38 individual patients at three time points over 24 h during and after the course of radiation treatment, a design allowing each patient to act as their own control and creatinine normalization. Creatinine-normalized concentrations for nine urinary acylcarnitine (acyl-CN) species are reported. Six acyl-CN species were reduced at the 6 h point. Acetylcarnitine (C2:0-CN) and valerylcarnitine (C5:0-CN) showed recovery at 24 h, but none of the other acyl-CN species showed recovery at that point. Levels of three acyl-CN species were not significantly altered by radiation. This rapid quantitative method for clinical samples covers the short- and medium-chain acylcarnitines and has the flexibility to be expanded to cover additional radiation-linked metabolites. The human data presented here indicates the utility of the current approach as a rapid, quantitative technique with potential applications by the medical community, by space research laboratories concerned with radiation exposure, and by disaster response groups.

DOI: https://doi.org/10.1021/jasms.9b00076
J Chromatogr A. 2020 Jan 23. pii: S0021-9673(20)30091-1. [Epub ahead of print] 460914

The use of a double coaxial electrospray ionization sprayer improves the peak resolutions of anionic metabolites in capillary ion chromatography-mass spectrometry.

Akiyoshi Hirayama, Sho Tabata, Ryuhei Kudo, Masako Hasebe, Kumi Suzuki, Masaru Tomita, Tomoyoshi Soga.

  Recently, ion chromatography coupled with mass spectrometry has been used for the determination of anionic metabolites. However, connection with a mass spectrometer in this method is not straightforward because backpressure produced by the addition of a make-up solution often affects the peak resolutions of the target metabolites. To overcome this problem, we developed a capillary ion chromatography-mass spectrometry method utilizing a double coaxial electrospray ionization sprayer. This method was not affected by backpressure and the number of theoretical plates was about three times that of a conventional sprayer. Under optimized conditions, 44 anionic metabolites, including organic acids, sugar phosphates, nucleotides, and cofactors, were successfully separated and selectively detected with a Q Exactive mass spectrometer. The calibration curves of the tested metabolites showed excellent linearity within the range of 1-100,000 nmol/L and the correlation coefficient was greater than 0.991. The detection limits for these metabolites were between 1 and 500 nmol/L (0.4 and 200 fmol). The developed method was applied to the quantitation of anionic metabolites in cultured cancer cell samples with tumor necrosis factor (TNF)-α stimulation. This allowed for the successful determination of 105 metabolites. The levels of tricarboxylic acid cycle intermediates changed significantly after TNF-α stimulation. These results demonstrate that the developed method is a promising new tool for comprehensive analysis of anionic metabolites.

Keywords:  Cancer cell culture; Capillary ion chromatography; Double coaxial electrospray ionization sprayer; Mass spectrometry; Tumor necrosis factor

DOI:  https://doi.org/10.1016/j.chroma.2020.460914
J Am Soc Mass Spectrom. 2020 Feb 05. 31(2): 319-325

Determination of Optimal Electrospray Parameters for Lipidomics in Infrared-Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry Imaging.

M Caleb Bagley, Måns Ekelöf, David C Muddiman.

  Infrared matrix-assisted laser desorption ionization (IR-MALDESI) is an ambient mass spectrometry imaging (MSI) technique that relies on electrospray ionization (ESI) for ion generation of desorbed neutrals. Although many mechanisms in IR-MALDESI have been studied in depth, there has not yet been a comprehensive study of how the ESI parameters change the profiles of tissue specific lipids. Acetonitrile (ACN)/water and methanol (MeOH)/water solvent systems and compositions were varied across a series of applied ESI voltages during IR-MALDESI analysis of rat liver tissue. Gradients of 12 min were run from 5 to 95% organic solvent in both positive and negative polarities across 11 voltages between 2.25 and 4.5 kV. These experiments informed longer gradients (25-30 min) across shorter solvent gradient ranges with fewer voltages. Optimal ESI parameters for lipidomics were determined by the number and abundance of detected lipids and the relative proportion of background ions. In positive polarity, the best solvent composition was 60-75% ACN/40-25% H2O with 0.2% formic acid at 3.2 kV applied voltage. The best parameters for negative polarity analysis are 45-55% ACN/55-45% H2O with 1 mM of acetic acid for voltages between 2.25 and 3.2 kV. Using these defined parameters, IR-MALDESI positive polarity lipidomics studies can increase lipid abundances 3-fold, with 15% greater coverage, while an abundance increase of 1.5-fold and 10% more coverage can be achieved relative to commonly used parameters in negative polarity.

Keywords:  IR-MALDESI; lipids; mass spectrometry imaging: electrospray ionization; metabolites

DOI:  https://doi.org/10.1021/jasms.9b00063
J Am Soc Mass Spectrom. 2020 Feb 05. 31(2): 379-385

Determination of Tryptophan and Kynurenine by LC-MS/MS by Using Amlodipine as an Internal Standard.

Burcu Eser, Yeşim Özkan, Aylin Sepici Dinçel.

  Tryptophan is an essential amino acid that plays an important role in cell metabolism, and kynurenine is its main metabolic pathway. By using ultra-high-performance liquid chromatography coupled to electrospray ionization triple-quadrupole mass spectrometry, tryptophan and kynurenine were determined using amlodipine as an internal standard. The analysis was carried out on an ACE-C18 (4.6 mm × 50 mm, 5 μm) reversed-phase analytical column using the gradient elution mode. For quantitative determination, amlodipine was used as an internal standard. Detection was performed using multiple reaction monitoring in electrospray ionization mode at m/z 205.1 → 117.7 and 187.9 for tryptophan, m/z 209.1 → 146 and 93.9 for kynurenine, and m/z 409.2 → 294.1 for the internal standard. Good linearity of the analyte to internal standard peak area ratios was seen in the concentration range 1.25-4000 ng/mL for tryptophan and 0.5-1600 ng/mL for kynurenine. The method showed excellent linearity with regression coefficients of 0.99 for kynurenine and 0.996 for tryptophan. The limits of quantification were 0.55 ng/mL for tryptophan and 0.47 ng/mL for kynurenine. The % RSD for all analytes ranged from 0.3 to 3.4% for intraday and 0.4 to 8.9% for interday experiments. A simple LC-MS/MS method has been developed and validated for measuring Kyn and Trp by using an affordable and more easily available internal standard, which is amlodipine.

Keywords:  LC−MS/MS; amlodipine; kynurenine; tryptophan

DOI:  https://doi.org/10.1021/jasms.9b00007
J Lipid Res. 2020 Feb 06. pii: jlr.D119000514. [Epub ahead of print]

Development of a sensitive and quantitative method for the identification of two major furan fatty acids in human plasma.

Long Xu, Changfeng Hu, Yongguo Liu, Siming Li, Walter Vetter, Huiyong Yin, Yonghua Wang.

  This paper focuses on the establishment of an accurate and sensitive quantitation method for analysis of furan fatty acids. Particularly, the sensitivity of GC-MS and UPLC-ESI-MS/MS was compared for the identification and quantification of furan fatty acids. Different methylation methods were tested with respect to GC-MS analysis. Special attention needs to be paid to the methylation of furan fatty acids as acidic catalysts might lead to the degradation of furan ring. GC-MS analysis in full scan mode demonstrated that the limit-of-quantitation (LOQ) was 10 μM. UPLC-ESI-MS/MS in multiple reaction monitoring (MRM) mode displayed a higher detection sensitivity than GC-MS. Moreover, identification of furan fatty acids with charge-reversal derivatization was tested in positive mode with two widely used pyridinium salts. Unexpectedly, significant oxidation was observed using N-(4-aminomethylphenyl) pyridinium (AMPP) as a derivatization agent. The formed 3-acyl-oxymethyl-1-methylpyridinium iodide (AMMP) derivatized by 2-bromo-1-methylpyridinium iodide and 3-carbinol-1-methylpyridinium iodide improved the sensitivity more than 2000 fold, compared with non-derivatization in negative mode by UPLC-ESI-MS/MS. This charge reversal derivatization enabled the targeted quantitation of furan fatty acids in human plasma. Thus, it is anticipated that this protocol could greatly contribute to the clarification of pathological mechanisms related to furan fatty acids and their metabolites.

Keywords:  Charge-reversal derivatization; Diabetes; Fatty acid; Furan fatty acids; Lipidomics; Lipids; Mass spectrometry; Multiple reaction monitoring; Precursor ion scan; UPLC-ESI-MS/MS

DOI:  https://doi.org/10.1194/jlr.D119000514
Front Chem. 2019 ;7 928

Advancement in Determination of Phthalate Metabolites by Gas Chromatography Eliminating Derivatization Step.

Maciej Tankiewicz, Ewa Olkowska, Andrzej Berg, Lidia Wolska.

  A gas chromatography-mass spectrometry (GC-MS) method to determine polar and thermally unstable phthalate metabolites [monomethyl phthalate-MMP, monoethyl phthalate-MEP, mono-n-butyl phthalate-MnBP, mono-(2-ethylhexyl) phthalate-MEHP] has been developed. This is the first report presenting the separation of monophthalates without derivatization step and any additional equipment or special injection port. Injection parameters (temperature, pressure, time, and volume of injection), chromatographic separation (retention gap, temperature program), and MS detection/identification (working parameters, ion selection) were investigated. Mechanisms and phenomena occurring under different conditions in the GC injector were evaluated and discussed. The limits of detection (LODs) of MMP, MEP, MnBP, MEHP in the protocol were 0.049, 0.036, 0.038, and 0.029 ng (per 2 μL of injection), respectively. The response of the monophthalates was found to be linear in the tested concentration range (for MMP: 0.15-100 ng, MEP and MnBP: 0.11-100 ng, MEHP: 0.087-100 ng per 2 μL) with the coefficient of determination higher than 0.9817 and inter-day precision in the range of 1.4-5.4%. The developed method is fast, easy and repeatable. Moreover, it allows for the elimination of derivatization agents, reduction of toxic waste production and simplification of analytical procedure.

Keywords:  analytical method; derivatization; gas chromatography; injection conditions; phthalate metabolites; separation; thermal stability

DOI:  https://doi.org/10.3389/fchem.2019.00928
Antioxidants (Basel). 2020 Jan 28. pii: E115. [Epub ahead of print]9(2):

Molecular Characterization of White Wines Antioxidant Metabolome by Ultra High Performance Liquid Chromatography High-Resolution Mass Spectrometry.

Remy Romanet, Florian Bahut, Maria Nikolantonaki, Régis D Gougeon.

  The knowledge about the molecular fraction contributing to white wines oxidative stability is still poorly understood. However, the role of S- and N-containing compounds, like glutathione and other peptides, as a source of reductant in many oxidation reactions, and acting against heavy metals toxicity, or lipid and polyphenol oxidation as ROS-scavenger is today very well established. In that respect, the aim of the present study is to introduce an original analytical tool for the direct determination of the available nucleophilic compounds in white wine under acidic pH conditions. One step derivatization of nucleophiles has been realized directly in wines using 4-methyl-1,2-benzoquinone (4MeQ) as an electrophilic probe. Derivatization conditions considering probe concentration, pH, reaction time, MS ionisation conditions and adducts stability, were optimized using model solutions containing standard sulfur and amino compounds (GSH, Cys, HCys and Ser-Aps-Cys-Asp-Ser, Asp-Met, Met and Glu). Ultra-high-performance liquid chromatography coupled to a quadrupole-time of flight mass spectrometer (UHPLC-QqTOF-MS) analysis of up to 92 white wines from different cultivars (Chardonnay, Sauvignon and Semillon) followed by Multivariate analysis (PLS DA) and Wilcoxon test allowed to isolate up to 141 putative wine relevant nucleophiles. Only 20 of these compounds, essentially thiols, were detectable in samples before derivatization, indicating the importance of the quinone trapping on the revelation of wine unknown nucleophiles. Moreover, annotation using online database (Oligonet, Metlin and KEGG) as well as elementary formula determined by isotopic profile, provided evidence of the presence of amino acids (Val, Leu, Ile, Pro, Trp, Cys and Met) and peptides with important antioxidant properties. The complimentary set of MS/MS spectral data greatly accelerated identification of nucleophiles and enabled peptides sequencing. These results show that probing wines with 4-methyl-1,2-benzoquinone enhances thiols ionisation capacity and gives a better screening of specific S- N- containing functional compounds as part of the white wines antioxidant metabolome.

Keywords:  Chardonnay wine oxidation; UHPLC-QqTOF-MS; nucleophiles; peptides; thiols; untargeted analysis

DOI:  https://doi.org/10.3390/antiox9020115
J Am Soc Mass Spectrom. 2020 Feb 05. 31(2): 355-365

Separation of Structurally Similar Anabolic Steroids as Cation Adducts in FAIMS-MS.

Michael S Wei, Robin H J Kemperman, Michelle A Palumbo, Richard A Yost.

  Novel synthetic anabolic androgenic steroids have been developed not only to dodge current antidoping tests at the professional sports level, but also for consumption by noncompetitive bodybuilders. These novel anabolic steroids are commonly referred to as "designer steroids" and pose a significant risk to users because of the lack of testing for toxicity and safety in animals or humans. Manufacturers of designer steroids dodge regulation by distributing them as nutritional or dietary supplements. Improving the throughput and accuracy of screening tests would help regulators to stay on top of illicit anabolic steroids. High-field asymmetric-waveform ion mobility spectrometry (FAIMS) utilizes an alternating asymmetric electric field to separate ions by their different mobilities at high- and low-fields as they travel through the separation space. When coupled to mass spectrometry (MS), FAIMS enhances the separation of analytes from other interfering compounds with little to no increase in analysis time. Here we investigate the effects of adding various cation species to sample solutions for the separation of structurally similar or isomeric anabolic androgenic steroids. FAIMS-MS spectra for these cation-modified samples show an increased number of compensation field (CF) peaks, some of which are confirmed to be unique for one steroid isomer over another. The CF peaks observed upon addition of cation species correspond to both monomer steroid-cation adduct ions and larger multimer ion complexes. Notably, the number of CF peaks and their CF shifts do not appear to have a straightforward relationship with cation size or electronegativity. Future directions aim at investigating the structures for these analyte-cation adduct ions for building a predictive model for their FAIMS separations.

Keywords:  FAIMS-MS; anabolic steroids

DOI:  https://doi.org/10.1021/jasms.9b00127
Anal Chim Acta. 2020 Mar 08. pii: S0003-2670(19)31447-3. [Epub ahead of print]1101 184-192

An automatic online solid-phase dehydrate extraction-ultra-high performance supercritical fluid chromatography-tandem mass spectrometry system using a dilution strategy for the screening of doping agents in human urine.

Yanshan Liang, Jiaqi Liu, Qisheng Zhong, Taohong Huang, Ting Zhou.

  An automatic online solid-phase dehydrate extraction (SPDE)-ultra-high performance supercritical fluid chromatography (UHPSFC)-MS/MS system was developed in this study, in which the automatic SPDE procedure was coupled with UHPSFC to allow UHPSFC to analyze aqueous samples directly. Moreover, a pre-column dilution strategy was employed, which focused the analytes in strong desorption solvent on the column head and helped to obtain narrow and symmetric peaks. The online SPDE-UHPSFC-MS/MS system was firstly applied to the screening of 45 prohibited substances in human urine for doping control, during which all the mechanisms and features of the online system were fully studied. The majority (91%) of the target compounds achieved weak matrix effects (80-120%), indicating that the online method was accurate and reliable thanks to the SPDE procedure and efficient UHPSFC separation. Owing to the reduction of the matrix effects, large volume injection and the pre-column dilution, the online system could achieve high sensitivity with the LODs ranging from 0.0380 ng L-1 to 1.24 μg L-1. Under the optimized conditions, the extraction recoveries of 66% target analytes were more than 50%. All the target compounds showed good linearity with linear correlation coefficients higher than 0.9928. The accuracy values of all the spiked prohibited substances were within 80.8-119.7%, while the RSDs% for the intra-/inter-day precision were within 10.8% and 15.4%. Compared with the dilute-and-shoot-ultra-high performance liquid chromatography-MS/MS method, in which the urine samples were simply diluted before analyzing, this online method was superior in sensitivity and reducing matrix effects, which demonstrated its utility in doping control. Compared with the previously reported online SPE-SFC system, the online SPDE-UHPSFC-MS/MS system showed advantages in automation, efficiency, sensitivity and chromatographic performance. In summary, the online SPDE-UHPSFC-MS/MS system is capable of analyzing complex aqueous samples.

Keywords:  Doping control; Human urine; Online sample preparation-chromatography system; Pre-column dilution; Supercritical fluid chromatography

DOI:  https://doi.org/10.1016/j.aca.2019.12.011
Bioanalysis. 2020 Feb 06.

A surrogate analyte-based LC-MS/MS method for the determination of 5-hydroxytryptamine, kynurenine and tryptophan.

Mengyu Cong, Jiayu Song, Fenglian Chen, Cheng Cao, Shuling Wang.

  Aim: The metabolism of tryptophan (TRP) through kynurenine (KYN) and 5-hydroxytryptamine (5-HT) pathways is linked to various diseases such as neurological diseases and cancer. The levels of 5-HT, KYN, TRP can be used as indicators for the diagnosis of various diseases in clinical and scientific research. Experimental: Since 5-HT, KYN, TRP are both endogenous molecules in biological samples, it is difficult to obtain a 'real blank sample'. A surrogate analyte-based LC-MS/MS method was chosen, using 5-HT-d4, KYN-d4 and TRP-d5 as surrogate analytes to replace the authentic analytes 5-HT, KYN and TRP, respectively. Theophylline was selected as the internal standard (IS). Results: The method was applied to quantification 5-HT, KYN and TRP of plasma, liver, colon, brain and verified to be acceptable in terms of linearity, precision, accuracy, matrix effect, recovery efficiency and stability.

Keywords:  LC–MS/MS; surrogate analyte; tryptophan and its metabolites

DOI:  https://doi.org/10.4155/bio-2019-0267
J Chromatogr A. 2020 Jan 27. pii: S0021-9673(20)30118-7. [Epub ahead of print] 460931

Evaluating MISER chromatography as a tool for characterizing HILIC column equilibration.

Kenneth D Berthelette, Thomas H Walter, Martin Gilar, Fabrice Gritti, Thomas S MacDonald, Miguel Soares.

Hydrophilic Interaction Liquid Chromatography (HILIC) is a technique for retaining polar analytes that uses polar stationary phases and acetonitrile-rich mobile phases. While this technique has several advantages over reversed-phase liquid chromatography (RPLC), one main drawback is the reported need for longer column equilibration. The reason for this is not fully understood and is a topic of current investigation. In order to better understand and reduce the equilibration needs, accurate characterization of column equilibration under varying conditions is required. The current method of characterizing HILIC column equilibration produces limited data points per test, or low time resolution, and is highly dependent on the column and probe compounds being used. There is a need for an improved method for characterizing HILIC column equilibration, especially if trends across stationary phases are to be observed. In this work, MISER, or Multiple Injections in a Single Experimental Run, is evaluated as a possible tool for characterizing HILIC column equilibration. MISER improves time resolution by allowing for replicate injections without interruption of data collection, enabling a more thorough evaluation of column equilibration compared to traditional techniques. Experimental results gathered using MISER show that equilibration of a BEH Amide column is notably shorter when equilibrating from acetonitrile to mobile phases containing higher percentages of water. Column equilibration to a 10% aqueous mobile phase was found to be approximately 5-fold faster than equilibration to a 3% aqueous mobile phase.

DOI: https://doi.org/10.1016/j.chroma.2020.460931
J Am Soc Mass Spectrom. 2020 Feb 05. 31(2): 463-466

Evaluation of Lipid In-Source Fragmentation on Different Orbitrap-based Mass Spectrometers.

Angela Criscuolo, Martin Zeller, Maria Fedorova.

Natural lipidomes represent a complex mixture of lipid molecular species with a variety of biological and signaling functions. Modern mass spectrometry (MS)-based analytical platforms are often used to resolve the complexity of natural lipidomes. The quantitative transfer of lipid molecular species in the gas phase during the electrospray ionization required for MS analysis might be challenged by lipid in-source fragmentation (ISF) hampering their accurate identification and quantification. Here we evaluated the effect of transmission radio frequency (RF) levels and ion transfer temperatures (ITTs) on the analysis of four different lipids (ceramide, cholesteryl ester, phosphatidylethanolamine, and triacylglyceride) ionized in positive ion mode on three different Orbitrap-based platforms. ITT and RF levels were ramped in a systematic way to determine the best settings, allowing the most sensitive detection accompanied by the lowest ISF of a lipid. The extent of the ISF was shown to depend on the configurations of the transmission devices (S-lens vs letterbox/ion funnel) at defined RF and ITT levels for each studied lipid class. We provide here the recommendations for reducing the extent of lipid ISF without a significant loss in sensitivity for Q Exactive HF, Q Exactive HF-X, and Orbitrap Fusion Lumos platforms.

DOI: https://doi.org/10.1021/jasms.9b00061
Anal Bioanal Chem. 2020 Feb 04.

Comparison of N-ethyl maleimide and N-(1-phenylethyl) maleimide for derivatization of biological thiols using liquid chromatography-mass spectrometry.

Mariana S T Russo, Alexander Napylov, Alexandra Paquet, Dajana Vuckovic.

  The ratio between reduced and oxidized thiols, mainly glutathione and oxidized glutathione, is one of the biomarkers for the evaluation of oxidative stress. The accurate measurement of thiol concentrations is challenging because reduced thiols are easily oxidized during sample manipulation. Derivatization is commonly used to protect thiols from oxidation. The objective of this work was to systematically compare two cell-permeable derivatizing agents: N-ethyl maleimide (NEM) and (R)-(+)-N-(1-phenylethyl)maleimide (NPEM) in terms of derivatization efficiency, ionization enhancement, side product formation, reaction selectivity for thiols, pH dependence of the reaction, and derivative stability. All thiol measurements and the characterization of side products were performed using a biphenyl reversed phase liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Four thiols, cysteine (CYS), homocysteine, N-acetylcysteine (NAC), and glutathione (GSH), were used for the evaluation. Using 1:10 ratio of thiol:derivatizing agent, complete derivatization was obtained within 30 min for both agents tested with the exception of CYS-NEM, where 97% efficiency was obtained. The more hydrophobic NPEM provided better ionization of the thiols, with enhancement ranging from 2.1x for GSH to 5.7x for CYS in comparison to NEM. NPEM derivatization led to more extensive side reactions, such as double derivatization and ring opening, which hindered the accurate measurement of the thiol concentrations. Both NEM and NPEM also showed poor stability of CYS derivative due to its time-dependent conversion to cyclic cysteine-maleimide derivative. Both reagents also showed significant reactivity with amine-containing metabolites depending on the pH used during derivatization, but overall NEM was found to be more selective towards thiol group than NPEM. Taking into account all evaluation criteria, NEM was selected as a more suitable reagent for the thiol protection and derivatization, but strict control of pH 7.0 is recommended to minimize the side reactions. This work illustrates the importance of the characterization of side products and derivative stability during the evaluation of thiol derivatizing agents and contributes fundamental understanding to improve the accuracy of thiol determinations. The key sources of errors during maleimide derivatization include the derivatization of amine-containing metabolites, poor derivative stability of certain thiols (CYS and NAC), and the side reactions especially if ring opening of the reagent is not minimized. Graphical abstract.

Keywords:  Derivatization; Glutathione; Liquid chromatography-mass spectrometry; N-(1-Phenylethyl) maleimide; N-Ethyl maleimide; Thiols

DOI:  https://doi.org/10.1007/s00216-020-02398-x
Int J Mol Sci. 2020 Feb 05. pii: E1049. [Epub ahead of print]21(3):

A Simplified Method for Simultaneous Determination of α-Lipoic Acid and Low-Molecular-Mass Thiols in Human Plasma.

Kamila Borowczyk, Patrycja Olejarz, Grażyna Chwatko, Marcin Szylberg, Rafał Głowacki.

  α-Lipoic acid, glutathione, cysteine, and cysteinylglycine can be applied as therapeutic agents in civilization diseases such as diabetes mellitus, cardiovascular diseases, and cancers. On the other hand, a higher concentration of homocysteine can result in health problems and has been indicated as an independent risk factor for cardiovascular disease and accelerated atherosclerosis. Here, the first simplified HPLC-UV assay that enables simultaneous determination of α-lipoic acid and low-molecular-mass thiols in plasma, reduces the number of steps, shortens the total time of sample preparation, and limits the amount of single-use polypropylene laboratory materials is described. The assay is based on reversed-phase high performance liquid chromatography with UV detection and simultaneous reduction of disulfide bound with tris(2-carboxyethyl)phosphine and the selective pre-column derivatization of the thiol group with 1-benzyl-2-chloropyridinium bromide. Linearity in the detector responses for plasma samples were observed in ranges: 0.12-5.0 nmol mL-1 for α-lipoic acid; 2.0-20.0 nmol mL-1 for glutathione, cysteinylglycine, and homocysteine; and 40.0-400.0 for cysteine. The LODs for α-lipoic acid and low-molecular-mass thiols were 0.08 and 0.12 nmol mL-1, respectively, while LOQs were 0.12 and 0.16 nmol mL-1, respectively. The usefulness of the proposed method has been proven by its application to real samples.

Keywords:  high performance liquid chromatography; low-molecular-mass thiols; simultaneous reduction and derivatization; ultraviolet detection; α-lipoic acid

DOI:  https://doi.org/10.3390/ijms21031049
Expert Opin Drug Discov. 2020 Feb 06. 1-24

Metabolomic tools used in marine natural product drug discovery.

Kevin Andrew Stuart, Keira Welsh, Molly Clare Walker, RuAngelie Edrada-Ebel.

  Introduction: The marine environment is a very promising resource for natural product research, with many of these reaching the market as new drugs, especially in the field of cancer therapy as well as the drug discovery pipeline for new antimicrobials. Exploitation for bioactive marine compounds with unique structures and novel bioactivity such as the isoquinoline alkaloid; trabectedin, the polyether macrolide; halichondrin B, and the peptide; dolastatin 10, requires the use of analytical techniques, which can generate unbiased, quantitative, and qualitative data to benefit the biodiscovery process. Metabolomics has shown to bridge this understanding and facilitate the development of new potential drugs from marine sources and particularly their microbial symbionts.Areas covered: In this review, articles on applied secondary metabolomics ranging from 1990-2018 as well as to the last quarter of 2019 were probed to investigate the impact of metabolomics on drug discovery for new antibiotics and cancer treatment.Expert opinion: The current literature review highlighted the effectiveness of metabolomics in the study of targeting biologically active secondary metabolites from marine sources for optimized discovery of potential new natural products to be made accessible to a R&D pipeline.

Keywords:  MS; NMR; Secondary metabolomics; anti-cancer drugs; antibiotics; drug discovery; marine natural products; molecular networking; multivariate analysis

DOI:  https://doi.org/10.1080/17460441.2020.1722636
Molecules. 2020 Feb 04. pii: E657. [Epub ahead of print]25(3):

Comparison of an Offline SPE-GC-MS and Online HS-SPME-GC-MS Method for the Analysis of Volatile Terpenoids in Wine.

Cody Williams, Astrid Buica.

  The aroma profile is an important marker for wine quality. Various classes of compounds are responsible for the aroma of wine, and one such class is terpenoids. In the context of this work, a validated gas chromatography-mass spectrometry (GC-MS) method for the quantitation of terpenoids in red and white wine using headspace solid-phase microextraction (HS-SPME) and solid-phase extraction (SPE) was established. Calibrations were performed in the respective base wine using both sample preparation methods. The linearity, precision and accuracy evaluated for the respective matrices were excellent for both sample preparations. However, the HS-SPME approach was more sensitive and more accurate. For both sample preparations, the quantification limits were lower than the odor thresholds in wine. The terpenoid concentrations (µg/L) were evaluated for 13 white wines using both sample preparation methods. Importantly, the online HS-SPME approach was more sensitive than the offline SPE method. The major terpenoids identified in the white wines evaluated were linalool (0.2-63 µg/L), geraniol (nd-66 µg/L) and α-terpineol (nd-85 µg/L).

Keywords:  gas chromatography; mass spectrometry.; solid-phase extraction; solid-phase microextraction; terpene; terpenoid; wine

DOI:  https://doi.org/10.3390/molecules25030657
Anal Chem. 2020 Feb 07.

An Interface for Reproducible, Multi-shot Direct Analysis of Solid-phase Microextraction Samples.

G Asher Newsome, Gwénaëlle Kavich, Alba Alvarez-Martin.

An enclosed interface that joins a direct analysis in real time (DART) probe, solid phase microextraction (SPME) fiber, and the inlet of a high-resolution mass spectrometer is described. Unlike other systems to couple SPME sampling to ambient mass spectrometry, the interface is able to perform discrete analyses on different areas of a single SPME fiber device for up to three technical replicate measurements of one sampling event. Inlet flow speed and desorption temperature are optimized, and reproducibility is demonstrated between replicate analyses on the same derivatized SPME fiber and with sequential fiber sampling events, yielding analyte measurement center of variance (CV) from 3-6%. Conditioning is also performed with the enclosed DART. The interface is a straightforward addition to commercially available technologies, and machine diagrams for custom components operated with SPME/DART/MS equipment are included.

DOI: https://doi.org/10.1021/acs.analchem.9b05691
Mass Spectrom (Tokyo). 2019 ;8(2): S0079

High-Temperature Liquid Chromatography and the Hyphenation with Mass Spectrometry Using High-Pressure Electrospray Ionization.

Lee Chuin Chen.

  Increasing the operating temperature of the liquid chromatography (LC) column has the same effect as reducing the diameter of the packing particles on minimizing the contribution of C-term in the van Deemter equation, flattening the curve of plate height vs. linear velocity in the high-speed region, thus allowing a fast LC analysis without the loss of plate count. While the use of smaller particles requires a higher pumping pressure, operating the column at higher temperature reduces the pressure due to lower liquid viscosity. At present, the adoption of high-temperature LC lags behind the ultra-high-pressure LC. Nevertheless, the availability of thermally stable columns has steadily improved and new innovations in this area have continued to emerge. This paper gives a brief review and updates on the recent advances in high-temperature liquid chromatography (HTLC). Recent efforts of hyphenating the capillary HTLC with mass spectrometry via a super-atmospheric pressure electrospray ionization is also reported.

Keywords:  capillary liquid chromatography; high-pressure electrospray ionization; high-temperature liquid chromatography; subcritical water LC-MS

DOI:  https://doi.org/10.5702/massspectrometry.S0079
Mass Spectrom (Tokyo). 2019 ;8(1): A0077

Influence of Solvent Composition and Surface Tension on the Signal Intensity of Amino Acids in Electrospray Ionization Mass Spectrometry.

Ami Kageyama Kaneshima, Akira Motoyama, Mitsuo Takayama.

  The influence of solvent composition and surface tension on the signal intensity of deprotonated molecules [M-H]- in electrospray ionization mass spectrometry (ESI MS) was evaluated using alanine (Ala), threonine (Thr) and phenylalanine (Phe), which have differing levels of hydrophobicity. The surface tension of the ESI solution was varied by changing the ratio of the organic solvents methanol (MeOH) and acetonitrile (MeCN) in water (H2O). In ESI MS, the signal intensity of all the amino acids was increased with decreasing surface tension for the two solutions, H2O/MeOH and H2O/MeCN. The use of H2O/MeCN was more favorable for achieving a strong signal for the analytes compared to H2O/MeOH. The smaller vaporization enthalpy of MeCN compared to MeOH was proposed as one of the most plausible explanation for this. The order of the signal intensity of amino acids was Phe>Thr>Ala, the same order as their hydrophobicity. It can be practically concluded that the use of solutions with lower surface tensions and lower vaporization enthalpies would result in higher signal intensities in ESI MS.

Keywords:  ESI MS; amino acid; signal intensity; solvent; surface tension

DOI:  https://doi.org/10.5702/massspectrometry.A0077
IEEE Trans Biomed Eng. 2020 Jan 30.

Disposable Paper-on-CMOS Platform for Real-time Simultaneous Detection of Metabolites.

Chunxiao Hu, Valerio Annese, Srinivas Velugotla, Mohammed A Al-Rawhani, Boon Chong Cheah, James Grant, Michael Barrett, David Cumming.

OBJECTIVE: Early stage diagnosis of sepsis without overburdening health services is essential to improving patient outcomes.
METHODS: A fast and simple-to-use platform that combines an integrated circuit with paper microfluidics for simultaneous detection of multiple-metabolites appropriate for diagnostics was presented. Paper based sensors are a primary candidate for widespread deployment of diagnostic or test devices. However, the majority of devices today use a simple paper strip to detect a single marker using the reflectance of light. However, for many diseases such as sepsis, one biomarker is not sufficient to make a unique diagnosis. In this work multiple measurements are made on patterned paper simultaneously. Using laser ablation to fabricate microfluidic channels on paper provides a flexible and direct approach for mass manufacture of disposable paper strips. A reusable photodiode array on a complementary metal oxide semiconductor chip is used as the transducer.
RESULTS: The system measures changes in optical absorbance in the paper to achieve a cost-effective and easily implemented system that is capable of multiple simultaneous assays. Potential sepsis metabolite biomarkers glucose and lactate have been studied and quantified with the platform, achieving sensitivity within the physiological range in human serum.
CONCLUSION: We have detailed a disposable paper-based CMOS photodiode sensor platform for real-time simultaneous detection of metabolites for diseases such as sepsis.
SIGNIFICANCE: A combination of a low-cost paper strip with microfluidic channels and a sensitive CMOS photodiode sensor array makes our platform a robust portable and inexpensive biosensing device for multiple diagnostic tests in many different applications.

DOI: https://doi.org/10.1109/TBME.2019.2962239
Anal Bioanal Chem. 2020 Feb 04.

Fast quantification of free amino acids in food by microfluidic voltage-assisted liquid desorption electrospray ionization-tandem mass spectrometry.

Hankun Hu, Shila Smith, Xiangtang Li, Zhengming Qian, Yaxia Su, Manting Lin, Jiancheng Tu, Yi-Ming Liu.

  A method based on microfluidic voltage-assisted liquid desorption electrospray ionization-tandem mass spectrometry (VAL-DESI-MS/MS) has been developed for fast quantification of free amino acids in food. Food extracts were transferred to the microfluidic platform and analyzed by liquid desorption ESI-MS/MS. Deuterated aspartic acid (i.e., 2,2,3-d3-Asp) was used as internal standard for analysis. The method had linear calibration curves with r2 values > 0.998. Limits of detection were at the level of sub μM for the amino acids tested, i.e., glutamic acid (Glu), arginine (Arg), tyrosine (Tyr), tryptophan (Trp), and phenylalanine (Phe). To validate the proposed method in food analysis, extracts of Cordyceps fungi were analyzed. Amino acid contents were found in the range from 0.63 mg/g (Tyr in Cordyceps sinensis) to 4.44 mg/g (Glu in Cordyceps militaris). Assay repeatability (RSD) was ≤ 5.2% for all the five amino acids measured in all the samples analyzed. Recovery was found in the range from 95.8 to 105.1% at two spiking concentrations of 0.250 mg/g and 1.00 mg/g. These results prove that the proposed microfluidic VAL-DESI-MS/MS method offers a quick and convenient means of quantifying free amino acids with accuracy and repeatability. Therefore, it may have potential in food analysis for nutritional and quality assessment purposes. Graphical abstract.

Keywords:  Amino acids; Desorption electrospray ionization; Direct analysis; Food; Mass spectrometry; Microfluidics

DOI:  https://doi.org/10.1007/s00216-020-02450-w