bims-metlip 2020-04-19 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2020–04–19
35 papers selected by
Sofia Costa, Cold Spring Harbor Laboratory

Nano-liquid chromatography combined with a sustainable microextraction based on natural deep eutectic solvents for analysis of phthalate esters.
Validation of a simplified procedure for convenient and rapid quantification of reduced and oxidized glutathione in human plasma by liquid chromatography tandem mass spectrometry analysis.
De novo discovery of metabolic heterogeneity with immunophenotype-guided imaging mass spectrometry.
Integration of Solid-Phase Extraction and Reversed-Phase Chromatography in Single Protein-Coated Columns for Direct Injection of Bupivacaine in Human Serum.
Dual mass spectrometry as a tool to improve annotation and quantification in targeted plasma lipidomics.
Simultaneous measurement of epinastine and its metabolite, 9,13b-dehydroepinastine, in human plasma by a newly developed ultra-performance liquid chromatography-tandem mass spectrometry and its application to pharmacokinetic studies.
ICBM-OCEAN: Processing ultrahigh-resolution mass spectrometry data of complex molecular mixtures.
Development and validation of an LC-MS/MS method for the quantification of flavonoid glucuronides (wogonoside, baicalin, and apigenin-glucuronide) in the bile and blood samples: application to a portal vein infusion study.
Metabolomics approach based on utra-performance liquid chromatography coupled to mass spectrometry with chemometrics methods for high-throughput analysis of metabolite biomarkers to exploring the abnormal metabolic pathways associated with myocardial dysfunction.
Analysis of Nicotine Metabolites in Hair and Nails Using QuEChERS Method Followed by Liquid Chromatography-Tandem Mass Spectrometry.
Development and validation of a simple online-SPE method coupled to high-resolution mass spectrometry for the analysis of stanozolol-N-glucuronides in urine samples.
Analysis of neurotransmitters in rat placenta exposed to flame retardants using IR-MALDESI mass spectrometry imaging.
[Fast HPLC-MS/MS screening method for identification of illegal drugs including new psychoactive substances and their metabolites in human urine].
Metabolomics and mass spectrometry imaging reveal channeled de novo purine synthesis in cells.
Exploration advantages of data combination and partition: First chemometric analysis of liquid chromatography-mass spectrometry data in full scan mode with quadruple fragmentor voltages.
UHPLC-MS/MS method to determine FP-208 in human plasma and its application to a pharmacokinetic study.
Simultaneous determination of five essential amino acids in plasma of Hyperlipidemic subjects by UPLC-MS/MS.
Ultrafast Photocatalytic Reaction Screening by Mass Spectrometry.
Retrospective screening of synthetic cannabinoids, synthetic opioids and designer benzodiazepines in data files from forensic post mortem samples analysed by UHPLC-QTOF-MS from 2014 to 2018.
Data-dependent normalization strategies for untargeted metabolomics-a case study.
A high-throughput SAMDI-mass spectrometry assay for isocitrate dehydrogenase 1.
Ozonization of tissue sections for MALDI MS imaging of carbon-carbon double bond positional isomers of phospholipids.
Simultaneous analysis of saturated and unsaturated oxylipins in 'ex vivo' cultured peripheral blood mononuclear cells and neutrophils.
A Two-Step Optimization Approach: Validated RP-HPLC Method for Determination of Gatifloxacin and Dexamethasone in Ophthalmic Formulation.
Development of a precise quantitative method for monitoring sirolimus in whole blood using LC/ESI-MS/MS.
Fast off-line FPSE-HPLC-PDA determination of six NSAIDs in saliva samples.
Development of retention mechanism for the separation of carboxylic acids and inorganic anions in cryptand-based ion chromatography.
Triple Resonance Methods to Improve Performance of Ion Trap Precursor and Neutral Loss Scans.
Boosting resolution in NMR spectroscopy by chemical shift upscaling.
Chromatographic detection of low-molecular-mass metal complexes in the cytosol of Saccharomyces cerevisiae.
Development and validation of an LC-MS/MS method for the quantification of imatinib and imatinib-d8 in human plasma for the support of an absolute bioavailability microdose trial.
Determination of iron in hair samples by slotted quartz tube-flame atomic absorption spectrometry after switchable solvent liquid phase extraction.
Long-term stability of ready-to-use norepinephrine solution at 0.2 and 0.5 mg/mL.
A mini mass spectrometer with a low noise Faraday detector.
Hollow fiber liquid-phase microextraction at-line coupled to capillary electrophoresis for direct analysis of human body fluids.

Electrophoresis. 2020 Apr 16.

Nano-liquid chromatography combined with a sustainable microextraction based on natural deep eutectic solvents for analysis of phthalate esters.

Álvaro Santana-Mayor, Ruth Rodríguez-Ramos, Bárbara Socas-Rodríguez, Miguel Ángel Rodríguez-Delgado, Giovanni D'Orazio.

  The separation of eleven phthalic acid ester (PAEs) was carried out by nano-liquid chromatography coupled to ultraviolet and MS detection. Preliminary experiments were achieved in order to select suitable stationary phases and chromatographic conditions. The baseline separation was obtained, for all compounds, with an XBridgeTM C18 column in less than 15 min, working in step gradient mode. The sensitivity of the method was improved by on-column focusing. PAEs were extracted from alcoholic and non-alcoholic beverages using vortex assisted-emulsification dispersive liquid-liquid microextration (VA-EDLLME) and natural deep eutectic solvents (NADESs). The whole method was validated in terms of linearity, sensitivity, precision, recovery and repeatability. Combination of both off-line sample preparation pre-concentration and large injection volume led to obtain LOQs in the range 5-47 ng/mL. The developed nano-LC-UV method was extended to MS detection to confirm the presence of PAEs in some beverages commercialised in different types of packaging. This article is protected by copyright. All rights reserved.

Keywords:  deep eutectic solvent; mass spectrometry; nano-LC; phthalates; wine

DOI:  https://doi.org/10.1002/elps.202000032
Biomed Chromatogr. 2020 Apr 17. e4854

Validation of a simplified procedure for convenient and rapid quantification of reduced and oxidized glutathione in human plasma by liquid chromatography tandem mass spectrometry analysis.

Addison C Enomoto, Erik Schneider, Toni McKinnon, Howard Goldfine, Mark A Levy.

  Endogenous glutathione (GSH) and glutathione disulfide (GSSG) status are highly sensitive to oxidative conditions and have wide applications as surrogate indicators of redox status in vivo. Established methods for GSH and GSSG quantification in whole blood display limited utility in human plasma, where GSH and GSSG levels are ~3-4 orders of magnitude below that observed in whole blood. This study presents simplified sample processing and analytical LC-MS/MS approaches exhibiting the sensitivity and accuracy required to measure GSH and GSSG concentrations in human plasma samples, which after 5-fold dilution to suppress matrix interferences, range from 200-500 nM (GSH) and 5-30 nM (GSSG). The utility of methods reported herein are evidenced by assay performance and validation parameters which indicate notable sensitivity (LLOQs of 4.99 nM (GSH) and 3.65 nM (GSSG)), high assay precision (intra-assay CVs 3.6% and 1.9%, and inter-assay CVs of 7.0% and 2.8% for GSH and GSSG, respectively). These methods also exhibited exceptional recovery of analyte-spiked plasma samples (98.0±7.64% for GSH and 98.5±12.7% for GSSG). Notable sample stability at -80°C was evident for GSH up to 55 weeks and GSSG up to 46 weeks, with average CVs < 15% and <10%, respectively.

Keywords:  GSH; GSSG; clinical analysis; glutathione; sample processing

DOI:  https://doi.org/10.1002/bmc.4854
Mol Metab. 2020 Feb 14. pii: S2212-8778(20)30025-9. [Epub ahead of print]36 100953

De novo discovery of metabolic heterogeneity with immunophenotype-guided imaging mass spectrometry.

Verena M Prade, Thomas Kunzke, Annette Feuchtinger, Maria Rohm, Birgit Luber, Florian Lordick, Achim Buck, Axel Walch.

   BACKGROUND: Imaging mass spectrometry enables in situ label-free detection of thousands of metabolites from intact tissue samples. However, automated steps for multi-omics analyses and interpretation of histological images have not yet been implemented in mass spectrometry data analysis workflows. The characterization of molecular properties within cellular and histological features is done via time-consuming, non-objective, and irreproducible definitions of regions of interest, which are often accompanied by a loss of spatial resolution due to mass spectra averaging.
METHODS: We developed a new imaging pipeline called Spatial Correlation Image Analysis (SPACiAL), which is a computational multimodal workflow designed to combine molecular imaging data with multiplex immunohistochemistry (IHC). SPACiAL allows comprehensive and spatially resolved in situ correlation analyses on a cellular resolution. To demonstrate the method, matrix-assisted laser desorption-ionization (MALDI) Fourier-transform ion cyclotron resonance (FTICR) imaging mass spectrometry of metabolites and multiplex IHC staining were performed on the very same tissue section of mouse pancreatic islets and on human gastric cancer tissue specimens. The SPACiAL pipeline was used to perform an automatic, semantic-based, functional tissue annotation of histological and cellular features to identify metabolic profiles. Spatial correlation networks were generated to analyze metabolic heterogeneity associated with cellular features.
RESULTS: To demonstrate the new method, the SPACiAL pipeline was used to identify metabolic signatures of alpha and beta cells within islets of Langerhans, which are cell types that are not distinguishable via morphology alone. The semantic-based, functional tissue annotation allows an unprecedented analysis of metabolic heterogeneity via the generation of spatial correlation networks. Additionally, we demonstrated intra- and intertumoral metabolic heterogeneity within HER2/neu-positive and -negative gastric tumor cells.
CONCLUSIONS: We developed the SPACiAL workflow to provide IHC-guided in situ metabolomics on intact tissue sections. Diminishing the workload by automated recognition of histological and functional features, the pipeline allows comprehensive analyses of metabolic heterogeneity. The multimodality of immunohistochemical staining and extensive molecular information from imaging mass spectrometry has the advantage of increasing both the efficiency and precision for spatially resolved analyses of specific cell types. The SPACiAL method is a stepping stone for the objective analysis of high-throughput, multi-omics data from clinical research and practice that is required for diagnostics, biomarker discovery, or therapy response prediction.

Keywords:  Imaging mass spectrometry; In situ metabolomics; Multiplex immunohistochemistry; Pixel-accurate analysis; Tissue annotation

DOI:  https://doi.org/10.1016/j.molmet.2020.01.017
J Chromatogr Sci. 2020 Apr 18. pii: bmaa014. [Epub ahead of print]

Integration of Solid-Phase Extraction and Reversed-Phase Chromatography in Single Protein-Coated Columns for Direct Injection of Bupivacaine in Human Serum.

Walaa Zarad, Heba El-Gendy, Ahmed Ali, Yasmine Aboulella, Samy Emara.

A rapid, reliable and precise integrated solid-phase extraction (SPE) and reversed-phase liquid chromatography method was developed and validated to determine bupivacaine in human serum using single protein-coated analytical columns. The protein-coated columns were packed with four different sorbents: TSK-ODS, LiChrosorb RP-8, LiChrosorb RP-2 and μ-Bondapak CN-bonded silica. The method involved direct injection of serum sample onto the columns for trapping of the analyte, clean-up from weakly retained serum endogenous components, as well as the final separation. The protein-coated columns operated in two different chromatographic modes. Serum proteins were extracted and cleaned up by SPE, whereas the final separation of bupivacaine was based on reversed-phase chromatography. The protein-coated TSK-ODS column resulted in more accurate peak integration and more reproducible results. A linear relationship between the concentrations of drug and peak areas was confirmed in the range of 100-2000 ng/mL. Detection and quantification limits were 24.85 and 85.36 ng/mL, respectively. The average recovery for bupivacaine ranged from 96.48% to 98.81%. The present methodology was successfully applied, with a high degree of confidence, to analyze clinical samples obtained from patient receiving 0.5% bupivacaine therapy.

DOI: https://doi.org/10.1093/chromsci/bmaa014
Metabolomics. 2020 Apr 17. 16(5): 53

Dual mass spectrometry as a tool to improve annotation and quantification in targeted plasma lipidomics.

Liang Gao, Amaury Cazenave-Gassiot, Bo Burla, Markus R Wenk, Federico Torta.

   INTRODUCTION: High quality data, based on reliable quantification and clear identification of the reported lipid species, are required for the clinical translation of human plasma lipidomic studies.
OBJECTIVE: Lipid quantification can be efficiently performed on triple quadrupole (QqQ) mass spectrometers in targeted multiple reaction monitoring (MRM) mode. However, a series of issues can be encountered when aiming at unambiguous identification and accurate quantification, including (i) resolving peaks of polyunsaturated species, (ii) discriminating between plasmanyl-, plasmenyl- and odd chain species and (iii) resolving the isotopic overlap between co-eluting lipid species.
METHODS: As a practical tool to improve the quality of targeted lipidomics studies, we applied a Dual MS platform by simultaneously coupling a reversed-phase liquid chromatography separation to a QqQ and a quadrupole-time of flight (Q-ToF) mass spectrometers. In one single experiment, this platform allows to correctly identify, by high-resolution MS and MS/MS, the peaks that are quantified by MRM.
RESULTS: As proof of concept, we applied the platform on glycerophosphocholines (GPCs) and sphingomyelins (SMs), which are highly abundant in human plasma and play crucial roles in various physiological functions. Our results demonstrated that Dual MS could provide a higher level of confidence in the identification and quantification of GPCs and SMs in human plasma. The same approach can also be applied to improve the study of other lipid classes and expanded for the identification of novel lipid molecular species.
CONCLUSIONS: This methodology might have a great potential to achieve a better specificity in the quantification of lipids by targeted lipidomics in high-throughput studies.

Keywords:  Glycerophosphocholine; Identification; Lipidomics; Liquid chromatography; Plasma; Quantification; Sphingomyelin; Tandem mass spectrometry

DOI:  https://doi.org/10.1007/s11306-020-01677-z
Biomed Chromatogr. 2020 Apr 17. e4848

Simultaneous measurement of epinastine and its metabolite, 9,13b-dehydroepinastine, in human plasma by a newly developed ultra-performance liquid chromatography-tandem mass spectrometry and its application to pharmacokinetic studies.

Seung-Hyun Jeong, Ji-Hun Jang, Hea-Young Cho, Yong-Bok Lee.

  Epinastine is an antiallergic drug with high selectivity for histamine receptors. It has been reported that 9,13b-dehydroepinastine is present as a metabolite in vivo in humans, but there was little information about their pharmacokinetics (PKs) in humans. Although several analytical methods have been reported for epinastine analysis in different matrices, none are available for its metabolite. Therefore, purpose of this study was to develop an analytical method to simultaneously measure epinastine and its metabolite, 9,13b-dehydroepinastine, in human plasma samples using ultra-performance liquid chromatography-tandem mass spectrometer. Analytes were separated in a C18 -column. Quantifying of this analysis was performed on a triple-quadrupole mass spectrometer. Chromatograms showed high sensitivity, selectivity, and resolution with no interference with plasma constituents. Calibration curves for both epinastine and 9,13b-dehydroepinastine in human plasma were 0.1-50 ng/mL and displayed excellent linearity with correlation coefficients (r2 ) greater than 0.99. Developed analytical method satisfied the criteria of international guidance and was validated. Method could be successfully applied to PK studies of epinastine and, for the first time, metabolite kinetics of epinastine to 9,13b-dehydroepinastine in humans after oral administration of 20 mg epinastine hydrochloride tablets. Our study is expected to be useful in future studies such as dosage settings and clinical pharmacotherapy.

Keywords:  9,13b-dehydroepinastine; Epinastine; Metabolite kinetics; Pharmacokinetics; Ultra-performance liquid chromatography-tandem mass spectrometer

DOI:  https://doi.org/10.1002/bmc.4848
Anal Chem. 2020 Apr 16.

ICBM-OCEAN: Processing ultrahigh-resolution mass spectrometry data of complex molecular mixtures.

Julian Merder, Jan Alfred Freund, Ulrike Feudel, Christian T Hansen, Jeffrey Alistair Hawkes, Benjamin Jacob, Katrin Klaproth, Jutta Niggemann, Beatriz Elizabeth Noriega-Ortega, Helena Osterholz, Pamela E Rossel, Michael Seidel, Gabriel Singer, Aron Stubbins, Hannelore Waska, Thorsten Dittmar.

Untargeted molecular analyses of complex mixtures are relevant for many fields of research, including geochemistry, pharmacology, and medicine. Ultrahigh-resolution mass spectrometry is one of the most powerful tools in this context. The availability of open scripts and online tools for specific data processing steps such as noise removal or molecular formula assignment is growing, but an integrative tool where all crucial steps are reproducibly evaluated and documented is lacking. We developed a novel, server-based tool (ICBM-OCEAN, "Institute for Chemistry and Biology of the Marine Environment, Oldenburg - complex molecular mixtures, evaluation and analysis") that integrates published and novel approaches for standardized processing of ultrahigh-resolution mass spectrometry data of complex molecular mixtures. Different from published approaches, we offer diagnostic and validation tools for all relevant steps. Among other features, we included objective and reproducible reduction of noise and systematic errors, spectra recalibration and alignment, and identification of likeliest molecular formulae. With 15 chemical elements, the tool offers high flexibility in formula attribution. Alignment of mass spectra among different samples prior to molecular formula assignment improves mass error and facilitates molecular formula confirmation with help of isotopologues. The online tool and the detailed instruction manual can be freely accessed at www.icbm.de/icbm-ocean.

DOI: https://doi.org/10.1021/acs.analchem.9b05659
Anal Biochem. 2020 Apr 13. pii: S0003-2697(20)30255-4. [Epub ahead of print] 113723

Development and validation of an LC-MS/MS method for the quantification of flavonoid glucuronides (wogonoside, baicalin, and apigenin-glucuronide) in the bile and blood samples: application to a portal vein infusion study.

Yifan Tu, Lei Zhou, Li Li, Lu Wang, Song Gao, Ming Hu.

  Glucuronidation is one of the major metabolic pathways for flavonoids. However, quantification of flavonoid glucuronides in biological samples, especially in the bile, is sometimes challenging due to signal suppression by bile acids. The purpose of this study is to establish a robust LC-MS/MS method for directly measuring flavonoid glucuronides in bile and blood. Wogonoside (wongonin-glucuronide), baicalin (baicalein-glucuronide) and apigenin-7-O-glucuronide were used as the model compounds and taurocholic acid (T-CA) were used as the model bile acid to establish the method. Bile samples were processed using solid phase extraction (SPE) and blood samples were prepared using protein precipitation method. The analytes were separated on a Resteck HPLC (50 mm × 2.1 mm ID, 1.7 μm) column using acetonitrile and 2.5mM ammonium acetate (pH=7.4) in water as the mobile phases. The mass analysis was performed in an AB Sciex 5500 Qtrap mass spectrometer via multiple reaction monitoring (MRM) in the positive mode. The results showed that the linear range of the above three analytes were 10 nM to 5000 nM in the bile and 1.56 nM to 4000 nM in the blood, respectively. The recoveries of three glucuronides were >85% and the matrix effects were < 20% at low, medium and high concentrations in the bile and the blood. The results also showed that >90% of these bile acids were removed by the selected SPE procedure to facilitate glucuronide analysis. The validated method was successfully applied to a portal vein infusion study using rats to quantify baicalin, wogonoside, and apigenin-glucuronide in bile and blood samples.

Keywords:  LC-MS/MS; SPE; Wogonoside; apigenin-7-O-glucuronide; baicalin; bile; blood; glucuronide quantification

DOI:  https://doi.org/10.1016/j.ab.2020.113723
Biomed Chromatogr. 2020 Apr 14. e4847

Metabolomics approach based on utra-performance liquid chromatography coupled to mass spectrometry with chemometrics methods for high-throughput analysis of metabolite biomarkers to exploring the abnormal metabolic pathways associated with myocardial dysfunction.

Ling-Kun Zhao, Yun-Bo Zhao, Peng-Cheng Yu, Peng-Xia Zhang.

  Ultra-performance liquid chromatography/mass spectrometry-based metabolomics can been used for discovery of metabolite biomarkers to exploring the metabolic pathway of diseases. Identification of metabolic pathways is key to understanding the pathogenesis and mechanism of disease. Myocardial dysfunction induced by sepsis (SMD) is a severe complication of septic shock and represents major causes of death in ICU, however its pathological mechanism is still not clear. In this study, ultrahigh-pressure liquid chromatography with mass spectrometry (UHPLC/MS)-based metabolomics with chemometrics anaylsis and multivariate pattern recognition analysis were used to detect the urinary metabolic profile changes of lipopolysaccharide (LPS)-induced SMD mouse model. Multivariate statistical analysis including principal component analysis and orthogonapartial least squares discriminant analysis for the discrimination of SMD was conducted to identify potential biomarkers. A total of 19 differential metabolites was discovered by high-resolution mass spectrometry-based urinary metabolomics strategy. The altered biochemical pathways based on these metabolites showed that tyrosine metabolism, and phenylalanine metabolism, ubiquinone biosynthesis, vitamin B6 metabolism were closely connected with the pathological processes of SMD. Consequently, integrated chemometrics analyses of these metabolic pathways are necessary to extract the information for the discovery of novel insights in the pathogenesis of disease.

Keywords:  Metabolomics; biomarkers; chemometrics; liquid chromatography; mass spectrometry; metabolic pathway

DOI:  https://doi.org/10.1002/bmc.4847
Molecules. 2020 Apr 11. pii: E1763. [Epub ahead of print]25(8):

Analysis of Nicotine Metabolites in Hair and Nails Using QuEChERS Method Followed by Liquid Chromatography-Tandem Mass Spectrometry.

Junhee Kim, Hyun-Deok Cho, Joon Hyuk Suh, Ji-Youn Lee, Eunyoung Lee, Chang Hwa Jin, Yu Wang, Sangwon Cha, Hosub Im, Sang Beom Han.

  Many studies have analyzed nicotine metabolites in blood and urine to determine the toxicity caused by smoking, and assess exposure to cigarettes. Recently, hair and nails have been used as alternative samples for the evaluation of smoking, as not only do they reflect long-term exposure but they are also stable and easy to collect. Liquid-liquid or solid-phase extraction has mainly been used to detect nicotine metabolites in biological samples; however, these have disadvantages, such as the use of toxic organic solvents and complex pretreatments. In this study, a modified QuEChERS method was proposed for the first time to prepare samples for the detection of nicotine metabolite cotinine (COT) and trans-3'-hydroxycotinine (3-HCOT) in hair and nails. High-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to analyze traces of nicotine metabolites. The established method was validated for selectivity, linearity, lower limit of quantitation, accuracy, precision and recovery. In comparison with conventional liquid-liquid extraction (LLE), the proposed method was more robust, and resulted in higher recoveries with favorable analytical sensitivity. Using this method, clinical samples from 26 Korean infants were successfully analyzed. This method is expected to be applicable in the routine analysis of nicotine metabolites for environmental and biological exposure monitoring.

Keywords:  LC–MS/MS; QuEChERS; bioanalysis; hair; nail; nicotine metabolites

DOI:  https://doi.org/10.3390/molecules25081763
Drug Test Anal. 2020 Apr 17.

Development and validation of a simple online-SPE method coupled to high-resolution mass spectrometry for the analysis of stanozolol-N-glucuronides in urine samples.

Lorenz Göschl, Günter Gmeiner, Valentin Enev, Nicolas Kratena, Peter Gärtner, Guro Forsdahl.

  Stanozolol is still the most commonly used illicit anabolic-androgenic steroid (AAS) in professional sports. Therefore, accurate and fast analysis and long detection windows are of high interest in the field of anti-doping analysis. In this work, a very simple, fast and highly sensitive online solid-phase extraction method coupled with liquid chromatography - high-resolution tandem mass spectrometry (HPLC-HRMSMS) for the analysis of stanozolol-N-glucuronides was developed. This fully validated procedure is characterized by only a few manual steps (dilution and addition of internal standard) in the sample preparation. A limit of identification (LOI) of 75 pg/ml, high accuracy (87.1% - 102.1%), precision (3.1% - 7.8%) and sensitivity was achieved. Furthermore, good linearity (> 0.99) and robustness, as well as no carry-over effects, could be observed. In addition to excellent confirmation analysis performance, this method shows sufficient potential for the identification and characterization of unknown metabolites. Using this method, it was possible to unambiguously confirm the presence of 1'N- and 2'N-stanozolol-glucuronide in human urine for the first time due to the access to reference material.

Keywords:  Anabolic androgenic steroids; mass spectrometry; online solid-phase extraction; phase-II metabolite; stanozolol

DOI:  https://doi.org/10.1002/dta.2805
Anal Bioanal Chem. 2020 Apr 16.

Analysis of neurotransmitters in rat placenta exposed to flame retardants using IR-MALDESI mass spectrometry imaging.

Crystal L Pace, Brian Horman, Heather Patisaul, David C Muddiman.

  Chemical exposures can adversely impact fetal development. For many compounds, including common flame retardants, the mechanisms by which this occurs remain unclear, but emerging evidence suggests that disruption at the level of the placenta may play a role. Understanding how the placenta might be vulnerable to chemical exposures is challenging due to its complex structure. The primary objective of this study was to develop a method for detecting placental neurotransmitters and related metabolites without chemical derivatization so changes in the abundance and spatial distribution of neurotransmitters in rat placenta following chemical exposure could be determined using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry imaging. Without chemical derivatization, 49 neurotransmitters and their related metabolites were putatively identified in untreated rat placenta sections using mass measurement accuracy and spectral accuracy. A few neurotransmitters were less abundant in placentas that were exposed to various flame retardants and were further investigated by KEGG metabolic pathway analysis. Many of these downregulated neurotransmitters shared the same enzyme responsible for metabolism, aromaticl-amino acid decarboxylase, suggesting a mechanistic role. These data constitute a new approach that could help identify novel mechanisms of toxicity in complex tissues. Graphical abstract.

Keywords:  Exposomics; IR-MALDESI; Mass spectrometry imaging; Neurotransmitters

DOI:  https://doi.org/10.1007/s00216-020-02626-4
Sud Med Ekspert. 2020 ;63(2): 32-40

[Fast HPLC-MS/MS screening method for identification of illegal drugs including new psychoactive substances and their metabolites in human urine].

S A Savchuk, K M Shestakova, L N Rizvanova, D A Burmykin, M V Ovcharov, N V Mesonzhnik, S A Appolonova.

  Aim of this study was to develop a fast screening method for identification of illegal drugs including new psychoactive substances and their metabolites in human urine by means of liquid chromatography mass-spectrometry HPLC-MS/MS screening method for identification of 138 psychoactive substances in urine was established, validated and tested. High-resolution mass-spectrometry was used for confirmation of the developed approach. The developed method was tested on 50 positive urine samples containing the target compounds, which proved reliability and accuracy of the established screening method.

Keywords:  mass-spectrometry; new psychoactive substances; screening method

DOI:  https://doi.org/10.17116/sudmed20206302132
Science. 2020 Apr 17. 368(6488): 283-290

Metabolomics and mass spectrometry imaging reveal channeled de novo purine synthesis in cells.

Vidhi Pareek, Hua Tian, Nicholas Winograd, Stephen J Benkovic.

Metabolons, multiprotein complexes consisting of sequential enzymes of a metabolic pathway, are proposed to be biosynthetic "hotspots" within the cell. However, experimental demonstration of their presence and functions has remained challenging. We used metabolomics and in situ three-dimensional submicrometer chemical imaging of single cells by gas cluster ion beam secondary ion mass spectrometry (GCIB-SIMS) to directly visualize de novo purine biosynthesis by a multienzyme complex, the purinosome. We found that purinosomes comprise nine enzymes that act synergistically, channeling the pathway intermediates to synthesize purine nucleotides, increasing the pathway flux, and influencing the adenosine monophosphate/guanosine monophosphate ratio. Our work also highlights the application of high-resolution GCIB-SIMS for multiplexed biomolecular analysis at the level of single cells.

DOI: https://doi.org/10.1126/science.aaz6465
Anal Chim Acta. 2020 May 08. pii: S0003-2670(20)30325-1. [Epub ahead of print]1110 158-168

Exploration advantages of data combination and partition: First chemometric analysis of liquid chromatography-mass spectrometry data in full scan mode with quadruple fragmentor voltages.

Xiao-Dong Sun, Hai-Long Wu, Jun-Chen Chen, An-Qi Chen, Yue Chen, Yang-Zi Ouyang, Yu-Jie Ding, Ru-Qin Yu.

  A novel soft strategy for combination and partition of mass spectra data recorded at different fragmentor voltages in full scan mode of a mass spectrometer was developed to generate abundant multi-way data. It is the first time that non-linear four-way and combined three-way LC-MS data have been obtained simultaneously in a single chromatographic run. This strategy ensures that each analyte can be ionized and detected at the most appropriate MS conditions (ionization modes, fragmentor voltages) and avoids a hard chromatographic segmentation in subsequent chemometric analysis. Two different experimental datasets were analyzed to validate the feasibility and applicability of this strategy. Some simple pretreatments were carried out before LC-MS analysis to prevent potential matrix effects. Proper chemometric tools were used to resolve three-way (partitioned data) and enhanced three-way LC-MS (combined data) data, respectively. The method was assessed by comparing the analytical results obtained from the same chemometric algorithm with both combined and partitioned datasets: (1) the combined data provided the best global overall resolution, higher sensitivity and more reliable results, (2) the partitioned data provided higher selectivity for some specific analytes. The results showed that the developed method could be a soft and ingenious tool to handle the unordered but information-rich raw LC-MS data. Moreover, the proposed strategy could take extra analytical advantages in terms of higher sensitivity and more reliable quantitative results when compared with LC-MS (with single fragmentor voltage) strategy and showed nearly the same capability in analytical quality as classic LC-MS/MS method.

Keywords:  Alternating trilinear decomposition; Data combination; Data partition; Liquid chromatography–mass spectrometry; Second-order calibration

DOI:  https://doi.org/10.1016/j.aca.2020.03.018
Bioanalysis. 2020 Apr 14.

UHPLC-MS/MS method to determine FP-208 in human plasma and its application to a pharmacokinetic study.

Huanhuan Wang, Teng Wang, Zhanqing Wang, Zhenjian Du, Qian Zhao, Ji Jiang, Pei Hu.

  Aim: FP-208 is a novel and effective small-molecule inhibitor blocking the mammalian target of rapamycin complex-1/mammalian target of rapamycin complex-2/PI3Ka. To investigate the pharmacokinetic profile of FP-208, a rapid and reliable analytical method was needed to be established to determine FP-208 in the plasma of patients with solid tumors. Materials & methods: FP208 was separated on a charged surface hybrid (CSH) C18 column (2.1 mm × 50 mm, 1.7 μm) after the plasma samples were purified using a protein precipitation method. Detection was performed on an AB Sciex 5500 mass spectrometer in the positive electrospray ionization mode. The established method was validated according to the bioanalytical guidelines. Conclusion: For the first time, the developed and validated method was successfully applied in the first-in-human study for FP-208 in patients with solid tumors after oral administration (Number: CTR20180683).

Keywords:  FP208; UHPLC–MS/MS; first-in-human; human plasma; mTOR inhibitor; mTORC1; mTORC2; pharmacokinetics; protein precipitation method; validation

DOI:  https://doi.org/10.4155/bio-2020-0008
Lipids Health Dis. 2020 Mar 23. 19(1): 52

Simultaneous determination of five essential amino acids in plasma of Hyperlipidemic subjects by UPLC-MS/MS.

Zhibin Chen, Feiyan Lin, Xuemei Ye, Yuqian Dong, Lufeng Hu, Aifang Huang.

   BACKGROUND: Millions of adults have been reported with hyperlipemia in the world. It is still unclear whether the plasma level of essential amino acids (AAs) will be influenced by the hyperlipemia. This study was aimed to investigate the AAs levels and the underlying metabolic relationship in hyperlipidemic subjects.
METHODS: An ultra-high performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method was developed for the determination of phenylalanine (Phe), valine (Val), histidine (His), tryptophan (Trp), and methionine (Met). Plasma samples (100 μL) were precipitated by acetonitrile (300 μL) and analyzed on a BEH C18 (2.1 mm × 100 mm, 1.7 μm) column at 40 °C by gradient elution. The mobile phase composed of 0.1% formic acid and acetonitrile was used with flow rate at 0.2-0.4 ml/0-3 min. Five AAs were determined at positive electrospray ionization (ESI+) at m/z 118.1/72.1 (Val), 150.12/104.02(Met), 156.06/110.05(His), 166.1/120.1(Phe), and 205.2/188.02 (Trp). A total of 75 healthy subjects and 83 hyperlipidemic subjects, who had blood routine test and plasma lipid test were determined by developed UPLC-MS/MS.
RESULTS: It was shown that there was good linearity for Val, Met, His, Phe, and Trp within 1-100 μg/mL. The relative standard deviations of precision and accuracy were all within 15%. The level of Val, Phe, Trp, His, and Met were 35.34 ± 15.64, 22.72 ± 9.13, 17.23 ± 4.94, 16.78 ± 13.64, and 6.24 ± 1.97 μg/mL in healthy subjects, while they were 38.04 ± 16.70, 22.41 ± 8.45, 15.62 ± 5.77, 18.35 ± 14.49, and 6.21 ± 1.97 μg/mL in hyperlipidemic subjects respectively. The Spearman's correlations analysis showed that there were high correlations between Val, Phe, Trp, His, Met and triglyceride in healthy subjects. While, those correlations decreased in hyperlipemia cases.
CONCLUSION: A convenient and sensitive method for simultaneous determination of Val, Phe, Trp, His, and Met in human plasma was developed. There was a high correlation between Val, Phe, Trp, His, Met and triglyceride. Hyperlipemia influences the metabolic balance of His, Phe, Trp, Met and Val.

Keywords:  Amino acid; Human; Hyperlipemia; Plasma; UPLC-MS/MS

DOI:  https://doi.org/10.1186/s12944-020-01216-8
Anal Chem. 2020 Apr 14.

Ultrafast Photocatalytic Reaction Screening by Mass Spectrometry.

Jie Sun, Huihui Liu, Xi Huang, Yuming Jiang, Caiqiao Xiong, Zongxiu Nie.

Here, we report a semiconductor assisted laser desorption/ionization mass spectrometry (SA-LDI MS) platform to monitor photocatalytic reactions on line and apply it for ultrafast reaction screening. In this method, we use photocatalytic nano-materials as the substrate for LDI, and then initiate and monitor the reactions simultaneously. The features of our method in-clude: i) It has reaction acceleration effect: only seconds are needed in our interfacial reactions vs hours in conventional bulk phase. ii) The reaction trend in our system agrees with that in bulk phase. iii) By adding a stable analogue of reactant as internal standard, the quantification of reaction can be achieved. iv) The sensitivity is high: for 500 amol reactant, the photo-catalytic reaction can still be initiated and detected. This platform has advantages in ultrafast reaction screening (e.g. 9 catalyst screening needs 24 h by UPLC-MS system but only 10 minutes by SA-LDI MS). Furthermore, the high specificity of MS enables the screening of catalytic selectivity of A-TiO2 nanoparticles for methyl red (MR) and acid yellow (AY) mixture, whose absorption wavelengths are overlapped and thus cannot be discriminated by conventional optical methods. Besides, by using SA-LDI MS, we also monitored the reductive debrominations during the degradation process of polybrominated diphenyl ethers (PBDEs), which is a sort of important pollutant hard to degrade and detect in liquid phase, and the photocatalytic reduction of CO2. Overall, SA-LDI MS realizes ultrafast photocatalytic reaction screening for the first time and provides prac-tical analytical value in the field of catalyst screening.

DOI: https://doi.org/10.1021/acs.analchem.0c00201
Forensic Sci Int. 2020 Apr 03. pii: S0379-0738(20)30136-5. [Epub ahead of print]311 110274

Retrospective screening of synthetic cannabinoids, synthetic opioids and designer benzodiazepines in data files from forensic post mortem samples analysed by UHPLC-QTOF-MS from 2014 to 2018.

Per Ole M Gundersen, Sebastian Broecker, Lars Slørdal, Olav Spigset, Martin Josefsson.

  The introduction of new psychoactive substances (NPS) on the illicit drug market has led to major challenges for the analytical laboratories. Keeping screening methods up to date with all relevant drugs is hard to achieve and the risk of missing important findings in biological samples is a matter of concern. Aiming for an extended retrospective data analysis, diagnostic fragment ions from synthetic cannabinoids (n=251), synthetic opioids (n=88) and designer benzodiazepines (n=26) not included in our original analytical method were obtained from the crowdsourced database HighResNPS.com and converted to a personalized library in a format compatible with the analytical instrumentation. Data files from the analysis of 1314 forensic post mortem samples with an Agilent 6540 ultra high pressure liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) performed in our laboratory from January 2014 to December 2018 were retrieved and retrospectively processed with the new personalized library. Potentially positive findings were grouped in two: The most confident findings contained MS/MS data for library match (category 1) whereas the less confident findings lacked such data (category 2). Five new category 1 findings were identified: Flubromazepam in two data files from 2015 and 2016, respectively, phenibut (4-amino-3-phenylbutyric acid) in one data file from 2015, fluorofentanyl in one data file from 2016 and cyclopropylfentanyl in one data file from 2018. Retention time matches with reference standards further strengthened these findings. A list of 35 presumably positive category 2 findings was generated. Of these, only one finding of phenibut was considered plausible after checking retention times and signal-to-noise ratios. This study shows that new compounds can be detected retrospectively in data files from QTOF-MS using an updated library containing diagnostic fragment ions. Automatic screening procedures can be useful, but a manual re-evaluation of positive findings will always be necessary.

Keywords:  Designer benzodiazepines; New psychoactive substances; Post mortem blood samples; Retrospective screening; Synthetic cannabinoids; Synthetic opioids; UHPLC-QTOF-MS

DOI:  https://doi.org/10.1016/j.forsciint.2020.110274
Anal Bioanal Chem. 2020 Apr 14.

Data-dependent normalization strategies for untargeted metabolomics-a case study.

Paula Cuevas-Delgado, Danuta Dudzik, Verónica Miguel, Santiago Lamas, Coral Barbas.

  Despite the recent advances in the standardization of untargeted metabolomics workflows, there is still a lack of attention to specific data treatment strategies that require deep knowledge of the biological problem and need to be applied after a well-thought out process to understand the effect of the practice. One of those strategies is data normalization. Data-driven assumptions are critical especially addressing unwanted variation present in the biological model as it can be the case in heterogeneous tissues, cells with different sizes or biofluids with different concentrations. Chronic kidney disease (CKD) is a widespread disorder affecting kidney structure and function. Animal models are being developed to be able to get valuable insights into the etiopathogenesis of the condition and effect of the treatments. Moreover, diagnosis and disease staging still require defining appropriate biomarkers. Untargeted metabolomics has the potential to deal with those challenges. Renal fibrosis is one of the consequences of kidney injury which greatly affects the concentration of metabolites in the same quantity of sample. To overcome this challenge, several data normalization strategies have been applied, following a multilevel normalization method with the overall aim of focussing on the relevant biological information and reducing the influence of disturbing factors. A comprehensive evaluation of the performance of the normalization strategies, both on methods assessing the intragroup variation and on the impact on differential analysis, is provided. Finally, we present evidence of the importance of biological-model-driven guided normalization methods and discuss multiple criteria that need to be taken into consideration to obtain robust and reliable data. Special concern is transmitted on the misleading conclusions that might be the consequence of inappropriate data pre-treatment solutions applied for untargeted methods. Graphical abstract.

Keywords:  Biomarker discovery; Capillary electrophoresis mass spectrometry; Data pre-treatment; Normalization; Tissue samples; Unwanted variation

DOI:  https://doi.org/10.1007/s00216-020-02594-9
Analyst. 2020 Apr 16.

A high-throughput SAMDI-mass spectrometry assay for isocitrate dehydrogenase 1.

Sarah E Anderson, Natalie S Fahey, Jungsoo Park, Patrick T O'Kane, Chad A Mirkin, Milan Mrksich.

The enzyme isocitrate dehydrogenase 1 (IDH1) catalyzes the conversion of isocitrate to alpha-ketoglutarate (αKG) and has emerged as an important therapeutic target for glioblastoma multiforme (GBM). Current methods for assaying IDH1 remain poorly suited for high-throughput screening of IDH1 antagonists. This paper describes a high-throughput and quantitative assay for IDH1 that is based on the self-assembled monolayers for matrix-assisted laser desorption/ionization-mass spectrometry (SAMDI-MS) method. The assay uses a self-assembled monolayer presenting a hydrazide group that covalently captures the αKG product of IDH1, where it can then be detected by MALDI-TOF mass spectrometry. Co-capture of an isotopically-labeled αKG internal standard allows the αKG concentration to be quantitated. The assay was used to analyze a series of standard αKG solutions and produced minimal error in measured αKG concentration values. The suitability of the assay for high-throughput analysis was evaluated in a 384-sample biochemical IDH1 screen. Cells expressing IDH1 were lysed and the lysate was applied to the monolayer to capture αKG, which was then quantitated using the SAMDI-MS assay. Cells in which IDH1 expression was reduced by small-interfering RNA exhibited a corresponding decrease in αKG concentration as measured by the assay. Application of the assay toward the high-throughput screening of IDH1 inhibitors or knockdown agents may facilitate the discovery of treatments for GBM.

DOI: https://doi.org/10.1039/d0an00174k
Anal Chem. 2020 Apr 14.

Ozonization of tissue sections for MALDI MS imaging of carbon-carbon double bond positional isomers of phospholipids.

Antonín Bednařík, Jan Preisler, Dominika Bezdeková, Markéta Machálková, Michal Hendrych, Jarmila Navratilova, Lucia Knopfová, Eugene Moskovets, Jens Soltwisch, Klaus Dreisewerd.

Visualizing the differential distribution of carbon-carbon double bond (C=C db) positional isomers of unsaturated phospholipids (PL) in tissue sections by use of refined matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) technologies offers a high promise to deeper understand PL metabolism and isomer-specific functions in health and disease. Here we introduce an on-tissue ozonization protocol that enables a particular straightforward derivatization of unsaturated lipids in tissue sections. Collision-induced dissociation (CID) of MALDI-generated ozonide ions (with yields in the several ten percent range) produced the Criegee fragment ion pairs, which are indicative of C=C db position(s). We used our technique for visualizing the differential distribution of 9 and 11 isomers of phosphatidylcholines in mouse brain and in human colon samples with the desorption laser spot size 15 µm emphasizing the potential of the technique to expose local isomer-specific metabolism of PLs.

DOI: https://doi.org/10.1021/acs.analchem.0c00641
J Pharm Biomed Anal. 2020 Apr 12. pii: S0731-7085(20)30126-6. [Epub ahead of print]186 113258

Simultaneous analysis of saturated and unsaturated oxylipins in 'ex vivo' cultured peripheral blood mononuclear cells and neutrophils.

X Capó, M D Ferrer, R A Olek, E Salaberry, R M Gomila, G Martorell, A Sureda, J A Tur, A Pons.

  Oxylipins are a family of saturated and unsaturated fatty acids peroxidation products with bioactive properties. We have developed an improved method for the measurement of ex vivo oxylipin production by peripheral blood mononuclear cells (PBMCs) and neutrophils. We aimed to develop an analytical method to determine the production rates of polyunsaturated fatty acids (PUFAs), PUFA-oxylipin, and saturated-oxylipins by stimulated PBMCs and neutrophils based on solid phase extraction and HPLC-MS/MS technology. A UHPLC system coupled to a Q-Exactive Hybrid Quadrupole-Orbitrap mass spectrometer was used to identify and quantify oxylipin production. For each oxylipin and PUFA their differential response was calculated with respect to a deuterated internal standard factor (ISF). To calculate oxylipin and PUFAs in the culture samples, the individual ISF was used for each oxylipin and PUFA with respect to the deuterated internal standard. PBMCs and neutrophils showed a different pattern of oxylipin production and fatty acid secretion. Lipopolysaccharide (LPS) did not stimulate oxylipin production or fatty acids secretion in PBMCs, whereas phorbol myristate acetate (PMA) stimulation increased the production rate of 5-HETE, 15-HETE, 15-HEPE, 17-DoHE, PGE2, AA, and DHA. LPS stimulation decreased 16-hydroxyl-palmitatte (16-OHPAL) production and DHA secretion in neutrophils, while PMA stimulation increased the production rate of AA and its derivate oxylipins, 5-HETE, 15-HETE, and PGE2. In conclusion, we have developed a new method to determine oxylipins derived from both saturated and unsaturated fatty acids in culture cell media. This method has enough sensitivity, and accuracy, to determine oxylipin production and fatty acid secretion by PBMCs and neutrophils.

Keywords:  Bioanalytical methods; HPLC; Mass-spectrometry; Oxylipins

DOI:  https://doi.org/10.1016/j.jpba.2020.113258
J Chromatogr Sci. 2020 Apr 13. pii: bmaa013. [Epub ahead of print]

A Two-Step Optimization Approach: Validated RP-HPLC Method for Determination of Gatifloxacin and Dexamethasone in Ophthalmic Formulation.

Martin N Saad, Hebatallah M Essam, Eman S Elzanfaly, Sawsan M Amer.

The growing technology of stationary phase chemistry has a great impact on the chromatographic system performance and analysis economics. In this context, a simple rapid reversed phase high-performance liquid chromatography method development is presented for the analysis of gatifloxacin (GFN) and dexamethasone sodium phosphate (DSP) in their ophthalmic formulation. A two-step optimization approach has been conducted using optimum chromatographic conditions as well as proper selection of stationary phase. The chromatographic separation was carried out using sodium phosphate buffer pH 3.0 ± 0.1 and acetonitrile 72:28 v/v, respectively, with flow rate 1 mL min-1 and simultaneous detection at 243 nm. Three different column technologies were investigated at the optimum set of the chromatographic conditions: Xbridge® bridged ethylene hybrid silica, Kinetex™ Core-Shell and the Onyx™ Monolithic stationary phase. The monolithic column has shown better chromatographic separation, based on system suitability testing as well as shorter analysis time and sensitivity. The proposed method was validated according to International Conference on Harmonization guidelines. The linearity was achieved for GFN and DSP in the range 0.58-120 μg mL-1 and 0.50-120 μg mL-1, respectively, with acceptable accuracy, precision and selectivity.

DOI: https://doi.org/10.1093/chromsci/bmaa013
Biomed Chromatogr. 2020 Apr 17. e4853

Development of a precise quantitative method for monitoring sirolimus in whole blood using LC/ESI-MS/MS.

Kensuke Shigeta, Masafumi Kikuchi, Masaki Tanaka, Shinya Takasaki, Hisashi Oishi, Tetsu Sado, Yasushi Matsuda, Masafumi Noda, Yoshinori Okada, Nariyasu Mano, Hiroaki Yamaguchi.

  Sirolimus is used on patients after solid organ transplantation and on lymphangioleiomyomatosis (LAM) patients, and therapeutic drug monitoring is required in clinical practice. We have previously reported an accurate method for quantitative determination of sirolimus, but its sample preparation step was complicated. In this study, we developed a modified liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method for sirolimus quantification. A supported liquid extraction cartridge was used to purify sirolimus from whole blood and ion suppression was mostly prevented. The validation results fulfilled the acceptable criteria. This method was compared with the antigen conjugated magnetic immunoassay (ACMIA) and our previously reported method, using whole blood samples from LAM patients. Comparison of the Bland-Altman plots of the currently developed method and the previous method revealed no significant difference between the two methods (mean bias, -2.02%; 95% CI, -7.81 to 3.78). The values obtained by using ACMIA were significantly higher than those obtained by using the current method by 13.87% (95% CI, 6.49 to 21.25) due to cross-reactivity. The degree of cross reactivities in LAM patients and in organ transplant patients were similar, and our LC/ESI-MS/MS method precisely measured blood concentrations of sirolimus.

Keywords:  ACMIA; LC/ESI-MS/MS; lymphangioleiomyomatosis; sirolimus; therapeutic drug monitoring

DOI:  https://doi.org/10.1002/bmc.4853
J Chromatogr B Analyt Technol Biomed Life Sci. 2020 May 01. pii: S1570-0232(20)30060-X. [Epub ahead of print]1144 122082

Fast off-line FPSE-HPLC-PDA determination of six NSAIDs in saliva samples.

A Tartaglia, A Kabir, F D'Ambrosio, P Ramundo, S Ulusoy, H I Ulusoy, G M Merone, F Savini, C D'Ovidio, U De Grazia, K G Furton, M Locatelli.

  A fast off-line FPSE-HPLC-PDA method has been reported that allows simultaneous clean up and determination of six non-steroidal anti-inflammatory drugs (NSAIDs) in saliva samples from healthy volunteers. Particularly, furprofen, indoprofen, ketoprofen, fenbufen, flurbiprofen, and ibuprofen were chromatographically resolved. Benzyl paraben was chosen as the internal standard (BzPB, IS). These target compounds were successfully extracted from human saliva using fabric phase sorptive extraction (FPSE) and then analysed in the liquid chromatographic system by means of a short analytical column (Symmetry C18, 75 × 4.6 mm, 3.5 µm) using acetonitrile (AcN) and phosphate buffer (PBS, 30 mM; pH = 2.5) as the mobile phases. The method, validated through the calculation of all analytical parameters in accordance of International Guidelines, was applied to real saliva sample analysis collected from informed volunteers. The proposed approach that included the use of sol-gel polytetrahydrofuran (sol-gel PTHF) sorbent immobilized on cellulose support and C18 stationary phase used in HPLC, showed high potential as a fast tool for future clinical and forensic applications. The herein reported results encourage potential future application of FPSE in the forensic field. Furthermore, the FPSE membrane was tested in dried saliva spot mode (DSS) in order to check its potential use as a sampling device, also for forensic applications.

Keywords:  Fabric phase sorptive extraction; HPLC-PDA; NSAIDs; Saliva samples; Validation

DOI:  https://doi.org/10.1016/j.jchromb.2020.122082
J Chromatogr A. 2020 Apr 01. pii: S0021-9673(20)30287-9. [Epub ahead of print] 461066

Development of retention mechanism for the separation of carboxylic acids and inorganic anions in cryptand-based ion chromatography.

Diána Lukács, Krisztián Horváth, Péter Hajós.

  The ion-exchange and complex forming equilibria were quantitatively described and demonstrated in order to understand major factors in the control of selectivity in the analytical separation of carboxylic acids and inorganic anions in cryptand based ion chromatography. A complex retention model has been developed for the separation on a non-conventional IC column. Changes in retention are treated both theoretically and experimentally. Retention mechanism is employed on a macrocycle-based (cryptand n-decyl-[2.2.2]) ion-exchange chromatographic phase to improve the selectivity for a mixture of model analytes. We introduced an alternative internal gradient method by mixed eluent (i.e. eluents formed by combination of two alkali hydroxide with different molar ratio). The effect of binary mixed eluent (Li/Na, Li/K) on the retention behavior and peak shape of carboxylic acids are also discussed in view of the proposed theory. It was shown that the effects of binary aqueous mobile phases, held isocratically behave very similar to the step gradient mode. The "internal gradient" separation system has advantages over traditional step gradient mode. Twenty-six anions of widely varying chemical character (mono-, di-, tri-valent inorganic anions, mono-, di-, tri-valent aliphatic carboxylic acids, aromatic- and haloacetic carboxylic acids) were investigated on the cryptand-based (D222) stationary phase using different methods by LiOH, NaOH and KOH eluent. The predicted vs measured retention data are in rather good agreement. High degree of linearity was obtained for inorganic anions, multivalent carboxylic acids, and for aromatic and haloacetic acids R2 = 0.992, 0.969, and 0.980, respectively.

Keywords:  Carboxylic acids; Complexation; Cryptand based anion chromatography; Inorganic anions; Ion-exchange; Retention modeling

DOI:  https://doi.org/10.1016/j.chroma.2020.461066
J Am Soc Mass Spectrom. 2020 Apr 13.

Triple Resonance Methods to Improve Performance of Ion Trap Precursor and Neutral Loss Scans.

Lucas J Szalwinski, Dalton T Snyder, Mitch Wells, R Graham Cooks.

Two experiments are described that extend the capabilities of quadrupole ion trap mass spectrometers operated in the precursor and neutral loss scan mode. The first experiment, a triple resonance precursor ion scan is used to enhance sensitivity, selectivity, and molecular coverage. This method augments the ion trap precursor ion scan with application of a second excitation frequency to selectively activate first generation (MS2) product ions as they are formed and produce second generation (MS3) product ions which are then mass-selectively ejected with a third auxiliary signal and detected. This single mass analyzer experiment can be equated to performing the sequential precursor ion scan in a multiple analyzer system (Anal. Chem. 1990, 62, 1809-1818). The second capability demonstrated is 'frequency tagging', a method used to differentiate between ions ejected due to inherent instability under given trapping conditions - which causes artifacts during these scans - and ions that are resonantly ejected by the product ion ejection frequency. Beat frequencies are used to modulate resonance ejection peaks but conveniently do not modulate boundary ejection peaks. Frequency tagging provides a mechanism to identify the artifact peaks that are a consequence of operating at high trapping voltage (i.e. low mass cutoff) for optimal precursor/product ion selectivity. The experiment is demonstrated for precursor and for neutral loss scans.

DOI: https://doi.org/10.1021/jasms.0c00048
Anal Chim Acta. 2020 May 08. pii: S0003-2670(20)30341-X. [Epub ahead of print]1110 109-114

Boosting resolution in NMR spectroscopy by chemical shift upscaling.

Qing Zeng, Jinyong Chen, Yanqin Lin, Zhong Chen.

  Resolution is an essential challenge in NMR spectroscopy. Narrow chemical shift range and extensive signal splittings due to scalar couplings often give rise to spectral congestion and even overlap in NMR spectra. Magnetic field strength is directly responsible for spectral resolution as higher magnetic field strength offers better signal dispersion. However, the process of further increasing magnetic field strength of NMR instruments is slow and expensive. Methodology aimed at resolution issue has long been developing. Here, we present a chemical shift upscaling method, in which chemical shifts are upscaled by a given factor while scalar couplings are unchanged. As a result, signal dispersion and hence the resolution are improved. Therefore, it is possible to separate multiplets which originally overlap with each other and to extract their integrals for quantitative analysis. Improved signal dispersion and the preservation of scalar couplings also facilitate multiplet analysis and signal assignment. Chemical shift upscaling offers a method for enhancing resolution limited by magnetic field strength.

Keywords:  Chemical shift; Nuclear magnetic resonance (NMR); Quantification; Scalar coupling; Spectral resolution

DOI:  https://doi.org/10.1016/j.aca.2020.03.032
Metallomics. 2020 Apr 17.

Chromatographic detection of low-molecular-mass metal complexes in the cytosol of Saccharomyces cerevisiae.

Trang Q Nguyen, Joshua E Kim, Hayley N Brawley, Paul A Lindahl.

Fluorescence-based chelators are commonly used to probe labile low-molecular-mass (LMM) metal pools in the cytosol of eukaryotic cells, but such chelators destroy the complexes of interest during detection. The objective of this study was to use chromatography to directly detect such complexes. Towards this end, 47 batches of cytosol were isolated from fermenting S. cerevisiae yeast cells and passed through a 10 kDa cut-off membrane. The metal contents of the cytosol and resulting flow-through solution (FTS) were determined. FTSs were applied to a size-exclusion LC column located in an anaerobic refrigerated glove box. The LC system was coupled to an online inductively-coupled-plasma mass spectrometer (ICP-MS) for detection of individual metals. Iron-detected chromatograms of cytosolic FTSs from WT cells exhibited 2-4 major species with apparent masses between 500-1300 Da. Increasing the iron concentration in the growth medium 40-fold increased the overall intensity of these peaks. Approximately 3 LMM cytosolic copper complexes with apparent masses between 300-1300 Da were also detected; their LC intensities were weak, but these increased with increasing concentrations of copper in the growth medium. Observed higher-mass copper-detected peaks were tentatively assigned to copper-bound metallothioneins Cup1 and Crs5. FTSs from strains in which Cup1 or the Cox17 copper chaperone were deleted altered the distribution of LMM copper complexes. LMM zinc- and manganese-detected species were also present in cytosol, albeit at low concentrations. Supplementing the growth medium with zinc increased the intensity of the zinc peak assigned to Crs5 but the intensities of LMM zinc complexes were unaffected. Phosphorus-detected chromatograms were dominated by peaks at apparent masses 400-800 Da, with minor peaks at 1000-1500 Da in some batches. Sulfur chromatograms contained a low-intensity peak that comigrated with a glutathione standard; quantification suggested a GSH concentration in the cytosol of ca. 13 mM. A second LMM sulfur peak that migrated at an apparent mass of 100 Da was also evident.

DOI: https://doi.org/10.1039/c9mt00312f
Pharmazie. 2020 Apr 06. 75(4): 136-141

Development and validation of an LC-MS/MS method for the quantification of imatinib and imatinib-d8 in human plasma for the support of an absolute bioavailability microdose trial.

J Roosendaal, N Venekamp, L Lucas, H Rosing, J H Beijnen.

Here we describe the development and validation of an LC-MS/MS method for the quantification of imatinib and imatinib-d8 in plasma for the support of a clinical absolute bioavailability microdosing trial. The focus lies on the technical aspects to analyse high concentrations of imatinib and low concentrations of imatinib-d8 that are present simultaneously in study samples, using a single sample processing and analytical method. With the validated assay, imatinib and imatinib-d8 can be quantified simultaneously in ranges from 25.0 - 5,000 ng/mL and 0.01 - 2.0 ng/mL, respectively. The method was successfully applied in an imatinib-d8 absolute bioavailability microdosing trial, where a 100 μg imatinib-d8 microdose was intravenously administered to a patient on oral imatinib treatment 400 mg once daily.

DOI: https://doi.org/10.1691/ph.2020.9150
J Pharm Biomed Anal. 2020 Apr 03. pii: S0731-7085(20)30261-2. [Epub ahead of print]186 113274

Determination of iron in hair samples by slotted quartz tube-flame atomic absorption spectrometry after switchable solvent liquid phase extraction.

Nihan Atsever, Tülay Borahan, Emine Gülhan Bakırdere, Sezgin Bakırdere.

  In this study, a green switchable solvent based liquid phase extraction (SS-LPE) method was developed for the determination of iron in human hair matrix at very low concentrations. The switchable solvent was synthesized from N,N-Dimethlybenzylamide (DMBA) and ultrapure water with the addition of dry ice for protonation, and used as the extraction solvent. The optimum conditions obtained for 8.0 mL aqueous solution after univariate optimization were 1.5 mL of pH 4.0 buffer solution, 0.75 mL of 0.015 % ligand solution, 0.75 mL of switchable solvent, 45 s vortex period for complexation and extraction, 1.25 mL of 1.50 M NaOH and 30 s vortex period after the addition of NaOH. Under the optimum conditions, a 92-fold improvement was obtained in the detection power of the flame atomic absorption spectrometry (FAAS). The limit of detection (LOD) and limit of quantification (LOQ) values were found to be 2.6 and 8.6 μg/L, respectively. Linear range of the developed method was between 11 and 75 μg/L, with a correlation coefficient (R2) value of 0.9992. Accuracy and the applicability of the proposed method were verified by recovery studies and the results obtained for 15, 20, 30 and 40 μg/L spiked concentrations were 113.3 ± 4.9, 90.6 ± 4.4, 102.8 ± 8.2 and 98.1 ± 7.5 %, respectively.

Keywords:  Flame atomic absorption spectrometry; Human hair; Iron; Liquid phase microextraction; Switchable solvent

DOI:  https://doi.org/10.1016/j.jpba.2020.113274
Eur J Hosp Pharm. 2020 Mar;27(e1): e93-e98

Long-term stability of ready-to-use norepinephrine solution at 0.2 and 0.5 mg/mL.

Sixtine Gilliot, Morgane Masse, Stéphanie Genay, Damien Lannoy, Christine Barthélémy, Bertrand Décaudin, Pascal Odou.

   Objectives: Norepinephrine is a vasopressor frequently administered after dilution to treat hypotension and shocks in intensive care units. The stability of norepinephrine is known to be highly sensitive to storage conditions. Moreover, medication errors linked to the dilution step are frequent and may be deleterious for critically-ill patients, especially in intensive care units. This study aimed to evaluate the stability of ready-to-use diluted norepinephrine solutions prepared at two target concentrations (0.2 and 0.5 mg/mL), according to the summary of product characteristics, and stored for 365 days in two containers: AT-closed cyclic olefin copolymer vials, and polypropylene syringes.
Methods: A fast reversed-phase liquid chromatography method coupled with an ultra-violet detector was developed to assess the chemical stability of norepinephrine solutions. Validation was conducted according to the linearity of the calibration ranges, selectivity, sensitivity, accuracy and precision. Dosage, sub-visible particle contamination, pH monitoring and sterility assays were performed. Chemical stability was maintained if the measured concentration respected the lower limit of 90% of the initial concentration. Containers were stored at -20±5°C, +5±3°C and +25±2°C with 60±5% relative humidity in a dark closed enclosure.
Results: Stability was successfully maintained for every concentration and container tested when stored at -20±5°C and +5±3°C. In these storage conditions, particle contamination, pH monitoring and sterility assay respected the required criteria. Chemical degradation and colouring of solutions appeared before the end of the 1 year study period for most norepinephrine solutions stored at room temperature.
Conclusions: Ready-to-use solutions containing 0.2 and 0.5 mg/mL norepinephrine in polypropylene syringes or cyclic olefin copolymer vials must be stored at refrigerated or frozen temperatures to obtain acceptable 1 year shelf-stability. Exposure to higher temperatures significantly decreases shelf-stability. Our study protocol for compounding polypropylene syringes and cyclic olefin copolymer vials containing norepinephrine is adapted to implementation in centralised intravenous additive services.

Keywords:  IV administration; drug stability; intensive & critical care; shelf life; validation analytical procedure

DOI:  https://doi.org/10.1136/ejhpharm-2019-002146
Analyst. 2020 Apr 16.

A mini mass spectrometer with a low noise Faraday detector.

Yang Tang, Qian Xu, Dayu Li, Wei Xu.

An ion trap mass spectrometer is conventionally featured with an electron multiplier as its detector. However, an electron multiplier can typically work at pressures below 20 mTorr with a high voltage applied, which limits the further miniaturization of ion trap mass spectrometers. In this work, a low noise Faraday detector was developed and integrated in our miniature mass spectrometer instrument, and a post data processing method was applied to improve its performance. A limit of detection of 1 ng mL-1 was achieved, and quantitation performance and mass resolution were characterized. This technology could be useful in the further development of miniature mass spectrometers by increasing background pressures.

DOI: https://doi.org/10.1039/d0an00420k
Anal Chem. 2020 Apr 14.

Hollow fiber liquid-phase microextraction at-line coupled to capillary electrophoresis for direct analysis of human body fluids.

Blanka Miková, Miloš Dvořák, Lenka Ryšavá, Pavel Kuban.

A simple and cheap all-in-one concept for at-line coupling of hollow fiber liquid-phase microextraction (HF-LPME) to commercial capillary electrophoresis (CE) is demonstrated, which enables direct analysis of complex samples. A disposable microextraction device compatible with injection systems of Agilent CE instruments is proposed that consists of a short segment of a porous HF attached to a tapered polypropylene holder. The holder maintains constant position of the HF in a CE vial during extraction and simultaneously it guides the injection end of a separation capillary into the HF lumen for automated CE injection and analysis. In a typical analytical procedure, the HF is impregnated with a water-immiscible solvent, its lumen is filled with 5 μL of an aqueous acceptor solution and the microextraction device is placed in a 2-mL glass CE vial containing 550 μL of a donor solution. The vial is agitated at 750 rpm for 10 min and the resulting acceptor solution is injected directly from the HF lumen into the commercial CE. No additional manual handling is required except for the transfer of the CE vial to the CE autosampler. Multiple complex samples can be simultaneously pretreated in a multiple-well plate format, thus significantly reducing total analysis time. Suitability of the analytical method is demonstrated by the direct determination of model basic drugs (nortriptyline, haloperidol, loperamide, and papaverine) in physiological solutions, urine and dried blood spot (DBS) samples. Repeatability of the method is better than 12.8%, extraction recoveries range between 34% and 76% and enrichment factors are 37 to 84. The method is linear in a range of two orders of magnitude (r2 ≥ 0.9977) with limits of detection 0.7 - 1.55 μg/L. The method has a high potential for the direct analysis of DBS samples since DBS elution and HF-LPME are performed simultaneously during the 10 min agitation. The manual DBS handling is thus reduced to inserting the DBS punch into the CE vial only. Moreover, the universal character of the HF-LPME might extend the applicability of the method to a wide range of analytes/matrices, and combination with other commercial detectors might improve selectivity/sensitivity of the CE analysis.

DOI: https://doi.org/10.1021/acs.analchem.0c00697