Publications by authors named "Heiko Hayen"

86 Publications

Ion identity molecular networking for mass spectrometry-based metabolomics in the GNPS environment.

Nat Commun 2021 06 22;12(1):3832. Epub 2021 Jun 22.

Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR, USA.

Molecular networking connects mass spectra of molecules based on the similarity of their fragmentation patterns. However, during ionization, molecules commonly form multiple ion species with different fragmentation behavior. As a result, the fragmentation spectra of these ion species often remain unconnected in tandem mass spectrometry-based molecular networks, leading to redundant and disconnected sub-networks of the same compound classes. To overcome this bottleneck, we develop Ion Identity Molecular Networking (IIMN) that integrates chromatographic peak shape correlation analysis into molecular networks to connect and collapse different ion species of the same molecule. The new feature relationships improve network connectivity for structurally related molecules, can be used to reveal unknown ion-ligand complexes, enhance annotation within molecular networks, and facilitate the expansion of spectral reference libraries. IIMN is integrated into various open source feature finding tools and the GNPS environment. Moreover, IIMN-based spectral libraries with a broad coverage of ion species are publicly available.
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http://dx.doi.org/10.1038/s41467-021-23953-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8219731PMC
June 2021

Determination of specific urinary nonylphenol metabolites by online-SPE-LC-MS/MS as novel human exposure biomarkers.

J Chromatogr B Analyt Technol Biomed Life Sci 2021 Jul 25;1177:122794. Epub 2021 May 25.

Institute for Prevention and Occupational Medicine of the German Social Accident Insurance, Institute of the Ruhr-University Bochum (IPA), Bürkle-de-la-Camp-Platz 1, Bochum 44789, Germany. Electronic address:

Nonylphenol (NP) is an endocrine disrupting and ecotoxic substance that has been detected in a variety of environmental matrices. It is utilized for the production of non-ionic nonylphenol ethoxylate (NPEO) detergents and other high production volume chemicals. Human biomonitoring data are scarce and mostly limited to the non-oxidized NP, which is ubiquitous in the (laboratory) environment and susceptible to external contamination. Here, we describe a sensitive, precise, accurate and rugged analytical method for the determination of OH-NP and oxo-NP, two potential alkyl-chain-oxidized metabolites of NP in human urine. We used single isomer standards, obtained by custom synthesis, for the quantification of the sum of the respective isomers. After enzymatic hydrolysis of potential urinary phase II conjugates, urine samples were analyzed by online turbulent flow chromatography for analyte enrichment and matrix depletion coupled to reversed phase liquid chromatography with negative electrospray-ionization triple quadrupole tandem mass spectrometry detection (online-SPE-LC-MS/MS). Quantification was performed by stable isotope dilution analysis. Limits of quantification in urinary matrix were 0.5 µg/L for OH-NP and 0.25 µg/L for oxo-NP. Mean relative recoveries were 101-105% (OH-NP) and 112-117% (oxo-NP) and the method imprecision (CV) in matrix was below 5%. In spite of extensive use restrictions in the EU since 2003, we could quantify OH-NP and oxo-NP in 94% and 47% of spot urine samples from the general German population (n = 32) collected in 2014. Thus, both metabolites seem suitable as sensitive and specific urinary biomarkers of NP exposure for future human biomonitoring population studies. Currently this method is used to quantitatively investigate human NP metabolism and to derive urinary metabolite excretion fractions that can be used to calculate external doses based on urinary biomarker concentrations.
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http://dx.doi.org/10.1016/j.jchromb.2021.122794DOI Listing
July 2021

Human metabolism and urinary excretion kinetics of di-n-butyl adipate (DnBA) after oral and dermal administration in three volunteers.

Toxicol Lett 2021 Jun 25;343:11-20. Epub 2021 Feb 25.

Institute for Prevention and Occupational Medicine of the German Social Accident Insurance, Institute of the Ruhr-University Bochum (IPA), Bürkle-de-la-Camp-Platz 1, 44789, Bochum, Germany. Electronic address:

Di-n-butyl adipate (DnBA) is used as a plasticizer and in various consumer products (e.g. personal care products) replacing, in part, the endocrine disruptor di-n-butyl phthalate (DnBP). We provide quantitative in vivo data on human DnBA metabolism and excretion after oral dose (105-185 μg/kg bw) and dermal application to three volunteers each as a tool for exposure and risk assessment. Complete and consecutive urine samples were collected for two (oral) and four days (dermal), respectively, and analyzed for the metabolites mono-n-butyl adipate (MnBA), 3- and tentative 4-hydroxy-mono-n-butyl adipate (3OH-MnBA, 4OH-MnBA), and 3-carboxy-mono-n-propyl adipate (3cx-MnPrA), as well as the hydrolysis product adipic acid (AA) using stable isotope dilution quantification. Metabolites were excreted within 24 h after oral dose with one or two concentration maxima at 0.8-3.0 h (n = 3) and 4.8-6.3 h (n = 2). AA was the major but unspecific metabolite with urinary excretion fractions (Fs) of 14-26 %. Mean Fs (range) of 3cx-MnPrA, MnBA, 3OH-MnBA, and tentative 4OH-MnBA were low, but consistent between volunteers (0.47 % (0.35-0.63 %), 0.079 % (0.065-0.091 %), 0.012 % (0.006-0.016 %), and 0.005 % (0.002-0.009 %), respectively). MnBA and 3OH-MnBA seem to be suitable, specific exposure biomarkers for DnBA, whereas 3cx-MnPrA and 4OH-MnBA seem to originate also from other, unknown sources not related to DnBA. Compared to the oral study, metabolite excretion in the dermal study was delayed and MnBA excretion was somewhat higher compared to the oxidized metabolites. Based on urinary concentrations and the above excretion fractions, calculated uptakes in the dermal study did not exceed the adipate ester ADI of 5 mg/(kg bw*day).
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http://dx.doi.org/10.1016/j.toxlet.2021.02.012DOI Listing
June 2021

Complementing Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry Imaging with Chromatography Data for Improved Assignment of Isobaric and Isomeric Phospholipids Utilizing Trapped Ion Mobility-Mass Spectrometry.

Anal Chem 2021 02 8;93(4):2135-2143. Epub 2021 Jan 8.

Institute of Inorganic and Analytical Chemistry, University of Münster, Corrensstraße 30, Münster 48149, Germany.

Lipids, such for example the multifaceted category of glycerophospholipids (GP), play a major role in many biological processes. High-resolution mass spectrometry is able to identify these highly diverse lipid species in combination with fragmentation experiments (MS/MS) on the basis of the accurate / and fragmentation pattern. However, for the differentiation of isomeric lipids or isobaric interferences, more elaborate separation methods are required. Especially for imaging techniques, such as matrix-assisted laser desorption/ionization (MALDI)-MS imaging, the identification is often exclusively based on the accurate /. Fragmentation via MS/MS increases the confidence in lipid annotation in imaging approaches. However, this is sometimes not feasible due to insufficient sensitivity and significantly prolonged analysis time. The use of a separation dimension such as trapped ion mobility spectrometry (TIMS) after ionization strengthens the confidence of the identification based on the collision cross section (CCS). Since CCS libraries are limited, a tissue-specific database was initially generated using hydrophilic interaction liquid chromatography-TIMS-MS. Using this database, the identification of isomeric lipid classes as well as isobaric interferences in a lipid class was performed using a mouse spleen sample in a workflow described in this study. Besides a CCS-based identification as an additional identification criterion for GP in general, the focus was on the distinction of the isomeric GP classes phosphatidylglycerol and bis(monoacylglycero)phosphate, as well as the differentiation of possible isobaric interferences based on the formation of adducts by MALDI-TIMS-MS imaging on a molecular level.
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http://dx.doi.org/10.1021/acs.analchem.0c03942DOI Listing
February 2021

Application of large volume injection for sensitive LC-MS/MS analysis of seven artificial sweeteners in surface waters.

MethodsX 2020 7;7:101134. Epub 2020 Nov 7.

Institute of Inorganic and Analytical Chemistry, University of Münster, Corrensstraße 30, 48149 Münster, Germany.

The combination of large volume injection and mixed-mode chromatography was performed for direct ultra-trace LC-MS/MS analysis of seven artificial sweeteners with varying physicochemical properties in surface water samples.•The injection volume was raised from 10 µL to 500 µL, while the overall analysis time was only increased by ≈5 min compared to the initial method.•Online column head refocusing and concentration of analytes enabled detection in sub-ng L concentration range without elaborate sample preparation steps.•Relative standard deviations <7% despite multiple injection into the loop.
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http://dx.doi.org/10.1016/j.mex.2020.101134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7689179PMC
November 2020

Investigation of cardiolipin oxidation products as a new endpoint for oxidative stress in C. elegans by means of online two-dimensional liquid chromatography and high-resolution mass spectrometry.

Free Radic Biol Med 2021 01 27;162:216-224. Epub 2020 Oct 27.

Institute of Inorganic and Analytical Chemistry, University of Münster, Corrensstr. 30, 48149, Münster, Germany. Electronic address:

The investigation of neurodegenerative and age-related diseases is a highly relevant topic in current research. Especially oxidative stress is thought to be the common underlying mechanism in diseases such as Parkinson's or Alzheimer's disease. The nematode Caenorhabditis elegans (C. elegans) is a prominent model organism, which is often used for such investigations and has gained extensive recognition in research regarding the linkage of reactive oxygen species (ROS) and neurodegeneration. Not only studies regarding genomics and proteomics have been increasingly conducted, also the number of studies based on the lipidome is rising. The phospholipid class of cardiolipin (CL) is a unique lipid class, which is exclusively located in mitochondria and is therefore of great relevance regarding oxidative stress and associated diseases. CL oxidation products have become a prominent marker for oxidative stress in various organisms. However, the CL distribution in the nematode C. elegans is still scarcely known on the molecular level and oxidation products have not yet been identified. In this work, we demonstrate the importance of CL distribution and the applicability of CL oxidation products as a sensitive marker for oxidative stress in C. elegans. For this reason, the CL distribution was determined by means of online two-dimensional liquid chromatography hyphenated with high-resolution mass spectrometry (2D-LC/HRMS). Subsequently, worms were treated with tert-butyl hydroperoxide (tBOOH) in order to provoke oxidative stress and induce the artificial formation of oxidized CL. We were able to detect increasing amounts of CL oxidation products of highly unsaturated CL species in a concentration-dependent manner. This finding emphasizes the great potential of CL oxidation products as a sensitive marker substance of oxidative stress in C. elegans, which is not only directly linked to mitochondria function but also favourable to other oxidative stress markers in terms of the needed sample material, relative substance stability and specificity of the oxidation site.
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http://dx.doi.org/10.1016/j.freeradbiomed.2020.10.019DOI Listing
January 2021

Hyphenation of supercritical fluid chromatography with different detection methods for identification and quantification of liamocin biosurfactants.

J Chromatogr A 2020 Sep 24;1631:461584. Epub 2020 Sep 24.

Institute of Inorganic and Analytical Chemistry, University of Münster, D-48149 Münster, Germany. Electronic address:

Liamocins are a class of biosurfactants with growing interest. However, methods for identification and quantification of liamocins on the molecular level are lagging behind. Therefore, we developed a chromatographic separation based on supercritical fluid chromatography (SFC) for liamocins and structurally related exophilins. The different congeners could be separated on a charge modulated hydroxyethyl amide functionalized silica-based column. Coupling to high-resolution mass spectrometry (MS) revealed four exophilin species and four liamocin species with mannitol and arabitol as polyol head group in a sample of the yeast-like fungus Aureobasidium pullulans (A. pullulans). In contrast to a recently published reversed phase high-performance liquid chromatography (HPLC) method, the different subclasses (exophilins, mannitol liamocins and arabitol liamocins) were additionally separated by means of SFC. The structures were confirmed by their accurate masses and tandem mass spectrometry (MS/MS). A complementary quantification method was developed using SFC coupled to charged-aerosol detection (CAD) to overcome the disadvantages of quantification by means of MS without authentic standards. A flow compensation by varying the make-up flow was used to obtain a constant composition of the mobile phase during detection and to ensure a stable detector response. The concentrations of the individual liamocin species were determined using an external calibration with n-octyl-β-d-glycopyranoside. The total amount of these concentrations agrees with the dry weight of an aliquot of the heavy oil. The developed SFC-based method has the advantage of shorter analysis time and superior selectivity compared to the previously published LC separation. In brief, the here presented SFC hyphenations enable comprehensive analysis of liamocin biosurfactants providing identification and absolute quantification of individual congeners.
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http://dx.doi.org/10.1016/j.chroma.2020.461584DOI Listing
September 2020

Importance of oxidation products in coumarin-mediated Fe(hydr)oxide mineral dissolution.

Biometals 2020 12 5;33(6):305-321. Epub 2020 Oct 5.

Leibniz-Institut f. Analytische Wissenschaften - ISAS, Dortmund, Germany.

Due to the low iron solubility in alkaline soils, plants have evolved different iron acquisition strategies, which are either based on ferric iron reduction (strategy I) or complexation by phytosiderophores (strategy II). Recently, a prominent role of coumarins for iron acquisition has been discovered, but details of the respective mechanism remain unclear. Since coumarins may act as iron-binding ligands but also as reductants, various reaction sequences are possible, resulting in different iron species and oxidized coumarins. In this context, it is often overlooked that oxidized coumarins are not just byproducts of iron(III) reduction, but may be actively involved in further steps of iron mobilization. In order to verify this active role of oxidized coumarins in Fe(hydr)oxide dissolution, we complemented iron dissolution data with data of single coumarins (esculetin, scopoletin, fraxetin) and their oxidation products, as a function of time, pH, and mineral (goethite, lepidocrocite). Our results demonstrate that there are four different routes for coumarin oxidation, leading to quinones, dimers, hydroxylated coumarins, demethylated coumarins, and combinations of these. The time-dependent species pattern differs with respect to mineral, pH, and coumarin molecule. Oxidized coumarins are often more reactive than the original coumarins, explaining unexpected iron mobilization by scopoletin, which is demethylated to esculetin. Also oxidative hydroxylation and dimerization increase the number of phenolic groups and yield new chelating properties. Several iron-species are identified for the three coumarins. Since oxidation reactions are initiated directly at mineral surfaces, they are often very effective-but this does not always result in more iron mobilization.
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http://dx.doi.org/10.1007/s10534-020-00248-yDOI Listing
December 2020

Hydroperoxylated vs Dihydroxylated Lipids: Differentiation of Isomeric Cardiolipin Oxidation Products by Multidimensional Separation Techniques.

Anal Chem 2020 09 12;92(17):12010-12016. Epub 2020 Aug 12.

Institute of Inorganic and Analytical Chemistry, University of Münster, Corrensstr. 30, 48149 Münster, Germany.

In recent years, cardiolipin (CL) oxidation products were recognized as potential markers for mitochondrial dysfunction in conjunction with age related diseases. The analysis of oxidized CL requires powerful analysis techniques due to high structural diversity. In addition, low concentrations of partly labile compounds pose a special challenge, supplemented by the occurrence of isomeric compounds, e.g., hydroperoxylated vs dihydroxylated products. Therefore, we present a hyphenated method based on liquid chromatography coupled to trapped ion mobility spectrometry (TIMS) for separation and tandem mass spectrometry (MS/MS) for structural characterization. This enables comprehensive analysis of an artificially oxidized CL extract of bovine heart. Isomeric oxidation products could be differentiated by mobility-resolved MS/MS fragmentation experiments. Our developed method could help to better understand the physiological role of oxidized CL.
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http://dx.doi.org/10.1021/acs.analchem.0c02605DOI Listing
September 2020

Comprehensive liamocin biosurfactants analysis by reversed phase liquid chromatography coupled to mass spectrometric and charged-aerosol detection.

J Chromatogr A 2020 Sep 15;1627:461404. Epub 2020 Jul 15.

Institute of Inorganic and Analytical Chemistry, University of Münster, D-48149 Münster, Germany. Electronic address:

Liamocin biosurfactants and structurally related exophilins secreted by the Aureobasidium pullulans (A. pullulans) strain NRRL62031 were firstly analyzed by hyphenation of high-performance liquid chromatography (HPLC) with high-resolution mass spectrometry (HRMS). Ten different analytes were detected and identified by their accurate masses and divided into subclasses according to their different head groups: three liamocins with arabitol as head group, three mannitol liamocins, and four exophilins. A baseline separation of congeners within the subclasses was achieved by reversed phase HPLC on a C18 stationary phase, whereas an overlap of subclasses occurred. The structures were simultaneously confirmed by online tandem mass spectrometry (MS/MS) experiments in positive and negative ionization mode. The assigned polyol head groups and thus the feasibility of this method were confirmed by gas chromatography (GC)-MS data obtained after hydrolysis and derivatization of the liamocins. Based on the varying structural characteristics of liamocins, e.g. the polyol head group (or even none for exophilins) and the degree of acetylation, different detector response in LC-MS was expected, impairing relative quantification of congeners. Therefore, a complementary quantification method was developed using HPLC coupled to charged-aerosol detection (CAD), which allows the determination of the amount of the individual liamocin species without authentic liamocin standards. Hence, the here presented hyphenated techniques facilitate comprehensive analysis of liamocin biosurfactants.
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http://dx.doi.org/10.1016/j.chroma.2020.461404DOI Listing
September 2020

Identification and structural characterization of lipid A from Escherichia coli, Pseudomonas putida and Pseudomonas taiwanensis using liquid chromatography coupled to high-resolution tandem mass spectrometry.

Rapid Commun Mass Spectrom 2020 Nov;34(21):e8897

Institute of Inorganic and Analytical Chemistry, University of Münster, Corrensstraße 30, Münster, 48149, Germany.

Rationale: Lipid A is a part of the lipopolysaccharide layer, which is a main component of the outer membrane from Gram-negative bacteria. It can be sensed by mammalians to identify the presence of Gram-negative bacteria in their tissues and plays a key role in the pathogenesis of bacterial infections. Lipid A is also used as an adjuvant in human vaccines, emphasizing the importance of its structural analysis.

Methods: In order to distinguish and characterize various lipid A species, a liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) method was developed. Isolation of lipid A from different bacteria was carried out using a modified Bligh and Dyer extraction following a mild acid hydrolysis. Chromatography was performed using a bifunctional reversed-phase-based stationary phase. High-resolution MS using negative electrospray ionization was applied and MS/MS experiments utilizing high-energy collisional dissociation generated diagnostic product ions, which allowed the assignment of the side chains to distinct positions of the lipid A backbone.

Results: The method was applied to lipid A isolations of Escherichia coli (E. coli), Pseudomonas putida (P. putida) and Pseudomonas taiwanensis (P. taiwanensis). Various lipid A species were identified by their accurate masses and their structures were characterized using MS/MS experiments. Previously described lipid A structures from E. coli were identified and their structures confirmed by MS/MS. For the biotechnologically relevant strains P. putida and P. taiwanensis, we confirmed species by MS/MS, which have previously only been analyzed using MS. In addition, several lipid A species were discovered that have not been previously described in the literature.

Conclusions: The combination of LC and MS/MS enabled the selective and sensitive identification and structural characterization of various lipid A species from Gram-negative bacteria. These species varied in their substituted side chains, speaking of fatty acids and phosphate groups. Characteristic product ions facilitated the assignment of side chains to distinct positions of the lipid A backbone.
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http://dx.doi.org/10.1002/rcm.8897DOI Listing
November 2020

Double bond localization in unsaturated rhamnolipid precursors 3-(3-hydroxyalkanoyloxy)alkanoic acids by liquid chromatography-mass spectrometry applying online Paternò-Büchi reaction.

Anal Bioanal Chem 2020 Sep 5;412(23):5601-5613. Epub 2020 Jul 5.

Institute of Inorganic and Analytical Chemistry, University of Münster, Corrensstraße 30, 48149, Münster, Germany.

Lipids are biomolecules with a broad variety of chemical structures, which renders them essential not only for various biological functions but also interestingly for biotechnological applications. Rhamnolipids are microbial glycolipids with surface-active properties and are widely used biosurfactants. They are composed of one or two L-rhamnoses and up to three hydroxy fatty acids. Their biosynthetic precursors are 3-hydroxy(alkanoyloxy)alkanoic acids (HAAs). The latter are also present in cell supernatants as complex mixtures and are extensively studied for their potential to replace synthetically derived surfactants. The carbon chain lengths of HAAs determine their physical properties, such as their abilities to foam and emulsify, and their critical micelle concentration. Despite growing biotechnological interest, methods for structural elucidation are limited and often rely on hydrolysis and analysis of free hydroxy fatty acids losing the connectivity information. Therefore, a high-performance liquid chromatography-mass spectrometry method was developed for comprehensive structural characterization of intact HAAs. Information is provided on chain length and number of double bonds in each hydroxy fatty acid and their linkage by tandem mass spectrometry (MS/MS). Post-column photochemical derivatization by online Paternὸ-Büchi reaction and MS/MS fragmentation experiments generated diagnostic fragments allowing structural characterization down to the double bond position level. Furthermore, the presented experiments demonstrate a powerful approach for structure elucidation of complex lipids by tailored fragmentation.
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http://dx.doi.org/10.1007/s00216-020-02776-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7413879PMC
September 2020

Determination of di-n-butyl adipate (DnBA) metabolites as possible biomarkers of exposure in human urine by online-SPE-LC-MS/MS.

J Chromatogr B Analyt Technol Biomed Life Sci 2020 Mar 13;1141:122029. Epub 2020 Feb 13.

Institute for Prevention and Occupational Medicine of the German Social Accident Insurance - Institute of the Ruhr-University Bochum (IPA), Bürkle-de-la-Camp-Platz 1, 44789 Bochum, Germany. Electronic address:

Di-n-butyl adipate (DnBA) is an alternative to the anti-androgenic and strictly regulated di-n-butyl phthalate (DnBP) used as a cosmetic ingredient, plasticizer, and in various articles of everyday life. Hence, exposures of the general population have to be expected. Currently, biomarkers of DnBA exposure and methods for their determination are not available. Here, we describe a sensitive, rugged and precise analytical method for the determination of the DnBA monoester metabolite mono-n-butyl adipate (MnBA), as well as its potential downstream metabolites 3-hydroxy-mono-n-butyl adipate (3OH-MnBA) and 3-carboxy-mono-n-propyl adipate (3cx-MnPrA) in human urine. Glucuronic acid conjugates present in urine were deconjugated using a pure β-glucuronidase. The metabolites were then analyzed by liquid chromatography on a C18 column with superficially porous particles coupled to electrospray ionization-triple quadrupole-tandem mass spectrometry, applying online turbulent flow chromatography for analyte enrichment and matrix depletion (online-SPE-LC-MS/MS). The metabolites were quantified using stable isotope dilution analysis with limits of quantification of 0.05 µg/L (MnBA), 0.1 µg/L (3OH-MnBA), and 0.5 µg/L (3cx-MnPrA). Method imprecision in urinary matrix was below 7% (coefficient of variation) for all analytes. Mean relative recoveries were between 93% and 107%. The suitability of the DnBA metabolites as biomarkers of exposure was demonstrated after dermal application of a commercially available sunscreen containing DnBA. Maximum concentrations were reached 6.5 h after dose (219 µg/L 3cx-MnPrA, 91 µg/L MnBA, and 3.9 µg/L 3OH-MnBA). Elimination kinetics were similar for all three metabolites. We were able to quantify 3cx-MnPrA and MnBA until 4 d after sunscreen application. In a sample set of 35 urine samples from the general German population, 3cx-MnPrA was quantified in 94% (median 2.54 µg/L, maximum 78.3 µg/L) and MnBA in 3% (median < LOQ, maximum 0.18 µg/L) of the samples. The method will be applied in future human metabolism and human biomonitoring population studies.
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http://dx.doi.org/10.1016/j.jchromb.2020.122029DOI Listing
March 2020

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

J Chromatogr A 2020 May 25;1619:460918. Epub 2020 Jan 25.

Institute of Inorganic and Analytical Chemistry, University of Münster, Corrensstr. 30, 48149 Münster, Germany. Electronic address:

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.
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http://dx.doi.org/10.1016/j.chroma.2020.460918DOI Listing
May 2020

Digging deeper - A new data mining workflow for improved processing and interpretation of high resolution GC-Q-TOF MS data in archaeological research.

Sci Rep 2020 01 21;10(1):767. Epub 2020 Jan 21.

Department of Anthropology and Archaeology, University of Bristol, 43 Woodland Road, Bristol, BS81UU, UK.

Gas chromatography-mass spectrometry profiling is the most established method for the analysis of organic residues, particularly lipids, from archaeological contexts. This technique allows the decryption of hidden chemical information associated with archaeological artefacts, such as ceramic pottery fragments. The molecular and isotopic compositions of such residues can be used to reconstruct past resource use, and hence address major questions relating to patterns of subsistence, diet and ritual practices in the past. A targeted data analysis approach, based on previous findings reported in the literature is common but greatly depends on the investigator's prior knowledge of specific compound classes and their mass spectrometric behaviour, and poses the risk of missing unknown, potentially diagnostic compounds. Organic residues from post-prehistoric archaeological samples often lead to highly complex chromatograms, which makes manual chromatogram inspection very tedious and time consuming, especially for large datasets. This poses a significant limitation regarding the scale and interpretative scopes of such projects. Therefore, we have developed a non-targeted data mining workflow to extract a higher number of known and unknown compounds from the raw data to reduce investigator's bias and to vastly accelerate overall analysis time. The workflow covers all steps from raw data handling, feature selection, and compound identification up to statistical interpretation.
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http://dx.doi.org/10.1038/s41598-019-57154-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6972930PMC
January 2020

Exploiting the Natural Diversity of RhlA Acyltransferases for the Synthesis of the Rhamnolipid Precursor 3-(3-Hydroxyalkanoyloxy)Alkanoic Acid.

Appl Environ Microbiol 2020 03 2;86(6). Epub 2020 Mar 2.

RWTH Aachen University, iAMB (Institute of Applied Microbiology, ABBt), Aachen Biology and Biotechnology, Aachen, Germany

While rhamnolipids of the type are commercially available, the natural diversity of rhamnolipids and their origin have barely been investigated. Here, we collected known and identified new genes encoding the acyltransferase responsible for the synthesis of the lipophilic rhamnolipid precursor 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA). Generally, all homologs were found in and A likely horizontal gene transfer event into is the only identified exception. The phylogeny of the RhlA homologs from and species is consistent with the organism phylogeny, and genes involved in rhamnolipid synthesis are located in operons. In contrast, RhlA homologs from the do not follow the organisms' phylogeny but form their own branch. Furthermore, in many and from the , an isolated homolog can be found in the genome. The RhlAs from PA01, LMG 05825, LMG 20103, PG1, LMG 19182, sp. strain R57-5, Ech586, and PRI-2C were expressed in and tested for HAA production. Indeed, except for the RhlA, HAAs were produced with the engineered strains. A detailed analysis of the produced HAA congeners by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) highlights the congener specificity of the RhlA proteins. The congener length varies from 4 to 18 carbon atoms, with the main congeners consisting of different combinations of saturated or monounsaturated C, C, and C fatty acids. The results are discussed in the context of the phylogeny of this unusual enzymatic activity. The RhlA specificity explains the observed differences in 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) congeners. Whole-cell catalysts can now be designed for the synthesis of different congener mixtures of HAAs and rhamnolipids, thereby contributing to the envisaged synthesis of designer HAAs.
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http://dx.doi.org/10.1128/AEM.02317-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054101PMC
March 2020

Sensing of nutrients by CPT1C regulates late endosome/lysosome anterograde transport and axon growth.

Elife 2019 12 23;8. Epub 2019 Dec 23.

Basic Sciences Department, Faculty of Medicine and Health Sciences, Universitat Internacional de Catalunya, Sant Cugat del Vallès, Spain.

Anterograde transport of late endosomes or lysosomes (LE/Lys) is crucial for proper axon growth. However, the role of energetic nutrients has been poorly explored. Malonyl-CoA is a precursor of fatty acids, and its intracellular levels highly fluctuate depending on glucose availability or the energy sensor AMP-activated protein kinase (AMPK). We demonstrate in HeLa cells that carnitine palmitoyltransferase 1C (CPT1C) senses malonyl-CoA and enhances LE/Lys anterograde transport by interacting with the endoplasmic reticulum protein protrudin and facilitating the transfer of Kinesin-1 from protrudin to LE/Lys. In cultured mouse cortical neurons, glucose deprivation, pharmacological activation of AMPK or inhibition of malonyl-CoA synthesis decreases LE/Lys abundance at the axon terminal, and shortens axon length in a CPT1C-dependent manner. These results identify CPT1C as a new regulator of anterograde LE/Lys transport in response to malonyl-CoA changes, and give insight into how axon growth is controlled by nutrients.
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http://dx.doi.org/10.7554/eLife.51063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6927751PMC
December 2019

Expanding the Kendrick Mass Plot Toolbox in MZmine 2 to Enable Rapid Polymer Characterization in Liquid Chromatography-Mass Spectrometry Data Sets.

Anal Chem 2020 01 19;92(1):628-633. Epub 2019 Dec 19.

Dow Deutschland Anlagengesellschaft mbH , Postfach 1120 , 21677 Stade , Germany.

Technological advances in mass spectrometry (MS) toward more accurate and faster data acquisition result in highly informative but also more complex data sets. Especially the hyphenation of liquid chromatography (LC) and MS yields large data files containing a high amount of compound specific information. Using electrospray-ionization for compounds such as polymers enables highly sensitive detection, yet results in very complex spectra, containing multiply charged ions and adducts. Recent years have seen the development of novel or updated data mining strategies to reduce the MS spectra complexity and to ultimately simplify the data analysis workflow. Among other techniques, the Kendrick mass defect analysis, which graphically highlights compounds containing a given repeating unit, has been revitalized with applications in multiple fields of study, such as lipids and polymers. Especially for the latter, various data mining concepts have been developed, which extend regular Kendrick mass defect analysis to multiply charged ion series. The aim of this work is to collect and subsequently implement these concepts in one of the most popular open-source MS data mining software, i.e., MZmine 2, to make them rapidly available for different MS based measurement techniques and various vendor formats, with a special focus on hyphenated techniques such as LC-MS. In combination with already existing data mining modules, an example data set was processed and simplified, enabling an ever faster evaluation and polymer characterization.
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http://dx.doi.org/10.1021/acs.analchem.9b03863DOI Listing
January 2020

Analysis of artificially oxidized cardiolipins and monolyso-cardiolipins via liquid chromatography/high-resolution mass spectrometry and Kendrick mass defect plots after hydrophilic interaction liquid chromatography based sample preparation.

Rapid Commun Mass Spectrom 2020 ;34(1):e8566

Institute of Inorganic and Analytical Chemistry, University of Münster, Corrensstraße 30, 48149, Münster, Germany.

Rationale: Cardiolipins (CL) are a special lipid class which plays a main role in energy metabolism in mitochondria and is involved in apoptosis. In contrast to other glycerophospholipids, they contain four fatty acyl residues which results in a high structural diversity. Oxidation, for example by reactive oxygen species, or lyso forms such as monolyso-CL (MLCL), increases this diversity. Mass spectrometric analysis and computational identification of CL, MLCL and their oxidation products is therefore a challenging task.

Methods: In order to distinguish CL, MLCL and their oxidation products, a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed. A hydrophilic interaction liquid chromatography (HILIC)-based solid-phase extraction (SPE) clean-up approach was developed for CL enrichment. Graphical analysis of CL, MLCL and their oxidation products was carried out by a three-dimensional Kendrick mass defect (3D-KMD) plot module, as well as a refined lipid search module of the open-source metabolomics data mining software MZmine 2.

Results: The HILIC-based SPE clean-up enabled complete separation of polar and nonpolar lipid classes. A yeast (Saccharomyces cerevisiae) lipid extract, which was artificially oxidized by means of the Fenton reaction, was analyzed by the developed LC/MS/MS method. CL species with differences in chain length and degree of unsaturation have been separated by high-performance liquid chromatography (HPLC). In total 66 CL, MLCL and oxidized species have been identified utilizing 3D-KMD plots in combination with database matching using MZmine 2. For further characterization of annotated species, MS/MS experiments have been utilized.

Conclusions: 3D-KMD plots capturing chromatographic and high-resolution mass spectrometry data have been successfully used for graphical identification of CL, MLCL as well as their oxidized species. Therefore, we chose multiple KMD bases such as hydrogen and oxygen to visualize the degree of unsaturation and oxidation capturing chromatographic data by means of a color-coded paint scale as the third dimension. In combination with database matching, the analysis of low concentrated lipid species in complex samples has been significantly improved.
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http://dx.doi.org/10.1002/rcm.8566DOI Listing
January 2020

A pH shift induces high-titer liamocin production in Aureobasidium pullulans.

Appl Microbiol Biotechnol 2019 Jun 25;103(12):4741-4752. Epub 2019 Apr 25.

iAMB-Institute of Applied Microbiology, ABBt-Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, D-52074, Aachen, Germany.

Liamocins are biosurfactants produced by the fungus Aureobasidium pullulans. A. pullulans belongs to the black yeasts and is known for its ability to produce pullulan and melanin. However, the production of liamocins has not been investigated intensively. Initially, HPLC methods for the quantification of liamocin and the identification of liamocin congeners were established. Eleven congeners could be detected, differing in the polyol head groups arabitol or mannitol. In addition, headless molecules, so-called exophilins, were also identified. The HPLC method reported here allows quick and reliable quantification of all identified congeners, an often-overlooked prerequisite for the investigation of valuable product formation. Liamocin synthesis was optimized during cultivation in lab-scale fermenters. While the pH can be kept constant, the best strategy for liamocin synthesis consists of a growth phase at neutral pH and a subsequent production phase induced by a manual pH shift to pH 3.5. Finally, combining increased nitrogen availability with a pulsed fed-batch fermentation, cell growth, and liamocin titers could be enhanced. Here, the maximal titers of above 10 g/L that were reached are the highest reported to date for liamocin synthesis using A. pullulans in lab-scale fermenters.
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http://dx.doi.org/10.1007/s00253-019-09677-3DOI Listing
June 2019

Lipid Species Annotation at Double Bond Position Level with Custom Databases by Extension of the MZmine 2 Open-Source Software Package.

Anal Chem 2019 04 29;91(8):5098-5105. Epub 2019 Mar 29.

Institute of Inorganic and Analytical Chemistry , University of Münster , Corrensstraße 30 , 48149 Münster , Germany.

In recent years, proprietary and open-source bioinformatics software tools have been developed for the identification of lipids in complex biological samples based on high-resolution mass spectrometry data. These existent software tools often rely on publicly available lipid databases, such as LIPID MAPS, which, in some cases, only contain a limited number of lipid species for a specific lipid class. Other software solutions implement their own lipid species databases, which are often confined regarding implemented lipid classes, such as phospholipids. To address these drawbacks, we provide an extension of the widely used open-source metabolomics software MZmine 2, which enables the annotation of detected chromatographic features as lipid species. The extension is designed for straightforward generation of a custom database for selected lipid classes. Furthermore, each lipid's sum formula of the created database can be rapidly modified to search for derivatization products, oxidation products, in-source fragments, or adducts. The versatility will be exemplified by a liquid chromatography-high resolution mass spectrometry data set with postcolumn Paternò-Büchi derivatization. The derivatization reaction was performed to pinpoint the double bond positions in diacylglyceryltrimethylhomoserine lipid species in a lipid extract of a green algae ( Chlamydomonas reinhardtii) sample. The developed Lipid Search module extension of MZmine 2 supports the identification of lipids as far as double bond position level.
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http://dx.doi.org/10.1021/acs.analchem.8b05493DOI Listing
April 2019

Lipid profiling and analytical discrimination of seven cereals using high temperature gas chromatography coupled to high resolution quadrupole time-of-flight mass spectrometry.

Food Chem 2019 Jun 3;282:27-35. Epub 2019 Jan 3.

Department of Anthropology and Archaeology, University of Bristol, Bristol BS8 1UU, United Kingdom.

Minor lipids in cereals (such as phytosterols and alkylresorcinols) can be important for human nutrition and/or be used as biomarkers for cereal intake. However, the analysis of cereal lipids is very challenging due to the complex lipidome comprising several hundred individual compounds present over a wide range of concentrations. Here we present a method for the profiling of cereal lipids using high temperature gas chromatography coupled to high resolution mass spectrometry (GC/Q-TOF MS). The method was used to investigate the lipid profiles of 77 samples of bread wheat, spelt, einkorn, emmer, barley, rye and oats. Distinct differences in the patterns of alkylresorcinols, free and conjugated sterols and tocopherols between the cereals could be observed. Furthermore, traces of tocomonoenols and diunsaturated and methyl-alkylresorcinols (not previously reported in cereals) could be detected. Finally, the lipid patterns in the cereals could be used to separate the cereals by Principal Component Analysis.
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http://dx.doi.org/10.1016/j.foodchem.2018.12.109DOI Listing
June 2019

LIPG-promoted lipid storage mediates adaptation to oxidative stress in breast cancer.

Int J Cancer 2019 08 7;145(4):901-915. Epub 2019 Feb 7.

Department of Toxicology, Leibniz-Research Centre for Working Environment and Human Factors at the TU Dortmund (IfADo), Dortmund, Germany.

Endothelial lipase (LIPG) is a cell surface associated lipase that displays phospholipase A1 activity towards phosphatidylcholine present in high-density lipoproteins (HDL). LIPG was recently reported to be expressed in breast cancer and to support proliferation, tumourigenicity and metastasis. Here we show that severe oxidative stress leading to AMPK activation triggers LIPG upregulation, resulting in intracellular lipid droplet accumulation in breast cancer cells, which supports survival. Neutralizing oxidative stress abrogated LIPG upregulation and the concomitant lipid storage. In human breast cancer, high LIPG expression was observed in a limited subset of tumours and was significantly associated with shorter metastasis-free survival in node-negative, untreated patients. Moreover, expression of PLIN2 and TXNRD1 in these tumours indicated a link to lipid storage and oxidative stress. Altogether, our findings reveal a previously unrecognized role for LIPG in enabling oxidative stress-induced lipid droplet accumulation in tumour cells that protects against oxidative stress, and thus supports tumour progression.
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http://dx.doi.org/10.1002/ijc.32138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6618071PMC
August 2019

Localization of double-bond positions in lipids by tandem mass spectrometry succeeding high-performance liquid chromatography with post-column derivatization.

Rapid Commun Mass Spectrom 2019 05 27;33 Suppl 1:86-94. Epub 2018 Sep 27.

University of Münster, Institute of Inorganic and Analytical Chemistry, Corrensstraße 30, 48149, Münster, Germany.

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http://dx.doi.org/10.1002/rcm.8262DOI Listing
May 2019

Separation and identification of phospholipids by hydrophilic interaction liquid chromatography coupled to tandem high resolution mass spectrometry with focus on isomeric phosphatidylglycerol and bis(monoacylglycero)phosphate.

J Chromatogr A 2018 Aug 30;1565:105-113. Epub 2018 Jun 30.

Institute of Inorganic and Analytical Chemistry, University of Münster, Corrensstr. 30, 48149 Münster, Germany. Electronic address:

Changes in lipid composition of cells or tissue are often linked to various diseases. Studies indicate alterations of bis(monoacylglycero)phosphate (BMP) species in diseases such as cancer. Therefore, an extended phospholipid profiling method based on hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution mass spectrometry (MS) and data-dependent MS/MS acquisition was developed to separate and unambiguously identify BMP species. Lipid species identification was based on retention time, accurate mass and specific MS/MS fragments. The developed method was applied in a proof of concept study to lipid extracts of a cell culture model of conditional oncogene overexpression in MCF-7/NeuT breast cancer cells. Comparison of control and oncogene-induced MCF-7/NeuT breast cancer cells showed changes in BMP species distribution. Thereby, a shift from long-chain to shorter-chain fatty acid composition in BMP species was detected.
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http://dx.doi.org/10.1016/j.chroma.2018.06.039DOI Listing
August 2018

Mass spectrometric characterization of siderophores produced by Pseudomonas taiwanensis VLB120 assisted by stable isotope labeling of nitrogen source.

Biometals 2018 10 28;31(5):785-795. Epub 2018 Jun 28.

Institute of Inorganic and Analytical Chemistry, University of Münster, Corrensstraße 30, 48149, Münster, Germany.

The structures of three previously unknown siderophores produced by the fluorescent, biotechnologically relevant Pseudomonas taiwanensis (P. taiwanensis) VLB120 bacteria were elucidated by means of hydrophilic interaction liquid chromatography (HILIC) hyphenated to high-resolution tandem mass spectrometry (HRMS/MS). They could be verified as iron(III)- and aluminum(III) complexes as well as the protonated molecules of the siderophores formed by in-source fragmentation. The siderophores were separated according to their different acyl side chains and additionally according their central ions. One of the siderophores was identified as pyoverdine with a malic acid (hydroxy succinic acid) amide side chain and a peptide moiety consisting of Orn-Asp-OHAsn-Thr-AcOHOrn-Ser-cOHOrn. The other analytes were assigned to an azotobactin with the identical peptide chain linked to the characteristic chromophoric unit and a pyoverdine with a variation in the amino acid sequence. Proline is directly linked to the pyoverdine chromophore instead of ornithine. Acidic and enzymatic hydrolyses were carried out to analyze the individual amino acids. Beside OHAsn, each amino acid of the peptide part was identified by HILIC-HRMS and comparison to authentic standards. Additionally, N-labeled cellular supernatants were analyzed by means of HRMS/MS. The results of the MS/MS experiments complemented by accurate mass data facilitated elucidation of the structures studied in this work and provided further confirmation of the three recently described pyoverdines of P. taiwanensis VLB120 (Baune et al. in Biometals 30:589-597, 2017. https://doi.org/10.1007/s10534-017-0029-7 ).
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http://dx.doi.org/10.1007/s10534-018-0122-6DOI Listing
October 2018

LC/MS analysis of vitamin D metabolites by dielectric barrier discharge ionization and a comparison with electrospray ionization and atmospheric pressure chemical ionization.

Anal Bioanal Chem 2018 Aug 26;410(20):4905-4911. Epub 2018 May 26.

Institute of Inorganic and Analytical Chemistry, University of Münster, Corrensstraße 30, 48149, Münster, Germany.

Serum vitamin D metabolite levels are of interest as biomarkers for vitamin D status, which has influence on numerous body functions and pathologies. The determination of vitamin D metabolite levels by liquid chromatography/mass spectrometry (LC/MS) is challenging due to their low concentrations and relatively low ionization efficiencies. Three ionization sources, dielectric barrier discharge ionization (DBDI), atmospheric pressure chemical ionization (APCI), and electrospray ionization (ESI), were compared regarding achievable limits of detection and occurring matrix effects. The latter were mainly caused by phospholipids. Therefore, in addition to a conventional solid phase extraction (SPE) stationary phase, a material for selective removal of phospholipids was examined. The selective removal of phospholipids significantly reduced observed matrix effects, especially when ESI was applied. Achievable limits of detection and observed matrix effects were lowest for APCI and with some limitations, also for DBDI. Graphical abstract.
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http://dx.doi.org/10.1007/s00216-018-1137-0DOI Listing
August 2018

Ambient desorption/ionization mass spectrometry: evolution from rapid qualitative screening to accurate quantification tool.

Anal Bioanal Chem 2018 Jul 21;410(17):4061-4076. Epub 2018 Apr 21.

Institute of Inorganic and Analytical Chemistry, University of Münster, Corrensstr. 30, 48149, Münster, Germany.

In this article, some recent trends and developments in ambient desorption/ionization mass spectrometry (ADI-MS) are reviewed, with a special focus on quantitative analyses with direct, open-air sampling. Accurate quantification with ADI-MS is still not routinely performed, but this aspect is considered of utmost importance for the advancement of the field. In fact, several research groups are devoted to the development of novel and optimized ADI-MS approaches. Some key trends include novel sample introduction strategies for improved reproducibility, tailored sample preparation protocols for removing the matrix and matrix effects, and multimode ionization sources. In addition, there is significant interest in quantitative mass spectrometry imaging. Graphical abstract Conceptual diagram of the ambient desorption/ionization mass spectrometry approach with different desorption/ionization probes.
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http://dx.doi.org/10.1007/s00216-018-1023-9DOI Listing
July 2018

Screening of semifluorinated n-alkanes by gas chromatography coupled to dielectric barrier discharge ionization mass spectrometry.

Rapid Commun Mass Spectrom 2018 Jul 27;32(13):1092-1098. Epub 2018 May 27.

University of Münster, Institute of Inorganic and Analytical Chemistry, Corrensstraße 30, 48149, Münster, Germany.

Rationale: The potential of an atmospheric pressure ionization source based on a dielectric barrier discharge in helium for the hyphenation of gas chromatography and mass spectrometry (GC/DBDI-MS) has been demonstrated only recently and for a limited range of compounds. Due to its 'soft' ionization properties and the possibility to choose from a variety of atmospheric pressure ionization MS instruments, GC/DBDI-MS has the potential to be an interesting alternative to 'classic' GC/MS techniques.

Methods: The hyphenation of GC with DBDI-MS at atmospheric pressure is used for the determination of semifluorinated n-alkanes in ski wax samples.

Results: Different to perfluorinated n-alkanes, which are typically detected as [M - F + O] and [M - F] , semifluorinated n-alkanes can be detected both in positive mode as [M - 3H + nO] and [M - H + nO] (n = 0, 1, 2, and 3) ions, as well as in negative mode as a fragment ion representing the fluorinated part of the respective semifluorinated n-alkane. The method allowed the sensitive detection of semifluorinated n-alkanes with achievable limits of detection (LODs) in the single digit pg range injected on column. To examine the applicability of the GC/DBDI-MS method, semifluorinated n-alkanes were determined in fluorinated ski waxes. Results were confirmed by complimentary GC/electron ionization MS measurements.

Conclusions: The unique SFA ionization patterns serve for complementary unambiguous identification of semifluorinated n-alkane species in positive mode and screening of contained n-alkanes fluorinated chain lengths in negative mode.
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http://dx.doi.org/10.1002/rcm.8139DOI Listing
July 2018

Three-dimensional Kendrick mass plots as a tool for graphical lipid identification.

Rapid Commun Mass Spectrom 2018 Jun;32(12):981-991

Institute of Inorganic and Analytical Chemistry, University of Münster, Corrensstraße 30, 48149, Münster, Germany.

Rationale: The rising field of lipidomics strongly relies on the identification of lipids in complex matrices. Recent technical advances regarding liquid chromatography (LC) and high-resolution mass spectrometry (HRMS) enable the mapping of the lipidome of an organism with short data acquisition times. However, interpretation and evaluation of resulting multidimensional datasets are challenging and this is still the bottleneck regarding overall analysis times.

Methods: A novel adaption of Kendrick mass plot analysis is presented for a rapid and accurate analysis of lipids in complex matrices. Separation of lipids by their respective head group was achieved via hydrophilic interaction liquid chromatography (HILIC) coupled to HRMS. The resulting LC/HRMS datasets are processed to a list of chromatographically separated features by applying an optimized MZmine 2 workflow. All features are plotted in a three-dimensional Kendrick mass plot, which allows a fast identification of present lipid classes, based on equidistant features with fitting retention times and the same Kendrick mass defect. Suspected lipid classes are used for exact mass database matching to annotate features. A second three-dimensional Kendrick mass plot of annotated features of a single lipid class helps to reveal potential database mismatches, resulting in a curated list of identified lipid species.

Results: The use of the novel adaption of the Kendrick mass plot has accelerated the identification of the relevant lipid species in the green alga Chlamydomonas reinhardtii. A total of 106 species were identified within the lipid classes: phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and sulfoquinovosyldiacylglycerol.

Conclusions: This work shows how the addition of chromatographic information, i.e. the retention time, to a classical two-dimensional Kendrick mass plot enables rapid and accurate analysis of LC/HRMS datasets, exemplified on a green alga (C. reinhardtii) sample. Three-dimensional Kendrick mass plots have improved lipid class identification and fast spotting of falsely annotated lipid species.
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http://dx.doi.org/10.1002/rcm.8117DOI Listing
June 2018
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