bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2020–06–21
seventeen papers selected by
Sofia Costa, Cold Spring Harbor Laboratory
  1. Machine Learning Applications for Mass Spectrometry-Based Metabolomics.
  2. Determination of steroid profile in hair by liquid chromatography tandem mass spectrometry.
  3. 8-Plex stable isotope labeling absolute quantitation strategy combined with dual-targeted recognizing function material for simultaneous separation and determination of glucosylsphingosine and galactosylsphingosine in human plasma.
  4. An Analytical System for Single-Cell Metabolomics of Typical Mammalian Cells Based on Highly Sensitive Nano-Liquid Chromatography Tandem Mass Spectrometry.
  5. Separation of enantiomers of chiral basic drugs with amylose- and cellulose- phenylcarbamate-based chiral columns in acetonitrile and aqueous-acetonitrile in high-performance liquid chromatography with a focus on substituent electron-donor and electron-acceptor effects.
  6. Enantioselective ultra-high performance liquid chromatography-tandem mass spectrometry method based on sub-2µm particle polysaccharide column for chiral separation of oxylipins and its application for the analysis of autoxidized fatty acids and platelet releasates.
  7. A lipidome atlas in MS-DIAL 4.
  8. Development and Optimization of Simultaneous Determinatıon of Fat Soluble Vitamins by Liquid Chromatography Tandem Mass Spectrometry.
  9. Analysis of Serotonin in Human Feces Using Solid Phase Extraction and Column-Switching LC-MS/MS.
  10. Spatial Localization of Vitamin D Metabolites in Mouse Kidney by Mass Spectrometry Imaging.
  11. Selective and fast methylation of free fatty acids directly in plasma for their individual analysis by gas chromatography- mass spectrometry.
  12. Fast and ergonomic extraction of adherent mammalian cells for NMR-based metabolomics studies.
  13. Enantiomeric separation and quantification of R/S-amphetamine in serum using semi-automated liquid-liquid extraction and ultra-high performance supercritical fluid chromatography-tandem mass spectrometry.
  14. Miniaturized extraction method for analysis of synthetic opioids in urine by microextraction with packed sorbent and liquid chromatography-tandem mass spectrometry.
  15. Optimization of DropletProbe-Mass Spectrometry for Whole-Body Tissue Distribution Analysis of Drug-Like Molecules.
  16. Rendering A Chiral Screening Step In Supercritical Fluid Chromatography Mass-Spectrometry Compatible.
  17. Estimation of the content of lipids composing endothelial lipid droplets based on Raman imaging.
  1. Metabolites. 2020 Jun 13. pii: E243. [Epub ahead of print]10(6):
    Machine Learning Applications for Mass Spectrometry-Based Metabolomics.
    Ulf W Liebal, An N T Phan, Malvika Sudhakar, Karthik Raman, Lars M Blank.
      The metabolome of an organism depends on environmental factors and intracellular regulation and provides information about the physiological conditions. Metabolomics helps to understand disease progression in clinical settings or estimate metabolite overproduction for metabolic engineering. The most popular analytical metabolomics platform is mass spectrometry (MS). However, MS metabolome data analysis is complicated, since metabolites interact nonlinearly, and the data structures themselves are complex. Machine learning methods have become immensely popular for statistical analysis due to the inherent nonlinear data representation and the ability to process large and heterogeneous data rapidly. In this review, we address recent developments in using machine learning for processing MS spectra and show how machine learning generates new biological insights. In particular, supervised machine learning has great potential in metabolomics research because of the ability to supply quantitative predictions. We review here commonly used tools, such as random forest, support vector machines, artificial neural networks, and genetic algorithms. During processing steps, the supervised machine learning methods help peak picking, normalization, and missing data imputation. For knowledge-driven analysis, machine learning contributes to biomarker detection, classification and regression, biochemical pathway identification, and carbon flux determination. Of important relevance is the combination of different omics data to identify the contributions of the various regulatory levels. Our overview of the recent publications also highlights that data quality determines analysis quality, but also adds to the challenge of choosing the right model for the data. Machine learning methods applied to MS-based metabolomics ease data analysis and can support clinical decisions, guide metabolic engineering, and stimulate fundamental biological discoveries.
    Keywords:  MS-based metabolomics; machine learning; metabolic engineering; metabolic flux analysis; multi-omics; synthetic biology
    DOI:  https://doi.org/10.3390/metabo10060243
  2. J Chromatogr A. 2020 Aug 02. pii: S0021-9673(20)30431-3. [Epub ahead of print]1624 461179
    Determination of steroid profile in hair by liquid chromatography tandem mass spectrometry.
    Alex Gomez-Gomez, Oscar J Pozo.
      The simultaneous determination of a large number of steroids (a.k.a. steroid profile) is a powerful tool that provides useful information about the status of steroid hormones. Steroid profile evaluated in matrices such as urine, saliva and plasma provide one-off moment information about the hormonal status and is highly affected by different factors such as circadian rhythm or apprehension to needles. In contrast, the determination of the steroid profile in hair would provide information about the chronic status of the steroid hormones. The objective of the current research was to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology for the determination of 11 steroids in hair, including 6 hormones and 5 metabolites. We have optimized different parts of the analytical procedure such as (i) hair shredding, (ii) hair amount, (iii) extraction from hair, (iv) extraction time, (v) required extractions and (vi) analytes preconcentration. MS parameters such as the inclusion of ESI- transitions were also evaluated. The optimization of these parameters was found to be critical to achieve the required sensitivity for the determination of steroids in hair. The method was validated with appropriate linearity in the endogenous range, intra- and inter-assay accuracies and matrix effect between 80% and 120% and intra- and inter-assay precisions below 20% for all analytes. Most of the analytes showed to be stable up to 10 months at room temperature. The suitability of the method was evaluated by obtaining the endogenous concentration range of steroids in 30 healthy volunteers. Results agreed with the scarce data previously reported for some steroids. For others, endogenous concentration ranges in hair were reported for the first time. Additionally, the method was used to compare intraindividual levels of steroids in beard and hair. Results revealed that with the exception of testosterone, beard is a suitable alternative to the hair determination of the steroid profile. In summary, the present strategy to evaluate the steroid profile in hair may be a useful tool with a high potential for a wide range of clinical purposes.
    Keywords:  Cortisol; Cortisone; Hair analysis; LC-MS/MS; Steroid profile; Validation
    DOI:  https://doi.org/10.1016/j.chroma.2020.461179
  3. Anal Chim Acta. 2020 Aug 08. pii: S0003-2670(20)30567-5. [Epub ahead of print]1124 40-51
    8-Plex stable isotope labeling absolute quantitation strategy combined with dual-targeted recognizing function material for simultaneous separation and determination of glucosylsphingosine and galactosylsphingosine in human plasma.
    Shi-En Chen, Shuyun Zhu, Jingwen Hu, Jing Sun, Zhenjia Zheng, Xian-En Zhao, Huwei Liu.
      Glucosylsphingosine (GlcS) in plasma is considered to be a reliable biomarker of Gaucher disease. The detection difficulty of GlcS is that it is difficult to achieve simultaneous separation and quantification with its isomer galactosylsphingosine (GalS), a biomarker of Krabbe disease. In this work, a multiplexed stable isotope labeling absolute quantization strategy coupled with magnetic dispersive solid phase extraction using new prepared dummy magnetic molecularly imprinted polymers (DMMIPs) has been developed for this purpose by ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). 8-Plex Amine-reactive Mass Difference Tags (M360/361/362/363/373/375/376/378-AMDTs), were designed, synthesized and used to label GalS and GlcS in different 8 plasma samples, respectively. Synchronously, M359-AMDTs was prepared and used to label mixed standards of GalS and GlcS, which served as internal standards in UHPLC-MS/MS quantitation. Then DMMIPs possessing dual recognition function were applied for specific enrichment and purification of all GlcS and GalS derivatives from a combined solution of labeled 8-plex plasma samples and mixed standards before UHPLC-MS/MS injection. The labeling efficiency, chromatographic retention and mass spectrometry responses of all the 9 AMDTs reagents were consistent for GlcS and GalS. The established and validated method enabled 8-plex plasma samples quantification in a single UHPLC-MS/MS run (<2.0 min). Good linearity of AMDTs-GlcS/GalS derivatives was obtained in the range of 0.02-800 nM. LODs of GlcS and GalS were both 0.005 nM. The recoveries were in the range of 96.1-107.2%. The method was successfully applied for multiplex quantitative analysis of GlcS and GalS in human plasma samples. The results indicated that this method was capable of better realizing the simultaneous separation and quantification of GalS and GlcS compared to reported methods.
    Keywords:  Chemical isotope labeling; Derivatization; Dummy magnetic molecularly imprinted polymers; Galactosylsphingosine; Glucosylsphingosine; Liquid chromatography-tandem mass spectrometry
    DOI:  https://doi.org/10.1016/j.aca.2020.05.032
  4. Mass Spectrom (Tokyo). 2020 ;9(1): A0080
    An Analytical System for Single-Cell Metabolomics of Typical Mammalian Cells Based on Highly Sensitive Nano-Liquid Chromatography Tandem Mass Spectrometry.
    Kohta Nakatani, Yoshihiro Izumi, Kosuke Hata, Takeshi Bamba.
      The rapid development of next-generation sequencing techniques has enabled single-cell genomic and transcriptomic analyses, which have revealed the importance of heterogeneity in biological systems. However, analytical methods to accurately identify and quantify comprehensive metabolites from single mammalian cells with a typical diameter of 10-20 μm are still in the process of development. The aim of this study was to develop a single-cell metabolomic analytical system based on highly sensitive nano-liquid chromatography tandem mass spectrometry (nano-LC-MS/MS) with multiple reaction monitoring. A packed nano-LC column (3-μm particle-size pentafluorophenylpropyl Discovery HSF5 of dimensions 100 μm i.d.×180 mm) was prepared using a slurry technique. The optimized nano-LC-MS/MS method showed 3-132-fold (average value, 26-fold) greater sensitivity than semimicro-LC-MS/MS, and the detection limits for several hydrophilic metabolites, including amino acids and nucleic acid related metabolites were in the sub-fmol range. By combining live single-cell sampling and nano-LC-MS/MS, we successfully detected 18 relatively abundant hydrophilic metabolites (16 amino acids and 2 nucleic acid related metabolites) from single HeLa cells (n=22). Based on single-cell metabolic profiles, the 22 HeLa cells were classified into three distinct subclasses, suggesting differences in metabolic function in cultured HeLa cell populations. Our single-cell metabolomic analytical system represents a potentially useful tool for in-depth studies focused on cell metabolism and heterogeneity.
    Keywords:  hydrophilic metabolites; metabolome; multiple reaction monitoring; nano-LC-MS/MS; single-cell analysis
    DOI:  https://doi.org/10.5702/massspectrometry.A0080
  5. J Chromatogr A. 2020 Aug 02. pii: S0021-9673(20)30479-9. [Epub ahead of print]1624 461218
    Separation of enantiomers of chiral basic drugs with amylose- and cellulose- phenylcarbamate-based chiral columns in acetonitrile and aqueous-acetonitrile in high-performance liquid chromatography with a focus on substituent electron-donor and electron-acceptor effects.
    Iza Matarashvili, Aluda Chelidze, Gizo Dolidze, Giorgi Kobidze, Nino Zaqashvili, Alexandre Dadianidze, Ivett Bacskay, Attila Felinger, Tivadar Farkas, Bezhan Chankvetadze.
      In this study, amylose- and cellulose-phenylcarbamate-based chiral columns with different chiral-selector (CS) chemistries were compared to each other for the separation of enantiomers of basic chiral analytes in acetonitrile and aqueous-acetonitrile mobile phases in HPLC. For two chemistries the amylose-based columns with coated and immobilized CSs were also compared. The comparison of CSs containing only electron-donating or electron-withdrawing substituents with those containing both electron-donating and electron-withdrawing substituents showed opposite results for the studied set of chiral analytes in the case of amylose and cellulose derivatives. Along with the chemistry of CS the focus was on the behavior of polysaccharide phenylcarbamates in acetonitrile versus aqueous acetonitrile as eluents. In agreement with earlier results, it was found that in contrast to the commonly accepted view, polysaccharide phenylcarbamates do not behave as typical reversed-phase materials for basic analytes either. In the range of water content in the mobile phase of up to 20-30% v/v the behavior of these CSs is similar to hydrophilic interaction liquid chromatography (HILIC)-type adsorbents. This means that with increasing water content in the mobile phase up to 20-30% v/v, the retention of analytes mostly decreases. The important finding of this study is that the separation efficiency improves for most analytes when switching from pure acetonitrile to aqueous acetonitrile. Therefore, in spite of reduced retention, the separation of enantiomers improves and thus, the HILIC-range of mobile phase composition, offering shorter analysis time and better peak resolution, is advantageous over pure polar-organic solvent mode. Interesting examples of enantiomer elution order (EEO) reversal were observed for some analytes based on the content of water in the mobile phase on Lux Cellulose-1 and Lux Amylose-2 columns.
    Keywords:  Chiral HPLC; Enantiomer elution order reversal; Enantioseparations; Mobile phase effect; Polysaccharide phenylcarbamate-based chiral stationary phases
    DOI:  https://doi.org/10.1016/j.chroma.2020.461218
  6. J Chromatogr A. 2020 Aug 02. pii: S0021-9673(20)30469-6. [Epub ahead of print]1624 461206
    Enantioselective ultra-high performance liquid chromatography-tandem mass spectrometry method based on sub-2µm particle polysaccharide column for chiral separation of oxylipins and its application for the analysis of autoxidized fatty acids and platelet releasates.
    Malgorzata Cebo, Xiaoqing Fu, Meinrad Gawaz, Madhumita Chatterjee, Michael Lämmerhofer.
      Oxylipins, the oxidation products of polyunsaturated fatty acids, are important signaling molecules in living organisms. Some of them have pro-inflammatory properties, while others act as pro-resolving agents. Oxylipins also play a major role in platelet biology and the progression of thrombo-inflammation. Depending on their structure, they may be pro-thrombotic or anti-thrombotic. For an unbiased biological interpretation, a detailed analysis of a broad spectrum of oxylipins including their stereoisomers is necessary. In our work, we developed for the first time an enantioselective UHPLC-ESI-MS/MS assay which allows quantifying individual oxylipin enantiomers. The assay made use of a sub-2µm particle-based amylose-(3,5-dimethylphenylcarbamate) chiral stationary phase (Chiralpak IA-U) under MS-compatible reversed-phase conditions. It covered 19 enantiomeric pairs of oxylipins and one diasteromeric pair of a lipid mediator: 2 pairs of hydroxyoctadecadienoic acids (HODE), 6 pairs of hydroxyeicosatetraenoic acids (HETE), 5 pairs of hydroxyeicosapentaenoic acids (HEPE), 3 pairs of hydroxydocosahexaenoic acids (HDoHE) and one pair of each: resolvins D1, hydroxyeicosatrienoic acid (HETrE), hydroxyoctadecatrienoic acid (HOTrE) and dihydroxyeicosatetraenoic acid (DiHETE). The new method is fast and showed outstanding peak resolution for most of the isomeric pairs. Excellent method sensitivity (average LOD was equal to 2.7 pg on column) was obtained by using a triple quadrupole instrument as a detector in a targeted, selected reaction monitoring (SRM) mode. The applicability of the method was verified by preliminary validation. It was then applied to analyze oxylipins produced by autoxidation of polyunsaturated fatty acids (PUFA) in air. Multiple oxylipins were found in each of the samples as racemic mixtures and served as reference substances for identification. Finally, the new enantioselective UHPLC method was applied to analyze releasates from platelets in resting state, and following activation with thrombin. The highest abundant oxylipin in the platelet releasate was 12(S)-HETE, but many other oxylipins were found in the thrombin activated samples, usually as single enantiomers (e.g. 12(S)-HEPE, 11(R)-HETE, 9(R)-HODE, 13-(S)-HODE, 14(S)-HDoHE). The latter was detected at about similar concentration in resting platelet releasates as well. 15-HETE showed elevated levels for both R-and S-enantiomers in releasates of thrombin-activated platelets. 12-HETrE was found presumably as both enantiomers, however, retention time inconsistencies indicate that the R-enantiomer is actually a different compound, maybe another constitutional isomer with different double-bond configuration.
    Keywords:  Autoxidation; Chiral separation; Chiral stationary phase; Oxylipin enantiomer; Polyunsaturated fatty acid; Targeted lipidomics
    DOI:  https://doi.org/10.1016/j.chroma.2020.461206
  7. Nat Biotechnol. 2020 Jun 15.
    A lipidome atlas in MS-DIAL 4.
    Hiroshi Tsugawa, Kazutaka Ikeda, Mikiko Takahashi, Aya Satoh, Yoshifumi Mori, Haruki Uchino, Nobuyuki Okahashi, Yutaka Yamada, Ipputa Tada, Paolo Bonini, Yasuhiro Higashi, Yozo Okazaki, Zhiwei Zhou, Zheng-Jiang Zhu, Jeremy Koelmel, Tomas Cajka, Oliver Fiehn, Kazuki Saito, Masanori Arita, Makoto Arita.
      We present Mass Spectrometry-Data Independent Analysis software version 4 (MS-DIAL 4), a comprehensive lipidome atlas with retention time, collision cross-section and tandem mass spectrometry information. We formulated mass spectral fragmentations of lipids across 117 lipid subclasses and included ion mobility tandem mass spectrometry. Using human, murine, algal and plant biological samples, we annotated and semiquantified 8,051 lipids using MS-DIAL 4 with a 1-2% estimated false discovery rate. MS-DIAL 4 helps standardize lipidomics data and discover lipid pathways.
    DOI:  https://doi.org/10.1038/s41587-020-0531-2
  8. Chem Phys Lipids. 2020 Jun 10. pii: S0009-3084(20)30063-3. [Epub ahead of print] 104932
    Development and Optimization of Simultaneous Determinatıon of Fat Soluble Vitamins by Liquid Chromatography Tandem Mass Spectrometry.
    Sebla Ertugrul, Cigdem Yucel, Erdim Sertoglu, Yalcin Ozkan, Taner Ozgurtas.
       OBJECTIVE: Fat-soluble vitamins (A, D, E and K) are isoprene derived apolar molecules. While deficiencies of these vitamins have been associated with various diseases such as type 2 diabetes and cancer, high doses of Vitamin A and D can cause toxic effects. Accurate detection of serum levels of these vitamins is of critical importance. In this study, it is aimed to develop and validate a sensitive and specific Liquid Chromatography Tandem Mass Spectrometry (LC-MS / MS) method that allows simultaneous analysis of fat-soluble vitamins.
    MATERIALS AND METHODS: Serum samples were deproteinized with methanol and chromatographic separation of analytes were performed by LC-MS/MS system (AgilentTechnologies 6420 Triple Quadrapole LC-MS), Agilent Pursuit PFP column (100 mm × 3.0 mm; 3.0 μm), in gradient mode using Mobile phase A (milli-Q+0.1% formic acid) and Mobile phase B (Methanol+0.1% formic acid). Ion scan was performed in MRM (multiple reaction monitoring) mode with positive ion selectivity in ESI ion source.
    RESULTS: The retention times were 6.93 min, 6.94 min and 9.34 min while concentrations were linear in the ranges between 10-150 ng/mL, 3-90 μg /dL and 6-90 μg/mL for 25-hydroxy vitamin D3 (25-OHD3), Vitamin A and Vitamin E, respectively. Inter-day Coefficient Variation (CV%) values for Vitamin A, Vitamin E and 25-OHD3 were; 9.08%, 9.85% and 3.07% and intra-day CV% values were; 2.98%, 5.05% and 5.01%. LOD and LOQ results were 2.11 μg/dL and 3.50 μg/dL for Vitamin A; 1.71 μg/mL and 2.45 μg/mL for Vitamin E; 1.47 ng/mL and 2.50 ng/mL for 25-OHD3, respectively.
    CONCLUSION: In this study, a LC-MS/MS method that can analyze fat soluble vitamins in 13 minutes was developed and validated. This method will be useful for clinical purposes by replacing low specificity immunoassay methods and High Performance Liquid Chromatography (HPLC) methods that can not allow simultaneous analysis.
    Keywords:  Fat-SolubleVitamins; LC-MS/MS; Method Development; Validation
    DOI:  https://doi.org/10.1016/j.chemphyslip.2020.104932
  9. Mass Spectrom (Tokyo). 2020 ;9(1): A0081
    Analysis of Serotonin in Human Feces Using Solid Phase Extraction and Column-Switching LC-MS/MS.
    Yukiko Hirabayashi, Kiminori Nakamura, Tsuyoshi Sonehara, Daisuke Suzuki, Satoru Hanzawa, Yu Shimizu, Tomoyasu Aizawa, Koshi Nakamura, Akiko Tamakoshi, Tokiyoshi Ayabe.
      Serotonin, an important neurotransmitter, is produced mainly in intestines, and serotonin levels in feces can be an indicator of the intestinal environment. Human feces, however, contain a large amount of contaminants, which vary widely owing to food contents and the intestinal environment, and these contaminants would be expected to interfere with the determination of serotonin levels in human feces. To remove these contaminants and determine serotonin levels, we developed a new method using solid phase extraction (SPE) and column-switching LC-MS/MS. Serotonin, labeled with a stable isotope, was added to human feces samples prior to SPE as an internal standard to correct for individual differences in matrix effects. The recovery rate for SPE was 55.9-81.0% (intraday) and 56.5-78.1% (interday) for feces from two subjects. We analyzed 220 fecal samples from 96 subjects including 76 pregnant and post-delivery women. The endogenous serotonin content per unit weight of dried feces was 0.09-14.13 ng/mg for pregnant and post-delivery women and 0.30-9.93 ng/mg for the remaining subjects.
    Keywords:  column-switching LC-MS/MS; feces; serotonin; solid phase extraction
    DOI:  https://doi.org/10.5702/massspectrometry.A0081
  10. ACS Omega. 2020 Jun 09. 5(22): 13430-13437
    Spatial Localization of Vitamin D Metabolites in Mouse Kidney by Mass Spectrometry Imaging.
    Karl W Smith, Bryn Flinders, Paul D Thompson, Faye L Cruickshank, C Logan Mackay, Ron M A Heeren, Diego F Cobice.
      Vitamin D plays a key role in the maintenance of calcium/phosphate homeostasis and elicits biological effects that are relevant to immune function and metabolism. It is predominantly formed through UV exposure in the skin by conversion of 7-dehydrocholsterol (vitamin D3). The clinical biomarker, 25-hydroxyvitamin D (25-(OH)-D), is enzymatically generated in the liver with the active hormone 1,25-dihydroxyvitamin D then formed under classical endocrine control in the kidney. Vitamin D metabolites are measured in biomatrices by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In LC-MS/MS, chemical derivatization (CD) approaches have been employed to achieve the desired limit of quantitation. Recently, matrix-assisted laser desorption/ionization (MALDI) has also been reported as an alternative method. However, these quantitative approaches do not offer any spatial information. Mass spectrometry imaging (MSI) has been proven to be a powerful tool to image the spatial distribution of molecules from the surface of biological tissue sections. On-tissue chemical derivatization (OTCD) enables MSI to image molecules with poor ionization efficiently. In this technical report, several derivatization reagents and OTCD methods were evaluated using different MSI ionization techniques. Here, a method for detection and spatial distribution of vitamin D metabolites in murine kidney tissue sections using an OTCD-MALDI-MSI platform is presented. Moreover, the suitability of using the Bruker ImagePrep for OTCD-based platforms has been demonstrated. Importantly, this method opens the door for expanding the range of other poor ionizable molecules that can be studied by OTCD-MSI by adapting existing CD methods.
    DOI:  https://doi.org/10.1021/acsomega.0c01697
  11. J Chromatogr A. 2020 Aug 02. pii: S0021-9673(20)30537-9. [Epub ahead of print]1624 461259
    Selective and fast methylation of free fatty acids directly in plasma for their individual analysis by gas chromatography- mass spectrometry.
    Cristian I Ciucanu, Daliborca C Vlad, Ionel Ciucanu, Victor Dumitraşcu.
      An analytical procedure for the rapid and selective derivatization of free fatty acids into methyl esters directly in plasma without transmethylation of lipid-bound fatty acids was developed for their analysis by gas chromatography-mass spectrometry. The methyl esters of free fatty acids were obtained by reaction with methyl iodide in the solution of dipolar aprotic solvents and in the presence of solid bases. The mechanism of the methylation reaction with these reagents was investigated. Optimal conditions for the selective methylation of free fatty acids were established using different dipolar aprotic solvents and different solid bases. The possible transmethylation of covalently bonded fatty acids from plasma lipids has been investigated under different experimental conditions in order to be avoided. Total methylation of free fatty acids was achieved in 1 min at room temperature using methyl iodide and anhydrous potassium carbonate or sodium carbonate in dimethyl sulfoxide. Under these conditions, transmethylation of lipid-bound fatty acids was avoided. The methyl esters can be injected directly from the reaction solvents. A plasma volume of 50 μL was used without special purification. The detection limits were around 0.1 ng/μL. The proposed method avoids the drawbacks of the previous methods used for the one-step analysis of individual free fatty acids in human plasma.
    Keywords:  Free fatty acids analysis; Gas chromatography-mass spectrometry; Human plasma; Selective methylation
    DOI:  https://doi.org/10.1016/j.chroma.2020.461259
  12. Anal Bioanal Chem. 2020 Jun 18.
    Fast and ergonomic extraction of adherent mammalian cells for NMR-based metabolomics studies.
    Manhal Mili, Baptiste Panthu, Anne-Marie Madec, Marie-Agnès Berger, Gilles J P Rautureau, Bénédicte Elena-Herrmann.
      Cellular metabolomics has become key to elucidate mechanistic aspects in various fields such as cancerology or pharmacology, and is rapidly becoming a standard phenotyping tool accessible to the broad biological community. Acquisition of reliable spectroscopic datasets, such as nuclear magnetic resonance (NMR) spectra, to characterize biological systems depends on the elaboration of robust methods for cellular metabolites extraction. Previous studies have addressed many issues raised by these protocols, however with little pondering on ergonomic and practical aspects of the methods that impact their scalability, reproducibility and hence their suitability to high-throughput studies or their use by non-metabolomics experts. Here, we optimize a fast and ergonomic protocol for extraction of metabolites from adherent mammalian cells for NMR metabolomics studies. The proposed extraction protocol, including cell washing, metabolism quenching and actual extraction of intracellular metabolites, was first optimized on HeLa cells. Efficiency of the protocol, in its globality and for the different individual steps, was assessed by NMR quantification of 27 metabolites from cellular extracts. We show that a single PBS wash provides a seemly compromise between contamination from growth medium and leakage of intracellular metabolites. In HeLa cells, extraction using pure methanol, without cell scraping, recovered a higher amount of intracellular metabolites than the reference methanol/water/chloroform method with cell scraping, with yields varying across metabolite classes. Optimized and reference protocols were further tested on eight cell lines of miscellaneous nature, and inter-operator reproducibility was demonstrated. Our results stress the need for tailored extraction protocols and show that fast protocols minimizing time-consuming steps, without compromising extraction yields, are suitable for high-throughput metabolomics studies. Graphical abstract.
    Keywords:  Adherent cells; Extraction; Metabolic profiles; Metabolomics; NMR; Quenching
    DOI:  https://doi.org/10.1007/s00216-020-02764-9
  13. Drug Test Anal. 2020 Jun 14.
    Enantiomeric separation and quantification of R/S-amphetamine in serum using semi-automated liquid-liquid extraction and ultra-high performance supercritical fluid chromatography-tandem mass spectrometry.
    H Havnen, M Hansen, O Spigset, S Hegstad.
      The amphetamine molecule contains a chiral center and its enantiomers exhibit differences in pharmacological effects, with the S-enantiomer mediating most of the central nervous system stimulating activity. The majority of prescribed amphetamine consists of the pure S-enantiomer, but therapeutic formulations containing the R-enantiomer in various proportions are also available. Illegal amphetamine remains available mainly as a racemic mixture of the R- and S-enantiomers. To distinguish between legal and illegal consumption of amphetamine a method for enantiomeric separation and quantification of R/S-amphetamine in serum was developed and validated using ultra-high performance supercritical fluid chromatography-tandem mass spectrometry (UHPSFC-MS/MS). Sample preparation prior to UHPSFC-MS/MS analysis was performed by a semi-automated liquid-liquid extraction method. The UHPSFC-MS/MS method used a Chiralpak AD-3 column with a mobile phase consisting of CO2 and 0.1% ammonium hydroxide in 2-propanol/methanol (50/50, v/v). The injection volume was 2 μL and run time was 4 minutes. MS/MS detection was performed with positive electrospray ionization and two multiple reaction monitoring transitions (m/z 136.1 > 119.0 and m/z 136.1 > 91.0). The calibration range was 12.5-1000 nM for each analyte. The between-assay relative standard deviations were in the range of 1.3-3.0%. Recovery was 73% and matrix effects ranged from 95 to 100% when corrected with internal standard. After development and validation, the method has been successfully implemented in our laboratory for both separation and quantification of R/S-amphetamine and has proved to be a reliable and useful tool for distinguishing intake of R- and S-amphetamine in authentic patient samples.
    Keywords:  R/S-amphetamine; UHPSFC-MS/MS; drugs of abuse; serum; therapeutic drug monitoring
    DOI:  https://doi.org/10.1002/dta.2879
  14. J Chromatogr A. 2020 Aug 02. pii: S0021-9673(20)30519-7. [Epub ahead of print]1624 461241
    Miniaturized extraction method for analysis of synthetic opioids in urine by microextraction with packed sorbent and liquid chromatography-tandem mass spectrometry.
    Kelly Francisco da Cunha, Leonardo Costalonga Rodrigues, Marilyn A Huestis, Jose Luiz Costa.
      Synthetic opioids are responsible for numerous overdoses and fatalities worldwide. Currently, fentanyl and its analogs are also mixed with heroin, cocaine and methamphetamine, or sold as oxycodone, hydrocodone and alprazolam in counterfeit medications. Microextraction techniques became more frequent in analytical toxicology over the last decade. A method to simultaneously quantify nine synthetic opioids, fentanyl, sufentanil, alfentanil, acrylfentanyl, thiofentanyl, valerylfentanyl, furanylfentanyl, acetyl fentanyl and carfentanil, and two metabolites, norfentanyl and acetyl norfentanyl, in urine samples by microextraction with packed sorbent (MEPS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated. A multivariate optimization was performed to establish the number and speed (stroke) of draw-eject sample cycles and the extraction solvent. The best extraction condition was eight draw-eject sample cycles, with a velocity of 3.6 µL/sec and acetonitrile as elution solvent. Linearity was achieved between 1 to 100 ng/mL, with a limit of detection (LOD) of 0.1 ng/mL and limit of quantification (LOQ) of 1 ng/mL. Imprecision (% relative standard deviation) and bias (%) were less than 12.8% and 5.7%, respectively. The method had good specificity and selectivity when challenged with 10 different matrix sources and 36 pharmaceuticals and drugs of abuse at concentrations of 100 or 500 ng/mL. The method was successfully applied to authentic urine samples. MEPS was an efficient semi-automatic extraction technique, requiring small volumes of organic solvents (640 µL) and sample (200 µL). The cartridges can be cleaned and reused (average of 150 sample extractions/barrel inside and needle).
    Keywords:  Fentanyl; LC–MS/MS; Microextraction with packed sorbent (MEPS); Synthetic opioids; Urine
    DOI:  https://doi.org/10.1016/j.chroma.2020.461241
  15. J Am Soc Mass Spectrom. 2020 Jun 15.
    Optimization of DropletProbe-Mass Spectrometry for Whole-Body Tissue Distribution Analysis of Drug-Like Molecules.
    Bingming Chen, Marissa Vavrek, Mark T Cancilla.
      DropletProbe mass spectrometry (MS) is an emerging tool for the rapid ex vivo analysis of drugs in tissues and whole-body sections. Its use has been demonstrated to better understand a drug's absorption, distribution, metabolism and excretion (ADME) properties. To further optimize the overall utility of this technique, it is important to characterize and understand the various tissue matrix effects and extraction solvents on the overall performance of dropletProbe MS analyses. Herein, we systematically evaluated the impact of extraction solvents and various tissues on the relative detected signal intensities of a test set of diverse drugs. It was observed that the tissue matrix had a minimal effect on the performance of dropletProbe MS for the limited set of tested compounds once an optimized extraction solvent was identified. A general starting extraction solvent of 1:1 acetonitrile:water (v:v) was identified to efficiently extract the test set of compounds from various tissues. Next, the optimized conditions were used to map the distribution of the drug diclofenac and its metabolites in whole-body mouse sections. The relative tissue distribution of diclofenac and its metabolites, including the phase II acyl-glucuronide metabolite, were successfully determined with the technique. It is recommended these conditions are used as a general guideline when initiating dropletProbe MS studies of therapeutic drug-like compounds.
    DOI:  https://doi.org/10.1021/jasms.0c00168
  16. J Chromatogr A. 2020 Aug 02. pii: S0021-9673(20)30465-9. [Epub ahead of print]1624 461201
    Rendering A Chiral Screening Step In Supercritical Fluid Chromatography Mass-Spectrometry Compatible.
    Sven Declerck, Yvan Vander Heyden, Debby Mangelings.
      Nowadays, sensitive chiral methods are required for the determination of chiral impurities and for assays in biological samples. Supercritical fluid chromatography (SFC), one of the main techniques to separate chiral molecules, can be coupled to MS to provide such sensitive methods. Moreover, chiral separation strategies are very useful to reduce the development time and cost of such methods. This study investigates the transfer of an existing non-MS compatible screening step (as part of a separation strategy) into an MS-compatible one. The initial step had a cumulative success rate of 100 % for 57 tested compounds using methanol or 2-propanol as mobile phase modifier on one of four chiral stationary phases. The additives applied in the original mobile phases, i.e. isopropylamine and trifluoroacetic acid, negatively affect the ionization in SFC-MS and thus need to be replaced. Formic acid, acetic acid, water, ammonia, ammonium acetate and ammonium formate were investigated as MS-compatible additives in different combinations and concentrations. Only methanol-based mobile phases were considered in this study because high system pressures were obtained with isopropanol. The other experimental parameters remained the same as in the initial screening step. The effects of the alternative additives on the obtained resolutions as well as on the global success rate were investigated. The best alternative MS-compatible mobile phase contained 0.5 % CH3COOH and 40 mM NH3 as additives. This mobile phase provided the highest number of separations and rather high resolutions. An MS-compatible screening step was defined with this alternative mobile phase. Compared to the original additives, a similar success rate was obtained.
    Keywords:  Additive screening; Chiral separations; MS-compatible chiral screening step; Supercritical fluid chromatography; Transfer
    DOI:  https://doi.org/10.1016/j.chroma.2020.461201
  17. Biochim Biophys Acta Mol Cell Biol Lipids. 2020 Jun 11. pii: S1388-1981(20)30150-5. [Epub ahead of print] 158758
    Estimation of the content of lipids composing endothelial lipid droplets based on Raman imaging.
    Marta Z Pacia, Katarzyna Majzner, Krzysztof Czamara, Magdalena Sternak, Stefan Chlopicki, Malgorzata Baranska.
      Lipid droplets (LDs) are dynamic organelles involved in intracellular lipid metabolism, and the biogenesis of LDs in endothelium is triggered by the excess of lipids in the environment. In this paper we present the methodology aimed to define the composition of endothelial LDs formed upon stimulation with oleic acid (OA) in two models: endothelial cells cultured in vitro and in isolated blood vessel ex vivo. The biochemical composition of LDs was determined using Raman imaging, followed by the lipid unsaturation calibration analysis and modelling of spectral bands based on individual spectra of selected lipids. Among LDs formed in response to OA in vitro or ex vivo conditions there were two types of LDs; those with more unsaturated (average number of CC bonds equalled 1.40) or saturated (average number of CC bonds equalled 0.95) lipids. The modelling of endothelial LDs composition revealed the OA represented a major component of LDs (80.6-91.3%) with an important content of arachidonic acid (8.7-19.4%). In conclusion, endothelial LDs consist of exogenous oleic acid uptaken from the extracellular space, and the endogenous arachidonic acid released from plasma membranes.
    Keywords:  Endothelium; Fluorescence imaging; Lipid droplets; Oleic acid; Raman imaging
    DOI:  https://doi.org/10.1016/j.bbalip.2020.158758
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