bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2020‒03‒08
33 papers selected by
Sofia Costa
Cold Spring Harbor Laboratory

  1. Anal Chim Acta. 2020 Apr 08. pii: S0003-2670(20)30061-1. [Epub ahead of print]1105 105-111
      Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are an important family of endogenous lipids, possessing antidiabetic and anti-inflammatory functions. Therefore, analysis of FAHFAs in biological samples obtained under healthy and disease states can uncover underlying mechanisms of various relevant disorders (e.g., diabetes and autoimmune diseases). Up to now, due to their extremely low abundance, the determination of the changed levels of these species is still a huge challenge, even though great efforts have been made by utilizing liquid chromatography-tandem mass spectrometry with or without derivatization. Herein, we described a novel method for analysis of FAHFAs present in lipid extracts of biological examples after solid-phase extraction and chemical derivatization with one authentic FAHFA specie as an internal standard based on the principles of multi-dimensional mass spectrometry-based shotgun lipidomics. The approach possessed marked sensitivity, high specificity, and broad linear dynamic range of over 3 orders without obvious matrix effects. Moreover, after chemical derivatization, the molecular masses of FAHFAs shift from an overlapped region with ceramide species to a new region without overlaps, removing these contaminating signals from ceramides, and thereby reducing the false results of FAHFAs. Finally, this novel method was successfully applied for determining FAHFAs levels in varieties of representative biological samples, including plasma from lean and overweight/obese individuals of normoglycemia, and tissue samples (such as liver and white adipose tissue from diabetic (db/db) mice). We revealed significant alterations of FAHFAs in samples under patho(physio)logical conditions compared to their respective controls. Taken together, the developed method could greatly contribute to studying altered FAHFA levels under a variety of biological/biomedical conditions, and facilitate the understanding of these lipid species in the patho(physio)logical process.
    Keywords:  Diabetes; FAHFAs; Multi-dimensional mass spectrometry; Shotgun lipidomics; Solid-phase extraction
  2. Anal Bioanal Chem. 2020 Mar 04.
      In this work, a lipidomics workflow based on offline semi-preparative lipid class-specific fractionation by supercritical fluid chromatography (SFC) followed by high-resolution mass spectrometry was introduced. The powerful SFC approach offered separation of a wide polarity range for lipids, enabled enrichment (up to 3 orders of magnitude) of lipids, selective fractionation of 14 lipid classes/subclasses, and increased dynamic range enabling in-depth characterization. A significantly increased coverage of low abundant lipids improving lipid identification by numbers and degree (species and molecular level) was obtained in Pichia pastoris when comparing high-resolution mass spectrometry based lipidomics with and without prior fractionation. Proof-of-principle experiments using a standard reference material (SRM 1950, NIST) for human plasma showed that the proposed strategy enabled quantitative lipidomics. Indeed, for 70 lipids, the consensus values available for this sample could be met. Thus, the novel workflow is ideally suited for lipid class-specific purification/isolation from milligram amounts of sample while not compromising on omics type of analysis (identification and quantification). Finally, compared with established fractionation/pre-concentration approaches, semi-preparative SFC is superior in terms of versatility, as it involved only volatile modifiers and salt additives facilitating any follow-up use such as qualitative or quantitate analysis or further purification down to the single lipid species level. Graphical Abstract.
    Keywords:  Human plasma; Lipid fractionation; Lipidomics; Pichia pastoris; Preparative supercritical fluid chromatography; SRM 1950
  3. Clin Chem Lab Med. 2020 Mar 02. pii: /j/cclm.ahead-of-print/cclm-2019-1237/cclm-2019-1237.xml. [Epub ahead of print]
      Background Our goal was to develop a simple, rapid and precise ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the determination of retinol and α-tocopherol in serum. Currently published LC-MS/MS methods either require complex extraction procedures (liquid-liquid or solid-phase) or do not meet desirable specifications for imprecision in serum (coefficient of variation [CV] <6.8% and 6.9%, respectively). Methods Sample preparation consisted of a simple protein precipitation with ethanol and acetonitrile. Stable isotope-labeled internal standards (IS) and a homemade calibration curve were used for quantification. The analysis was performed using an Acquity I-class Xevo TQ XS LC-MS/MS. Chromatographic runtime was 6.0 min using a reversed phase gradient elution. UniSpray (US) as an ionization technique was compared to electrospray ionization (ESI). Analytical validation included matrix effect, recovery and trueness compared to National Institute of Standards and Technology (NIST) standards and United Kingdom National External Quality Assessment Service (UK NEQAS) samples. Results Intra- and inter-run CVs were <4.9% for retinol and <1.7% for α-tocopherol, both complying with desirable specifications for imprecision. Bias compared to NIST standards was <3.1% for both compounds. The method was linear over the entire tested range. The lower limit of quantification (LLOQ) with US was lower than with ESI for both retinol (0.022 vs. 0.043 mg/L) and α-tocopherol (0.22 vs. 0.87 mg/L). Matrix effects were not significant (<15%) for retinol. However, for α-tocopherol matrix effects of on average 54.0% were noted using ESI, but not with US. Conclusions We developed a fast, precise and accurate UPLC-MS/MS method for the determination of retinol and α-tocopherol in human serum using a single-step sample pretreatment. Ionization using US eliminated the matrix effects for α-tocopherol.
    Keywords:  UniSpray; liquid chromatography; retinol; tandem mass spectrometry; α-tocopherol
  4. J Sep Sci. 2020 Mar 07.
      Metabolite identification is a crucial step in non-targeted metabolomics, but also represents one of its current bottlenecks. Accurate identifications are required for correct biological interpretation. To date, annotation and identification are usually based on the use of accurate mass search or tandem MS analysis, but neglect orthogonal information such as retention times obtained by chromatographic separation. While several tools are available for the analysis and prediction of tandem MS data, prediction of retention times for metabolite identification are not widespread. Here, we review the current state of retention time prediction in liquid chromatography-mass spectrometry-based metabolomics, with a focus on publications published after 2010. This article is protected by copyright. All rights reserved.
    Keywords:  Liquid Chromatography-Mass Spectrometry; Metabolite identification; Metabolomics; Retention time prediction
  5. Anal Chim Acta. 2020 Apr 08. pii: S0003-2670(20)30062-3. [Epub ahead of print]1105 112-119
      Perturbation of thiol homeostasis in biological fluids are thought to be associated with several diseases, and reliable analytical methods for the determination of low molecular weight (LMW) thiols in human plasma or serum are thus required. In this study, a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method is described for high throughput determination of four LMW thiols (glutathione, cysteine, homocysteine and cysteinylglycine) in human serum. It is based on the use of a bromoacetyl functionalized C60 (Br-C60) as a derivatization reagent to label thiols. The Br-C60 labeling can add an 832-Da tag to thiols, which moves thiol signals to high mass region and effectively avoids the signal interference generated by the traditional MALDI matrix below 800 Da. The labeling can be completed within 5 min under microwave-assisted condition. Thereby, the Br-C60 labeling based MALDI-TOF MS analytical method can achieve high throughput analysis of LMW thiols in serum. Good linearities of the method for the thiols in human serum were obtained in the range of 0.5-500.0 μM with correlation coefficient (R) greater than 0.9960. The limit of detection is in the range of 0.07-0.18 μM for the investigated thiols in human serum with relative standard deviations of lower than 13.5% and recoveries ranging from 81.9 to 117.1%. Using the method, four thiols in microliter serum samples of breast cancer (BC) patients were determined. The result showed that the contents of the four thiols in BC serum samples significantly changed compared to the healthy control (HC).
    Keywords:  Breast cancer; Br–C60; Derivatization; Low molecular weight thiols; MALDI-TOF MS
  6. Anal Chem. 2020 Mar 03.
      A large proportion of the complexity and redundancy of LC-MS metabolomics data comes from adduct formation. To reduce such redundancy, many tools have been developed to recognize and annotate adduct ions. These tools rely on predefined adduct lists that are generated empirically from reversed-phase LC-MS studies. In addition, hydrophilic interaction chromatography (HILIC) is gaining popularity in metabolomics studies due to its enhanced performance over other methods for polar compounds. HILIC methods typically use high concentrations of buffer salts to improve chromatographic performance. Therefore, it is necessary to analyze adduct formation in HILIC metabolomics. To this end, we developed covariant ion analysis (COVINA) to investigate metabolite adduct formation. Using this tool, we completely annotated 201 adduct and fragment ions from 10 metabolites. Many of the metabolite adduct ions were found to contain cluster ions corresponding to mobile phase additives. We further utilized COVINA to find the major ionized forms of metabolites. Our results show that for some metabolites, the adduct ion signals can be >200-fold higher than the signals from the deprotonated form, offering better sensitivity for targeted metabolomics analysis. Finally, we developed an in-source CID ramping (InCIDR) method to analyze the intensity changes of the adduct and fragment ions from metabolites. Our analysis demonstrates a promising method to distinguish the protonated and deprotonated ions of metabolites from the adduct and fragment ions.
  7. J Chromatogr Sci. 2020 Mar 05. pii: bmaa010. [Epub ahead of print]
      The aim of this study was to develop and validate a new, rapid, sensitive, selective and reliable liquid chromatography-tandem mass spectrometry method for simultaneous determination of 3-O-Acetyl-11-keto-β-boswellic acid (AKBA) and its active metabolite 3-O-Acetyl-11-hydroxy-β-boswellic acid (Ac-11-hydroxy-BA) in rat plasma. Both analytes (AKBA and Ac-11-hydroxy-BA) and the internal standard (IS, ursolic acid) were extracted from 100 μL of rat plasma by protein precipitation. Chromatographic separation was achieved on PRP-H1 RP-C18 column (75 mm × 2 mm, 1.6 μm) using acetonitrile-water (95.5 v/v) as the mobile phase. Mass detection was conducted by electrospray ionization in positive ion multiple reaction monitoring (MRM) mode. A linear dynamic range of 1-1,000 ng/mL for both AKBA and Ac-11-hydroxy-BA was established with mean correlation coefficient (r (1)) of 0.999. Intra- and inter-day precision (% CV) of analysis were found in the range of 1.9-7.4%. The accuracy determined for these analytes ranged from 92.4 to 107.2%. The extraction recoveries for both analytes ranged from 92.6 to 97.3% for spiked plasma samples and were consistent. The % change in stability samples compared to nominal concentration ranged from 0.4 to 4.2%. This method was successfully tested to a pharmacokinetic (PK) study for estimation of AKBA and acetyl-11-hydroxy-BA in rat plasma following oral administration of AKBA. This method has been validated with the advantage of shorter run time that can be used for high-throughput analysis and has been successfully applied to the pharmacokinetic study of AKBA in rats.
  8. Anal Chem. 2020 Mar 05.
      About ten years ago most of the liquid chromatography (LC) electrospray (ESI) mass spectrometry (MS) analysis of environmental, metabolomics, pharmaceutical samples were predominantly carried out as targeted analysis. Targeted analyses allow detection and quantification a few selected analytes with the aid of standard substances. Today, the centre of gravity is shifting towards non-targeted methods which utilize high-resolution mass spectrometry (HRMS). Furthermore, the targeted and non-targeted methods are merging into each other. LC/HRMS based non-targeted methods allow detecting compounds recovered from the sample preparation and ionizing in the electrospray ionization (ESI) source. New possibilities arising from applying the machine learning tools to LC/HRMS data have already transformed the process of identifying the compounds. The computer-aided identification process is not compatible with traditional calibration graph based quantification methods. The main obstacle arises from the fact that in ESI different compounds ionize to a very different extent. The differences up to 100 million times have been reported. This phenomenon results in a vastly different response of different compounds at the same concentration and complicates the quantification for compounds without standard substances. However, decision making is hindered without quantitative information. Therefore, the need to obtain quantitative information from the non-targeted analysis is triggering an emerging field of research. This review aims at giving an overview of different possibilities for quantitatively comparing the results obtained from LC/HRMS based non-targeted analysis. More specifically, quantification via structurally similar internal standards, different isotope labelling strategies, radiolabelling, and predicted ionization efficiencies are reviewed.
  9. Anal Chim Acta. 2020 Apr 08. pii: S0003-2670(19)31532-6. [Epub ahead of print]1105 28-44
      Untargeted metabolomics is now widely recognized as a useful tool for exploring metabolic changes taking place in biological systems under different conditions. By its nature, this is a highly interdisciplinary field of research, and mastering all of the steps comprised in the pipeline can be a challenging task, especially for those researchers new to the topic. In this tutorial, we aim to provide an overview of the most widely adopted methods of performing LC-HRMS-based untargeted metabolomics of biological samples. A detailed protocol is provided in the Supplementary Information for rapidly implementing a basic screening workflow in a laboratory setting. This tutorial covers experimental design, sample preparation and analysis, signal processing and data treatment, and, finally, data analysis and its biological interpretation. Each section is accompanied by up-to-date literature to guide readers through the preparation and optimization of such a workflow, as well as practical information for avoiding or fixing some of the most frequently encountered pitfalls.
    Keywords:  Biological samples; High-resolution mass spectrometry; Liquid chromatography; Metabolomics; Methodology; Untargeted
  10. Forensic Sci Int. 2020 Feb 11. pii: S0379-0738(20)30055-4. [Epub ahead of print]309 110193
      The aim of this work was to develop and validate a liquid chromatography tandem mass spectrometry method for detecting sixty drugs and metabolites that are most commonly encountered in postmortem whole blood analysis. Although a large number of drugs were included in the panel, acceptance criteria for method validation were achieved. All calibration curves were found to be linear with coefficients of determination greater than 0.99. The limits of detection ranged from 0.2ng/mL to 1.0ng/mL and the limits of quantification range from 1.0ng/mL to 5.0ng/mL. Using three controls, within-run precision was 0.7%-10.3% and between-run precision was 0.6%-9.0%. Accuracy was ranged from 95.0%-104.1%. Matrix effects ranged from -15% to +22%. After excluding matrix effects, analytical recoveries ranged from 76% to 100%. Coefficients of variation for matrix effects ranged from 0.5%-13% and coefficients of variation for recovery ranged from 0.9%-13.0%. Over 1000 postmortem blood samples were analyzed. Among them, 435 cases (45%) tested positive for at least one analyte of interest. In conclusion, this study presents a technique for multianalyte screening of sixty drugs and metabolites that are commonly encountered in postmortem toxicology. This technique was then applied in routine analysis of autopsy blood samples in order to assess the applicability of this method. Data from postmortem cases is rarely reported from Saudi Arabia, and one of the current study goals is to present new information from postmortem cases to help prevent wide-spread drug use.
    Keywords:  Drug abuse; Forensic toxicology; LC–MS/MS; Postmortem
  11. J Am Soc Mass Spectrom. 2020 Mar 04. 31(3): 517-526
      Imaging mass spectrometry (IMS) is becoming an essential technique in lipidomics. Still, many questions remain open, precluding it from achieving its full potential. Among them, identification of species directly from the tissue is of paramount importance. However, it is not an easy task, due to the abundance and variety of lipid species, their numerous fragmentation pathways, and the formation of a significant number of adducts, both with the matrix and with the cations present in the tissue. Here, we explore the fragmentation pathways of 17 lipid classes, demonstrating that in-source fragmentation hampers identification of some lipid species. Then, we analyze what type of adducts each class is more prone to form. Finally, we use that information together with data from on-tissue MS/MS and MS3 to refine the peak assignment in a real experiment over sections of human nevi, to demonstrate that statistical analysis of the data is significantly more robust if unwanted peaks due to fragmentation, matrix, and other species that only introduce noise in the analysis are excluded.
    Keywords:  lipid fragmentation; lipid identification; mass spectrometry imaging; nevus
  12. Anal Chem. 2020 Mar 02.
      In the past decade, the field of LC-MS based metabolomics has transformed from an obscure specialty into a major "-omics" platform for studying metabolic processes and biomolecular characterization. However, as a whole the field is still very fractured, as the nature of the instrumentation and of the information produced by the platform essentially creates incompatible "islands" of datasets. This lack of data coherency results in the inability to accumulate a critical mass of metabolomics data that has enabled other -omics platforms to make impactful discoveries and meaningful advances. As such, we have developed a novel algorithm, called Disparate Metabolomics Data Reassembler (DIMEDR), which attempts to bridge the inconsistencies between incongruent LC-MS metabolomics datasets of the same biological sample type. A single "primary" dataset is postprocessed via traditional means of peak identification, alignment, and grouping. DIMEDR utilizes this primary dataset as a progenitor template by which data from subsequent disparate datasets are reassembled and integrated into a unified framework that maximizes spectral feature similarity across all samples. This is accomplished by a novel procedure for universal retention time correction and comparison via identification of ubiquitous features in the initial primary dataset, which are subsequently utilized as endogenous internal standards during integration. For demonstration purposes, two human and two mouse urine metabolomics datasets from four unrelated studies acquired over 4 years were unified via DIMEDR, which enabled meaningful analysis across otherwise incomparable and unrelated datasets.
  13. J Anal Toxicol. 2020 Feb 29. pii: bkz117. [Epub ahead of print]
      Antidepressant drugs are one of the most widely used medicines for treating major depressive disorders for long time periods. Oral fluid (OF) testing offers an easy and non-invasive sample collection. Detection of antidepressants in OF is important in clinical and forensic settings, such as therapeutic drug monitoring and roadside testing for driving under influence. We developed and validated a comprehensive liquid chromatography-tandem mass spectrometry method for 18 antidepressants (amitriptyline, bupropion, citalopram, clomipramine, cyclobenzaprine, desipramine, desvenlafaxine, doxepin, duloxetine, fluoxetine, imipramine, mirtazapine, nortriptyline, paroxetine, sertraline, trazodone, trimipramine, venlafaxine) in oral fluid collected by Quantisal® oral collection devices. One-half milliliter of Quantisal® OF (125 μL of neat OF) was submitted to solid-phase extraction. The chromatographic separation was performed employing a biphenyl column in gradient mode with a total run time of 5 min. The MS detection was achieved by multiple-reaction monitoring with two transitions per compound. The range for linearity of all analytes was from 10 to 1,000 ng/mL, with a limit of detection of 10 ng/mL. Intra and inter-day accuracy and precision (n = 15) were all within acceptable limits, ±20% error and ±15% relative standard deviation. Analyte recovery at 400 ng/mL concentration (n = 15) ranged from 91 to 129%. Matrix effect ranged from 73.7 to 157%. The internal proficiency test detected all antidepressants with accuracy ranging from 83.1 to 112.1%. The authentic patient sample showed a percentage difference compared to the previously calculated concentration of 86.3-111%. This method provides for the rapid detection of 18 antidepressants and metabolites in OF, which is readily applicable to a routine laboratory.
    Keywords:  Antidepressant; LC–MS-MS; Oral fluid
  14. Anal Bioanal Chem. 2020 Mar 04.
      Pheochromocytoma and paraganglioma (PPGL) are rare neuroendocrine tumors arising from adrenal and extra-adrenal chromaffin cells. They produce excessive amounts of catecholamines and their metabolites. A newly analytical procedure based on the semi-automated microextraction by packed sorbent (MEPS) technique, using a digitally controlled syringe (eVol) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS), was developed to quantify free urinary catecholamines and metanephrines. The important parameters affecting MEPS performance, namely the type of sorbent material (porous graphitized carbon (PGC), polar enhanced polymer (PEP), cation-exchange (CX) and C18), number of extraction cycles, and elution solvent system, were evaluated. The optimal experimental conditions involved the loading of sample mixture in seven extraction cycles through a C18 sorbent in a MEPS syringe, followed by using elution solutions (water/acetonitrile/formic acid, 95/4.75/0.25). The entire sample preparation took about 4 min. Chromatographic separation was well achieved with an HSS PFP column using the gradient elution. The linearity range of the method was 0.167-33.4 ng/mL for epinephrine, 0.650-130 ng/mL for norepinephrine, 1.53-306 ng/mL for dopamine, 1.34-268 ng/mL for metanephrine, 3.43-686 ng/mL for normetanephrine, and 1.33-265 ng/mL for 3-methoxytyramine. The intra- and interassay precisions were ≤ 12.8%, and the respective accuracies were 88.4-112.0% and 89.0-109.5%. The carryover and sample stability without acidification were also investigated. Validation using clinical urine specimens showed that the proposed method had higher sensitivity compared with other urinary biochemical tests. The developed MEPS-LC-MS/MS method was simple, fast, and cost-effective; it helped to obtain information about multiple metabolites. It is applicable in routine clinical laboratories for the screening of PPGL. Graphical abstract.
    Keywords:  Catecholamines; Human urine samples; Liquid chromatography-tandem mass spectrometry; Metanephrines; Microextraction by packed sorbent; Pheochromocytoma and paraganglioma
  15. Clin Chem. 2020 Mar 03. pii: hvaa022. [Epub ahead of print]
      BACKGROUND: While thyroxine (T4), 3,3',5-triiodothyronine (T3), and 3,3',5'-triiodothyronine (rT3) have routine methods available for evaluating patients with suspected thyroid disease, appropriate methods for the measurement of other thyroid hormone metabolites (THMs) are lacking. The effects of other iodothyronines or iodothyroacetic acids are therefore less explored. To better understand the (patho)physiological role of THMs, a robust method to measure iodothyronines and iodothyroacetic acids in serum in a single analysis is needed, including associated reference intervals.METHODS: Clinical and Laboratory Standards Institute guidelines, European Medicines Agency guidelines, and the National Institute of Standards and Technology protocol were used for the method validation and reference intervals. Reference intervals were determined in 132 healthy males and 121 healthy females. Serum samples were deproteinized with acetonitrile, followed by anion-exchange solid phase extraction and analysis with LC-MS/MS, using eight 13C6-internal standards.
    RESULTS: The analytical method validation was performed for all nine THMs. Reference intervals (2.5th to 97.5th percentile) were determined for L-thyronine (4.9-11.3 ng/dL), 3-monoiodothyronine (0.06 --0.41 ng/dL), 3,5-diiodothyronine (<0.13 ng/dL), 3,3'-diiodothyronine (0.25--0.77 ng/dL), T3 (66.4--129.9 ng/dL), rT3 (15.0--64.1 ng/dL), T4 (4.3--10.0 µg/dL), triac/3,3',5-triiodothyroacetic acid (not detected), and tetrac/3,3',5,5'-tetraiodothyroacetic acid (2.2--27.2 ng/dL).
    CONCLUSIONS: A broad dynamic concentration range exists among the nine THMs. This method should help to develop a better understanding of the clinical relevance of other THMs, as well as an understanding of thyroid hormone metabolism in health and disease.
    Keywords:  Iodothyronines; iodothyroacetic acids; mass spectrometry; reference interval; tetrac; thyroxine; triac
  16. Carbohydr Res. 2020 Feb 25. pii: S0008-6215(19)30635-4. [Epub ahead of print]490 107959
      Glycosphingolipids (GSLs) exist exclusively in the outer leaflet of plasma membrane in mammalian cells and have diverse structures including different classes of sugars and various molecular species of ceramide moieties. Establishing methods that measure each molecular species in GSL classes should aid functional characterization of GSLs and reveal details about the mechanism of pathogenesis in glycosphingolipidoses. Using an IF-3 chiral column that has never been used for lipid analyses, we developed a liquid chromatography-mass spectrometry (LC-MS) method to separate various GSLs based on sugar and ceramide moieties. To examine GSLs in detail a multichannel-multiple reaction monitoring (multichannel-MRM) mode was used and covered a range of 500-2000 Da. Common fragment ions detected with higher collision energy in the positive ion mode were m/z 264 and 292, and are derived from d18:1 and d20:1 ions, respectively. Both species were used as product ions in the multichannel-MRM for the simultaneous measurement of neutral GSLs, gangliosides and sulfatides. Comprehensive analysis of GSLs in mouse brain using this method revealed that for gangliosides and LacCer, d18:1-C18:0 and d20:1-C18:0 were the major molecular species, whereas d18:1-C24:0 and d18:1-C24:1 were the major molecular species of sulfatides. The results revealed a diverse GSL fatty acid profile. In conclusion, by combining IF-3 chiral column and the multichannel-MRM method various molecular species of GSLs were detected successfully, and a metabolomics approach based on this LC-MS method should facilitate functional analysis of GSLs and the discovery of early biomarkers of glycosphingolipidoses at the molecular level.
    Keywords:  Chiral column; Glycosphingolipid; Glycosphingolipidoses; Lipidomics; Liquid chromatography-mass spectrometry (LC-MS); Mouse brain
  17. J Pharm Biomed Anal. 2020 Jan 28. pii: S0731-7085(19)32572-5. [Epub ahead of print]184 113123
      Hypoglycemia deprives the brain of its primary energy source glucose. Reductions in whole-brain amino acid energy substrate levels suggest that these non-glucose fuels may be metabolized during glucose shortage. Recurring hypoglycemia can cause mal-adaptive impairment of glucose counter-regulation; yet, it is unclear if amplified reliance upon alternative metabolic substrates impedes detection of continuing neuro-glucopenia. This research aimed to develop high-sensitivity UHPLC-electrospray ionization mass spectrometric (LC-ESI-MS) methodology, for complementary use with high-neuroanatomical resolution microdissection tools, for measurement of glucogenic amino acid, e.g. glutamine (Gln), glutamate (Glu), and aspartate (Asp) content in the characterized glucose-sensing ventromedial hypothalamic nucleus (VMN) during acute versus chronic hypoglycemia. Results show that VMN tissue Gln, Glu, and Asp levels were significantly decreased during a single hypoglycemic episode, and that Gln and Asp measures were correspondingly normalized or further diminished during renewed hypoglycemia. Results provide proof-of-principle that LC-ESI-MS has requisite sensitivity for amino acid energy substrate quantification in distinctive brain gluco-regulatory structures under conditions of eu- versus hypoglycemia. This novel combinatory methodology will support ongoing efforts to determine how amino acid energy yield may impact VMN metabolic sensory function during persistent hypoglycemia.
    Keywords:  9-fluorenylmethyl; Aspartate; Chloroformate; Glutamine; Insulin-induced hypoglycemia; Ventromedial hypothalamic nucleus
  18. Analyst. 2020 Mar 03.
      A methodology is presented for the determination of four pyrethroid (PYR) preservatives in wooden food contact materials (FCMs) using direct analysis in real time (DART) coupled with quadrupole-Orbitrap high-resolution mass spectrometry (Q-Orbitrap HRMS). The sampling mode and critical parameters of the DART-Q-Orbitrap HRMS protocol were systematically investigated. Good linearity was achieved for the four analytes with correlation coefficients all greater than 0.99. The limits of detection (LODs) and limits of quantitation (LOQs) of the method were in the range of 0.04-0.20 mg kg-1 and 0.10-0.50 mg kg-1, respectively. The mean recoveries ranged from 72.1% to 82.7% with relative standard deviations (RSDs) from 5.2% to 11.8% at three spiked levels. The developed method was proved to be suitable for rapid screening of PYRs in complex wooden FCM samples to ensure product safety and consumer health.
  19. Anal Biochem. 2020 Feb 28. pii: S0003-2697(20)30202-5. [Epub ahead of print] 113670
      Testosterone regulates the male reproductive system and acts directly or indirectly on nearly all systems during fetal, pubertal and adult life. Testosterone homeostasis depends on its synthesis and degradation. The major biotransformation reactions are hydroxylation by different cytochrome P450 (CYP) isoforms. There are no described methods to determine the profile of testosterone-hydroxylated metabolites in human urine. The aim of this study was to develop an analytical method to determine testosterone-hydroxylated metabolites in human urine using UPLC-MS. Seven testosterone-hydroxylated metabolites, androstenedione, and testosterone, were identified by comparison of their tret and positive electrospray ionization (ESI+) data, with those of analytical standards. The method developed is sensitive, specific, repeatable, and precise. Limits of detection and quantitation for all compounds ranged from 1.360 to 13.054 ng/ml and 4.234 to 39.679 ng/ml, respectively. The percentages of recovery were between 81.2 and 128.8%. The applicability of the analytical method was confirmed by analysis of urine samples obtained from two groups of healthy men (25-30 and 50-75 years old). All analytes were identified with slightly different metabolites profiles in both groups. In conclusion, the UPLC-MS method developed here was validated for the analysis of testosterone-hydroxylated metabolites in human urine.
    Keywords:  Testosterone-hydroxylated metabolites; UPLC-MS method; human urine; testosterone biotransformation
  20. Hepatol Commun. 2020 Mar;4(3): 425-433
      Carbon-13 magnetic resonance spectroscopy (MRS) following oral intake of 13C-labeled glucose is the gold standard for imaging glycogen metabolism in humans. However, the temporal resolution of previous studies has been >13 minutes. Here, we describe a high-sensitivity 13C MRS method for imaging hepatic glycogen synthesis with a temporal resolution of 1 minute or less. Nuclear magnetic resonance spectra were acquired from the liver of 3 healthy volunteers, using a 13C clamshell radiofrequency transmit and paddle-shaped array receive coils in a 3 Tesla magnetic resonance imaging system. Following a 15-minute baseline 13C MRS scan of the liver, [1-13C]-glucose was ingested and 13C MRS data were acquired for an additional 1-3 hours. Dynamic change of the hepatic glycogen synthesis level was analyzed by reconstructing the acquired MRS data with temporal resolutions of 30 seconds to 15 minutes. Plasma levels of 13C-labeled glucose and lactate were measured using gas chromatography-mass spectrometry. While not detected at baseline 13C MRS, [1-13C]-labeled α-glucose and β-glucose and glycogen peaks accumulated rapidly, beginning as early as ~2 minutes after oral administration of [1-13C]-glucose. The [1-13C]-glucose signals peaked at ~5 minutes, whereas [1-13C]-glycogen peaked at ~25 minutes after [1-13C]-glucose ingestion; both signals declined toward baseline levels over the next 1-3 hours. Plasma levels of 13C-glucose and 13C-lactate rose gradually, and approximately 20% of all plasma glucose and 5% of plasma lactate were 13C-labeled by 2 hours after ingestion. Conclusion: We observed rapid accumulation of hepatic [1-13C]-glycogen following orally administered [1-13C]-glucose, using a dynamic 13C MRS method with a temporal resolution of 1 minute or less. Commercially available technology allows high temporal resolution studies of glycogen metabolism in the human liver.
  21. J Sep Sci. 2020 Mar 02.
      Recently the connection between oxidative stress and various diseases, including cancer and Alzheimer's, attracts notice as a pathway suitable for diagnostic purposes. 8-oxo-deoxyguanosine and 8-oxo-deoxyadenosine produced from the interaction of reactive oxygen species with DNA become prominent as biomarkers. Several methods have been developed for their determination in biofluids, including solid-phase extraction and enzyme-linked immunosorbent assays. However, still, there is a need for reliable and fast analytical methods. In this context, solid-phase microextraction offers many advantages such as flexibility in geometry and applicable sample volume, as well as high adaptability to high-throughput sampling. In this study, a solid-phase microextraction method was developed for the determination of 8-oxo-deoxyguanosine and 8-oxo-deoxyadenosine in biofluids. The extractive phase of solid-phase microextraction consisted of hydrophilic-lipophilic balanced polymeric particles. In order to develop a solid-phase microextraction method suitable for the determination of the analytes in saliva and urine, several parameters, including desorption solvent, desorption time, sample pH, and ionic strength, were scrutinized. Analytical figures of merit indicated that the developed method provides reasonable inter-day and intra-day precision (<15% in both biofluids) with acceptable accuracy. The method provides LOQ for both biomarkers at 5.0 ng mL-1 and 10.0 ng mL-1 levels in saliva and urine matrices, respectively. This article is protected by copyright. All rights reserved.
    Keywords:  Biofluid Analysis; Biomarkers; Oxidative stress; Sample preparation; Solid-Phase Microextraction
  22. Chirality. 2020 Mar 05.
      The supercritical fluid chromatographic separation of underivatized amino acids was explored using immobilized chiral crown ether column CROWNPAK CR-I (+) and mass spectrometric detection. The type of modifier, acidic additives, and the role of water were investigated. Enantioseparation was achieved for all 18 amino acids investigated with short retention times (less than 3 minutes) and average resolution of greater than 5.0. Analysis of enantiomerically pure standards demonstrated the D enantiomer eluted first for all amino acids using a CROWNPAK CR-I (+) column.
    Keywords:  amino acids; chiral stationary phase; enantioseparations; supercritical fluid chromatography
  23. J Am Soc Mass Spectrom. 2020 Mar 04.
      The zebrafish (Danio rerio) is an ideal model for whole animal studies of lipid metabolism and lipid-related disease. In this work, infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry imaging (MSI) was applied for direct visualization of lipid and metabolite distributions across various organs in whole-body zebrafish tissue sections. Detailed methods for overcoming the challenges of cryosectioning adult male zebrafish for MSI and complementary histological imaging are described. Representative two-dimensional ion maps demonstrated organ specific localization of lipid analytes allowing for visualization of areas of interest including the brain, liver, intestines, and skeletal muscle. A high resolving power mass spectrometer was utilized for accurate mass measurements, which permitted the use of open-source, web-based tools for MS1 annotations including METASPACE and METLIN. Whole-body MSI with IR-MALDESI allowed for broad lipid coverage with high spatial resolution, illustrating the potential of this technique for studying lipid-related diseases using zebrafish as a model organism.
    Keywords:  IR-MALDESI; cryosectioning; lipids; mass spectrometry imaging; zebrafish
  24. Metabolites. 2020 Feb 27. pii: E82. [Epub ahead of print]10(3):
      Dried blood spots (DBS) have proven to be a powerful sampling and storage method for newborn screening and many other applications. However, DBS methods have not yet been optimized for broad-spectrum targeted metabolomic analysis. In this study, we developed a robust, DBS-based, broad-spectrum, targeted metabolomic method that was able to measure over 400 metabolites from a 6.3 mm punch from standard Whatman 903TM filter paper cards. The effects of blood spot volumes, hematocrit, vacutainer chemistry, extraction methods, carryover, and comparability with plasma and fingerstick capillary blood samples were analyzed. The stability of over 400 metabolites stored under varying conditions over one year was also tested. No significant impacts of blood volume and hematocrit variations were observed when the spotted blood volume was over 60 µL and the hematocrit was between 31% and 50%. The median area under the curve (AUC) of metabolites in the DBS metabolome declined by 40% in the first 3 months and then did not decline further for at least 1 year. All originally detectable metabolites remained within detectable limits. The optimal storage conditions for metabolomic analysis were -80 °C with desiccants and without an O2 scavenger. The method was clinically validated for its potential utility in the diagnosis of the mitochondrial disease mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS). Our method provides a convenient alternative to freezing, storing, and shipping liquid blood samples for comparative metabolomic studies.
    Keywords:  MELAS; broad-Spectrum; dried blood spots; metabolomics; targeted
  25. Analyst. 2020 Mar 04.
      Rapid analysis of metabolites in biofluids is of great importance for disease diagnosis or new-born disease screening. Herein, we introduce an agarose hydrogel conditioning method to enhance the performance of paper spray ionization mass spectrometry. With facile and fast hydrogel conditioning, the signal intensity of therapeutic drugs spiked in urine was 5 to 15 fold higher than that in direct paper spray ionization mass spectrometry analysis. Consequently, the sensitivity of metabolites in urine was improved via hydrogel conditioning, resulting in 9 to 15 fold decrease in the possibility of detection (POD) levels. These results show that agarose hydrogel conditioning coupled with paper spray ionization mass spectrometry could serve as a facile ionization method for ambient mass spectrometry, which might be useful in fast screening of metabolites and therapeutic drugs in raw biofluids.
  26. Talanta. 2020 May 15. pii: S0039-9140(20)30094-1. [Epub ahead of print]212 120803
      Some compounds of arsenic are extremely toxic to the human body even at low levels. Long-term exposure from drinking contaminated water causes adverse health effects. This has led the European Union and other global organizations to reduce the maximum residue limit for the total arsenic up to 10 μg L-1 in water for human consumption. The toxicity of arsenic depends on the oxidation state, chemical structure and solubility in the biological environment. Main analytical procedures report on the determination of arsenic species in water samples which have been developed based on atomic spectroscopy -speciation performed in the sample treatment step- and inductively coupled plasma-mass spectrometry (ICP-MS). In such cases, speciation is performed by coupling to high-performance liquid chromatography (HPLC). As far as we know, the determination of arsenic species in water samples at low concentration levels by capillary electrophoresis (CE) coupled to electrospray mass spectrometry (ESI-MS) has not been described up to now. In this research CE-ESI(-)-MS is proposed for the identification and simultaneous quantification of organic and inorganic arsenic species in water samples for human consumption. The target compounds were dimethylarsinate (DMA), mono-methylarsonate (MMA), arsenite (i-As(III)) and arsenate (i-As(V)). Optimization of the composition and nature of both the electrophoretic separation medium -using hexafluoro-2-propanol (HFIP) as an additive- and the sheath liquid was performed and aimed at achieving the best sensitivity and separation efficiency for the CE-ESI(-)-MS coupling. Two simple sample treatments were proposed: one based on dispersive liquid-liquid micro-extraction (DLLME) intended for a rapid determination of the total content of inorganic arsenic in the water sample; and the second based on partial evaporation of the water sample to detect each of the four main arsenic species. For the second, limits of detection between 0.02 and 0.04 μg L-1 were found, which are similar results as the ones achieved with methods based on HPLC-ICP-MS. The DLLME + CE-ESI(-)-MS method was applied to the determination of total inorganic arsenic in eleven water samples in the province of Salamanca (Spain). Five of the analyzed samples had values close to, or superior to the maximum residue limit for the total of arsenic in water intended for human consumption (10 μg L-1). The validated method CE-ESI(-)-MS for arsenic speciation was applied with success to the analysis of the weakly mineralized water sample (dry residue < 50 mg L-1) of both groundwater and bottled water.
    Keywords:  Arsenic; Capillary electrophoresis-mass spectrometry; Electrospray ionization; Hexafluoro-2-propanol; Speciation; Water samples
  27. Analyst. 2020 Mar 05.
      The use of synthetic stimulants, including designer cathinones, remains a significant concern worldwide. Thus, the detection and identification of synthetic cathinones in biological matrices is of paramount importance for clinical and forensic laboratories. In this study, distribution of mephedrone and its metabolites was investigated in fingerprints. Following a controlled human mephedrone administration (100 mg nasally insufflated), two mass spectrometry-based methods for fingerprint analysis have been evaluated. The samples deposited on triangular pieces of chromatography paper were directly analysed under ambient conditions by paper spray-mass spectrometry (PS-MS) while those deposited on glass cover slips were extracted and analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The LC-MS/MS method was 5-6 times more sensitive than PS-MS but required sample preparation and longer analysis time. Mephedrone was detected in 62% and in 38% of all post-administration samples analysed by LC-MS/MS and PS-MS, respectively. Nor-mephedrone was the only metabolite detected in 3.8% of all samples analysed by LC-MS/MS. A large inter- and intra-subject variation was observed for mephedrone which may be due to several factors, such as the applied finger pressure, angle and duration of contact with the deposition surface and inability to control the 'amount' of collected fingerprint deposits. Until these limitations are addressed, we suggest that the sole use of fingerprints can be a useful diagnostic tool in qualitative rather than quantitative analysis, and requires a confirmatory analysis in a different biological matrix.
  28. Biomed Opt Express. 2020 Feb 01. 11(2): 963-970
      Methyglyoxal (MGO) is an important pathological factor for diabetic cardiovascular complications. Conventional methods for MGO detection in biological samples, such as high performance liquid chromatography (HPLC)-UV spectrometry, LC-fluorescence spectrometry, and HPLC-mass spectrometry, are time-consuming, high-cost, and complicated. Here, we present a method for MGO quantitative detection based on far-IR spectral analyses. Our method uses o-phenylenediamine (OPD) to produce a chemical reaction with MGO, which results in multiple fingerprint feature changes associated with the molar ratio of MGO and OPD. We use the linear relationship between MGO concentration and peak intensity of the reaction product to quantitatively determine MGO concentration. The corresponding linear detectable range is 5∼2500 nmol/mL nmol per mL with a correlation coefficient of 0.999. This quantitative method is also tested by blood samples with adjusted MGO concentrations, and shows 95% accuracy with only 30s testing time. Our method provides a fast, simple and economical approach to determining MGO concentration in blood.
  29. J Chromatogr A. 2020 Feb 20. pii: S0021-9673(20)30185-0. [Epub ahead of print] 460987
      It is critical to determine the chiral impurity profile of pharmaceutical compounds. The rising trend of drug candidates bearing multiple chiral centers has aggravated the analytical challenges. The traditional chiral HPLC methods can take gruelingly long time to develop yet may not offer sufficient resolution for all stereoisomers. A fast analytical strategy with a high success rate is in urgent demand for compounds with multiple chiral centers. In this study, we have developed an effective and fast multiple heart-cutting (MHC) multicolumn two-dimensional liquid chromatography (LC-mLC) platform approach. The m in the name of LC-mLC highlights the employment of multiple chiral columns with different chiral selectors and mobile phases in the second dimension (2D) within the same run. A short achiral HPLC method in the first dimension (1D) allows the separation of diastereomers and other achiral impurities, followed by 2D analysis enabling different chiral columns and different mobile phases on each coeluted 1D peak for maximum resolution. This LC-mLC strategy breaks down the complex multiple-chiral-center separation problems into simple individual one-chiral-center separation, which dramatically reduces chiral method development time and sample analysis turnaround. Its versatile nature and fast turnaround approach have made it a highly efficient strategy to enable quick stereoselective synthetic route development. This platform LC-mLC strategy has been successfully demonstrated in separating eight stereoisomers for a pharmaceutical compound with 3 chiral centers, within total method development time of less than 2 hours and a final analysis time of less than 24 min, including column equilibration time. It was also proved highly efficient in separating multiple chiral and achiral components in an in-process sample containing structurally similar starting materials, intermediates, side products and multiple stereoisomers of the product with 3 chiral centers, with minimal method development time.
    Keywords:  2D-LC; Chiral impurity; Chiral separation; Multicolumn; Multiple chiral centers; Multiple heart-cutting
  30. Food Chem. 2020 Feb 24. pii: S0308-8146(20)30331-9. [Epub ahead of print]318 126469
      In this study, the occurrence of cannabinoids in hemp-based food products was investigated. For that purpose, a new liquid chromatography tandem mass spectrometry method for the quantification of fifteen cannabinoids was developed and validated for multiple matrices. Method performances were good, fulfilling the SANTE/11813/2017 requirements, and allowing for products compliance testing with various national legislations on cannabinoids levels in food products. The limit of quantification of each analyte was 0.15 mg/kg for hemp seed and hemp protein, 0.6 mg/kg for hemp seed oil, and 0.005 mg/kg for raw milk and milk powder. The applicability of the method was further demonstrated by conducting a limited survey on twenty hemp-based food products. The survey revealed that products from the same category can have very different cannabinoids profiles and levels. These results highlighted the importance of cannabinoids testing of food products in view of the current heterogeneous and fast evolving regulatory landscape worldwide.
    Keywords:  Cannabinoids; Cannabis sativa L.; Food; Hemp; LC-MS/MS; QuEChERS
  31. Molecules. 2020 Feb 29. pii: E1092. [Epub ahead of print]25(5):
      Human mental disorders can be currently classified as one of the most relevant health topics. Including in this are depression and anxiety, which can affect us at any stage of life, causing economic and social problems. The treatments involve cognitive psychotherapy, and mainly the oral intake of pharmaceutical antidepressants. Therefore, the development of analytical methods for monitoring the levels of these drugs in biological fluids is critical. Considering the current demand for sensitive and automated analytical methods, the coupling between liquid chromatography and mass spectrometry, combined with suitable sample preparation, becomes a useful way to improve the analytical results even more. Herein we present an automated multidimensional method based on high-performance liquid chromatography-tandem mass spectrometry using a lab-made, graphene-based capillary extraction column connected to a C8 analytical column to determined five pharmaceutical drugs in urine. A method enhancement was performed by considering the chromatographic separation and the variables of the loading phase, loading time, loading flow, and injection volume. Under optimized conditions, the study reports good linearity with R2 > 0.98, and limits of detection in the range of 0.5-20 µg L-1. Afterward, the method was applied to the direct analysis of ten untreated urine samples, reporting traces of citalopram in one of them. The results suggest that the proposed approach could be a promising alternative that provides direct and fully automated analysis of pharmaceutical drugs in complex biological matrices.
    Keywords:  antidepressants; automation; extraction column; liquid chromatography; mass spectrometry; multidimensional; on-line; pharmaceutical drugs; sample preparation; urine
  32. J Pharm Biomed Anal. 2020 Feb 25. pii: S0731-7085(19)32737-2. [Epub ahead of print]185 113204
      Allopurinol is the most commonly used drug for the treatment of hyperuricemia in people, and in view of the risks of fatal hypersensitivity in patients with renal dysfunction, doses based on the glomerular filtration rate are proposed. In veterinary medicine, allopurinol is used in the treatment of canine leishmaniasis (CanL) caused by Leishmania infantum owing to the drug action of inhibiting the parasite's RNA synthesis. However, renal dysfunction frequently ensues from disease progression in dogs. The purpose of the present study was to standardize and validate a sensitive high-performance liquid chromatography-mass spectrometric (HPLC-MS/MS) method to determine the concentration of allopurinol and its active metabolite oxypurinol in canine urine for clinical pharmacokinetic investigation. Urine samples of eleven (11) dogs with naturally occurring CanL and in the maintenance phase of the treatment with alopurinol were used. For the chromatographic analysis of urine, the mobile phase consisted of a solution of 0.1 % formic acid (88 %) in 10 mM ammonium acetate. Separation of allopurinol and oxypurinol occurred in a flow of 0.8 mL/min on a C8 reverse phase column 5 μm, and acyclovir was the internal standard. The HPLC-MS/MS method was validated by reaching the limits of detection and quantification, reproducibility and linearity. The lower limit of quantification achieved by the method was 10 μg/mL for both allopurinol and oxypurinol. Calibration curves were prepared in blank urine added with allopurinol at concentrations of 10-1000 μg/mL, and oxypurinol at 10-200 μg/mL. Coefficients of variation of less than 15 % between intracurrent and intercurrent accuracy values were observed for both allopurinol and oxypurinol. Urine test samples remained stable after being subjected to freeze-thaw cycles and remaining at room temperature for 4 h. The method proved to be adequate to quantify allopurinol and oxypurinol in urine samples from dogs under treatment.
    Keywords:  Canine model; Leishmaniasis; Translational medicine; Urinary analysis; Validation
  33. J Sep Sci. 2020 Mar 07.
      In this study, a green mode of solidification of floating organic droplet-based dispersive liquid-liquid microextraction has been developed for the extraction of sixteen polycyclic aromatic hydrocarbons from honey samples before their determination by gas chromatography-mass spectrometry. In this method, an appropriate volume of menthol:decanoic acid deep eutectic solvent (as an extraction solvent) is added on a sugar cube (as a disperser agent). In the following, the cube is released into the diluted honey sample placed in a tube. After manual shaking a cloudy state is obtained as a result of dispersing the extraction solvent droplets throughout the sample solution and the analytes are extracted into them. After placing the tube in an ice bath, the droplet of the extractant is solidified on the top of the solution. This drop is taken and after dissolving in acetonitrile, an aliquot of the solution is injected into the separation system. Under optimum conditions, the suggested approach had high extraction recoveries (76-93%) and enrichment factors (380-465), low limits of detection (14-52 ng kg-1 ) and quantification (47-173 ng kg-1 ), and satisfactory repeatability (relative standard deviation ≤ 9%). This article is protected by copyright. All rights reserved.
    Keywords:  Deep eutectic solvent; Gas chromatography; Honey; Polycyclic aromatic hydrocarbon; Sugar cube