bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2020‒05‒17
thirty-four papers selected by
Sofia Costa
Cold Spring Harbor Laboratory

  1. Anal Chem. 2020 May 10.
      Unidentified peaks remain a major problem in untargeted metabolomics by LC-MS/MS. Confidence in peak annotations increases by combining MS/MS matching and retention time. We here show how retention times can be predicted from molecular structures. Two large, publicly available datasets were used for model training in machine learning: the Fiehn hydrophilic interaction liquid chromatography dataset (HILIC) of 981 primary metabolites and biogenic amines, and the RIKEN Plant Specialized Metabolome Annotation (PlaSMA) database of 852 secondary metabolites that uses reversed-phase liquid chromatography (RPLC). Five different machine learning algorithms have been integrated into the Retip R package: the random forest, Bayesian-regularized neural network, XGBoost, light gradient-boosting machine (LightGBM) and Keras algorithms for building the retention time prediction models. A complete workflow for retention time prediction was developed in R. It can be freely downloaded from the GitHub repository ( Keras outperformed other machine learning algorithms in the test set with minimum overfitting, verified by small error differences between training, test and validation sets. Keras yielded a mean absolute error (MAE) of 0.78 minutes for HILIC and 0.57 minutes for RPLC. Retip is integrated into the mass spectrometry software tools MS-DIAL and MS-FINDER, allowing a complete compound annotation workflow. In a test application on mouse blood plasma samples, we found a 68% reduction in the number of candidate structures when searching all isomers in MS-FINDER compound identification software. Retention time prediction increases the identification rate in liquid chromatography and subsequently leads to an improved biological interpretation of metabolomics data.
  2. Bioanalysis. 2020 May 15.
      Fat-soluble vitamins (FSVs) are micronutrients essential in maintaining normal physiological function, metabolism and human growth. Ongoing increased awareness regarding FSVs concentrations and their impact on human growth along with disease progression warrant the need of developing selective and sensitive analytical methods. LC-MS/MS is currently the method of choice for accurate quantitation of FSVs. However, there are multiple approaches for extraction, separation and calibration of FSVs in biological matrices. This review discusses recent Liquid chromatography-tandem mass spectrometry methods for the simultaneous quantification of FSVs in biological matrices and summarizes sample pretreatment procedures, chromatographic conditions and calibration approaches. Current challenges and clinical applications in various disease states are also highlighted.
    Keywords:  biological samples; fat-soluble vitamins ; high performance liquid chromatography; liquid chromatography-tandem mass spectrometry
  3. Anal Chem. 2020 May 13.
      Full-scan, data-dependent acquisition (DDA), and data-independent acquisition (DIA) are the three common data acquisition modes in high resolution mass spectrometry-based untargeted metabolomics. It is an important yet underrated research topic on which acquisition mode is more suitable for a given untargeted metabolomics application. In this work, we compared the three data acquisition techniques using a standard mixture of 134 endogenous metabolites and a human urine sample. Both hydrophilic interaction and reversed-phase liquid chromatographic separation along with positive and negative ionization modes were tested. Both the standard mixture and urine samples generated consistent results. Full-scan mode is able to capture the largest number of metabolic features, followed by DIA and DDA (53.7% and 64.8% respective features fewer on average in urine than full-scan). Comparing the MS2 spectra in DIA and DDA, spectra quality is higher in DDA with average dot product score 83.1% higher than DIA in Urine(H), and the number of MS2 spectra (spectra quantity) is larger in DIA (on average 97.8% more than DDA in urine). Moreover, a comparison of relative standard deviation distribution between modes shows consistency in the quantitative precision, with the exception of DDA showing a minor disadvantage (on average 19.8% and 26.8% fewer features in urine with RSD < 5% than full-scan and DIA). In terms of data preprocessing convenience, full-scan and DDA data can be processed by well-established software. In contrast, several bioinformatic issues remain to be addressed in processing DIA data and the development of more effective computational programs is highly demanded.
  4. J Sep Sci. 2020 May 13.
      In the following work, a new method for the analysis of the phthalate monoesters in human urine was reported. Phthalate monoesters are metabolites generated as a result of phthalate exposure. In compliance with the dictates of Green Analytical Chemistry, a rapid and simple protocol was developed and optimized for the quantification of phthalate monoesters (i.e., monoethyl phthalate, monoisobutyl phthalate, mono-n-butyl phthalate, mono-(2-ethylhexyl) phthalate, mono-n-octyl phthalate, monocyclohexyl phthalate, mono-isononyl phthalate) in human urine, which entails preceding derivatization with methyl chloroformate combined with the use of commercial solid phase microextraction and the analysis by gas chromatography-triple quadrupole mass spectrometry (GC-QqQ-MS). The affinity of the derivatized analytes toward five commercial coatings was evaluated, and in terms of analyte extraction, the best results were reached with the use of the divinylbenzene/carboxen/polydimethylsiloxane fiber. The multivariate approach of experimental design was used to seek for the best working conditions of the derivatization reaction and the solid phase microextraction extraction, thus obtaining the optimum response values. The proposed method was validated according to the guidelines issued by the Food and Drug Administration achieving satisfactory values in terms of linearity, sensitivity, matrix effect, intra- and inter-day accuracy, and precision. This article is protected by copyright. All rights reserved.
    Keywords:  gas chromatography; human urine; phthalate metabolites; solid phase microextraction; tandem mass spectrometry
  5. Metabolites. 2020 May 12. pii: E190. [Epub ahead of print]10(5):
      Metabolomics is increasingly important for biomedical research, but large-scale metabolite identification in untargeted metabolomics is still challenging. Here, we present Jumbo Mass spectrometry-based Program of Metabolomics (JUMPm) software, a streamlined software tool for identifying potential metabolite formulas and structures in mass spectrometry. During database search, the false discovery rate is evaluated by a target-decoy strategy, where the decoys are produced by breaking the octet rule of chemistry. We illustrated the utility of JUMPm by detecting metabolite formulas and structures from liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) analyses of unlabeled and stable-isotope labeled yeast samples. We also benchmarked the performance of JUMPm by analyzing a mixed sample from a commercially available metabolite library in both hydrophilic and hydrophobic LC-MS/MS. These analyses confirm that metabolite identification can be significantly improved by estimating the element composition in formulas using stable isotope labeling, or by introducing LC retention time during a spectral library search, which are incorporated into JUMPm functions. Finally, we compared the performance of JUMPm and two commonly used programs, Compound Discoverer 3.1 and MZmine 2, with respect to putative metabolite identifications. Our results indicate that JUMPm is an effective tool for metabolite identification of both unlabeled and labeled data in untargeted metabolomics.
    Keywords:  algorithm; database search; mass spectrometry; metabolite formula; metabolite identification; metabolite structure; metabolome; metabolomics; software; yeast
  6. J Pharm Biomed Anal. 2020 Apr 23. pii: S0731-7085(19)32529-4. [Epub ahead of print]186 113320
      Pethidine is an opiate agonist used orally and parenterally. Pethidine-containing drugs abuse is frequently encountered on health workers and patients. The analysis methods used to determine the abuse of pethidine are important for forensic toxicology. Pethidine is metabolized to norpethidine by the liver. Therefore, the determination of pethidine and norpethidine in urine is one of the methods to determine the abuse of pethidine. In this study, we have developed a precise, simple and rapid ultra-performance liquid chromatography-tandem mass spectrometer method for the determination of pethidine and norpethidine simultaneously. The developed method was validated in terms of selectivity and linearity which was in the range of 9-1800 ng/mL for both pethidine and norpethidine. The intra-assay and inter-assay accuracy and precision were found within acceptable limits of the EMA guideline. Lower limits of quantitation were 9 ng/mL for both pethidine and norpethidine. The developed method was successfully applied for the determination of both analytes in the real samples.
    Keywords:  Abuse of the drug; Method development and validation; Pethidine and norpethidine; Ultra-Performance liquid chromatography−tandem mass spectrometry; Urine samples
  7. Nucleic Acids Res. 2020 May 11. pii: gkaa332. [Epub ahead of print]
      The amount of biological data, generated with (single cell) omics technologies, is rapidly increasing, thereby exacerbating bottlenecks in the data analysis and interpretation of omics experiments. Data mining platforms that facilitate non-bioinformatician experimental scientists to analyze a wide range of experimental designs and data types can alleviate such bottlenecks, aiding in the exploration of (newly generated or publicly available) omics datasets. Here, we present BIOMEX, a browser-based software, designed to facilitate the Biological Interpretation Of Multi-omics EXperiments by bench scientists. BIOMEX integrates state-of-the-art statistical tools and field-tested algorithms into a flexible but well-defined workflow that accommodates metabolomics, transcriptomics, proteomics, mass cytometry and single cell data from different platforms and organisms. The BIOMEX workflow is accompanied by a manual and video tutorials that provide the necessary background to navigate the interface and get acquainted with the employed methods. BIOMEX guides the user through omics-tailored analyses, such as data pretreatment and normalization, dimensionality reduction, differential and enrichment analysis, pathway mapping, clustering, marker analysis, trajectory inference, meta-analysis and others. BIOMEX is fully interactive, allowing users to easily change parameters and generate customized plots exportable as high-quality publication-ready figures. BIOMEX is open source and freely available at
  8. J Anal Methods Chem. 2020 ;2020 2359397
      Isoflavones and isoflavandiols have shown many health benefits, such as reducing cardiovascular disease, cancer, age-related disease, and osteoporosis. However, to investigate the relationships between consumption of isoflavones and their health benefits, it is important to be able to accurately quantify exposure in the large numbers of samples typically produced in association studies (i.e., several thousands). Current methods rely on solid-phase extraction protocols for sample cleanup, resulting in protracted extraction and analysis times. Here, we describe a fast and easy sample preparation method of human urine samples for subsequent quantification of daidzein, genistein (isoflavones), and equol (isoflavandiol) using LC-MS/MS. Sample preparation involves only the addition of dimethylformamide (DMF) and formic acid (FA) after enzymatic hydrolysis of their metabolites by a β-glucuronidase and sulfatase mixture. The method was validated by precision, linearity, accuracy, recoveries, limit of detection (LOD), and limit of quantification (LOQ). Linear calibration curves have been shown by daidzein, genistein, and equol. The correlation coefficients values are r 2 > 0.99 for daidzein, genistein, and equol. LOD for daidzein and genistein was 1 ng/ml and equol was 2 ng/ml. Recoveries were >90%, and the relative standard deviation for intraday (<10%) and interday (≤20% over 10 days) was good. This method is suitable for quantification of isoflavones and the microbial metabolite equol in human urine and is particularly useful where large numbers of samples require analysis.
  9. Nat Protoc. 2020 May 13.
      Global Natural Product Social Molecular Networking (GNPS) is an interactive online small molecule-focused tandem mass spectrometry (MS2) data curation and analysis infrastructure. It is intended to provide as much chemical insight as possible into an untargeted MS2 dataset and to connect this chemical insight to the user's underlying biological questions. This can be performed within one liquid chromatography (LC)-MS2 experiment or at the repository scale. GNPS-MassIVE is a public data repository for untargeted MS2 data with sample information (metadata) and annotated MS2 spectra. These publicly accessible data can be annotated and updated with the GNPS infrastructure keeping a continuous record of all changes. This knowledge is disseminated across all public data; it is a living dataset. Molecular networking-one of the main analysis tools used within the GNPS platform-creates a structured data table that reflects the molecular diversity captured in tandem mass spectrometry experiments by computing the relationships of the MS2 spectra as spectral similarity. This protocol provides step-by-step instructions for creating reproducible, high-quality molecular networks. For training purposes, the reader is led through a 90- to 120-min procedure that starts by recalling an example public dataset and its sample information and proceeds to creating and interpreting a molecular network. Each data analysis job can be shared or cloned to disseminate the knowledge gained, thus propagating information that can lead to the discovery of molecules, metabolic pathways, and ecosystem/community interactions.
  10. Chem Soc Rev. 2020 May 12.
      Microbial and plant specialized metabolites constitute an immense chemical diversity, and play key roles in mediating ecological interactions between organisms. Also referred to as natural products, they have been widely applied in medicine, agriculture, cosmetic and food industries. Traditionally, the main discovery strategies have centered around the use of activity-guided fractionation of metabolite extracts. Increasingly, omics data is being used to complement this, as it has the potential to reduce rediscovery rates, guide experimental work towards the most promising metabolites, and identify enzymatic pathways that enable their biosynthetic production. In recent years, genomic and metabolomic analyses of specialized metabolic diversity have been scaled up to study thousands of samples simultaneously. Here, we survey data analysis technologies that facilitate the effective exploration of large genomic and metabolomic datasets, and discuss various emerging strategies to integrate these two types of omics data in order to further accelerate discovery.
  11. J Chromatogr A. 2020 Apr 27. pii: S0021-9673(20)30361-7. [Epub ahead of print] 461132
      A novel and simple derivatization method using a series of amide acetals as derivatization reagents, along with gas chromatography tandem mass spectrometry (GC-MS/MS) analysis, were developed and validated for simultaneous determination of 9 perfluoroalkyl carboxylic acids (PFCAs) in this study. The structures and fragmentation pathway of PFCAs derivatives were deduced and verified. The derivatization method developed in this study improved the sensitivity of the detection of PFCAs by GC. The applicability of 6 amide acetals for the derivatization of PFCAs was demonstrated. Derivatization conditions for 9 PFCAs were optimized with addition of 20 μL of derivatization reagent and reaction at 35 °C for 30 min. 9 PFCAs derivatives were confirmed to be stable over 15 days. The instrument detection limits (IDLs) were lower than 0.01 pg/μL. The intra and inter-day precisions were below 4.06% and 5.48%, respectively. To demonstrate the utility of the derivatization method, the level of PFCAs in the marine products were detected. The alkaline digestion followed by solid-phase extraction (SPE) cleanup method was used for pretreatment. The method detection limits (MDLs) ranged from 0.04 to 0.10 ng/g, and the spiked recoveries ranged between 54.72% and 107.29%, with relative standard deviation (RSD) of 1.53%-11.89%. Five PFCAs were detected in the range of 0.66 to 499.03 ng/g dry weight.
    Keywords:  Amide acetals; Derivatization; Gas chromatography tandem mass spectrometry; Perfluoroalkyl carboxylic acids
  12. Rapid Commun Mass Spectrom. 2020 May 15.
      RATIONALE: Fatty acid esters of hydroxy fatty acids (FAHFAs) are recently discovered endogenous lipids with outstanding health benefits. FAHFAs are known to exhibit antioxidant, antidiabetic and anti-inflammatory properties. The number of known long chain FAHFAs in mammalian tissues and dietary resources increased recently because of the latest developments in high-resolution tandem mass spectrometry techniques. However, there are no reports on the identification of short chain fatty acid esterified hydroxy fatty acids (SFAHFAs).METHODS: Intestinal contents, tissues, and plasma of rats fed with high-fat diet (HFD) and normal diet (ND) were analysed for fatty acids, hydroxy fatty acids, and FAHFAs using ultra-high-performance liquid chromatography (UHPLC) and linear trap quadrupole-Orbitrap mass spectrometry (LTQ Orbitrap MS) with negative heated electrospray ionization.
    RESULTS: Untargeted analysis of total lipid extracts from murine samples (male 13-week old WKAH/HKmSlc rats) led to the identification of several new SFAHFAs of acetic acid or propanoic acid esterified long chain (>C20)-hydroxy fatty acids. Furthermore, MS3 analysis revealed the position of the hydroxyl group in the long chain fatty acid as C-2. The relative amounts of SFAHFAs were quantified in intestinal contents and their tissues (caecum, small intestine, and large intestine), liver, and plasma of rats fed with HFD and ND. The large intestine showed the highest abundance of SFAHFAs with a concentration range from 0.84 to 57 pmoles/mg followed by the cecum with a range of 0.66 to 28.6 pmoles/mg. The SFAHFAs were significantly altered between the HFD and ND groups, with a strong decreasing tendency under HFD conditions.
    CONCLUSIONS: Identification of these novel SFHFAs can contribute to better understanding the chemical and biological properties of individual SFHFAs and their possible sources in the gut, which in turn helps us tackle the role of these lipids in various metabolic diseases.
  13. J Chromatogr A. 2020 Jan 16. pii: S0021-9673(20)30072-8. [Epub ahead of print] 460895
      Baseline separation and analysis of multicomponent mixtures of closely related pharmaceuticals using single column selectivity can often be challenging, requiring the combination of orthogonal stationary and mobile phase methods to monitor all the species and optimize reaction outcomes. In recent years, two-dimensional liquid chromatography (2D-LC) has become a valuable tool for improving peak capacity and selectivity. Though powerful, standard 2D-LC instrumentation and software can often lead to tedious method development and has a requirement for very specific expertise that is poorly suited for a fast-paced industrial environment. In this regard, the introduction of an automated online 2D-LC setup that could screen multiple columns in both dimensions without manual intervention will undeniably serve to streamline column/mobile phase selection and secure the viability of 2D-LC as a mainstay instrument for industrial applications. Herein, we introduce and investigate a multicolumn online 2D-LC approach that simplifies column screening and method development dramatically. This setup incorporates 6-position column selection valve technology whose functionality enables us to combine multiple columns in the first and second dimensions. This strategy in conjunction with diode array detection (DAD) in both dimensions and mass spectrometry (MS) acquisition in the second dimension serves to explore different columns and mobile phases as a framework for screening targeted compounds in multicomponent mixtures without having to perform chromatographic purification. Multiple online heart cutting achiral RPLC - achiral RPLC and achiral RPLC - chiral RPLC coupled to DAD and ESI-MS methods combining several stationary phase selectivity in an automated fashion are successfully applied to the separation and analysis of complex mixtures of drug substances, where in many instances, traditional 1D-ultra-high performance liquid chromatography (UHPLC) fails or delivers sub-optimal results. This automated online multicolumn 2D-LC workflow enables rapid and efficient identification of column/eluent combinations, as well as sample analysis across multiple columns in both dimensions overnight with a single click.
    Keywords:  Achiral and chiral separations; Automated multicolumn screening; Closely related species; Diode array detection- Mass spectrometry; Method development; Multicomponent mixtures; Two-dimensional liquid chromatography
  14. Methods Mol Biol. 2020 ;2144 111-123
      Gas chromatography-mass spectrometry (GC-MS) enables sensitive detection and relative quantification of fatty acids. In Caenorhabditis elegans, the use of GC-MS can corroborate findings from common staining methodologies, providing great resolution on the lipid species altered in abundance in aging, genetic mutants, or with dietary or pharmacologic manipulation. Here we describe a method to quantitate relative abundance of fatty acids in total worm lipid extracts, as well as a method that quantitates fatty acids following separation into neutral lipid pools (triacylglycerols and cholesteryl esters) versus more polar lipids (phospholipids) by solid-phase extraction (SPE).
    Keywords:  Aging; C. elegans; Fat; Fatty acids; Gas chromatography–mass spectrometry; Lipids; Phospholipids; Solid-phase chromatography; Triglycerides
  15. Food Chem. 2020 Apr 16. pii: S0308-8146(20)30697-X. [Epub ahead of print]325 126835
      The impact of secondary metabolites on fruit quality, plant growth and human health has led to an increased demand for analytical methods to characterize and quantify these metabolites in recent years. A versatile, sensitive and rapid method based on UHPLC-QqQ-MS/MS was developed for simultaneous qualitation and quantitation of coumarins, furocoumarins, flavonoids and phenolic acids. The chromatographic elution and multiple reaction monitoring mode transitions were optimized to achieve good separation and accurate quantitation of 47 analytes, including 13 groups of isomers, during a single 13 min chromatographic run. This method was validated with good precision and recoveries, wide linear ranges and low limits of detection and quantitation (0.014-1.50 μg L-1). The validated method was further applied to quantify the analytes in flavedo, albedo and pulp from two pummelo varieties, C. grandis 'Shatianyu' and C. grandis 'Guanximiyu'. This method combines high sensitivity, good selectivity, and short chromatographic run time.
    Keywords:  Coumarins; Flavonoids; Phenolic acids; Pummelo; UHPLC-QqQ-MS/MS
  16. Biotechnol Prog. 2020 May 10. e3015
      The first step of many metabolomics studies is quenching, a technique vital for rapidly halting metabolism and ensuring that the metabolite profile remains unchanging during sample processing. The most widely used approach is to plunge the sample into pre-chilled cold methanol, however, this led to significant metabolite loss in Synecheococcus sp. PCC 7002. Here we describe our analysis of the impacts of cold methanol quenching on the model marine cyanobacterium Synechococcus sp. PCC 7002, as well as our brief investigation of alternative quenching methods. We tested several methods including cold methanol, cold saline, and two filtration approaches. Targeted central metabolites were extracted and metabolomic profiles were generated using liquid chromatography mass spectrometry (LC-MS/MS). The results indicate that cold methanol quenching induces dramatic metabolite leakage in Synechococcus, resulting in a majority of central metabolites being lost prior to extraction. Alternatively, usage of a chilled saline quenching solution mitigates metabolite leakage and improves sample recovery without sacrificing rapid quenching of cellular metabolism. Finally, we illustrate that metabolite leakage can be assessed, and subsequently accounted for, in order to determine absolute metabolite pool sizes; however, our results show that metabolite leakage is inconsistent across various metabolite pools and therefore must be determined for each individually measured metabolite.
    Keywords:  Synechococcus; intracellular; metabolite leakage; metabolomics; methanol quenching; sampling techniques
  17. Curr Opin Plant Biol. 2020 May 06. pii: S1369-5266(20)30029-7. [Epub ahead of print]55 84-92
      The exact mechanics of specialized metabolism and its importance throughout plant evolution remain mysterious. Specialized metabolites and their corresponding biosynthetic genes are crucial to understand the reason for the prevalence of certain metabolism. Even though mass spectrometry-based metabolomics has enabled us to acquire data about the structural properties of unknown specialized metabolites as well as known metabolites and their corresponding isomers/analogs, extensive analytical approaches are still required. Herein, we review the most advanced analytical approaches using stable isotope labeling that can be used to identify the unknown specialized metabolites.
  18. Prostaglandins Leukot Essent Fatty Acids. 2020 Apr 15. pii: S0952-3278(20)30054-5. [Epub ahead of print]157 102096
      The blood fraction most commonly used for the assessment of fatty acid status is the plasma or serum phospholipids, since these provide a measure of long term dietary intake. However, conventional assays of fatty acid status in human plasma and serum require labour intensive, multi-step approaches, which are impractical for high-throughput analyses. This study reports a system capable of selectively separating plasma phospholipids from other lipid classes in just a few minutes. We demonstrate that compositional analysis of the fatty acids in plasma phospholipids using our plasma spot method resulted in greater than 97% of neutral lipid standards had been eluted from the paper, whereas more than 96% of the PC remained on the paper. These results were almost identical to conventional methods involving liquid/liquid extraction and thin layer chromatography separation. Evaluation of our plasma spot fractionation and assay using plasma from 110 human subjects (75 males, 35 females), provides confirmation of significant correlations between the fatty acid measures and those obtained from conventional measures for all fatty acids (r > 0.97, P<0.0001), including the omega (n)-6 (r = 0.988, P<0.0001) and n-3 long chain polyunsaturated fatty acids (r = 0.997, P<0.0001). These results establish our newly developed plasma spot separation technique as a rapid and reliable method for the assessment of plasma phospholipid fatty acid composition.
    Keywords:  Dried plasma spot; LCPUFA; Silica gel coated paper; phospholipids
  19. J Sep Sci. 2020 May 12.
      Magnetic dispersive solid-phase extraction followed by dispersive liquid-liquid microextraction coupled with GC/MS was applied for the quantitative analysis of phenazopyridine in urinary samples. Magnetic dispersive solid-phase extraction was carried out using magnetic graphene oxide nanoparticles modified by poly(thiophene-pyrrole) co-polymer. The eluting solvent of this step was used as the disperser solvent for the dispersive liquid-liquid microextraction procedure. To reach the maximum efficiency of the method, effective parameters including sorbent amount, adsorption time, type and volume of disperser and extraction solvents, pH of the sample solution, and ionic strength as well as desorption time and approach were optimized, separately. Characterization of the synthesized sorbent was studied by utilizing infrared spectroscopy, scanning electron microscopy, and energy-dispersive X-ray analysis. Calibration curve was linear in the range of 0.5-250 ng/mL (R2 = 0.9988) with LOD and LOQ of 0.1 and 0.5 ng/mL, respectively. Intra- and inter-day precisions (RSD%, n = 3) of the method were in the range of 4.6-5.4% and 4.0-5.5%, respectively, at three different concentration levels. Under the optimal condition, this method was successfully applied for the determination of phenazopyridine in human urine samples. The relative recoveries were obtained in the range of 85.0-89.0%. This article is protected by copyright. All rights reserved.
    Keywords:  dispersive solid phase extraction; magnetic graphene oxide; microextraction; phenazopyridine; urine
  20. Anal Chim Acta. 2020 Jun 22. pii: S0003-2670(20)30105-7. [Epub ahead of print]1117 74-88
      Ambient mass spectrometry imaging (AMSI) is a molecular imaging technique developed in recent years for in situ and real time visualization of the distribution of chemical compounds in biological tissues without the need of labeling or staining. With the development for more than one decade, AMSI becomes a powerful molecular imaging technique in variousfields such as forensics, metabolomics, cancer diagnosis, and drug monitoring. In this review, we describe the recent advances of AMSI for imaging biological tissues in details. Three types of AMSI techniques based on different ionization mechanisms and analytical strategies are summarized, i.e., direct desorption/ionization of analytes for AMSI, desorption and then ionization of analytes for AMSI, and extraction of analytes for AMSI, and the features of them are presented from the aspects of tissue origin, target image molecule, and spatial resolution among others. In addition, future development directions for AMSI are discussed.
    Keywords:  Ambient ionization; Biological tissue; Imaging; Mass spectrometry; Molecular distribution
  21. J Appl Lab Med. 2020 May 14. pii: jfaa022. [Epub ahead of print]
      OBJECTIVES: Sphingolipids have been demonstrated to be involved in many human diseases. However, measurement of sphingolipids, especially of sphingosine 1-phosphate (S1P) and dihydro-sphingosine 1-phosphate (dhS1P), in blood samples requires strict sampling, since blood cells easily secrete these substances during sampling and storage, making it difficult to introduce measurement of sphingolipids in clinical laboratory medicine. On the other hand, cerebrospinal fluid (CSF) contains few blood cells. Therefore, we attempted to establish a system based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the measurement of sphingolipids in the CSF, and applied it for the diagnosis of carcinomatous meningitis.METHODS: We developed and validated a LC-MS/MS-based measurement system for S1P and dhS1P and for ceramides and sphingosines, used this system to measure the levels of these sphingolipids in the CSF collected from the subjects with cancerous meningitis, and compared the levels with those in normal routine CSF samples.
    RESULTS: Both the measurement systems for S1P/dhS1P and for ceramides/sphingosines provided precision with the coefficient of variation below 20% for sphingolipids in the CSF samples. We also confirmed that the levels of S1P, as well as ceramides/sphingosines, in the CSF samples did not increase after the sampling. In the CSF samples collected from patients with cancerous meningitis, we observed that the ratio of S1P to ceramides/sphingosine and that of dhS1P to dihydro-sphingosine were higher than those in control samples.
    CONCLUSIONS: We established and validated a measurement system for sphingolipids in the CSF. The system offers promise for being introduced into clinical laboratory testing.
    Keywords:  carcinomatous meningitis; ceramides; cerebrospinal fluid; sphingosine 1-phosphate
  22. Chem Commun (Camb). 2020 May 15.
      We report for the first time the integration of ultra-high-pressure liquid chromatography-tandem mass spectrometry with machine learning for identifying fatty acid metabolite biomarkers of ischemic stroke. In particular, we develop an optimal model to discriminate ischemic stroke patients from healthy persons with 100% sensitivity and 93.18% specificity. This research may facilitate understanding the roles of fatty acid metabolites in stroke occurrence, holding great potential in clinical stroke diagnosis.
  23. Cells. 2020 May 12. pii: E1197. [Epub ahead of print]9(5):
      Altered lipid metabolism has been associated to cystic fibrosis disease, which is characterized by chronic lung inflammation and various organs dysfunction. Here, we present the validation of an untargeted lipidomics approach based on high-resolution mass spectrometry aimed at identifying those lipid species that unequivocally sign CF pathophysiology. Of n.13375 mass spectra recorded on cystic fibrosis bronchial epithelial airways epithelial cells IB3, n.7787 presented the MS/MS data, and, after software and manual validation, the final number of annotated lipids was restricted to n.1159. On these lipids, univariate and multivariate statistical approaches were employed in order to select relevant lipids for cellular phenotype discrimination between cystic fibrosis and HBE healthy cells. In cystic fibrosis IB3 cells, a pervasive alteration in the lipid metabolism revealed changes in the classes of ether-linked phospholipids, cholesterol esters, and glycosylated sphingolipids. Through functions association, it was evidenced that lipids variation involves the moiety implicated in membrane composition, endoplasmic reticulum, mitochondria compartments, and chemical and biophysical lipids properties. This study provides a new perspective in understanding the pathogenesis of cystic fibrosis and strengthens the need to use a validated mass spectrometry-based lipidomics approach for the discovery of potential biomarkers and perturbed metabolism.
    Keywords:  OMICS; biomarker; cell structure; cystic fibrosis; lipidomics; membrane composition; sphingolipid; untargeted analysis
  24. Analyst. 2020 May 12.
      Metabolites with ketone or aldehyde functionalities comprise a large proportion of the human metabolome, most notably in the form of sugars. However, these reactive molecules are also generated through oxidative stress or gut microbiota metabolism and have been linked to disease development. The discovery and structural validation of this class of metabolites over the large concentration range found in human samples is crucial to identify their links to pathogenesis. Herein, we have utilized an advanced chemoselective probe methodology alongside bioinformatic analysis to identify carbonyl-metabolites in urine and fecal samples. In total, 99 metabolites were identified in urine samples and the chemical structure for 40 metabolites were unambiguously validated using a co-injection procedure. We also describe the preparation of a metabolite-conjugate library of 94 compounds utilized to efficiently validate these ketones and aldehydes. This method was used to validate 33 metabolites in a pooled fecal sample extract to demonstrate the potential for rapid and efficient metabolite detection over a wide metabolite concentration range. This analysis revealed the presence of six metabolites that have not previously been detected in either sample type. The constructed library can be utilized for straightforward, large-scale, and expeditious analysis of carbonyls in any sample type.
  25. Trends Biochem Sci. 2020 Jun;pii: S0968-0004(20)30059-1. [Epub ahead of print]45(6): 545-546
  26. Molecules. 2020 May 10. pii: E2250. [Epub ahead of print]25(9):
      Two scan modes of the triple quadrupole tandem mass spectrometer, namely Collision Induced Dissociation Precursor Ion scan and Neutral Loss scan, allow selectively pinpointing, in a complex mixture, compounds that feature specific chemical groups, which yield characteristic fragment ions or are lost as distinctive neutral fragments. This feature of the triple quadrupole tandem mass spectrometer allows the non-target screening of mixtures for classes of components. The effective (center-of-mass) energy to achieve specific fragmentation depends on the inter-quadrupole voltage (laboratory-frame collision energy) and on the masses of the precursor molecular ion and of the collision gas, through a non-linear relationship. Thus, in a class of homologous compounds, precursor ions activated at the same laboratory-frame collision energy face different center-of-mass collision energy, and therefore the same fragmentation channel operates with different degrees of efficiency. This article reports a linear equation to calculate the laboratory-frame collision energy necessary to operate Collision-Induced Dissociation at the same center-of-mass energy on closely related compounds with different molecular mass. A routine triple quadrupole tandem mass spectrometer can operate this novel feature (iso-energetic collision-induced dissociation scan; i-CID) to analyze mixtures of endogenous metabolites by Precursor Ion and Neutral Loss scans. The latter experiment also entails the hitherto unprecedented synchronized scanning of all three quadrupoles of the triple quadrupole tandem mass spectrometer. To exemplify the application of this technique, this article shows two proof-of-principle approaches to the determination of biological mixtures, one by Precursor Ion analysis on alpha amino acid derivatized with a popular chromophore, and the other on modified nucleosides with a Neutral Fragment Loss scan.
    Keywords:  HPLC; MS/MS; NTS techniques (separation, ionization, and detection); amino acids; equation; nucleosides; open access software; target gas; triple quadrupole
  27. Foods. 2020 May 09. pii: E609. [Epub ahead of print]9(5):
      Monitoring of biogenic amines in food is important for quality control, in terms of freshness evaluation and even more for food safety. A novel and cost-effective method was developed and validated for the determination of the main biogenic amines: histamine, putrescine, cadaverine, spermidine and spermine in fish tissues. The method includes extraction of amines with perchloric acid, pre-column derivatization with Pyrene Sulfonyl Chloride (PSCl), extraction of derivatives with toluene, back-dissolution in ACN after evaporation and determination by reversed phase high performance liquid chromatography with UV and intramolecular excimer fluorescence detection. The structure of the pyrene-derivatives was confirmed by liquid chromatography-mass spectrometry with electrospray ionization. The standard addition technique was applied for the quantitation due to significant matrix effect, while the use of 1,7-diaminoheptane as internal standard offered an additional confirmation tool for the identification of the analytes. Method repeatability expressed as %RSD ranged between 7.4-14% for the different amines and recovery ranged from 67% for histamine up to 114% for spermine. The limits of detection ranged between 0.1-1.4 mg kg-1 and the limits of quantification between 0.3-4.2 mg kg-1. The method was applied to canned fish samples and the concentrations of the individual biogenic amines were below the detection limit up to 40.1 mg kg-1, while their sum was within the range 4.1-49.6 mg kg-1.
    Keywords:  HPLC; bioamines; dietary exposure; excimer fluorescence; intramolecular excitement; polyamines; pyrene probe; scombroid poisoning; seafood
  28. Metabolites. 2020 May 07. pii: E186. [Epub ahead of print]10(5):
      Liquid chromatography coupled to high-resolution mass spectrometry platforms are increasingly employed to comprehensively measure metabolome changes in systems biology and complex diseases. Over the past decade, several powerful computational pipelines have been developed for spectral processing, annotation, and analysis. However, significant obstacles remain with regard to parameter settings, computational efficiencies, batch effects, and functional interpretations. Here, we introduce MetaboAnalystR 3.0, a significantly improved pipeline with three key new features: (1) efficient parameter optimization for peak picking; (2) automated batch effect correction; and 3) more accurate pathway activity prediction. Our benchmark studies showed that this workflow was 20~100X faster compared to other well-established workflows and produced more biologically meaningful results. In summary, MetaboAnalystR 3.0 offers an efficient pipeline to support high-throughput global metabolomics in the open-source R environment.
    Keywords:  batch effects; global metabolomics; pathway activity prediction; peak detection
  29. Anal Chem. 2020 May 11.
      The present project studied the signal drift in liquid chromatography tandem mass spectrometry (LC-MS/MS) and proposed a strategy for compensating such drift. In the study, four 4-component groups were repeatedly run on different LC-MS/MS systems for over 12 hours to investigate the dependence of signal drift on time and hardware systems. The 4-component groups each consisted of 1) an analyte, 2) a stable isotope labeled analyte, 3) a compound with similar structure to the analyte, and 4) a compound with dissimilar structure. All the species showed significant signal drift, generally more than 25% over 12 hours. The analyte and its stable isotope labeled analog always have the same drifting pattern including the trends and direction from one LC-MS/MS system to another. Signal drift was also found to be concentration dependent. Our experiments further proved that conventional stable isotope labeled internal standard in LC-MS/MS quantification would not compensate the variations caused by concentration-dependent signal drift. An ideal internal standard for LC-MS/MS has both identical structure and similar concentration to the analyte. For that, we proposed a new internal standard strategy, pseudo internal standard (Pseudo IS), for LC-MS/MS quantification. Pseudo IS could effectively compensate signal drift in spite of its significant time, system, and concentration dependencies.
  30. Molecules. 2020 May 07. pii: E2189. [Epub ahead of print]25(9):
      The properties of teak wood, such as natural durability and beautiful color, are closely associated with wood extractives. In order to further understand the performance differences between teak heartwood and sapwood, we analyzed the chemical components of extractives from 12 wood samples using an ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS)-based metabolomics approach. In total, 691 metabolites were identified, and these were classified into 17 different categories. Clustering analysis and principal component analysis of metabolites showed that heartwood samples could be clearly separated from sapwood samples. Differential metabolite analysis revealed that the levels of primary metabolites, including carbohydrates, amino acids, lipids, and nucleotides, were significantly lower in the heartwood than in the sapwood. Conversely, many secondary metabolites, including flavonoids, phenylpropanoids, and quinones, had higher levels in the heartwood than in the sapwood. In addition, we detected 16 specifically expressed secondary metabolites in the heartwood, the presence of which may correlate with the durability and color of teak heartwood. Our study improves the understanding of differential metabolites between sapwood and heartwood of teak and provides a reference for the study of heartwood formation.
    Keywords:  Tectona grandis; UPLC–ESI–MS/MS-based metabolomics; heartwood; sapwood
  31. Forensic Sci Int. 2019 ;1 214-220
      Paper spray mass spectrometry is a rapid and sensitive tool for explosives detection but has so far only been demonstrated using high resolution mass spectrometry, which bears too high a cost for many practical applications. Here we explore the potential for paper spray to be implemented in field applications with portable mass spectrometry. This involved (a) replacing the paper substrate with a swabbing material (which we call "swab spray") for compatibility with standard collection materials; (b) collection of explosives from surfaces; (c) an exploration of interferences within a ± 0.5 m/z window; and (d) demonstration of the use of high-field assisted waveform ion mobility spectrometer (FAIMS) for enhanced selectivity. We show that paper and Nomex® are viable collection materials, with Nomex providing cleaner spectra and therefore greater potential for integration with portable mass spectrometers. We show that sensitive detection using swab spray will require a mass spectrometer with a mass resolving power of 4000 or more. We show that by coupling the swab spray ionisation source with FAIMS, it is possible to reduce background interferences, thereby facilitating the use of a low resolving power (e.g. quadrupole) mass spectrometer.
    Keywords:  Explosives; FAIMS; Mass spectrometry; Swab spray
  32. Mikrochim Acta. 2020 May 12. 187(6): 326
      A highly oriented crystalline metal-organic framework (MOF) nanofilm array was prepared and used for surface-assisted laser desorption ionization mass spectrometry (SALDI-MS) for determination of small molecules. The MOF nanofilm was characterized using scanning/transmission electron microscopy, X-ray diffraction, and ultraviolet-visible spectroscopy. Different small molecules (anthracene, n-eicosanoic acid, Rhodamine B) were successfully determined by this MOF nanofilm array with limits of detection (LOD) between 0.1-5 ng·mL-1 and limits of quantification (LOQ) between 1 and 10 ng·mL-1. Compared to previously reported MOF-based SALDI-MS, this array exhibits better reproducibility (3.3-5.2%) and recovery (89-105%). The intensity of the MS peak remains the same after 25 repeated cycles. This indicates good repeatability. This MOF nanofilm-based SALDI-MS array can be used for determination of fatty acid and Rhodamine B in real samples with good recovery (83-106%). Graphical abstract Schematic representation of the principle of crystalline MOFs nanofilm-based SALDI-MS array.
    Keywords:  Fatty acids; Laser desorption ionization; Nanosheet; PAHs; Rhodamine B
  33. Anal Chem. 2020 May 15.
      The profile of cholesteryl esters (CEs) is increasingly used in metabolic disease monitoring due to the roles of CE in regulating the cholesterol level. While electrospray ionization-tandem mass spectrometry is routinely applied for the identification and quantitation of CE, it has a limitation of not being able to provide the location of carbon-carbon double bond (C = C) within unsaturated fatty acyls. In this study, we paired offline 2-acetylpyridine (2-AP) Paternò-Büchi (PB) reaction and reversed-phase liquid chromatography tandem mass spectrometry (RPLC-MS/MS) to achieve highly sensitive and structural informative CE analysis from complex mixtures. The 2-AP PB reactions of CE standards provided 20-30% conversion but resulted in enhanced ion signal relative to that of intact CE detected as ammonium adduct ions. MS/MS of 2-AP derivatized CE via collision-induced dissociation produced two abundant diagnostic ions for each C = C in a fatty acyl, leading to both sensitive identification and quantitation of C = C location isomers. Twelve saturated and twenty-seven unsaturated CEs were profiled in pooled human plasma; of the latter group, relative quantitation of 6 groups of C=C location isomers was achieved. A dehydrocholesteryl ester, DHE 18:2(Δ9,12), was confidently differentiated from co-existing compositional isomers: CE 18:3 (Δ9,12,15) and CE 18:3 (Δ6,9,12). The above results represented improved CE coverage at C = C location level than those reported by gas-chromatography MS or acetone PB-MS/MS methods.
  34. J Proteome Res. 2020 May 13.
      Breast cancer (BC) is a heterogeneous malignancy that is responsible for a great portion of female cancer cases and cancer-related deaths in the United States. In comparison to other major BC subtypes, triple negative breast cancer (TNBC) presents with a relatively low survival rate and a high rate of metastasis. This has led to a strong, though largely unmet, need for more sensitive and specific methods of early stage TNBC (ES-TNBC) detection to combat its high-grade pathology and relatively low survival rate. The current study employs a liquid chromatography-tandem mass spectrometry assay capable of targeted, highly specific and sensitive detection of lipids to propose two diagnostic biomarker panels for TNBC/ES-TNBC. Using this approach, 110 lipids were reliably detected in 166 human plasma samples, 45 controls and 121 BC (96 non-TNBC and 25 TNBC) subjects. Univariate and multivariate analyses allowed the construction and application of a 19-lipid biomarker panel capable of distinguishing TNBC (and ES-TNBC) from controls, as well as, a 5-lipid biomarker panel capable of differentiating TNBC from non-TNBC and ES-TNBC from ES-non-TNBC. Receiver operating characteristic curves with notable classification performances were generated from the biomarker panels according to their orthogonal partial least squares-discrimination analysis models. TNBC was distinguished from controls with an area under the receiving operating characteristic curve (AUROC) = 0.93, sensitivity = 0.96, specificity = 0.76, and ES-TNBC from controls with an AUROC = 0.96, sensitivity = 0.95, and specificity = 0.89. TNBC was differentiated from non-TNBC with an AUROC = 0.88, sensitivity = 0.88, specificity = 0.79, and ES-TNBC from ES-non-TNBC with an AUROC = 0.95, sensitivity = 0.95, and specificity = 0.87. A pathway enrichment analysis between TNBC and controls also revealed significant disturbances in choline metabolism, sphingolipid signaling, and glycerophospholipid metabolism. To the best of our knowledge, this is the first study to propose a diagnostic lipid biomarker panel for TNBC detection. All raw mass spectrometry data have been deposited to MassIVE (dataset identifier: MSV000085324).