bims-metlip 2019-10-13 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2019–10–13
29 papers selected by
Sofia Costa

The Power of LC-MS Based Multiomics: Exploring Adipogenic Differentiation of Human Mesenchymal Stem/Stromal Cells.
In Silico Optimization of Mass Spectrometry Fragmentation Strategies in Metabolomics.
[UPLC-Q-TOF-MS-based metabolomics study of celastrol].
Uniformly 15N-Labeled Recombinant Ricin A-chain as an Internal Retention Time Standard for Increased Confidence in Forensic Identification of Ricin by Untargeted Nanoflow Liquid Chromatography-Tandem Mass Spectrometry.
A simultaneously quantitative profiling method for 40 endogenous amino acids and derivatives in cell lines using hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry.
Rapid Quantitation of 25-Hydroxyvitamin D2 and D3 in Human Serum Using Liquid Chromatography/drift Tube Ion Mobility-Mass Spectrometry.
Expanding the mass range for UVPD-based native top-down mass spectrometry.
Simultaneous determination of cardiovascular drugs in dried blood spot by liquid chromatography-tandem mass spectrometry.
Identification of metabolites from tandem mass spectra with a machine learning approach utilizing structural features.
Unsupervised machine learning for exploratory data analysis in imaging mass spectrometry.
Sensitive analysis of multiple low-molecular-weight thiols in a single human cervical cancer cell by chemical derivatization-liquid chromatography-mass spectrometry.
Hybrid Search: A Method for Identifying Metabolites Absent from Tandem Mass Spectrometry Libraries.
Derivatization-free determination of short-chain volatile amines in human plasma and urine by headspace gas chromatography-mass spectrometry.
Metabolomics: a search for biomarkers of visceral fat and liver fat content.
Regiospecific analysis of triacylglycerols by ultra-high-performance-liquid chromatography-electrospray ionization-tandem mass spectrometry.
High-resolution capillary zone electrophoresis and mass spectrometry for distinction of undersialylated and hypoglycosylated transferrin glycoforms in body fluids.
Identification of metabolomic changes in horse plasma after racing by liquid chromatography-high resolution mass spectrometry as a strategy for doping testing.
A Validated Ultra-Performance Liquid Chromatographic Method for the Simultaneous Determination of Nadifloxacin, Mometasone Furoate and Miconazole Nitrate in Their Combined Dosage Form and Spiked Human Plasma Samples.
Ultra-performance liquid chromatography-tandem mass spectrometry quantitative profiling of tryptophan metabolites in human plasma and its application to clinical study.
MetumpX - A Metabolomics Support Package for Untargeted Mass Spectrometry.
Family-wide Annotation of Enzymatic Pathways by Parallel In Vivo Metabolomics.
Identification of a biomarker panel for improvement of prostate cancer diagnosis by volatile metabolic profiling of urine.
An LCMS-based untargeted metabolomics protocol for cochlear perilymph: highlighting metabolic effects of hydrogen gas on the inner ear of noise exposed Guinea pigs.
High Performance Molecular Imaging with MALDI Trapped Ion Mobility Time-of-Flight (timsTOF) Mass Spectrometry.
Dispersive solid phase extraction combined with dispersive liquid-liquid microextraction for simultaneous determination of seven succinate dehydrogenase inhibitor fungicides in watermelon by ultra-performance liquid chromatography/tandem mass spectrometry.
Random forest-based imputation outperforms other methods for imputing LC-MS metabolomics data: a comparative study.
[Determination of 5 kinds of fish anesthetics residues in fish by ultra-high performance liquid chromatography-tandem mass spectrometry].
Quantitative analysis of underivatized 17 β-estradiol using a high-throughput LC-MS/MS assay - Application to support a pharmacokinetic study in ovariectomized guinea pigs.
Metabolomics of the aqueous humor in patients with primary congenital glaucoma.

Molecules. 2019 Oct 08. pii: E3615. [Epub ahead of print]24(19):

The Power of LC-MS Based Multiomics: Exploring Adipogenic Differentiation of Human Mesenchymal Stem/Stromal Cells.

Evelyn Rampler, Dominik Egger, Harald Schoeny, Mate Rusz, Maria Pires Pacheco, Giada Marino, Cornelia Kasper, Thomas Naegele, Gunda Koellensperger.

  The molecular study of fat cell development in the human body is essential for our understanding of obesity and related diseases. Mesenchymal stem/stromal cells (MSC) are the ideal source to study fat formation as they are the progenitors of adipocytes. In this work, we used human MSCs, received from surgery waste, and differentiated them into fat adipocytes. The combination of several layers of information coming from lipidomics, metabolomics and proteomics enabled network analysis of the biochemical pathways in adipogenesis. Simultaneous analysis of metabolites, lipids, and proteins in cell culture is challenging due to the compound's chemical difference, so most studies involve separate analysis with unimolecular strategies. In this study, we employed a multimolecular approach using a two-phase extraction to monitor the crosstalk between lipid metabolism and protein-based signaling in a single sample (~105 cells). We developed an innovative analytical workflow including standardization with in-house produced 13C isotopically labeled compounds, hyphenated high-end mass spectrometry (high-resolution Orbitrap MS), and chromatography (HILIC, RP) for simultaneous untargeted screening and targeted quantification. Metabolite and lipid concentrations ranged over three to four orders of magnitude and were detected down to the low fmol (absolute on column) level. Biological validation and data interpretation of the multiomics workflow was performed based on proteomics network reconstruction, metabolic modelling (MetaboAnalyst 4.0), and pathway analysis (OmicsNet). Comparing MSCs and adipocytes, we observed significant regulation of different metabolites and lipids such as triglycerides, gangliosides, and carnitine with 113 fully reprogrammed pathways. The observed changes are in accordance with literature findings dealing with adipogenic differentiation of MSC. These results are a proof of principle for the power of multimolecular extraction combined with orthogonal LC-MS assays and network construction. Considering the analytical and biological validation performed in this study, we conclude that the proposed multiomics workflow is ideally suited for comprehensive follow-up studies on adipogenesis and is fit for purpose for different applications with a high potential to understand the complex pathophysiology of diseases.

Keywords:  LC-MS; fat differentiation; lipidomics; mathematical modelling; mesenchymal stem cells; metabolomics; multiomics; network analysis; proteomics; stromal cells

DOI:  https://doi.org/10.3390/molecules24193615
Metabolites. 2019 Oct 09. pii: E219. [Epub ahead of print]9(10):

In Silico Optimization of Mass Spectrometry Fragmentation Strategies in Metabolomics.

Joe Wandy, Vinny Davies, Justin J J van der Hooft, Stefan Weidt, Rónán Daly, Simon Rogers.

  Liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) is widely used in identifying small molecules in untargeted metabolomics. Various strategies exist to acquire MS/MS fragmentation spectra; however, the development of new acquisition strategies is hampered by the lack of simulators that let researchers prototype, compare, and optimize strategies before validations on real machines. We introduce Virtual Metabolomics Mass Spectrometer (ViMMS), a metabolomics LC-MS/MS simulator framework that allows for scan-level control of the MS2 acquisition process in silico. ViMMS can generate new LC-MS/MS data based on empirical data or virtually re-run a previous LC-MS/MS analysis using pre-existing data to allow the testing of different fragmentation strategies. To demonstrate its utility, we show how ViMMS can be used to optimize N for Top-N data-dependent acquisition (DDA) acquisition, giving results comparable to modifying N on the mass spectrometer. We expect that ViMMS will save method development time by allowing for offline evaluation of novel fragmentation strategies and optimization of the fragmentation strategy for a particular experiment.

Keywords:  data-dependent acquisition (DDA); fragmentation (MS/MS); in silico; liquid chromatography–mass spectrometry (LC/MS); simulator

DOI:  https://doi.org/10.3390/metabo9100219
Zhongguo Zhong Yao Za Zhi. 2019 Aug;44(16): 3562-3568

[UPLC-Q-TOF-MS-based metabolomics study of celastrol].

Ting Zhang, Yi-Kun Wang, Qi Zhao, Xue-Rong Xiao, Fei Li.

  The mass spectrometry-based metabolomics method was used to systematically investigate the formation of celastrol metabolites,and the effect of celastrol on endogenous metabolites. The mice plasma,urine and feces samples were collected after oral administration of celastrol. Ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry( UPLC-QTOF-MS) was applied to analyze the exogenous metabolites of celastrol and its altered endogenous metabolites. Mass defect filtering was adopted to screen for the exogenous metabolites of celastrol. Multivariate statistical analysis was used to identify the endogenous metabolites affected by celastrol. Celastrol and its eight metabolites were detected in urine and feces of mice,and 5 metabolites of them were reported for the first time. The hydroxylated metabolites were observed in the metabolism of both human liver microsomes and mouse liver microsomes. Further recombinant enzyme experiments revealed CYP3 A4 was the major metabolic enzyme involved in the formation of hydroxylated metabolites. Urinary metabolomics revealed that celastrol can affect the excretion of intestinal bacteria-related endogenous metabolites,including hippuric acid,phenylacetylglycine,5-hydroxyindoleacetic acid,urocanic acid,cinnamoylglycine,phenylproplonylglycine and xanthurenic acid. These results are helpful to elucidate the metabolism and disposition of celastrol in vivo,and its mechanism of action.

Keywords:  UPLC-Q-TOF-MS; celastrol; in vitro metabolism; in vivo metabolism; metabolites of intestinal bacteria

DOI:  https://doi.org/10.19540/j.cnki.cjcmm.20190606.502
Anal Chem. 2019 Oct 09.

Uniformly 15N-Labeled Recombinant Ricin A-chain as an Internal Retention Time Standard for Increased Confidence in Forensic Identification of Ricin by Untargeted Nanoflow Liquid Chromatography-Tandem Mass Spectrometry.

Eric D Merkley, Kristin E Burnum-Johnson, Lindsey N Anderson, Sarah C Jenson, Karen L Wahl.

Ricin, a toxic protein from the castor plant, is of forensic and biosecurity interest because of its high toxicity and common occurrence in crimes and attempted crimes. Qualitative methods to detect ricin are therefore needed. Untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics methods are well-suited because of their high speci-ficity. Specificity in LC-MS/MS comes from both the LC and MS components. However, modern untargeted proteomics methods often use nanoflow LC, which has less reproducible retention times than standard-flow LC, making it challenging to use retention time as a point of identification in a forensic assay. We address this challenge by using retention times rela-tive to a standard, namely uniformly 15N-labeled ricin A-chain produced recombinantly in a bacterial expression system. This material, added as an internal standard prior to trypsin digestion, produces a stable isotope labeled standard for every ricin tryptic peptide in the sample. We show that the MS signals for 15N and natural isotopic abundance ricin peptides are distinct, with mass shifts that correspond to the numbers of nitrogen atoms in each peptide or fragment. We also show that, as expected, labeled and unlabeled peptides coelute, with relative retention time differences of less than 0.2%.

DOI: https://doi.org/10.1021/acs.analchem.9b03389
Talanta. 2020 Jan 15. pii: S0039-9140(19)30889-6. [Epub ahead of print]207 120256

A simultaneously quantitative profiling method for 40 endogenous amino acids and derivatives in cell lines using hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry.

Bangjie Zhu, Li Li, Hai Wei, Wenbin Zhou, Wentao Zhou, Fugang Li, Peiyuan Lin, Jiaqiang Sheng, Qingjiang Wang, Chao Yan, Yu Cheng.

  Endogenous metabolites of amino acids and their derivatives in biosamples are frequently highlighted as the most differential metabolites in recent metabolomics studies. The method for the detection of amino acid derivatives such as N-acetyl amino acids and oligopeptides is rarely reported. We developed a rapid, high-throughput, sensitive and reliable quantitative method to simultaneously profile 40 underivatized amino acids and their derivatives including N-acetyl amino acids and oligopeptides in cell lines, based on ultra-high-performance liquid chromatography-electrospray tandem mass spectrometry (UHPLC- MS/MS) by using a hydrophilic interaction liquid chromatography (HILIC) column. The optimized method was successfully validated with satisfactory linearity, sensitivity, accuracy, precision, matrix effects, recovery and stability for all analytes. Only one compound (cysteine-glutathione disulfide) showed relatively low recoveries at three concentration levels (60.8-74.3%). The limit of quantification (LOQ) for most compounds was in the range of 0.6-10 ng/mL (6-100 pg on column). This method was successfully applied to the analysis of amino acids and their derivatives in breast cancer cell samples. Principal component analysis (PCA) and the orthogonal projections to latent structures (OPLS) showed a clear discrimination of the non-tumorigenic breast epithelial cell line MCF-10A from the breast cancer cell line HCC 1806. Characteristic metabolic changes in amino acid metabolism were observed in the breast cancer cell line. This quantified analytical method of 40 endogenous amino acids and their derivatives in cell lines meets the requirement of quantification in specific expanded metabolomics studies with good sensitivity.

Keywords:  Amino acid; Amino acid derivatives; Cancer cell; HILIC; Target metabolomics; UHPLC-MS/MS

DOI:  https://doi.org/10.1016/j.talanta.2019.120256
Anal Chem. 2019 Oct 08.

Rapid Quantitation of 25-Hydroxyvitamin D2 and D3 in Human Serum Using Liquid Chromatography/drift Tube Ion Mobility-Mass Spectrometry.

Nick Oranzi, Robin H J Kemperman, Jiajun Lei, Brett Holmquist, Christopher D Chouinard, Timothy J Garrett, Richard A Yost.

Ion mobility was integrated with liquid chromatography/high resolution mass spectrometry (LC/IM-HRMS) to quantify 25-hydroxyvitamin D(25OHD) in human serum. It has previously been shown that 25OHD adopts two gas-phase conformations which are resolved using ion mobility; in contrast, the inactive epimer, 3-epi-25-hydroxyvitamin D (epi25OHD) only adopts one. Interference from epi25OHD was eliminated by filtering the chromatogram to retain the drift time that corresponds to the unique gas-phase conformation of 25OHD. Although ion mobility separates the epimers, some chromatography is required to separate compounds which interfere with ionization or fall at the same nominal m/z. Standards were prepared in 4% albumin solutions and compared against commercial serum quality controls. Standards and quality controls were analyzed and validated using a two-minute LC/IM-MS method. 25-hydroxyvitamin D3 and D2 were quantified over the range between 2 and 500 ng/mL with bias and precision within 15%. When epi25OHD was spiked into quality control samples, no significant bias was introduced, and analysis of 30 patient samples shows good agreement between this LC/IM-MS and traditional LC/MS/MS methods. This work shows that ion mobility can be incorporated with liquid chromatography and mass spectrometry for rapid quantitation of 25OHD in human serum.

DOI: https://doi.org/10.1021/acs.analchem.9b02683
Chem Sci. 2019 Aug 14. 10(30): 7163-7171

Expanding the mass range for UVPD-based native top-down mass spectrometry.

Jean-François Greisch, Sem Tamara, Richard A Scheltema, Howard W R Maxwell, Robert D Fagerlund, Peter C Fineran, Stephan Tetter, Donald Hilvert, Albert J R Heck.

Native top-down mass spectrometry is emerging as a methodology that can be used to structurally investigate protein assemblies. To extend the possibilities of native top-down mass spectrometry to larger and more heterogeneous biomolecular assemblies, advances in both the mass analyzer and applied fragmentation techniques are still essential. Here, we explore ultraviolet photodissociation (UVPD) of protein assemblies on an Orbitrap with extended mass range, expanding its usage to large and heterogeneous macromolecular complexes, reaching masses above 1 million Da. We demonstrate that UVPD can lead not only to the ejection of intact subunits directly from such large intact complexes, but also to backbone fragmentation of these subunits, providing enough sequence information for subunit identification. The Orbitrap mass analyzer enables simultaneous monitoring of the precursor, the subunits, and the subunit fragments formed upon UVPD activation. While only partial sequence coverage of the subunits is observed, the UVPD data yields information about the localization of chromophores covalently attached to the subunits of the light harvesting complex B-phycoerythrin, extensive backbone fragmentation in a subunit of a CRISPR-Cas Csy (type I-F Cascade) complex, and sequence modifications in a virus-like proteinaceous nano-container. Through these multiple applications we demonstrate for the first time that UVPD based native top-down mass spectrometry is feasible for large and heterogeneous particles, including ribonucleoprotein complexes and MDa virus-like particles.

DOI: https://doi.org/10.1039/c9sc01857c
J Food Drug Anal. 2019 Oct;pii: S1021-9498(19)30051-1. [Epub ahead of print]27(4): 906-914

Simultaneous determination of cardiovascular drugs in dried blood spot by liquid chromatography-tandem mass spectrometry.

Hyung Min Kim, Ju-Hwan Park, Nguyen Phuoc Long, Dae-Duk Kim, Sung Won Kwon.

  A dried blood spot (DBS) sampling method was exploited to extract cardiovascular drugs using a small volume of whole blood of human and rodent. Thereafter, an analytical method using liquid chromatography with tandem mass spectrometry (LC-MS/MS) was developed and validated for the determination of 12 cardiovascular drugs. A 6 mm internal diameter disc containing 10 μL of blood was punched from a specifically designed card and analyzed by LC-MS/MS using a gradient elution method with a total run time of 16 min. For sample separation, a universal octadecyl-silica column was used with a flow rate of 0.2 mL/min. The developed method was validated in terms of linearity, accuracy, and precision, which showed satisfactory results. In addition, the matrix effects were closely investigated to confirm the extraction efficiency. Additionally, the stability was tested by storing DBSs at room temperature; the results showed that these drugs were stable for at least 30 days. Accordingly, the proposed LC-MS/MS method is capable to analyze several cardiovascular drugs in a single analysis. It can be applied to therapeutic drug monitoring in patients as well as in the in vivo settings.

Keywords:  Cardiovascular drugs; Dried blood spot; LC-MS/MS; Therapeutic drug monitoring

DOI:  https://doi.org/10.1016/j.jfda.2019.06.001
Bioinformatics. 2019 Oct 12. pii: btz736. [Epub ahead of print]

Identification of metabolites from tandem mass spectra with a machine learning approach utilizing structural features.

Yuanyue Li, Michael Kuhn, Anne-Claude Gavin, Peer Bork.

MOTIVATION: Untargeted mass spectrometry is a powerful method for detecting metabolites in biological samples. However, fast and accurate identification of the metabolites' structures from MS/MS spectra is still a great challenge.
RESULTS: We present a new analysis method, called SF-Matching, that is based on the hypothesis that molecules with similar structural features will exhibit similar fragmentation patterns. We combine information on fragmentation patterns of molecules with shared substructures and then use random forest models to predict whether a given structure can yield a certain fragmentation pattern. These models can then be used to score candidate molecules for a given mass spectrum. For rapid identification, we pre-compute such scores for common biological molecular structure databases. Using benchmarking datasets, we find that our method has similar performance to CSI:FingerID and that very high accuracies can be achieved by combining our method with CSI:FingerID. Rarefaction analysis of the training dataset shows that the performance of our method will increase as more experimental data become available.
AVAILABILITY: SF-Matching is available from http://www.bork.embl.de/Docu/sf_matching.

DOI: https://doi.org/10.1093/bioinformatics/btz736
Mass Spectrom Rev. 2019 Oct 11.

Unsupervised machine learning for exploratory data analysis in imaging mass spectrometry.

Nico Verbeeck, Richard M Caprioli, Raf Van de Plas.

  Imaging mass spectrometry (IMS) is a rapidly advancing molecular imaging modality that can map the spatial distribution of molecules with high chemical specificity. IMS does not require prior tagging of molecular targets and is able to measure a large number of ions concurrently in a single experiment. While this makes it particularly suited for exploratory analysis, the large amount and high-dimensional nature of data generated by IMS techniques make automated computational analysis indispensable. Research into computational methods for IMS data has touched upon different aspects, including spectral preprocessing, data formats, dimensionality reduction, spatial registration, sample classification, differential analysis between IMS experiments, and data-driven fusion methods to extract patterns corroborated by both IMS and other imaging modalities. In this work, we review unsupervised machine learning methods for exploratory analysis of IMS data, with particular focus on (a) factorization, (b) clustering, and (c) manifold learning. To provide a view across the various IMS modalities, we have attempted to include examples from a range of approaches including matrix assisted laser desorption/ionization, desorption electrospray ionization, and secondary ion mass spectrometry-based IMS. This review aims to be an entry point for both (i) analytical chemists and mass spectrometry experts who want to explore computational techniques; and (ii) computer scientists and data mining specialists who want to enter the IMS field. © 2019 The Authors. Mass Spectrometry Reviews published by Wiley Periodicals, Inc. Mass SpecRev 00:1-47, 2019.

Keywords:  DESI; LAESI; LAICP; MALDI; SIMS; clustering; data analysis; imaging mass spectrometry; machine learning; manifold learning; matrix factorization; unsupervised

DOI:  https://doi.org/10.1002/mas.21602
Analyst. 2019 Oct 09.

Sensitive analysis of multiple low-molecular-weight thiols in a single human cervical cancer cell by chemical derivatization-liquid chromatography-mass spectrometry.

Hua-Ming Xiao, Xian Wang, Quan-Lan Liao, Shuai Zhao, Wei-Hua Huang, Yu-Qi Feng.

Low-molecular-weight (LMW) thiols are important small molecules that regulate or maintain redox homeostasis in physiological and pathological processes. Assessing the concentrations of LMW thiols in biological systems may provide valuable information regarding physiological processes and the early diagnosis of some diseases. Here, we developed a method to simultaneously determine the concentrations of multiple LWM thiols in single cells by chemical derivatization assisted liquid chromatography-mass spectrometry (LC-MS). In this method, we synthesized a pair of stable isotope reagents, N-(acridin-9-yl)-2-bromoacetamide (AYBA) and N-(1,2,3,4-[2H4]-acridin-9-yl)-2-bromoacetamide ([2H4]AYBA). AYBA was used to derivatize LWM thiols in human cervical cancer (HeLa) cells, while [2H4]AYBA was used to derivatize standard LWM thiols to prepare internal standards for the LC-MS method development. The proposed AYBA derivatization greatly enhanced the detection sensitivity of LWM thiols by LC-MS, and thereby achieved the simultaneous detection of multiple LWM thiols by LC-MS in ∼1000 HeLa cells. Finally, the developed method was successfully utilized for the quantitative analysis of multiple LWM thiols in a single HeLa cell and the content changes of LWM thiols in a single HeLa cell before and after oxidative stress treatment. Accordingly, six LMW thiols were detected, including cysteamine, cysteine, glutathione, homocysteine, hydrogen sulfide, and pantetheine.

DOI: https://doi.org/10.1039/c9an01566c
Anal Chem. 2019 Oct 10.

Hybrid Search: A Method for Identifying Metabolites Absent from Tandem Mass Spectrometry Libraries.

Brian T Cooper, Xinjian Yan, Yamil Simón-Manso, Dmitrii V Tchekhovskoi, Yuri A Mirokhin, Stephen E Stein.

Metabolomics has a critical need for better tools for mass spectral identification. Common metabolites may be identified by searching libraries of tandem mass spectra, which offers important advantages over other approaches to identification. But tandem libraries are not nearly complete enough to represent the full molecular diversity present in complex biological sam-ples. We present a novel hybrid search method that can help identify metabolites not in the library by similarity to com-pounds that are. We call it "hybrid" searching because it combines conventional, direct peak matching with the logical equivalent of neutral-loss matching. A successful hybrid search requires the library to contain "cognates" of the unknown: similar compounds with a structural difference confined to a single region of the molecule, that does not substantially alter its fragmentation behavior. We demonstrate that the hybrid search is highly likely to find similar compounds under such circumstances.

DOI: https://doi.org/10.1021/acs.analchem.9b03415
J Clin Lab Anal. 2019 Oct 08. e23062

Derivatization-free determination of short-chain volatile amines in human plasma and urine by headspace gas chromatography-mass spectrometry.

Peter Neyer, Luca Bernasconi, Jens A Fuchs, Martina D Allenspach, Christian Steuer.

   BACKGROUND: Short-chain volatile amines (SCVA) are an interesting compound class playing crucial roles in physiological and toxicological human settings. Dimethylamine (DMA), trimethylamine (TMA), diethylamine (DEA), and triethylamine (TEA) were investigated in detail.
METHODS: Headspace gas chromatography coupled to mass spectrometry (HS-GC-MS) was used for the simultaneous qualitative and quantitative determination of four SCVA in different human body fluids. Four hundred microliters of Li-heparin plasma and urine were analyzed after liberation of volatile amines under heated conditions in an aqueous alkaline and saline environment. Target analytes were separated on a volatile amine column and detected on a Thermo DSQ II mass spectrometer scheduled in single ion monitoring mode.
RESULTS: Chromatographic separation of selected SCVA was done within 7.5 minutes. The method was developed and validated with respect to accuracy, precision, recovery and stability. Accuracy and precision criteria were below 12% for all target analytes at low and high levels. The selected extraction procedure provided recoveries of more than 92% from both matrices for TMA, DEA and TEA. The recovery of DMA from Li-heparin plasma was lower but still in the acceptable range (>75%). The newly validated method was successfully applied to plasma and urine samples from healthy volunteers. Detected concentrations of endogenous metabolites DMA and TMA are comparable to already known reference ranges.
CONCLUSION: Herein, we describe the successful development and validation of a reliable and broadly applicable HS-GC-MS procedure for the simultaneous and quantitative determination of SCVA in human plasma and urine without relying on derivatization chemistry.

Keywords:  derivatization-free multi-analyte procedure; headspace-GC-MS; method validation; volatile amines

DOI:  https://doi.org/10.1002/jcla.23062
Metabolomics. 2019 Oct 05. 15(10): 139

Metabolomics: a search for biomarkers of visceral fat and liver fat content.

Sebastiaan Boone, Dennis Mook-Kanamori, Frits Rosendaal, Martin den Heijer, Hildo Lamb, Albert de Roos, Saskia le Cessie, Ko Willems van Dijk, Renée de Mutsert.

   INTODUCTION: Excess visceral and liver fat are known risk factors for cardiometabolic disorders. Metabolomics might allow for easier quantification of these ectopic fat depots, instead of using invasive and costly tools such as MRI or approximations such as waist circumference.
OBJECTIVE: We explored the potential use of plasma metabolites as biomarkers of visceral adipose tissue (VAT) and hepatic triglyceride content (HTGC).
METHODS: We performed a cross-sectional analysis of a subset of the Netherlands Epidemiology of Obesity study. Plasma metabolite profiles were determined using the Biocrates AbsoluteIDQ p150 kit in 176 individuals with normal fasting plasma glucose. VAT was assessed with magnetic resonance imaging and HTGC with proton-MR spectroscopy. We used linear regression to investigate the associations of 190 metabolite variables with VAT and HTGC.
RESULTS: After adjustment for age, sex, total body fat, currently used approximations of visceral and liver fat, and multiple testing, three metabolite ratios were associated with VAT. The strongest association was the lysophosphatidylcholines to total phosphatidylcholines (PCs) ratio [- 14.1 (95% CI - 21.7; - 6.6) cm2 VAT per SD of metabolite concentration]. Four individual metabolites were associated with HTGC, especially the diacyl PCs of which C32:1 was the strongest at a 1.31 (95% CI 1.14; 1.51) fold increased HTGC per SD of metabolite concentration.
CONCLUSION: Metabolomics may be a useful tool to identify biomarkers of visceral fat and liver fat content that have added diagnostic value over current approximations. Replication studies are required to validate the diagnostic value of these metabolites.

Keywords:  Biomarkers; Liver fat; Metabolomics; Visceral adipose tissue

DOI:  https://doi.org/10.1007/s11306-019-1599-x
Anal Chem. 2019 Oct 10.

Regiospecific analysis of triacylglycerols by ultra-high-performance-liquid chromatography-electrospray ionization-tandem mass spectrometry.

Marko T Tarvainen, Heikki P Kallio, Baoru Yang.

An ultra-high-performance-liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) method was developed for the analysis of AAB and ABC type triacylglycerol (TG) regioisomers. Excellent linear regressions were established between the ratio of [RR]+ product ions and the proportion of regioisomers of TGs. The method was further optimized by analysis of 18 regiospecific pairs of AAB type TGs and 5 triplets of regioisomers of ABC type TGs with acyl carbon number (ACN) ranging from 36 to 54 and number of double bonds (DB) from 0 to 7. Reverse linear relationships were recognized between the slope of the calibration curve and the number of double bonds of the sn-2 fatty acids. Negative linear regressions were found between the intercepts of the calibration curves and the sum of ACN + DB of sn-2 fatty acids. The method was highly repeatable as shown by the low deviation and high stability of the calibration curves at different concentrations and between different periods of analysis. This is the first time that calibration curves for the ABC type TGs are reported. The results provide crucial and novel information for reliable and quantitative determination of regioisomeric TGs in natural fats and oils.

DOI: https://doi.org/10.1021/acs.analchem.9b02968
J Sep Sci. 2019 Oct 11.

High-resolution capillary zone electrophoresis and mass spectrometry for distinction of undersialylated and hypoglycosylated transferrin glycoforms in body fluids.

Jitka Caslavska, Christof Schild, Wolfgang Thormann.

  High-resolution capillary zone electrophoresis is used to distinguish transferrin glycoforms present in human serum, cerebrospinal fluid and serum treated with neuraminidase and N-glycosidase F. The obtained data are compared to mass spectrometry data from the literature. The main focus is on the analysis of the various asialo-transferrin, monosialo-transferrin and disialo-transferrin molecules found in these samples. The features of capillary zone electrophoresis and mass spectrometry are reviewed and highlighted in the context of the analysis of undersialylated and hypoglycosylated transferrin molecules. High-resolution capillary zone electrophoresis represents an effective tool to assess the diversity of transferrin patterns whereas mass spectrometry is the method of choice to elucidate structural identification about the glycoforms. Hypoglycosylated transferrin glycoforms present in sera of alcohol abusers and normal subjects are structurally identical to those in sera of patients with a congenital disorder of glycosylation type I. Asialo-transferrin, monosialo-transferrin and disialo-transferrin observed in sera of patients with a type II congenital disorder of glycosylation or a hemolytic uremic syndrome, in cerebrospinal fluid and after treatment of serum with neuraminidase are undersialylated transferrin glycoforms with two N-glycans of varying structure. Undersialylated disialo-transferrin is also observed in sera with high levels of trisialo-transferrin. This article is protected by copyright. All rights reserved.

Keywords:  asialo-transferrin; capillary electrophoresis; congenital disorders of glycosylation; disialo-transferrin; mass spectrometry

DOI:  https://doi.org/10.1002/jssc.201900857
J Equine Sci. 2019 Sep;30(3): 55-61

Identification of metabolomic changes in horse plasma after racing by liquid chromatography-high resolution mass spectrometry as a strategy for doping testing.

Toshiki Ueda, Teruaki Tozaki, Satoshi Nozawa, Kenji Kinoshita, Hitoshi Gawahara.

  Recently, the illegal use of novel technologies, such as gene and cell therapies, has become a great concern for the horseracing industry. As a potential way to control this, metabolomics approaches that comprehensively analyze metabolites in biological samples have been gaining attention. However, it may be difficult to identify metabolic biomarkers for doping because physiological conditions generally differ between resting and exercise states in horses. To understand the metabolic differences in horse plasma between the resting state at training centres and the sample collection stage after racing for doping test (SAD), we took plasma samples from these two stages (n=30 for each stage) and compared the metabolites present in these samples by liquid chromatography-high resolution mass spectrometry. This analysis identified 5,010 peaks, of which 1,256 peaks (approximately 25%) were annotated using KEGG analysis. Principal component analysis showed that the resting state and SAD groups had entirely different metabolite compositions. In particular, the levels of inosine, xanthosine, uric acid, and allantoin, which are induced by extensive exercise, were significantly increased in the SAD group. In addition, many metabolites not affected by extensive exercise were also identified. These results will contribute to the discovery of biomarkers for detecting doping substances that cannot be detected by conventional methods.

Keywords:  doping control; gene and cell doping; horse racing; liquid chromatography-high resolution mass spectrometry; metabolomics

DOI:  https://doi.org/10.1294/jes.30.55
J Chromatogr Sci. 2019 Oct 10. pii: bmz082. [Epub ahead of print]

A Validated Ultra-Performance Liquid Chromatographic Method for the Simultaneous Determination of Nadifloxacin, Mometasone Furoate and Miconazole Nitrate in Their Combined Dosage Form and Spiked Human Plasma Samples.

Mohamed Tarek, Hebatallah A Wagdy, Eman S Elzanfaly, Sawsan M Amer.

  Nadifloxacin, mometasone furoate and miconazole nitrate are formulated together as a topical antifungal dosage form. In this work, a reversed-phase ultra-performance liquid chromatographic method coupled with a diode array detector (RP-UPLC-DAD) was developed and validated to determine nadifloxacin, mometasone furoate and miconazole nitrate simultaneously in their bulk powder, in pharmaceutical preparation and in spiked human plasma samples. Separation was achieved on an ACQUITY UPLC C18 column of 2.2 μm particle size (2.1 × 100 mm) via isocratic elution using a mobile phase consisting of methanol, acetonitrile and water with ratio (50:20:30; v/v/v) and 0.1 g ammonium acetate, then pH was adjusted to (7.00) using acetic acid, flow rate 0.6 mL/min, temperature 30°C and UV detection at 220 nm. The method is linear in a range from 5 to 400 μg/mL for both nadifloxacin and miconazole nitrate and from 20 to 500 μg/mL for mometasone furoate. The method was validated according to the ICH guidelines then applied successfully to determine the mentioned drugs in their pharmaceutical preparation and spiked human plasma samples. For plasma samples, the results showed that the method can determine nadifloxacin, mometasone furoate and miconazole nitrate in human plasma samples with high accuracy and precision.

Keywords:  Pharmaceutical preparation; Plasma sample; UPLC; Validation

DOI:  https://doi.org/10.1093/chromsci/bmz082
J Chromatogr B Analyt Technol Biomed Life Sci. 2019 Aug 03. pii: S1570-0232(19)30631-2. [Epub ahead of print]1128 121745

Ultra-performance liquid chromatography-tandem mass spectrometry quantitative profiling of tryptophan metabolites in human plasma and its application to clinical study.

Yun Chen, Hui Chen, Ganggang Shi, Min Yang, Fuchun Zheng, Zhijie Zheng, Shuyao Zhang, Shilong Zhong.

  A sensitive, rapid and reliable ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to assay tryptophan (TRP) and its nine metabolites, including kynurenine (KYN), kynurenic acid (KYNA), 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid (3-HAA), xanthurenic acid (XA), 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), 3-indolepropionic acid (IPA) and 3-indoleacetic acid (IAA) in human plasma. Tryptophan-d5 (TRP-d5) and carbamazepine (CAR) were applied to the method quantification, where TRP-d5 was the corresponding internal standard (IS) for TRP and KYN, and CAR was the corresponding IS for the other analytes. Plasma samples were processed by deproteinisation with acetonitrile, followed by separation on an Acquity UPLC HSS T3 column by using gradient elution with 0.1% (v/v) formic acid in water and acetonitrile and detection by electrospray ionisation tandem mass spectrometry in positive ion multiple reaction monitoring (MRM) within a total run time of 5 min. The calibration ranges were 3-600 ng/mL for 3-HK, 1.5-300 ng/mL for 5-HT, 25-5000 ng/mL for KYN, 1-200 ng/mL for XA, 100-20,000 ng/mL for TRP, 5-1000 ng/mL for KYNA, 2-400 ng/mL for 3-HAA, 2.5-500 ng/mL for 5-HIAA and 10-2000 ng/mL for IAA and IPA. All intra- and inter-day analytical variations were acceptable. Matrix effect and recovery evaluation proved that matrix effect can be negligible, and sample preparation approach was effective. The newly developed method can simultaneously determine a panel of TRP metabolites and was successfully applied in the clinical study characterising TRP metabolism in healthy volunteers.

Keywords:  5-HT; Human plasma; Indole; Kynurenine; Tryptophan; UPLC-MS/MS

DOI:  https://doi.org/10.1016/j.jchromb.2019.121745
Bioinformatics. 2019 Oct 09. pii: btz765. [Epub ahead of print]

MetumpX - A Metabolomics Support Package for Untargeted Mass Spectrometry.

Bilal Wajid, Hasan Iqbal, Momina Jamil, Hafsa Rafique, Faria Anwar.

MOTIVATION: Metabolomics is a data analysis and interpretation field aiming to study functions of small molecules within the organism. Consequently Metabolomics requires researchers in life sciences to be comfortable in downloading, installing and scripting of software that are mostly not user friendly and lack basic GUIs. As the researchers struggle with these skills, there is a dire need to develop software packages that can automatically install software pipelines truely speeding up the learning curve to build software workstations. Therefore this paper aims to provide MetumpX, a software package that eases in their installation of 103 software by automatically resolving their individual dependencies and also allowing the users to choose which software works best for them.
RESULTS: MetumpX is a Ubuntu based software package that facilitate easy download and installation of about 103 tools spread across the standard metabolomics pipeline. As far as the authors know MetumpX is the only solution of this kind where the focus lies on automating development of software workstations.
AVAILABILITY: https://github.com/hasaniqbal777/MetumpX-bin.
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

DOI: https://doi.org/10.1093/bioinformatics/btz765
Cell Chem Biol. 2019 Sep 30. pii: S2451-9456(19)30314-9. [Epub ahead of print]

Family-wide Annotation of Enzymatic Pathways by Parallel In Vivo Metabolomics.

Joon T Kim, Veronica L Li, Stephanie M Terrell, Curt R Fischer, Jonathan Z Long.

  Enzymes catalyze fundamental biochemical reactions that control cellular and organismal homeostasis. Here we present an approach for de novo biochemical pathway discovery across entire mammalian enzyme families using parallel viral transduction in mice and untargeted liquid chromatography-mass spectrometry. Applying this method to the M20 peptidases uncovers both known pathways of amino acid metabolism as well as a previously unknown CNDP2-regulated pathway for threonyl dipeptide catabolism. Ablation of CNDP2 in mice elevates threonyl dipeptides across multiple tissues, establishing the physiologic relevance of our biochemical assignments. Taken together, these data underscore the utility of parallel in vivo metabolomics for the family-wide discovery of enzymatic pathways.

Keywords:  acid; acy1; amino; cndp2; enzyme; in vivo; metabolomics; peptidase; pm20d1

DOI:  https://doi.org/10.1016/j.chembiol.2019.09.009
Br J Cancer. 2019 Oct 07.

Identification of a biomarker panel for improvement of prostate cancer diagnosis by volatile metabolic profiling of urine.

Ana Rita Lima, Joana Pinto, Ana Isabel Azevedo, Daniela Barros-Silva, Carmen Jerónimo, Rui Henrique, Maria de Lourdes Bastos, Paula Guedes de Pinho, Márcia Carvalho.

BACKGROUND: The lack of sensitive and specific biomarkers for the early detection of prostate cancer (PCa) is a major hurdle to improve patient management.
METHODS: A metabolomics approach based on GC-MS was used to investigate the performance of volatile organic compounds (VOCs) in general and, more specifically, volatile carbonyl compounds (VCCs) present in urine as potential markers for PCa detection.
RESULTS: Results showed that PCa patients (n = 40) can be differentiated from cancer-free subjects (n = 42) based on their urinary volatile profile in both VOCs and VCCs models, unveiling significant differences in the levels of several metabolites. The models constructed were further validated using an external validation set (n = 18 PCa and n = 18 controls) to evaluate sensitivity, specificity and accuracy of the urinary volatile profile to discriminate PCa from controls. The VOCs model disclosed 78% sensitivity, 94% specificity and 86% accuracy, whereas the VCCs model achieved the same sensitivity, a specificity of 100% and an accuracy of 89%. Our findings unveil a panel of 6 volatile compounds significantly altered in PCa patients' urine samples that was able to identify PCa, with a sensitivity of 89%, specificity of 83%, and accuracy of 86%.
CONCLUSIONS: It is disclosed a biomarker panel with potential to be used as a non-invasive diagnostic tool for PCa.

DOI: https://doi.org/10.1038/s41416-019-0585-4
Metabolomics. 2019 Oct 05. 15(10): 138

An LCMS-based untargeted metabolomics protocol for cochlear perilymph: highlighting metabolic effects of hydrogen gas on the inner ear of noise exposed Guinea pigs.

Kristian Pirttilä, Pernilla Videhult Pierre, Jakob Haglöf, Mikael Engskog, Mikael Hedeland, Göran Laurell, Torbjörn Arvidsson, Curt Pettersson.

   INTRODUCTION: Noise-induced hearing loss (NIHL) is an increasing problem in society and accounts for a third of all cases of acquired hearing loss. NIHL is caused by formation of reactive oxygen species (ROS) in the cochlea causing oxidative stress. Hydrogen gas (H2) can alleviate the damage caused by oxidative stress and can be easily administered through inhalation.
OBJECTIVES: To present a protocol for untargeted metabolomics of guinea pig perilymph and investigate the effect of H2 administration on the perilymph metabolome of noise exposed guinea pigs.
METHODS: The left ear of guinea pigs were exposed to hazardous impulse noise only (Noise, n = 10), noise and H2 (Noise + H2, n = 10), only H2 (H2, n = 4), or untreated (Control, n = 2). Scala tympani perilymph was sampled from the cochlea of both ears. The polar component of the perilymph metabolome was analyzed using a HILIC-UHPLC-Q-TOF-MS-based untargeted metabolomics protocol. Multivariate data analysis (MVDA) was performed separately for the exposed- and unexposed ear.
RESULTS: MVDA allowed separation of groups Noise and Noise + H2 in both the exposed and unexposed ear and yielded 15 metabolites with differentiating relative abundances. Seven were found in both exposed and unexposed ear data and included two osmoprotectants. Eight metabolites were unique to the unexposed ear and included a number of short-chain acylcarnitines.
CONCLUSIONS: A HILIC-UHPLC-Q-TOF-MS-based protocol for untargeted metabolomics of perilymph is presented and shown to be fit-for-purpose. We found a clear difference in the perilymph metabolome of noise exposed guinea pigs with and without H2 treatment.

Keywords:  In vivo; LCMS; Metabolomics; NIHL; Noise-induced hearing loss; Perilymph

DOI:  https://doi.org/10.1007/s11306-019-1595-1
Anal Chem. 2019 Oct 08.

High Performance Molecular Imaging with MALDI Trapped Ion Mobility Time-of-Flight (timsTOF) Mass Spectrometry.

Jeffrey M Spraggins, Katerina V Djambazova, Emilio S Rivera, Lukasz Migas, Elizabeth Kathleen Neumann, Arne Fuetterer, Juergen Suetering, Niels Goedecke, Alice Ly, Raf Van de Plas, Richard M Caprioli.

Imaging mass spectrometry (IMS) enables the spatially targeted molecular assessment of biological tissues at cellular resolutions. New developments and technologies are essential for uncovering the molecular drivers of native physiological function and disease. Instrumentation must maximize spatial resolution, throughput, sensitivity, and specificity, because tissue imaging experiments consist of thousands to millions of pixels. Here, we report the development and application of a matrix-assisted laser desorption/ionization (MALDI) trapped ion mobility spectrometry imaging platform. This prototype MALDI timsTOF instrument is capable of 10 µm spatial resolutions and 20 pixels/s throughput molecular imaging. The MALDI source utilizes a Bruker SmartBeam 3-D laser system that can generate a square burn pattern of <10 x 10 µm at the sample surface. General image performance was assessed using murine kidney and brain tissues and demonstrate that high spatial resolution imaging data can be generated rapidly with mass measurement errors < 5 ppm and ~40,000 resolving power. Initial TIMS-based imaging experiments were performed on whole body mouse pup tissue demonstrating the separation of closely isobaric [PC(32:0)+Na]+ and [PC(34:3)+H]+ (3 mDa mass difference) in the gas-phase. We have shown that the MALDI timsTOF platform can maintain reasonable data acquisition rates (>2 pixels/s) while providing the specificity necessary to differentiate components in complex mixtures of lipid adducts. The combination of high spatial resolution and throughput imaging capabilities with high-performance TIMS separations provides a uniquely tunable platform to address many challenges associated with advanced molecular imaging applications.

DOI: https://doi.org/10.1021/acs.analchem.9b03612
J Sep Sci. 2019 Oct 07.

Dispersive solid phase extraction combined with dispersive liquid-liquid microextraction for simultaneous determination of seven succinate dehydrogenase inhibitor fungicides in watermelon by ultra-performance liquid chromatography/tandem mass spectrometry.

Junxue Wu, Shenwei Zhi, Chunhong Jia, Xinghai Li, Xiaodan Zhu, Ercheng Zhao.

  In this study, a simple and accurate sample preparation method based on dispersive solid-phase extraction and dispersive liquid-liquid microextraction has been developed for the determination of seven novel succinate dehydrogenase inhibitor fungicides (isopyrazam, fluopyram, pydiflumetofen, boscalid, penthiopyrad, fluxapyroxad and thifluzamide) in watermelon. The watermelon samples were extracted with acetonitrile, cleanup by dispersive solid-phase extraction procedure using primary secondary amine, extracted and concentrated by the following dispersive liquid-liquid microextraction procedure with 1,1,2,2-tetrachloroethane, and then analyzed by ultra-performance liquid chromatography/tandem mass spectrometry. The main experimental factors affecting the performance of dispersive solid-phase extraction and dispersive liquid-liquid microextraction procedure on extraction efficiency were investigated. The proposed method had a good linearity in the range of 0.1-100 μg kg-1 with correlation coefficients (r) of 0.9979-0.9999. The limit of quantification of seven fungicides was 0.1 μg kg-1 in the method. The fortified recoveries of seven succinate dehydrogenase inhibitor fungicides at three levels ranged from 72.0 to 111.6% with relative standard deviations of 3.4-14.1% (n = 5). The proposed method was successfully used for the rapid determination of seven SDHI fungicides in watermelon. This article is protected by copyright. All rights reserved.

Keywords:  determination; dispersive liquid-liquid mixroextraction; dispersive solid phase extraction; succinate dehydrogenase inhibitor fungicides

DOI:  https://doi.org/10.1002/jssc.201900862
BMC Bioinformatics. 2019 Oct 11. 20(1): 492

Random forest-based imputation outperforms other methods for imputing LC-MS metabolomics data: a comparative study.

Marietta Kokla, Jyrki Virtanen, Marjukka Kolehmainen, Jussi Paananen, Kati Hanhineva.

   BACKGROUND: LC-MS technology makes it possible to measure the relative abundance of numerous molecular features of a sample in single analysis. However, especially non-targeted metabolite profiling approaches generate vast arrays of data that are prone to aberrations such as missing values. No matter the reason for the missing values in the data, coherent and complete data matrix is always a pre-requisite for accurate and reliable statistical analysis. Therefore, there is a need for proper imputation strategies that account for the missingness and reduce the bias in the statistical analysis.
RESULTS: Here we present our results after evaluating nine imputation methods in four different percentages of missing values of different origin. The performance of each imputation method was analyzed by Normalized Root Mean Squared Error (NRMSE). We demonstrated that random forest (RF) had the lowest NRMSE in the estimation of missing values for Missing at Random (MAR) and Missing Completely at Random (MCAR). In case of absent values due to Missing Not at Random (MNAR), the left truncated data was best imputed with minimum value imputation. We also tested the different imputation methods for datasets containing missing data of various origin, and RF was the most accurate method in all cases. The results were obtained by repeating the evaluation process 100 times with the use of metabolomics datasets where the missing values were introduced to represent absent data of different origin.
CONCLUSION: Type and rate of missingness affects the performance and suitability of imputation methods. RF-based imputation method performs best in most of the tested scenarios, including combinations of different types and rates of missingness. Therefore, we recommend using random forest-based imputation for imputing missing metabolomics data, and especially in situations where the types of missingness are not known in advance.

Keywords:  High dimensional data; Imputation; MAR; MCAR; MNAR; Metabolomics; Missing values; RF

DOI:  https://doi.org/10.1186/s12859-019-3110-0
Wei Sheng Yan Jiu. 2019 Jul;48(4): 640-650

[Determination of 5 kinds of fish anesthetics residues in fish by ultra-high performance liquid chromatography-tandem mass spectrometry].

Ping Liu, Shuangying Xin, Sai Fan, Lili Wang, Rencai Luo, Guohua Wu, Rong Zhao, Yao Zhao.

OBJECTIVE: A method for the simultaneous determination of 5 kinds of fish anesthetics residues in fish has been developed by ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS). Eugenol, methyl-eugenol, methyl-isoeugenol, acetyl-isoeugenol and tricaine methanesulfonate(MS-222) were concerned.
METHODS: After homogenization fish samples were extracted by acetonitrile-water(80↿0, V/V), purified by Oasis PRiME HLB solid-phase extraction column. Then after centrifuged and concentrated, the samples were separated by Waters ACQUITY UPLC BEH Phenyl column(2. 1 mm×100 mm, 1. 7 μm). The detection was confirmed and quantified by mass spectrum of triple quadrupole in the multiple reaction monitoring(MRM) mode.
RESULTS: The calibration curves showed good linearity in each range with correlation coefficients greater than 0. 995. Three levels spiked recovery experiments were carried out using blank fish mud extraction as substrate, the recoveries ranged from 72. 6% to 106. 0%, the relative standard deviations(RSDs) ranged from 2. 2% to 20. 1%(n=6). The qualitative limits of detections(S/N>3) were 0. 14-0. 30 μg/kg and the quantitative limits(S/N>10) were 0. 5-1. 0 μg/kg.
CONSLUSION: The method is simple and easy to operate, with less organic reagent, high sensitivity and good stability. The isomers of methyl eugenol and methyl isoeugenol were successfully separated. It is suitable for the detection of 5 kinds of fish anesthetics in fish.

Keywords: UPLC-MS/MS; eugenol; fish anesthetic; food detection; tricaine methanesulfonate(MS-222)
J Pharm Biomed Anal. 2019 Sep 28. pii: S0731-7085(19)31160-4. [Epub ahead of print]178 112897

Quantitative analysis of underivatized 17 β-estradiol using a high-throughput LC-MS/MS assay - Application to support a pharmacokinetic study in ovariectomized guinea pigs.

Adarsh Gandhi, Murali K Matta, Sharron Stewart, Ashok Chockalingam, Alan Knapton, Rodney Rouse, Wendy Wu, Vikram Patel.

  Difference in female sex hormone, β-estradiol (E2), levels can contribute to sex differences in biological processes that underlie target tissue functions (QT interval), vulnerability to diseases (hepatitis or HIV), and response toward therapies. Accurate quantification of plasma E2 level is thus an important aspect in both basic science research examining hormone-regulated physiological mechanisms and in clinical settings to support patient care associated with altered E2 levels. Due to lack of a high-throughput high-sensitivity analytical method, we developed and validated a LC-MS/MS assay for accurate low-level quantification of E2 and demonstrated its application to a guinea pig pharmacokinetic study in which guinea pigs were treated with 10 or 40 μg/kg E2 subcutaneously and blood samples collected at 0 (pre-dose), 0.25, 0.5, 1, 2, 4, 8, 12 and 24 h post-dosing. E2 was extracted using 90 μL ovariectomized guinea pig plasma by liquid-liquid extraction. The method was robust, sensitive with linear range from 3.9 to 1000 pg/mL, and the assay met acceptance criteria for validation parameters listed in the current FDA Guidance on Bioanalytical Method Validation. Compared to the 10 μg/kg dose, more than dose proportional increase in maximum E2 plasma concentration (Cmax) and AUC0-∞ and correspondingly longer half-life were observed after 40 μg/kg dose. This assay is a significant improvement over existing E2 quantification methods in bioanalytical field, with high precision and accuracy, low sample and injection volumes, no derivatization, and short assay run time of 3 min. This assay is amenable in high-throughput settings requiring low-level E2 quantitation in basic science research and clinical settings.

Keywords:  17 β-estradiol; High-throughput; Liquid-liquid extraction; Mass spectrometry; Pharmacokinetics

DOI:  https://doi.org/10.1016/j.jpba.2019.112897
Mol Vis. 2019 ;25 489-501

Metabolomics of the aqueous humor in patients with primary congenital glaucoma.

Xueli Chen, Yuhong Chen, Li Wang, Xinghuai Sun.

Purpose: Primary congenital glaucoma (PCG) is an autosomal recessive eye disorder, accounting for 0.01%-0.04% of blindness around the world. Unfortunately, the molecular characteristics concerning the pathogenic mechanisms of the disease remain poorly understood.
Methods: Here, for the first time, we employed gas chromatography coupled to time-of-flight mass spectrometry (GC/TOF MS) to reveal comprehensively the metabolic characteristics of PCG.
Results: First, 363 metabolites were detected in 50 aqueous humor (AH) samples from 30 patients with PCG, 10 patients with congenital cataracts (CCs), and 10 patients with aged-related cataracts (ARCs). Second, 290 metabolites in total were found in another 15 patients with PCG and 10 patients with primary open angle glaucoma (POAG). A further analysis suggested that patients with PCG had a significantly distinct metabolomics profile. Three amino acid-associated metabolites, including glycine, urea, and phenylalanine, were identified to be significantly different (p≤0.05) in relation to PCG. Meanwhile, three glaucoma-associated single nucleotide polymorphisms (SNPs), rs7114303, rs9364602, and rs2165241, were determined to be related to these three metabolites. The results here indicate that certain amino acid-associated metabolites and their metabolisms are key regulatory elements and metabolic pathways in the pathogenesis of PCG.
Conclusions: Collectively, this work not only extended our understanding of the molecular characteristics of PCG, but also presented glycine as a potential biomarker for earlier diagnosis and may provide new therapeutic strategies for the disease.