bims-metlip 2020-08-23 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2020–08–23
fourteen papers selected by
Sofia Costa, Cold Spring Harbor Laboratory

Integration of GC-MS and LC-MS for untargeted metabolomics profiling.
Toward Comprehensive Per- and Polyfluoroalkyl Substances Annotation Using FluoroMatch Software and Intelligent High-Resolution Tandem Mass Spectrometry Acquisition.
Simultaneous Determination of 34 Amino Acids in Tumor Tissues from Colorectal Cancer Patients Based on the Targeted UHPLC-MS/MS Method.
A Comprehensive HPLC-MS-MS Screening Method for 77 New Psychoactive Substances, 24 Classic Drugs and 18 Related Metabolites in Blood, Urine and Oral Fluid.
Evaluation of Non-Target Long-term LC-HRMS Time Series Data using Multivariate Statistical Approaches.
Lipidome-wide characterization of phosphatidylinositols and phosphatidylglycerols on CC location level.
Simultaneous quantitation of four androgens and 17-hydroxyprogesterone in polycystic ovarian syndrome patients by LC-MS/MS.
ReDU: a framework to find and reanalyze public mass spectrometry data.
MS Analysis of DNA Modifications in Urinary/Body Fluids.
Detection and Quantification of RNA Phosphorothioate Modifications Using Mass Spectrometry.
Cellular Fatty Acid Analysis in Macrophage Using Stable Isotope Labeling.
Unraveling the Cyclization of L-argininosuccinic Acid in Biological Samples: A Study via Mass Spectrometry and NMR spectroscopy.
1D 1 H NMR as a Tool for Fecal Metabolomics.
Liquid Chromatography-Mass Spectrometry Analysis of Cytosine Modifications.

J Pharm Biomed Anal. 2020 Aug 02. pii: S0731-7085(20)31395-9. [Epub ahead of print]190 113509

Integration of GC-MS and LC-MS for untargeted metabolomics profiling.

Özge Cansın Zeki, Cemil Can Eylem, Tuba Reçber, Sedef Kır, Emirhan Nemutlu.

  Recently, metabolomics analyses have become increasingly common in the general scientific community as it is applied in several researches relating to diseases diagnosis. Identification and quantification of small molecules belonging to metabolism in biological systems have an important role in diagnosis of diseases. The combination of chromatography with mass spectrometry is used for the accurate and reproducible analysis of hundreds to thousands of metabolites in biological fluids or tissue samples. The number of metabolites that can be identified in biological fluids or tissue varies according to the gas (GC) or liquid (LC) chromatographic techniques used. The cover of these chromatographic techniques also differs from each other based on the metabolite group (polar, lipids, organic acid etc.). Consequently, some of the metabolites can only be analyzed using either GC or LC. However, more than one metabolite or metabolite group may be found altered in a particular disease. Thus, in order to find these alterations, metabolomics analyses that cover a wide range of metabolite groups are usually applied. In this regard, GC-MS and LC-MS techniques are mostly used together to identify completely all the altered metabolites during disease diagnosis. Using these combined techniques also allows identification of metabolite(s) with significantly altered phenotype. This review sheds light on metabolomics studies involving the simultaneous use of GC-MS and LC-MS. The review also discusses the coverage, sample preparation, data acquisition and data preprocessing for untargeted metabolomics studies. Moreover, the advantages and disadvantages of these methods were also evaluated. Finally, precautions and suggestions on how to perform metabolomics studies in an accurate, precise, complete and unbiased way were also outlined.

Keywords:  Data analysis; GC–MS; LC–MS; Metabolomics; Sample preparation

DOI:  https://doi.org/10.1016/j.jpba.2020.113509
Anal Chem. 2020 Aug 18. 92(16): 11186-11194

Toward Comprehensive Per- and Polyfluoroalkyl Substances Annotation Using FluoroMatch Software and Intelligent High-Resolution Tandem Mass Spectrometry Acquisition.

Jeremy P Koelmel, Matthew K Paige, Juan J Aristizabal-Henao, Nicole M Robey, Sara L Nason, Paul J Stelben, Yang Li, Nicholas M Kroeger, Michael P Napolitano, Tina Savvaides, Vasilis Vasiliou, Pawel Rostkowski, Timothy J Garrett, Elizabeth Lin, Chris Deigl, Karl Jobst, Timothy G Townsend, Krystal J Godri Pollitt, John A Bowden.

Thousands of per- and polyfluoroalkyl substances (PFAS) exist in the environment and pose a potential health hazard. Suspect and nontarget screening with liquid chromatography (LC)-high-resolution tandem mass spectrometry (HRMS/MS) can be used for comprehensive characterization of PFAS. To date, no automated open source PFAS data analysis software exists to mine these extensive data sets. We introduce FluoroMatch, which automates file conversion, chromatographic peak picking, blank feature filtering, PFAS annotation based on precursor and fragment masses, and annotation ranking. The software library currently contains ∼7 000 PFAS fragmentation patterns based on rules derived from standards and literature, and the software automates a process for users to add additional compounds. The use of intelligent data-acquisition methods (iterative exclusion) nearly doubled the number of annotations. The software application is demonstrated by characterizing PFAS in landfill leachate as well as in leachate foam generated to concentrate the compounds for remediation purposes. FluoroMatch had wide coverage, returning 27 PFAS annotations for landfill leachate samples, explaining 71% of the all-ion fragmentation (CF2)n related fragments. By improving the throughput and coverage of PFAS annotation, FluoroMatch will accelerate the discovery of PFAS posing significant human risk.

DOI: https://doi.org/10.1021/acs.analchem.0c01591
J Anal Methods Chem. 2020 ;2020 4641709

Simultaneous Determination of 34 Amino Acids in Tumor Tissues from Colorectal Cancer Patients Based on the Targeted UHPLC-MS/MS Method.

Yang Yang, Feng Zhang, Shouhong Gao, Zhipeng Wang, Mingming Li, Hua Wei, Renqian Zhong, Wansheng Chen.

A targeted ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was established and validated for the simultaneous determination of 34 amino acids in tissue samples from colorectal cancer (CRC) patients. The chromatographic separation was achieved on an Agilent ZORBAX SB-C18 column (3.0 × 150 mm, 5 μm) with a binary gradient elution system (A, 0.02% heptafluorobutyric acid and 0.2% formic acid in water, v/v; B, methanol). The run time was 10 min. The multiple reaction monitoring mode was chosen with an electrospray ionization source operating in the positive ionization mode for data acquisition. The linear correlation coefficients were >0.99 for all the analytes in their corresponding calibration ranges. The sample was pretreated based on tissue homogenate and protein precipitation with a 100 mg aliquot sample. The average recovery and matrix effect for 34 amino acids and 3 internal standards were 39.00%∼146.95% and 49.45%∼173.63%, respectively. The intra- and interday accuracy for all the analytes ranged from -13.52% to 14.21% (RSD ≤8.57%) and from -14.52% to 12.59% (RSD ≤10.31%), respectively. Deviations of stability under different conditions were within ±15% for all the analytes. This method was applied to simultaneous quantification of 34 amino acids in tissue samples from 94 CRC patients.

DOI: https://doi.org/10.1155/2020/4641709
J Anal Toxicol. 2020 Aug 20. pii: bkaa103. [Epub ahead of print]

A Comprehensive HPLC-MS-MS Screening Method for 77 New Psychoactive Substances, 24 Classic Drugs and 18 Related Metabolites in Blood, Urine and Oral Fluid.

Annagiulia Di Trana, Giulio Mannocchi, Filippo Pirani, Nunzia La Maida, Massimo Gottardi, Simona Pichini, Francesco Paolo Busardò.

  To date, more than 800 molecules are classified as New Psychoactive Substances (NPS), and it is reported that this number increases every year. Whereas several cases of polydrug consumption which led to acute intoxication and death are reported, a lack of effective analytical screening method to detect NPS and classical drug of abuse in human matrices affects the prompt identification of the probable cause of intoxication in emergency department of hospitals. In this concern, a fast, simple and comprehensive high-performance chromatography-tandem mass spectrometry (HPLC-MS-MS) screening method to detect and quantify 77 NPS, 24 classic drugs and 18 related metabolites has been successfully developed and validated in blood, urine and oral fluid. A small volume (100 µL) of whole blood samples spiked with internal standard deuterated mixture was added to 70 µL of M3® buffer and after precipitation of blood proteins, the supernatant was evaporated to dryness and reconstituted in 1 mL of mobile phase. Same volume (100 µL) of urine and oral fluid samples spiked with internal standard deuterated mix were only diluted with with 500 µL of M3 ® reagent. One microliter samples of each matrix was injected into HPLC-MS-MS equipment. The run time lasted 10 min with a gradient mobile phase. Mass spectrometric analysis was performed in positive ion MRM mode. The method was linear for all analytes under investigation with a determination coefficient always better than 0.99. The calibration range for blood and oral fluid was from limits of quantification (LOQs) to 200 ng/mL, whereas that for urine was LOQs to 1000 ng/mL. Recovery and matrix effect were always higher than 80%, whereas intra-assay and inter-assay precision was always better than 19% and accuracy was always within 19% of target in every matrix. Applicability of the method was verified by analysis of samples from real cases.

Keywords:  HPLC–MS-MS; New Psychoactive Substances; biological fluids

DOI:  https://doi.org/10.1093/jat/bkaa103
Anal Chem. 2020 Aug 19.

Evaluation of Non-Target Long-term LC-HRMS Time Series Data using Multivariate Statistical Approaches.

Kirsten Purschke, Maryam Vosough, Juri Leonhardt, Markus Weber, Torsten Claus Schmidt.

The use of liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS), has steadily increased in many application fields ranging from metabolomics to environmental science. HRMS data are frequently used for non-target screening (NTS), i.e., the search for compounds that are not previously known and where no reference substances are available. However, the large quantity of data produced by NTS analytical workflows makes data interpretation and a time dependent monitoring of samples very sophisticated and necessitates exploiting chemometric data processing techniques. Consequently, in this study a prioritisation method to handle time series in non-target data was established. As proof of concept, industrial wastewater was investigated. As routine industrial wastewater analyses monitor occurrence of a limited number of targeted water contaminants, NTS provides the opportunity to detect also unknown trace organic compounds (TrOCs) that are not in the focus of routine target analysis. The developed prioritisation method enables reducing raw data and including identification of prioritised unknown contaminants. To that end, a five-months' time series for industrial wastewaters was utilised, analysed by liquid chromatography time of flight mass spectrometry (LC qTOF MS) and evaluated by NTS. Following peak detection, alignment, grouping and blank subtraction, 3303 features were obtained of wastewater treatment plant (WWTP) influent samples. Subsequently, two complementary ways for exploratory time trend detection and feature prioritisation are proposed. Therefore, following a pre filtering step, principal component analysis (PCA; feature wise) and group wise PCA (GPCA) of the matrix (temporal wise) were used to annotate trends of relevant wastewater contaminants. With sparse factorisation of data matrices using GPCA, groups of correlated features/mass fragments or adducts were detected, recovered and prioritised. Similarities and differences in the chemical composition of wastewater samples were observed over time to reveal hidden factors accounting for the structure of the data. The detected features were reduced to 130 relevant time trends related to TrOCs for identification. Exemplarily, as proof of concept one non target pollutant was identified as N methylpyrrolidone. The developed chemometric strategies of this study are not only suitable for industrial wastewater but could also be efficiently employed for time trend exploration in other scientific fields.

DOI: https://doi.org/10.1021/acs.analchem.0c01897
Anal Chim Acta. 2020 Sep 01. pii: S0003-2670(20)30665-6. [Epub ahead of print]1128 107-115

Lipidome-wide characterization of phosphatidylinositols and phosphatidylglycerols on CC location level.

Tian Xia, Hanlin Ren, Wenpeng Zhang, Yu Xia.

  Phosphatidylglycerol (PG) and phosphatidylinositol (PI) are two essential classes of glycerophospholipids (GPs), playing versatile roles such as signalling messengers and lipid-protein interaction ligands in cell. Although a majority of PG and PI molecular species contain unsaturated fatty acyl chain(s), conventional tandem mass spectrometry (MS/MS) methods cannot discern isomers different in carbon-carbon double bond (CC) locations. In this work, we paired phosphate methylation with acetone Paternò-Büchi (PB) reaction, aiming to provide a solution for sensitive and structurally informative analysis of these two important classes of GPs down to the location of CC. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflow was established. Offline methylated PG or PI mixtures were subjected to hydrophilic interaction chromatographic separation, online acetone PB reaction, and MS/MS via collision-induced dissociation (CID) for CC location determination in positive ion mode. This method was sensitive, offering limit of identification at 5 nM for both PG and PI standards down to CC locations. On molecular species level, 49 PI and 31 PG were identified from bovine liver, while 61 PIs were identified from human plasma. This workflow also enabled ratiometric comparisons of CC location isomers (C18:1 Δ9 vs. Δ11) of a series of PIs from type 2 diabetes (T2D) plasma to that of normal plasma samples. PI 16:0_18:1 and PI 18:0_18:1 were found to exhibit significant changes in CC isomeric ratios between T2D and normal plasma samples. The above results demonstrate that the developed LC-PB-MS/MS workflow is applicable to different classes of lipids and compatible with other established lipid derivatization methods to achieve comprehensive lipid analysis.

Keywords:  Lipid isomers identification; Methylation derivatization; Paternò–Büchi reaction; Phosphatidylglycerol; Phosphatidylinositol

DOI:  https://doi.org/10.1016/j.aca.2020.06.017
J Clin Lab Anal. 2020 Aug 21. e23539

Simultaneous quantitation of four androgens and 17-hydroxyprogesterone in polycystic ovarian syndrome patients by LC-MS/MS.

Zheng Cao, Yifan Lu, Yuting Cong, Ying Liu, Youran Li, Husheng Wang, Qiaoli Zhang, Wenxi Huang, Jingrui Liu, Ying Dong, Guodong Tang, Yiqi R Luo, Chenghong Yin, Yanhong Zhai.

   BACKGROUND: Due to the low concentration of androgens in women and the limitation of immunoassays, it remains a challenge to accurately determine the levels of serum androgens in polycystic ovary syndrome (PCOS) patients for clinical laboratories. In this report, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous quantitation of testosterone (T), androstenedione (A4), dehydroepiandrosterone sulfate (DHEAS), dihydrotestosterone (DHT), and 17-hydroxyprogesterone (17-OHP) that are associated with PCOS.
METHODS: The serum samples were processed by protein precipitation and solid phase extraction before analysis with the in-house developed LC-MS/MS. The chromatographic separation was achieved with a C18 column, using a linear gradient elution with two mobile phases: 0.02% formic acid in water (phase A) and 0.1% formic acid in methanol (phase B). The separated analytes were detected by positive or negative electrospray ionization mode under multiple reaction monitoring (MRM).
RESULTS: The assay for all the five analytes was linear, stable, with imprecision less than 9% and recoveries within ±10%. The lower limits of quantification were 0.05, 0.05, 5, 0.025, and 0.025 ng/mL for T, A4, DHEAS, DHT, and 17-OHP, respectively. In the receiver operating characteristic curve (ROC) analyses with the PCOS (n = 63) and healthy (n = 161) subjects, the AUC of the four-androgen combined was greater than that of any single androgen tested in PCOS diagnosis.
CONCLUSIONS: The LC-MS/MS method for the four androgens and 17-OHP showed good performance for clinical implementation. More importantly, simultaneous quantitation of the four androgens provided better diagnostic power for PCOS.

Keywords:  LC-MS/MS; PCOS; androgen; diagnosis; validation

DOI:  https://doi.org/10.1002/jcla.23539
Nat Methods. 2020 Aug 17.

ReDU: a framework to find and reanalyze public mass spectrometry data.

Alan K Jarmusch, Mingxun Wang, Christine M Aceves, Rohit S Advani, Shaden Aguirre, Alexander A Aksenov, Gajender Aleti, Allegra T Aron, Anelize Bauermeister, Sanjana Bolleddu, Amina Bouslimani, Andres Mauricio Caraballo Rodriguez, Rama Chaar, Roxana Coras, Emmanuel O Elijah, Madeleine Ernst, Julia M Gauglitz, Emily C Gentry, Makhai Husband, Scott A Jarmusch, Kenneth L Jones, Zdenek Kamenik, Audrey Le Gouellec, Aileen Lu, Laura-Isobel McCall, Kerry L McPhail, Michael J Meehan, Alexey V Melnik, Riya C Menezes, Yessica Alejandra Montoya Giraldo, Ngoc Hung Nguyen, Louis Felix Nothias, Mélissa Nothias-Esposito, Morgan Panitchpakdi, Daniel Petras, Robert A Quinn, Nicole Sikora, Justin J J van der Hooft, Fernando Vargas, Alison Vrbanac, Kelly C Weldon, Rob Knight, Nuno Bandeira, Pieter C Dorrestein.

We present ReDU (https://redu.ucsd.edu/), a system for metadata capture of public mass spectrometry-based metabolomics data, with validated controlled vocabularies. Systematic capture of knowledge enables the reanalysis of public data and/or co-analysis of one's own data. ReDU enables multiple types of analyses, including finding chemicals and associated metadata, comparing the shared and different chemicals between groups of samples, and metadata-filtered, repository-scale molecular networking.

DOI: https://doi.org/10.1038/s41592-020-0916-7
Methods Mol Biol. 2021 ;2198 109-122

MS Analysis of DNA Modifications in Urinary/Body Fluids.

Aleksandra Skalska, Agnieszka Siomek-Gorecka, Ryszard Olinski, Rafal Rozalski.

  Analytical techniques based on mass spectrometry allow to analyze DNA modifications in body fluids. Here we describe two chromatographic methods that can be used for the simultaneous determination of the modified DNA bases and nucleosides in the same urine sample: isotope-dilution automated online two-dimensional ultraperformance liquid chromatography with tandem mass spectrometry (2D-UPLC-MS/MS) and high-performance liquid chromatography coupled with gas chromatography and mass spectrometry (HPLC/GC/MS).

Keywords:  5-carboxycytosine; 5-formylcytosine; 5-hydroxymethylcytosine; 5-hydroxymethyluracil; DNA lesions; DNA modifications; Gas chromatography; Isotope-dilution mass spectrometry; Urinary excretion

DOI:  https://doi.org/10.1007/978-1-0716-0876-0_9
Curr Protoc Nucleic Acid Chem. 2020 Sep;82(1): e113

Detection and Quantification of RNA Phosphorothioate Modifications Using Mass Spectrometry.

Ying Wu, Ya Ying Zheng, Qishan Lin, Jia Sheng.

  This article describes a protocol for detecting and quantifying RNA phosphorothioate modifications in cellular RNA samples. Starting from solid-phase synthesis of phosphorothioate RNA dinucleotides, followed by purification with reversed-phase HPLC, phosphorothioate RNA dinucleotide standards are prepared for UPLC-MS and LC-MS/MS methods. RNA samples are extracted from cells using TRIzol reagent, then digested with a nuclease mixture and analyzed by mass spectrometry. UPLC-MS is employed first to identify RNA phosphorothioate modifications. An optimized LC-MS/MS method is then employed to quantify the frequency of RNA phosphorothioate modifications in a series of model cells. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Synthesis, purification, and characterization of RNA phosphorothioate dinucleotides Basic Protocol 2: Digestion of RNA samples extracted from cells Basic Protocol 3: Detection and quantification of RNA phosphorothioate modifications by mass spectrometry.

Keywords:  LC-MS/MS; RNA phosphorothioate modification; UPLC-MS; nucleic acids

DOI:  https://doi.org/10.1002/cpnc.113
Methods Mol Biol. 2020 ;2184 47-60

Cellular Fatty Acid Analysis in Macrophage Using Stable Isotope Labeling.

Kevin J Williams, Steven J Bensinger.

  Fatty acids (FAs) are essential for building complex lipids, posttranslational modifications, and energetics. FAs can be imported from extracellular sources or synthesized by cells. The analysis of fatty acid methyl esters (FAMEs) by gas chromatography-mass spectrometry (GC-MS) allows for the quantitative analysis of long-chain and very-long-chain fatty acid content of cells. When coupled with isotopic labeling, this approach can elucidate the synthetic pathways being engaged by the cells, and the relative contribution of synthesis and import to maintain lipid content. Here, we describe a method for total cellular fatty acid analysis in macrophages.

Keywords:  FAME analysis; Fatty acids; GC–MS; Macrophages; Stable isotope labeling

DOI:  https://doi.org/10.1007/978-1-0716-0802-9_4
Anal Chem. 2020 Aug 21.

Unraveling the Cyclization of L-argininosuccinic Acid in Biological Samples: A Study via Mass Spectrometry and NMR spectroscopy.

Maricruz Mamani-Huanca, Ana Gradillas, Ángeles López-Gonzálvez, Coral Barbas.

Since L-argininosuccinic acid (ASA) is the characteristic biomarker for the diagnosis of certain diseases, its reliable detection in complex biological samples is necessary to obtain a complete evaluation with greater specificity and accuracy. ASA can undergo intramolecular cyclization, yielding an equilibrium with the resulting cyclic forms which can predominate under different analytical conditions. In this work, the appearance and transformation of the different forms of ASA have been studied and a strategy for targeted screening analysis of ASA and its cyclic forms using CE-ESI-TOF-MS has been developed. The data and spectra obtained allowed us to gain further insight into accurate identification, concluding that there is a dynamic equilibrium depending on the pH. Moreover, one- and two-dimensional NMR spectroscopy experiments have allowed us to determine the predominant tautomeric structure for the major cyclic ASA derivative, confirming the importance of intramolecular hydrogen bonds.

DOI: https://doi.org/10.1021/acs.analchem.0c01420
Curr Protoc Chem Biol. 2020 Sep;12(3): e83

1D 1 H NMR as a Tool for Fecal Metabolomics.

Caroline M Ganobis, M Sameer Al-Abdul-Wahid, Simone Renwick, Sandi Yen, Charley Carriero, Marc G Aucoin, Emma Allen-Vercoe.

  Metabolomic studies allow a deeper understanding of the processes of a given ecological community than nucleic acid-based surveys alone. In the case of the gut microbiota, a metabolic profile of, for example, a fecal sample provides details about the function and interactions within the distal region of the gastrointestinal tract, and such a profile can be generated in a number of different ways. This unit elaborates on the use of 1D 1 H NMR spectroscopy as a commonly used method to characterize small-molecule metabolites of the fecal metabonome (meta-metabolome). We describe a set of protocols for the preparation of fecal water extraction, storage, scanning, measurement of pH, and spectral processing and analysis. We also compare the effects of various sample storage conditions for processed and unprocessed samples to provide a framework for comprehensive analysis of small molecules from stool-derived samples. © 2020 Wiley Periodicals LLC Basic Protocol 1: Extracting fecal water from crude fecal samples Alternate Protocol 1: Extracting fecal water from small crude fecal samples Basic Protocol 2: Acquiring NMR spectra of metabolite samples Alternate Protocol 2: Acquiring NMR spectra of metabolite samples using Bruker spectrometer running TopSpin 3.x Alternate Protocol 3: Acquiring NMR spectra of metabolite samples by semiautomated process Basic Protocol 3: Measuring sample pH Support Protocol 1: Cleaning NMR tubes Basic Protocol 4: Processing raw spectra data Basic Protocol 5: Profiling spectra Support Protocol 2: Spectral profiling of sugars and other complex metabolites.

Keywords:  NMR; fecal water; metabolomics; metabonomics; spectrometry

DOI:  https://doi.org/10.1002/cpch.83
Methods Mol Biol. 2021 ;2198 67-78

Liquid Chromatography-Mass Spectrometry Analysis of Cytosine Modifications.

Nan Dai, Ivan R Corrêa.

  Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a widely used technique in the global analysis of epigenetic DNA modifications. The high-resolution chromatographic separation along with sensitive MS detection permits the identification and quantification of deoxyribonucleosides with precision and reliability. Although there have been tremendous advances in LC and MS instrumentation in recent years, sample preparation has not experienced a similar rate of development and is often a bottleneck to chemical analysis. Here we present a protocol for identification and quantification of cytosine modifications that combines a robust and efficient method to generate single nucleosides from genomic DNA samples followed by direct LC-MS/MS analysis.

Keywords:  Epigenetics; Methylome profiling; Multistage mass spectrometry; Nucleoside digestion; Nucleotide modifications

DOI:  https://doi.org/10.1007/978-1-0716-0876-0_6