The analyses were performed using a Ultra High Performance Liquid Chromatograpy-UV/Vis-High Resolution Mass Spectrometer (UHPLC-DAD-HRMS) on a maXis G3 oa Qq-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with an electrospray (ESI) source and connected to an Ultimate 3000 UHPLC system (Dionex, Sunnyvale, CA). The column used was a reverse-phase Kinetex 2.6 µm C18, 100×2.1 mm (Phenomenex, Torrance, CA) and the column temperature was maintained at 40°C. A linear water-acetonitrile gradient was used (both buffered with 20 mM formic acid) starting from 10% acetonitrile and increased to 100% in 10 minutes and maintaining this for 3 minutes before returning to the starting conditions in 0.1 minute and staying there for 2.4 minutes before the following run. A flow rate of 0.4 mL min−1 was used. HRMS was performed in ESI+ with a data acquisition range of 10 scans per second at m/z 100–1000. The MS was calibrated by use of the internal standard sodium formate, which was automatically infused prior to each run. UV spectra were collected at wavelengths from 200–700 nm. Data processing was performed using the Bruker software DataAnalysis and TargetAnalysis.
Target analysis
Manufactured by Bruker
Target Analysis is a specialized analytical instrument designed for the detection and quantification of specific target compounds in samples. It provides accurate and reliable results through advanced analytical techniques. The core function of Target Analysis is to enable users to identify and measure the presence of targeted substances with high precision.
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5 protocols using target analysis
1
UHPLC-DAD-HRMS Analysis of Compounds
2
Identification of Compounds in Crude Extracts
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The HPLC-HR-MS data of crude extracts were further analyzed to identify the known compounds present in the extracts using the software Target Analysis (Bruker Daltonik GmbH). The known compounds were identified on the basis of their high resolution mass, isotope pattern and retention time according to the known method45 (link). With this approach, re-isolation of known but less interesting compounds could be avoided whereas unknown compounds with potential bioactivity could be identified easily.
3
Mycotoxin Detection and Quantification
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Data files were processed using Target Analysis (Bruker Daltonics). Based on information on the elemental composition of the analytes the software extracts ion chromatograms of the exact mass ± 3 mDa of aflatoxin B1, B2, G1, and G2, citrinin, cyclopiazonic acid, deoxynivalenol, enniatin A1 and B1 (ammonium adducts), fumonisin B1 and B2, nivalenol, ochratoxin A, patulin, sterigmatocystin, T2 toxin and zearalenone followed by integration of the chromatograms and reporting of the results.
4
Quantitative Analysis of Metabolites in Cell Cultures
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Bruker Data Analysis was used to calibrate the raw MS spectra and extract retention time, m/z, and intensity of the seven standard metabolites from their chromatograms. The extracted information was subsequently used as a reference to analyze the culture of media samples. We used the Bruker software TargetAnalysis to identify and relatively quantify the seven metabolites in all the culture media samples.47 (link) The retention time, m/z, formula, and name of the seven metabolites were registered in the searching database of the software. The key searching parameter was set as follows: retention tolerance was 0.2–0.8 min; mass accuracy tolerance was 5–10 mDa; mSigma tolerance was 50–200. The chromatogram of each culture media sample was calibrated by internally injected NaFA (250 mM) before targeting the seven metabolites based upon retention time, m/z, and formula. The peak height and area of the corresponding identified metabolite were displayed on the result panel. In addition, the MS/MS spectra from the raw chromatogram were also manually validated for reassured identification.
5
Mass Spectrometry Analysis of Secondary Metabolites
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Extracts were analyzed using the UltiMate 3000 LC System (Dionex) coupled to an amaZon X ion trap with electrospray ionization (Bruker Daltonics). A water/acetonitrile gradient with 0.1% formic acid as mobile phase with 0.4 mL/min flow separated the compounds on a C18 column (ACQUITY UPLC BEH, 1.7 mm, Waters). Relative amounts were quantified either directly by integration of the area peak in DataAnalysis 4.2 Software (Bruker Daltonics) or by using Target Analysis (Bruker Daltonics) with the recently described setup (Ahrendt et al., 2015 (link)). The m/z ratios that were used for this procedure are as follows: m/z 215.2 [M+H]+ (desmethylphurealipid A), m/z 586.4 [M+H]+ (GameXPeptide A), m/z 552.4 [M+H]+ (GameXPeptide C) [M+H]+, m/z 255.1 [M+H]+ (IPS), m/z 334.7 [M+2H]2+ (mevalagmapeptide), m/z 273.2 [M+H]+ (nematophin), m/z 229.2 [M+H]+ (phurealipid A), m/z 295.2 [M+H]+ (photopyrone D), m/z 574.4 [M+H]+ (rhabdopeptide 1), m/z 838.4 [M+H]+ (szentiamide), m/z 663.3 [M+H]+ (xenematide A), m/z 466.3 [M+H]+ (xenocoumacin 1), m/z 281.1 [M+H]+ (xenofuranone A), and m/z 410.3 [M+H]+ (xenortide A). All analyses were performed in triplicate. Base peak chromatograms and extracted ion chromatograms (EICs) displayed in some figures were also created with DataAnalysis 4.2 (Bruker Daltonics).
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