D-2HG and L-2HG were extracted from CSF according to protocols described earlier [21 (link)] and modified as described below. The extracted samples were dried, and derivatized with 100 μL of 50 mg/mL DATAN in a 4:1 solution of dichloromethane and acetic acid. Isotopically labeled (13C5) D-α-HG was spiked into the samples as internal standard to quantify the absolute concentration for 2-HG. The samples were incubated at 80 °C for 40 min and then cooled at room temperature before drying by speed vacuum. The dried samples were reconstituted with 2 mM ammonium formate (pH 3.1, adjusted by formic acid). The data were acquired via multiple reaction monitoring in negative ionization mode using a 6495 Triple Quadrupole mass spectrometer coupled to an HPLC system (Agilent Technologies, Santa Clara, CA). The acquired mass spectrometry data were analyzed, integrated the peaks using Agilent Mass Hunter Quantitative Analysis software, and manually reviewed. The concentration of 2-HG was absolutely quantified using a linear regression curve in Agilent Mass Hunter Quantitative Analysis software.
Agilent masshunter quantitative analysis software
Manufactured by Agilent Technologies
Sourced in United States
Agilent MassHunter Quantitative Analysis software is a data analysis tool designed for mass spectrometry applications. It provides users with capabilities to quantify target analytes in complex samples.
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Lab products found in correlation
Agilent mass hunter qualitative analysis software, by Agilent Technologies (3 mentions)
6495 triple quadrupole ms, by Agilent Technologies (2 mentions)
1290 infinity 2 uhplc, by Agilent Technologies (2 mentions)
Masshunter qualitative analysis software, by Agilent Technologies (2 mentions)
Hplc system, by Agilent Technologies (1 mentions)
6495 triple quadrupole mass spectrometer, by Agilent Technologies (1 mentions)
Eclipse plus c18 column, by Agilent Technologies (1 mentions)
6495 qqq triple quadrupole mass spectrometer, by Agilent Technologies (1 mentions)
Agilent mass hunter software, by Agilent Technologies (1 mentions)
Agilent masshunter quantitative analysis, by Agilent Technologies (1 mentions)
Zorbax eclipse plus c18 column, by Agilent Technologies (1 mentions)
Agilent 6540 quadrupole time of flight mass spectrometer, by Agilent Technologies (1 mentions)
Agilent 1290 uhplc system, by Agilent Technologies (1 mentions)
Simca p 14, by Sartorius (1 mentions)
Sb c18 column, by Agilent Technologies (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Isoproterenol, by Merck Group (1 mentions)
Formic acid, by Merck Group (1 mentions)
Isoproterenol, by Nacalai Tesque (1 mentions)
Cosmospin filter g, by Nacalai Tesque (1 mentions)
22 protocols using agilent masshunter quantitative analysis software
1
CSF 2-Hydroxyglutarate Quantification
2
Profiling Tumor Metabolites in Mice
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We injected 6–8-week-old BALB/c mice subcutaneously with a single cell suspension of 100,000 CT26 cells in the right flank. Once the tumor volume reached 100 mm3, we started treating the mice either with 20 mg/kg PEG-GGT or vehicle on a biweekly basis. On the 7th day after treatment initiation (3 PEG-GGT doses) we euthanized the mice. We isolated and washed small sections of tumors (20 mg) in phosphate buffer saline (PBS) and then splash-froze them in liquid nitrogen. We collected the mouse blood from cardiac puncture and left the blood to coagulate at room temperature for 10 min. We then centrifuged the blood at 2000g for 10 min, isolated the serum and froze it in −80°C. The amino acids were extracted from mouse tissue and serum using the liquid-liquid extraction method as explained earlier.94 (link),95 (link) Pooled samples were used as quality control samples during MS acquisition. Agilent 6495 triple quadrupole MS coupled to Infinity 1290 LC was used for data acquisition via multiple reaction monitoring (MRM) mode through Agilent Mass Hunter Data Acquisition Software (ver. 10.1) as described previously.96 (link) Peak integration and data analysis were performed using Agilent Mass Hunter Quantitative Analysis Software. Peak areas were normalized with Tryptophan-15N2 spike internal standard.
3
Quantitative Analysis of Endogenous Metabolites
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The extracted ion chromatogram height for lactic acid, thymidine, arachidonic acid, and 2′-deoxycytidine, and the internal standards (ISTDs) were determined using the Agilent Mass Hunter Quantitative Analysis software, version B.05.00 or newer (Agilent Technologies). The height of each endogenous metabolite was normalized to the corresponding ISTD by dividing the endogenous metabolite height by the corresponding isotopically labelled ISTD height. Relative fold changes were then calculated for each ISTD-normalized metabolite in each sample by dividing the sample response by the median ISTD-normalized response of the reference treatment (0.1% DMSO) samples, producing a reference-normalized value for each metabolite in each sample within a plate of cell culture samples. A Grubbs’ test was then used to identify outlier samples within each treatment and exposure level, and outlier samples were then removed from further analyses.
4
N-Linked Glycopeptide Analysis by LC-MS/MS
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For N-linked glycopeptide analysis, samples were digested with trypsin to obtain glycopeptides. Before trypsin digestion, the samples were first reduced with 2 µL of 550 mM dithiothreitol (DTT) at 65 °C for 50 min in 50 mM ammonium bicarbonate (NH4HCO3) solution. Then 4 µL of 450 mM iodine acetamide (IAA) was used to alkylate samples for 25 min in the dark to prevent the re-formation of disulfide bonds between cysteines. One µg of trypsin was added to digest samples in a 37 °C water bath for 18 h overnight. To stop the digestion, samples were stored at −20 °C for 1 h. The instrument used was an Agilent 1290 infinity ultra-high-pressure liquid chromatography (UHPLC) system coupled to an Agilent 6495 triple quadrupole (QQQ) mass spectrometer. Solvents of the 10 min LC gradient include solvent A of 3% acetonitrile and solvent B of 90% acetonitrile in nanopore water. Samples were first separated with an Agilent Eclipse plus C18 column (RRHD 1.8 µm, 2.1 × 150 mm) connected to an Agilent Eclipse plus C18 trap column (RRHD 1.8 µm, 2.1 × 5 mm). The tandem mass spectrometry mode operated on the instrument was dynamic multiple reaction monitoring (MRM). Data collected from the instrument were analyzed with the Agilent MassHunter Quantitative Analysis software (B.05.02, Agilent, Santa Clara, CA, USA).
5
Bile Acid Profiling by LC-MS/MS
6
CD73 Enzyme Activity Assay in HTR-8/SVneo Cells
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We assessed the CD73 enzyme activity of HTR-8/SVneo cells by measuring the conversion rate from AMP to adenosine. In brief, HTR-8/SVneo cells were incubated with Hank's balanced salt solution (HBSS) in the presence or absence of APCP for 30 min. After washing 3 times with HBSS, cells were stimulated with 100 μM AMP for 10 min. Then, the supernatants (100 μl) were subjected to liquid chromatograph-mass spectrometry (LC-MS) analysis (Agilent 1290–6495C, California, USA). An Ultimate® HILIC Amphion column (2.1 × 100 mm, 3 μm, Welch Materials, China) was used for separation. The mobile phase consisted of (A) 0.1 % formic acid solution and (B) acetonitrile, 0–2.0 min, 2 % A; 2.0–6.0 min, 70 % A; 6.0–7.0 min, 70 % A; 7.0–8.0 min, 2 % A. The flow rate was 0.3 ml/min, and the injection volume was 2 μl. Adenosine was monitored with an MRM transition of 268.11 to 136.1 m/z, fragmentor of 166 V and collision energy of 17 V. AMP was monitored with an MRM transition of 348.07 to 136.1 m/z, fragmentor of 166 V and collision energy of 21 V. Data were processed with the Agilent MassHunter Quantitative Analysis software (version 10.1, Agilent, USA). The enzyme activity of CD73 was expressed as adenosine production per mg protein for 10 min.
7
Determining LOD and LOQ Accurately
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Limit of detection (LOD) and limit of quantitation (LOQ) were determined using the LINEST function of excel and data from the Agilent Mass Hunter Quantitative Analysis where a signal ratio of 3.3:1 from baseline was used for LOD and LOQ was determined using signal ratio of 10:1 from baseline. R2 values and equations were calculated using Agilent Mass Hunter Quantitative Analysis software, where the calibration curve fit origins were forced through zero.
8
Oxidative Stress Lipid Mediator Profiling
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For peak determination and integration, signaling lipid mediators profiled by the oxidative stress platform were pre-processed by LabSolutions (Shimadzu, Version 5.65); peak-picking of oxylipins was performed with Agilent MassHunter Quantitative Analysis software (Agilent, Version B.05.01) and amines with MultiQuant Software for Quantitative Analysis (AB SCIEX, Version 3.0.2). For all metabolites, raw data correction was accomplished using selected internal standards by calculating the ratio of peak area of the target compound to the peak area of assigned internal standard from which a response ratio for each analyte was obtained. QC samples were used for evaluating the quality of the targeted compounds according to the in-house written protocol and the data were hereafter ready to be used for statistical analyses.
9
Global Semiquantitative Analysis of SHL Preparations
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Global semiquantitative analysis was carried out using the (.d) files with the same polarity of the replicate measurements of each SHL preparation form obtained by the Agilent MassHunter Acquisition software and the corresponding combined data files (.cef) with the identities obtained by the Agilent MPP software. The (.d) and (.cef) files were imported into Agilent MassHunter Quantitative Analysis software (Version: B.06.00). The retention time window was set at 0.6 min in the method setup task. The m/z of IS adducts, [IS+NH4]+ and [M−H]−, were chosen for the positive and negative ionization modes and flagged. Other chemical components were set as targets relative to the IS, and the ionization polarities were identified. After validating the method setup, global semiquantitative analysis was performed based on the peak area ratio of each target to the IS. The results were exported as an Excel file for reporting.
10
Phenolic Profiling of Lemon Verbena Infusions
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The phenolic composition of the infusions was analyzed as previously described [13 (link)]. Briefly, after lyophilization, dry residues of lemon verbena infusions were resuspended in methanol/water (50:50, v/v) and filtered (0.22 µm). The obtained extracts were analyzed in an Agilent 1290 UHPLC system (Agilent Technologies, Santa Clara, CA, USA) coupled to an Agilent 6540 quadrupole-time-of-flight mass spectrometer (Q-TOF MS) equipped with an orthogonal ESI source. The chromatographic separation was performed using a Zorbax Eclipse Plus C18 column (2.1 × 100 mm, 1.8 μm of particle diameter; Agilent Technologies, Santa Clara, CA, USA). The phenolic compounds were then tentatively identified using the Agilent Mass Hunter Qualitative analysis software (version B.07.00, Agilent Technologies, Santa Clara, CA, USA), making use of accurate mass data, ion source fragmentation, MS/MS fragmentation patterns, MS databases (e.g., HMDB, Metlin, PubChem), and bibliographic search. For quantitative comparison in terms of relative abundance, peak area values were obtained using the Agilent Mass Hunter Quantitative analysis software (for Q-TOF, version B.08.00, Agilent Technologies, Santa Clara, CA, USA).
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