The plasma metabolome analysis was conducted by MxP® Global Profiling (Metanomics-Health GmbH, Berlin, Germany) and an MxP® Lipids (Metanomics-Health GmbH, Berlin, Germany) (Figure 1 A). For the global profiling, two types of mass spectrometry analyses were applied, namely gas chromatography-mass spectrometry (GC-MS; Agilent 6890 GC coupled to an Agilent 5973 MS System, Agilent, Waldbronn, Germany) and liquid chromatography-MS/MS (LC-MS/MS; Agilent 1100 HPLC-System, Agilent, Waldbronn, Germany, coupled to an Applied Biosystems API4000 MS/MS-System, Applied Biosystems, Darmstadt, Germany). For lipid profiling, the lipids were fractionated [16 (link)] and analyzed by LC-MS/MS using electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), with detection of specific multiple reaction monitoring (MRM) transitions for cholesterol esters (CE), sphingomyelins (SM) and ceramides (CER), respectively. Other lipid classes were measured by cas chromatography flame ionization detection (GC-FID). Sphingolipids were determined semi-quantitatively by ultra high-performance liquid chromatography (UHPLC)-MS/MS. The sample preparation for global profiling and lipids is described in detail in [12 (link)].
Agilent 5973 ms system
Manufactured by Agilent Technologies
Sourced in Germany
The Agilent 5973 MS System is a gas chromatography-mass spectrometry (GC-MS) instrument used for the identification and quantification of chemical compounds. It features a 5973 series mass selective detector (MSD) for high-performance mass analysis.
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4 protocols using agilent 5973 ms system
1
Plasma Metabolomic Profiling by Mass Spectrometry
2
Broad Metabolite and Hormone Profiling
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Broad metabolite profiling as well as quantification of catecholamines and steroids from human plasma samples were performed at metanomics GmbH (Berlin, Germany) as previously described.13 , 14 Briefly, MxP® Broad profiling was performed using gas chromatography‐mass spectrometry (Agilent 6890 GC coupled to an Agilent 5973 MS‐System) and LC‐MS/MS (Agilent 1100 HPLC‐System coupled to a MS/MS‐System from Applied Biosystems API4000). For determination of catecholamines and steroids, solid phase extraction (SPE; Spark Symbiosis Pharma) coupled to LC‐MS/MS was used.
3
GC-MS Analysis of Phytochemicals
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The phytochemical analysis of LRC was performed with an Agilent 6890 GC apparatus interfaced to an Agilent 5973 MS system equipped with an electron ionization (EI) source and autoinjector (Agilent Technologies, Inc.). GC/MS was conducted according to a previous study (21 (link)). The GC system was built with a DB-5 column (30.0 m x 0.25 mm x 0.25 µm). The temperature varied from 80°C (5 min) to 200°C at 5°C/min, and the injection volume was 2 µl. Injection was performed in the split mode adjusted to 1:5. The carrier gas was helium at 1.0 ml/min. The inlet, source and quadrupole temperatures were set at 290, 230 and 190°C, respectively. For MS detection, the EI mode with an ionization energy of 70 eV was used, with a mass range at m/z 50-800. Agilent ChemStation software (Agilent Technologies, Inc.) was used for data processing. The phytochemical compounds were identified by mass fragmentation patterns compared via Wiley Spectral Library search.
4
Metabolomics Analysis of Fish Muscle
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Twelve muscle samples containing pooled muscle tissue of five individual fish from the same tank were analysed in an MxP ® Global Profiling by Metanomics Health GmbH. Samples were weighed, freeze dried and homogenised, and dry weight was determined before extraction. After extraction and protein precipitation, samples were separated into lipid and polar fractions before further analysis using the GC-MS (Agilent 6890 GC coupled to an Agilent 5973 MS System, Agilent) and liquid chromatography-MS/MS (LC-MS/MS; Agilent 1100 HPLC-System, Agilent, coupled to an Applied Biosystems API4000 MS/MS-System, Applied Biosystems). Integration and validation of chromatographic data were performed by Metanomics Health GmbH. Metabolite levels were normalised against the median of the pool reference samples (derived from aliquots of all samples) to give pool-normalised ratios performed for each sample per metabolite. GraphPad Prism (version 8.3.0) was used for illustration of pool-normalised metabolite data. Evaluation of all metabolites was semi-quantitative and metabolite levels were reported as pool-normalised ratios (online Supplementary Table S4). Results were given for free (non-covalently bound) metabolites as sample preparation did not involve hydrolysation.
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