Sample preparation and metabolite extraction were performed according to Izumi et al. [58 ]. For sample preparation, culture broth was filtered under vacuum suction. Filter-bound cells were frozen in liquid nitrogen to quench metabolism. Cell metabolites were extracted using methanol–water–chloroform (5:2:2) extraction, polar and nonpolar phase metabolites were separated and finally lyophilized before analysis. A detailed description is given in [58 ] and Additional file 6.5 . Cell extracts were analyzed according to Huang et al. [59 (link)] by (1) pentafluorophenylpropyl (PFPP) stationary phase liquid chromatography (Discovery HS F5, 150 mm × 2.1 mm, particle size 3 µm, Sigma-Aldrich Corp., Germany) coupled with electrospray ionization (ESI) in positive and negative modes; and (2) reversed phase ion pairing liquid chromatography with a C18 column (CERI L-column 2 ODS, 150 mm × 2.1 mm, particle size 3 μm, Chemicals Evaluation and Research Institute, Kyoto, Japan) coupled with ESI in negative mode, to a triple-quadrupole mass spectrometer (LCMS 8030 plus; Shimadzu, Japan). A detailed description of mobile phases, concentration gradients, flow rates, injection volumes, column oven temperatures, as well as MS parameters is given in Additional file 6.5 .
Discovery hs f5
Manufactured by Merck Group
Sourced in United States
The Discovery HS F5 is a laboratory equipment product by Merck Group. It is designed for general laboratory use, providing core functionality for various applications. The device specifications and detailed technical information are available upon request.
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4 protocols using discovery hs f5
1
Targeted Metabolomic Analysis of Cellular Extracts
2
Metabolite Profiling of Serum in RIPK1-tKO Mice
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The liquid chromatography–tandem mass spectrometry analysis of metabolites in serum from 10-month-old control or RIPK1-tKO mice was performed using a Nexera X2 system (Shimadzu Corp.) equipped with two LC-30 AD pumps, a DGU-20A5R degasser, an SIL-30 AC autosampler, a CTO-20 AC column oven, and a CBM-20A control module, coupled with an LCMS-8060 triple quadrupole mass spectrometer (Shimadzu Corp.). A pentafluorophenylpropyl column (Discovery HS F5; 150 mm by 2.1 mm, 3 μm; Sigma-Aldrich) was used for the separation of metabolites. The mobile phase was composed of A, 0.1% (v/v) formic acid in water, and B, 0.1% (v/v) formic acid in acetonitrile. The flow rate, column temperature, and injection volume were set as 0.25 ml/min, 40°C, and 3 μl, respectively. The gradient program for mobile phase B was as follows: 0 min, 0%; 2 min, 0%; 5 min, 25%; 11 min, 35%; 15 min, 95%; 20 min, 95%; 20.1 min, 0%; and 25 min, 0%. The mass spectrometer was equipped with an electrospray ionization source.
3
Quantification of Monoamines and Metabolites
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An Agilent 1290 Liquid Chromatography (LC) system equipped with an autosampler and coupled to AB Sciex QTRAP 6500 mass spectrometer (MS) was used to quantify the corresponding monoamine and metabolites. Chromatographic separation was achieved on a Discovery HS F5 (150 mm x 4 mm, 3 µm, Sigma-Aldrich, St. Louis, MO, USA) pentafluorophenyl column thermostated at 37 o C. The mobile phase was water (A) and methanol (B) with 0.1% of HCOOH in both solvents. An increasing linear gradient (v/v) of B was used (t(min), %B), as follows, (0, 0), (0.5, 0), (5.90, 30), (6, 100), (9, 100), (9.10, 0), (10.0, 0) at a constant flow rate (500 µL/min). The flow was directed to waste for the first 2 min to prevent the inorganic ions of aCSF solution to A negative ion mode was used in the analysis of HVA (m/z 181 → 122).
Six standards (from 0.1 nM to 10 nM for DA or from 10 nM to 1 µM for metabolites)
were prepared daily in a solution composed by aCSF/antioxidant mixture (2:1) to obtain the calibration curve. The method showed linearity within the concentration range studied and the detection limit (signal-to-noise ratio=3) for DA was 0.05 nM and for DOPAC and HVA was 1 nM. The accuracy of the assay was 85 -115% and the intraand inter-assay coefficients of variation were less than 15%. Analyst v1.4.2 software was used to calculate the areas of chromatographic peaks.
Six standards (from 0.1 nM to 10 nM for DA or from 10 nM to 1 µM for metabolites)
were prepared daily in a solution composed by aCSF/antioxidant mixture (2:1) to obtain the calibration curve. The method showed linearity within the concentration range studied and the detection limit (signal-to-noise ratio=3) for DA was 0.05 nM and for DOPAC and HVA was 1 nM. The accuracy of the assay was 85 -115% and the intraand inter-assay coefficients of variation were less than 15%. Analyst v1.4.2 software was used to calculate the areas of chromatographic peaks.
4
Quantification of Neurotransmitters and Metabolites
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An Agilent 1290 Liquid Chromatography (LC) system equipped with an autosampler and coupled to AB Sciex QTRAP 6500 mass spectrometer (MS) was used to quantify DA, 5-HT and metabolites. Chromatographic separation was achieved in a Discovery HS F5 (150 mm x 4 mm, 3 µm, Sigma-Aldrich, St. Louis, MO, USA) pentafluorophenyl column thermostated at 37 o C. The mobile phase was water (A) and methanol (B) with 0.1% of formic acid in both solvents. An increasing linear gradient (v/v) of B was used (t(min), %B), as follows, (0, 0), (0.5, 0), (5.90, 30), (6, 100), (9, 100), (9.10, 0), (10.0, 0) at a constant flow rate (500 µl/min). The flow was directed to waste for the first 2 min to prevent the inorganic ions of aCSF solution to enter the mass spectrometer. The microdialysate samples were refrigerated at 4 ºC and 20 µL was injected, without sample pretreatment, into the LC-MS/MS system. Mass spectrometric quantification in positive ion mode was carried out using the following transitions: DA (m/z 154 → 137 and 154 → 91, collision energies (CE) of 15 and 31 V, respectively), DOPAC (m/z 123 → 77, CE of 24 V), 5-HT (m/z 177 → 160, CE of 13 V) and 5-HIAA (m/z 192 → 146, CE of 23 V). A negative ion mode was used in the analysis of HVA (m/z 181 → 122, CE of -20 V).
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