After animal sacrifice, the blood was harvested by cardiac puncture with a disposable syringe, immediately transferred to blood collection tubes (Covidien, Mansfield, MA), and centrifuged (1,500 g, 10 min, 4 °C) to yield plasma. The plasma was stored at −80 °C until analysis. To extract lipid metabolites from plasma, deuterated internal standards (d11-14,15-DHET, d11-11,12-EET, d4-9-HODE, d8-5-HETE, d4-6-keto PGF1a, d4-LTB4, d4-PGE2, d4-TXB2, 500 nM) were added into ~200 μL plasma. The mixture was then loaded onto pre-washed Waters® Oasis solid phase extraction (SPE) cartridges, and washed with 5% methanol containing 0.1% acetic acid, and the analytes were eluted with methanol and ethyl acetate, dried by a centrifugal vacuum evaporator, and then dispersed in methanol for LC-MS/MS analysis. The LC-MS/MS analysis was performed on an Agilent 1200SL HPLC coupled with a 4000 QTRAP MS/MS (AB Sciex) as previously14 (link). The peaks were identified based on retention time and specific multiple reaction monitoring (MRM) transitions of the standards of lipid metabolite. The concentrations of the lipid metabolites were calculated according to calibration curves of standards.
4000 qtrap ms ms
Manufactured by AB Sciex
The 4000 QTRAP MS/MS is a high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. It combines a triple quadrupole mass analyzer with a linear ion trap to provide sensitive and selective quantitation and structural characterization of analytes in complex samples.
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6 protocols using 4000 qtrap ms ms
1
Plasma Lipid Metabolite Extraction and Quantification
2
Lipid Metabolite Extraction from Colon Tissues
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To extract lipid metabolites from colon tissues, ~100 mg tissues were mixed with an antioxidant solution (0.2 mg/mL butylated hydroxytoluene and 0.2 mg/mL triphenylphosphine in methanol), deuterated internal standards, and 400 μL extract solution (0.1% acetic acid with 0.2 mg/mL butylated hydroxytoluene in a methanol solution), and then homogenized; the resulting homogenates were kept in −80 °C overnight. After centrifugation of the homogenates, the pellets were washed with methanol (containing 0.1% butylated hydroxytoluene and 0.1% acetic acid) and then combined with the supernatant. The combined solutions were loaded onto pre-washed Waters® Oasis solid phase extraction (SPE) cartridges, washed with a solution of 95:5 water/methanol with 0.1% acetic acid, the analytes were eluted with methanol and ethyl acetate, dried using a centrifugal vacuum evaporator, then reconstituted in methanol for LC-MS/MS analysis. The LC-MS/MS analysis was carried out using an Agilent 1200SL HPLC system (Agilent, Santa Clara, CA) coupled to a 4000 QTRAP MS/MS (AB Sciex, Foster City, CA) as described in our previous report [7 ].
3
Quantification of Lipid Metabolites by LC-MS/MS
4
Oxylipinomics Analysis of Small Intestine
5
Adipose Tissue Lipid Metabolite Extraction and Analysis
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To extract lipid metabolites from adipose tissues, ~100 mg tissues were mixed with an antioxidant solution (0.2 mg/mL butylated hydroxytoluene and 0.2 mg/mL triphenylphosphine in methanol), 10 µL of deuterated internal standards (500 nM of d4-6-keto PGF1a, d4-TXB2, d4-PGE2, d4-LTB4, d11-14,15-DHET, d4-9-HODE, d8-5-HETE, d11-11,12-EET), and 400 µL extract solution (0.1% acetic acid with 0.2 mg/mL butylated hydroxytoluene in methanol), and then homogenized; the resulting homogenates were kept in −80 °C overnight. After centrifugation of the homogenates, the pellets were washed with methanol (containing 0.1% butylated hydroxytoluene and 0.1% acetic acid) and then combined with the supernatant. The lipid metabolites in the combined solutions were loaded onto pre-washed Waters® Oasis solid phase extraction (SPE) cartridges, washed with 95:5 v/v water/methanol with 0.1% acetic acid, the analytes were eluted with methanol and ethyl acetate, dried using a centrifugal vacuum evaporator, then reconstituted in methanol for LC-MS/MS analysis. The LC-MS/MS analysis was carried out on an Agilent 1200SL HPLC system (Agilent, Santa Clara, CA) coupled to a 4000 QTRAP MS/MS (AB Sciex, Foster City, CA) as described in our previous report (24 (link)). The lipid mediators whose levels were above the detection limit of LC-MS/MS were reported.
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Adipose Tissue Lipid Metabolite Extraction and Analysis
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