Dry AT lipid extract corresponding to 20 mg of AT dissolved in CHCl3/MeOH (150 μL; 2:1, v/v). Formic acid (0.1% v/v) acidified MeCN (900 μL) was added. Solution was loaded onto dry SPE cartridge (HybridSPE® – Phospholipid, 30 mg/1 ml, Lot: 4800102, Supelco), washed subsequently with formic acid (0.1% v/v) acidified MeCN (1 mL), MeCN (1 mL), and eluted with MeCN containing 5% (w/v) NH4OH (2 × 1 mL). Eluates were dried in vacuo (Eppendorf concentrator 5301, 1 mbar).
Hybridspe phospholipid
Manufactured by Merck Group
Sourced in United States
HybridSPE®-Phospholipid is a laboratory equipment product developed by Merck Group. It is designed for the selective extraction and removal of phospholipids from samples during sample preparation processes.
Automatically generated - may contain errors
3 protocols using hybridspe phospholipid
1
Phospholipid Extraction and Purification
2
Extraction and Analysis of Milk Phospholipids
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To a 1.5-mL polypropylene tube, 100 μL milk sample, 890 μL 1% formic acid in methanol and 10 μL internal standard (1) 15:0-18:1-d7-PE were added. The contents were mixed for 30 s at 2000 rpm, followed by centrifugation for 10 min at 10,000 rpm. After that, 900 μL of the supernatant was loaded onto a HybridSPE-Phospholipid (bed weight, 20 mg) cartridge (Supelco, Sigma Aldrich, St. Louis, MO, USA), washed in sequence with 2 × 1 mL methanol and 2 × 1 mL 2-propanol. Finally, the phospholipids were eluted with 2 mL 5% ammonia in methanol. The extract was evaporated under a nitrogen stream at 35 °C. Next, 90 μL of the diluted LLE extract was used to dissolve the dried phospholipid extract. Additionally, 10 μL internal standard (2) 15:0-18:1-d7-15:0 TG was added to the final extract, which was then transferred to a 1.5-mL chromatographic vial and subsequently analysed by LC–Q-TOF–MS.
3
Plasma Metabolomics Using LC-TOFMS
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80μL of plasma was added to 240μL of 1% formic acid/acetonitrile containing the internal standard (same as CE-FTMS) at 0°C. The mixture was centrifuged at 2,300× g, 4°C for 5 min and then filtered using a hybrid SPE phospholipid cartridge (Hybrid SPE - Phospholipid 30 mg/mL, SUPELCO) to remove phospholipids. The filtrate was then evaporated to dryness under nitrogen, dissolved in 80μL of 50% isopropanol (v/v), and conducted a metabolomic analysis using liquid chromatography time-of-flight mass spectrometry (LC-TOFMS) based on the methods previously described (HMT’s LC package) [24, 25 (link)]. Briefly, LC-TOFMS analysis was performed using an Agilent 1200 HPLC pump, an ODS column (2 mm×50 mm, two μm i.d.), and an Agilent 6210 time-of-flight mass spectrometer (Agilent Technologies, USA). The system was controlled by MassHunter (Agilent Technologies, USA), and the spectrometer was scanned from m/z 50 to 1,000.
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