LC/MS Analysis of Phospholipids in Milk on Ascentis® Express HILIC after SPE using Supelclean™ Si, Shotgun HPLC Analysis

LC/MS Analysis of Phospholipids in Milk on Ascentis® Express HILIC after SPE using Supelclean™ Si, Shotgun HPLC Analysis

Conditions

sample/matrix Phospholipids from 10 mL cow's milk (Folch method extraction)
SPE tube/cartridge Supelclean LC-Si, 1 g/6 mL (57051)
condition hexane
sample addition 1 mL extract in chloroform/methanol (2:1, v/v)
washing 3 mL of hexane/diethylether (8:2, v/v) and 3 mL of hexane/diethylether (1:1, v/v) to remove nonpolar lipids
elution 1 x 4 mL methanol, followed by 1 x 2 mL methanol, followed by 1 x 2 mL chloroform/methanol/water (3:5:2, v/v/v).
eluate post-treatment dry under a gentle stream of nitrogen, redissolved residue in chloroform/methanol (2:1, v/v)
column Ascentis Express HILIC, 15 cm x 4.6 mm I.D., 2.7 µm particles (53981-U)
column temp. ambient
mobile phase [A] acetonitrile; [B] acetonitrile:water (2:1)
gradient 0% B held for 5 min; to 30% B in 5 min; to 100% B in 20 min; held at 100% B for 10 min; to 0% B in 1 min
flow rate 0.7 mL/min
injection 5 µL
detector ELSD. nebulizing gas (nitrogen) flow: 2 mL/min (180 KPa); drift tube temperature: 50 °C

Description

Analysis Note Extraction of the lipid fraction was carried out from 10 mL of the cow’s milk sample, according to the Folch method to ensure the exhaustive extraction of the whole lipid content. The total extract was evaporated under vacuum, and the final dry residue (400 mg) was re-dissolved in chloroform/methanol 2:1 (v/v). Lipid extract (100 mg) was afterward dissolved in 1 mL mixture of chloroform/methanol (2:1, v/v). After the cartridge has been conditioned with hexane, the non-polar lipids were eluted with 3 mL of hexane/diethyl-ether (8:2, v/v) and 3 mL of hexane/diethyl-ether (1:1, v/v). Recovery of PLs from the cartridge was obtained by two-step elution, using 4 mL of methanol as first extraction solvent, and subsequently 2 mL of methanol followed by 2 mL of chloroform/methanol/water (3:5:2, v/v/v). The recovered fraction was dried under a gentle stream of nitrogen, yielding 49.7 mg dry residue. The residue was finally re-dissolved in chloroform/methanol (2:1, v/v).
Categories Analytical Chromatography, Food & Beverage Analysis, Flavors, Fragrances, Beverage Testing, Milk
Featured Industry Food and Beverages
Legal Information Ascentis is a registered trademark of Sigma-Aldrich Co. LLC, Supelclean is a trademark of Sigma-Aldrich Co. LLC
suitability application for HPLC

Materials

     
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