LC/MS Analysis of Phospholipids in Milk on Ascentis® Express HILIC after SPE using Supelclean™ Si, Shotgun HPLC Analysis
Materiais
coluna analítica
padrão
tubo ou placa SPE
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)
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
column temp.
ambient
detector
ELSD. nebulizing gas (nitrogen) flow: 2 mL/min (180 KPa); drift tube temperature: 50 °C
injection
5 μL
Descrição
Nota de análise
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).
Informações legais
Ascentis is a registered trademark of Merck KGaA, Darmstadt, Germany
Supelclean is a trademark of Sigma-Aldrich Co. LLC