New HPLC method for separation of blood plasma phospholipids

The aim of the present work was to develop a new HPLC method for separation of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and lysophosphatidylcholine (LPC) from small-volume samples of blood plasma. Human plasma glycerophospholipids were separated by liquid–liquid extraction method followed by solid phase extraction (SPE) on aminopropyl columns. Reversed-phase Sephasil C8 column (10 cm×2.1 mm, I.D. 5 μm) and micropreparative chromatograph “SMART” were used for separation of PC, PE, LPC and PI from SPE phospholipids extract. Binary-step gradient of eluent A: acetonitrile–methanol (130:5, v/v) and B (0.01% trifluoroacetic acid) provided good, fast and reproducible resolution of investigated phospholipids classes in 12 min at 30 °C. Eluted phospholipids were detected at wavelengths λ=235 and 254 nm. This method made it possible to determine quantitatively: 5 μg ml −1 PC, 1 μg ml −1 LPC, 4 μg ml −1 PE and 3 μg ml −1 PI in blood plasma samples.