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 x 2.1 mm, I.D. 5 mum) 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 degreesC. Eluted phospholipids were detected at wavelengths lambda = 235 and 254 nm. This method made it possible to determine quantitatively: 5 mug ml(-1) PC, 1 mug ml(-1) LPC, 4 mug ml(-1) PE and 3 mug m(-1) PI in blood plasma samples. (C) 2003 Elsevier Science B.V. All rights reserved.