On-line high speed lipid extraction for nanoflow liquid chromatography-tandem mass spectrometry

• Automated on-line lipid extractor for nanoflow LC-ESI–MS/MS.
• High speed (∼5 min) lipid extraction with a small volume of urine sample (∼20 μL).
• Minimization of lipid oxidation by elimination of air-exposure during extraction.

An on-line lipid extraction method is introduced by utilizing a short capillary extraction column using HILIC and C4 particles prior to nanoflow liquid chromatography-tandem mass spectrometry (nLC–MS/MS). The on-line extraction using a urine sample spiked with PL standards showed similar or slightly higher recovery values (86%–96%) of phospholipids (PLs) compared to those obtained by the conventional off-line extraction based on the Folch method with or without using the air-exposed drying process. In this study, we demonstrated that PL oxidation can occur during the air-exposed drying process of lipid extracts in standard liquid–liquid extraction procedures, which was confirmed by the oxidized PL (OxPL) molecules that were generated from an off-line extraction using a few PL standards. Quantitative comparison of these OxPL species between on- and off-line extraction followed by nLC–MS/MS with multiple reaction monitoring (MRM) analysis showed a significant decrease (2–10 fold) in unwanted OxPL species when on-line extraction was employed. While the number of identified PLs from a urine sample was somewhat lower than those by off-line extraction, the number of OxPLs was significantly reduced (from 70 to 22) with on-line extraction. The new method offers high speed (∼ 5 min) automated extraction of PLs with nLC–MS/MS analysis and presents the possibility of handling a biological sample with a very limited amount of lipids.