Determination of eight phosphatidylethanol homologues in blood by reversed phase liquid chromatography-tandem mass spectrometry - How to avoid co-elution of phosphatidylethanols and unwanted phospholipids.

Phosphatidylethanols (PEths) are specific, direct alcohol biomarkers with a substantially longer half-life than ethanol, and can be used to distinguish between heavy- and social drinking. More than forty PEth homologues have been detected in blood from heavy drinkers, and PEth 16:0/18:1 is the predominant one. Since PEths are phospholipids it can be difficult to isolate them from unwanted phospholipids during sample preparation. To minimize possible matrix effects it is therefore important to separate PEths from other phospholipids during LC-MS/MS analysis. In this study, we have investigated how the retention and chromatographic separation of eight PEth homologues and the phospholipid background are influenced by changes in mobile phase composition using two different LC columns, the Acquity BEH C column (50 × 2.1 mm ID, 1.7 µm particles) and the Kinetex biphenyl column (100 × 2.1 mm ID, 1.7 µm particles). Our findings show that the buffer concentration of the aqueous part of the mobile phase had a huge effect on the retention of PEth homologues and separation of PEths from unwanted phospholipids. By using a buffer-free mobile phase consisting of 0.025% ammonia in Type 1 water, pH 10.7, as solvent A and methanol as solvent B, all eight PEth homologues were separated from both the early eluting lyso-phospholipids and the later eluting phospholipids with two fatty chains using the BEH C column. The knowledge obtained in this study can be of great importance for those seeking to develop reliable and robust bioanalytical LC-MS/MS methods for determination of PEth homologues.