A method for determination of phosphatidylethanol from high density lipoproteins by reversed-phase HPLC with TOF-MS detection

Phosphatidylethanol (PEth) is a unique phospholipid that is formed in the body only in the presence of ethanol. According to a new hypothesis, blood high-density lipoprotein (HDL) particles may act as carriers of PEth and mediate part of the antiatherogenic effects of moderate alcohol drinking. Liquid chromatographic method using reversed-phase C8 column and negative ion mode electrospray ionization-mass spectrometry detection with time-of-flight (TOF) instrument was developed for the determination of very small amounts of PEth that might be present on blood HDL particles. The samples used in the current study were human HDL spiked with PEth and internal standard phosphatidylpropanol (PProp). The use of reversed-phase column enabled a short analysis time of 19 min/injection, which is only one-third of the earlier normal-phase methods reported. Because of the narrow bore column (2.1 mm i.d.) and short analysis time, the solvent consumption was decreased. The sensitivity of detection obtained with TOF-MS was better than that of previous methods, with the detection limit being as low as 1 ng/ml in injected sample (20 pg on-column ∼28 fmol PEth), corresponding to approximately 6.7 ng of PEth in milliliter of unprepared HDL. Good linearity of detection was obtained for a range of 1-100 ng/ml of PEth, whereas all of the deviations in precision and accuracy were less than 15%.