Quantitative Targeted Lipidomics in Urine: Phosphatidylserine by UPLC-MS/MS and other selected phospholipids

摘要

Liquid/Liquid extraction with MTBE was found to be very effective for each phospholipids class tested; Drying urine overnight and reconstituting in mobile phase was tested, but recovery was low, so this approach was not pursued further. Chromatography with C18 and C18 CSH was tested; C18 CSH offers more flexibility and hopefully more chances to fine tune separation down the road. Two different types of mobile phases were tested; initially the aim was to optimize sensitivity with a water/ acetonitrile (A) and methanol/isopropanol and acetonitrile (B) mixtures, both containing 0.05% ammonium hydroxide and 1mM ammonium formate. Superior separation and peak shape was achieved with a high ionic strength, mobile phase: mobile phase A consists of acetonitrile/ water 60/40 (v/v) and mobile phase B is isopropanol /acetonitrile 90/10 (v/v), both with 10 mM ammonium formate and 0.05% ammonium hydroxide. To obtain good peak shape a compromise between sensitivity and separation power had to be accepted and the second mobile phase combination was used for the study. Our preliminary data on phosphatidylserine show that a comprehensive quantitative method for human urinary phospholipids is possible; the assay development for other phospholipids classes is under development using the same approach.