SPECIFIC DETECTION AND QUANTIFICATION OF PALMITOYL-STEAROYL-PHOSPHATIDYLSERINE IN HUMAN BLOOD USING NORMAL-PHASE LIQUID-CHROMATOGRAPHY COUPLED WITH ELECTROSPRAY MASS-SPECTROMETRY

A narrow-bore normal-phase high-performance liquid chromatography (HPL C) method was developed for separation of phospholipid classes using a n HPLC diol column and a gradient of chloroform and methanol with 0.2% formic acid titrated to pH 5.3 with ammonia. The HPLC system was coup led on-line with an electrospray mass spectrometry (ES-MS) or electros pray tandem mass spectrometry (ES-MS-MS) system and the separation of several major phospholipid classes was shown. The molecular species of some phospholipid classes in human blood were qualitatively determine d. A method was further developed for specific determination of a mole cular species from phosphatidylserine, palmitoyl-stearoyl-phosphatidyl serine (PSPS), in human blood using HPLC-ES-MS. The analyses were perf ormed by single ion monitoring of the [M-H](-) molecular ions of PSPS and an internal standard, dipalmitoyl-phosphatidylserine. The limit of quantification of the method was 1.2 ng of PSPS. The calibration curv e ranged from 0.12 to 5.81 mu g/ml of PSPS dissolved in the mobile pha se. The curve was fitted to a second-order polynomial equation and fou nd to be highly reproducible. Analysis of control samples was found to be reproducible with a between-series precision below 9.2% R.S.D. The amount of endogenous PSPS in human blood was determined in 13 subject s and found to range from 1.73 to 3.09 mu g/ml. The identity of endoge nous PSPS was confirmed by HPLC-ES-MS-MS. (C) 1998 Elsevier Science B. V. All rights reserved.