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 (HPLC) method was developed for separation of phospholipid classes using an 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 coupled on-line with an electrospray mass spectrometry (ES-MS) or electrospray 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 determined. A method was further developed for specific determination of a molecular species from phosphatidylserine, palmitoyl-stearoyl-phosphatidylserine (PSPS), in human blood using HPLC-ES-MS. The analyses were performed 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 curve ranged from 0.12 to 5.81 microg/ml of PSPS dissolved in the mobile phase. The curve was fitted to a second-order polynomial equation and found 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 subjects and found to range from 1.73 to 3.09 microg/ml. The identity of endogenous PSPS was confirmed by HPLC-ES-MS-MS.
Hvattum
E
E
Nycomed Imaging AS, Oslo, Norway.
Larsen
A
A
Uran
S
S
Michelsen
P M
PM
Skotland
T
T
D000641:Ammonia; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004355:Drug Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry / Q000379:methods*; D000432:Methanol; D010718:Phosphatidylserines / Q000097:blood*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
C116995:1-palmitoyl-2-stearoyl-3-phosphoserine; D010718:Phosphatidylserines; D000641:Ammonia; D000432:Methanol