title: Quantification of phosphatidylserine, phosphatidic acid and free fatty acids in an ultrasound contrast agent by normal-phase high-performance liquid chromatography with evaporative light scattering detection.
abstract: Sonazoid is a new contrast agent for ultrasound imaging. The product is an aqueous suspension of perfluorobutane microbubbles coated with phospholipids obtained from hydrogenated egg phosphatidylserine (H-EPS). A normal-phase high-performance liquid chromatographic (HPLC) method with evaporative light scattering detection was developed for quantification of free fatty acids, phosphatidylserine and phosphatidic acid in H-EPS and Sonazoid. Separation of the lipids was carried out on an HPLC diol column and a gradient of chloroform and methanol with 0.2% formic acid titrated to pH 7.5 with ammonia. The calibration standards contained stearic acid, distearoyl-phosphatidic acid (DSPA) and distearoyl-phosphatidylserine (DSPS) in the concentration range of 0.016-1.0mg/ml (0.4-25microg injected). The method was validated with a limit of quantification of the three lipids set to 0.4microg (approximately 20-60microM). The best fit of the three calibration curves were obtained when the logarithmic transformed theoretical lipid concentration was plotted against the logarithmic transformed area under the peak and fitted to a second order polynomial equation. Stearic acid, DSPA and DSPS were analysed with an intermediate precision ranging from 4.4% to 5.3% R.S.D. and they were extracted from an aqueous suspension with a recovery ranging from 103.3% to 113.3%. The sum of total phospholipid concentration determined in H-EPS ranged from 96.4% to 103.2% of the theoretical values. The lipids in the ultrasound product were quantitated with a repeatability ranging from 6.2% to 11.7% R.S.D.
authors.affiliation: GE Healthcare Medical Diagnostics, Nycoveien 2, N-0401 Oslo, Norway.
authors.forename: Erlend
authors.initials: E
authors.lastname: Hvattum
authors.forename: Steinar
authors.initials: S
authors.lastname: Uran
authors.forename: Anne Gunvor
authors.initials: AG
authors.lastname: Sandbaek
authors.forename: Anders A
authors.initials: AA
authors.lastname: Karlsson
authors.forename: Tore
authors.initials: T
authors.lastname: Skotland
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003287:Contrast Media / Q000737:chemistry*; D005230:Fatty Acids, Nonesterified / Q000032:analysis*; D005290:Ferric Compounds / Q000737:chemistry*; D007501:Iron / Q000737:chemistry*; D008027:Light; D010087:Oxides / Q000737:chemistry*; D010712:Phosphatidic Acids / Q000032:analysis*; D010718:Phosphatidylserines / Q000032:analysis*; D015203:Reproducibility of Results; D012542:Scattering, Radiation; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D003287:Contrast Media; D005230:Fatty Acids, Nonesterified; D005290:Ferric Compounds; D010087:Oxides; D010712:Phosphatidic Acids; D010718:Phosphatidylserines; C069727:Sonazoid; D007501:Iron