QUANTITATIVE-ANALYSIS OF PHOSPHATIDYLCHOLINE MOLECULAR-SPECIES USING HPLC AND LIGHT-SCATTERING DETECTION

A number of HPLC chromatographic procedures can be used to separate in tact molecular species of phosphatidylcholine (PC), but on-line quanti fication has remained problematic due to insensitivity of UV-detection for saturated species. Here, a new method is presented, separating al l major PC molecular species from a variety of biological samples in i ntact form using a single, short and isocratic run. Species were separ ated on two RP18 reverse-phase columns in series and all species displ ayed an exponential relation between retention time and the percentage of acetonitrile or triethylamine in the mobile phase, allowing optimi zation of the mobile phase on a theoretical base, rather than on time- consuming test-runs. The use of triethylamine as a volatile additive i nstead of choline chloride allowed the use of light scattering detecti on. On a molar base, the response of the detector was invariant betwee n species and allowed quantification of as little as 50 pmoles. The me thod was tested using phosphatidylcholines with widely different molec ular species patterns, such a PC from rat liver, porcine pulmonary sur factant, bovine heart, boar sperm cells, and the parasite Schistosoma mansoni. As only volatile components are present in the solvents, indi vidual molecular species can easily be recovered in pure form from the column effluent, enabling their further analysis (e.g., scintillation counting).