Jfhm. Brouwers et al., QUANTITATIVE-ANALYSIS OF PHOSPHATIDYLCHOLINE MOLECULAR-SPECIES USING HPLC AND LIGHT-SCATTERING DETECTION, Journal of lipid research, 39(2), 1998, pp. 344-353
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).