Improved determination of individual molecular species of phosphatidylcholine in biological samples by high-performance liquid chromatography with internal standards.

Phosphatidylcholine isolated from samples of bile, liver and plasma was converted into 1,2-diradylglycerobenzoate molecular species by hydrolysis with phospholipase C and reaction with benzoic anhydride. Up to seventeen molecular species were separated and determined by reversed-phase high-performance liquid chromatography with detection at 230 nm. The major improvement introduced here was the use of distearoylphosphatidylcholine as the internal standard, which corrected the results for incomplete hydrolysis and benzoylation. Other improvements concerned the clean-up of benzoyl derivatives and the chromatographic separation. The analytical results obtained were validated by comparison with the results of either lipid phosphorus or gas chromatographic determinations.