The purpose of this research was to adapt a colorimetric, phospholipase D-based serum-phospholipid assay for the quantification of phosphatidylcholine (PC) in liposomes using a microtitre plate reader. PC from natural egg PC liposomes was quantified reliably. In contrast, poor sensitivity was found for liposomes composed of saturated PCs (di-palmitoyl-phosphatidylcholine [DPPC], hydrogenated egg PC). Triton X-100 was then added to the liposomes followed by heating above the phase transition temperature. This modified sample preparation resulted in recoveries of 102.6% +/- 1.0%, 104.4% +/- 7.6%, and 109.4% +/- 3.2% for E80, E80-3/cholesterol, and DPPC liposomes, respectively. Absolute quantification of unknown PCs against a choline chloride standard is feasible, but relative measurements against the very same PC are recommended whenever possible. Validation experiments revealed an absolute quantification limit of 1.25 microg per assay, a good linearity in the range of 25 to 1000 microg/mL PC (r2> or = 0.9990) and a quite high accuracy (99.8%-101.4% of theory) and precision (relative standard deviation < or = 3.2%) for all 3 PCs studied. The method is thus regarded as suitable for sensitive, rapid, and reliable routine quantification of PCs in liposomes.