Quantitation of individual phospholipid molecular species by UV absorption measurements

To validate the utility of on-line measurements of UV absorption for the direct quantitation of individual phospholipid molecular species isolated by reverse-phase HPLC, we synthesized 37 different individual molecular species of diacyl choline glycerophospholipids. UV absorbance response factors (integrate UV absorbance/nmole phospholipid) were calculated for all diacyl, alkenylacyl, and alkylacyl species by injecting varying amounts of the purified molecular species in the 2-100 nmole range and integrating the UV absorbance at 203 nm. There was excellent agreement between the results of the quantitation of individual molecular species determined by measurements of phospholipid mass in HPLC fractions with that based on measurements of total integrated UV absorption and the use of absorbance response factors. The on-line quantitation of individual phospholipid molecular species by UV absorption measurement should prove useful as an adjunct to other techniques of phospholipid quantitation, as a means to assist in the identification of individual molecular species in complex biologic mixtures, and as a stand-alone approach to phospholipid quantitation to facilitate studies of the metabolism of individual species particularly when coupled with radiolabeling techniques.