[A rapid, isocratic method for analysis of phospholipids by reversed-phase high performance liquid chromatography]

Linear calibration curves were obtained for 4.5-10.5 micrograms of phosphatidylcholine(PC), 1.5-3.5 micrograms of phosphatidic acid(PA), 9.0-21.0 micrograms of cardiolipin(CL), 2.25-5.25 micrograms of phosphatidylglycerol(PG) by reversed-phase high performance liquid chromatography with UV detector at 205 nm, P-E C18 column (15 cm x 0.46 cm i.d., 5 microns) and isocratic elution with mobile phase of methanol-acetonitrile-water (79:8:13, V/V/V) at a flow rate of 2.0 mL/min. Separation and detection of these four standards were achieved reproducibly and easily in less than 12 minutes. The RSDs of peak area measurements were from 0.77% to 8.86%. Average recovery of PC was 89.21% (RSD = 2.1%). The air-dried bear biles were ground into powder and extracted continuously with chloroform-methanol (1:1, V/V) to a final dilution 25 folds the volume of the sample. The extract was washed by addition of half its volume of 0.58% aqueous NaCl. The resulting mixture separated into two phases. The lower phase was the total pure lipid extract and was dried at room temperature under nitrogen. The lipid residue was dissolved in methanol to the desired volume and injected into the HPLC system for analysis. The results indicated that six kinds of bear biles had differences in the phospholipid composition. The method enables a simple, rapid and reproducible quantification of PC, PA, PG and CL.