[Determination of phospholipids in bear bile by isocratic high performance liquid chromatography]

A high performance liquid chromatographic method for the determination of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) in the bear bile was established. The air-dried bile of bear was ground into powder and extracted continuously with V(chloroform):V(methanol) = 1:1 to a final dilution 25-fold the volume of the sample. The extract was washed by addition to it of 0.5 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 diluted to the desired volume by addition of V(chloroform):V(methanol) = 1:1 mixture and then injected into the HPLC system. The conditions of HPLC were P-E silica column (150 mm x 4.6 mm i.d., 10 microns); V(acetonitrile):V(methanol) = 76:24 mobile phase with a flow rate of 1.5 mL/min and UV detector at 205 nm. The linear regression equation was Y = 5,917X + 1,709(r = 0.9970) for PI, Y = 7,362.1X-6,608.2(r = 0.9949) for PE and Y = 2,457X-2,460.5(r = 0.9972) for PC. The linear range were 1.0-12.0 micrograms, 2.0-24.0 micrograms and 9.5-114.0 micrograms respectively, The relative standard deviation of peaks were 1.6%, 0.89% and 2.6% respectively. The average recovery of PC was 89.30% (RSD = 2.0%). The method enables a simple, rapid and reproducible quantification of PI, PE and PC.