Fluorometric determination of phospholipids by high-performance liquid chromatography with postcolumn on-line hydrolysis under UV-irradiation, phosphomolybdic acid and thiamine-thiochrome reactions

A postcolumn phosphate-selective fluorescence detection system was developed for the determination of phospholipids by high-performance liquid chromatography. Phospholipids were separated on a silica gel column and then on-line hydrolyzed by photolysis in the presence of titanium dioxide under UV-irradiation (253.7 nm). The resultant orthophosphate was reacted with a molybdenum reagent to form phosphomolybdic acid, which was then reacted with the alkaline thiamine reagent. Finally, the fluorescence intensity of the resultant thiochrome converted from thiamine by phosphomolybdic acid was measured by setting the excitation wavelength at 380 nm and the emission wavelength at 442 nm. The calibration curves for phospholipids under the gradient elution program were linear (correlation coefficient, 0.984-0.991) in the range 0.02 - 0.5 mg ml(-1) (injection volume, 10 mu l). When every 2 mu g each of five phospholipids was repeatedly injected into this system, the relative standard deviations were between 4.14 and 4.76% (n=5).