Analytical Sciences

Fluorometric Determination of Phospholipids byHigh-Performance Liquid Chromatography with Postcolumn On-Line Hydrolysisunder UV-Irradiation, Phosphomolybdic Acid and Thiamine-ThiochromeReactions

Yoshihiko IKEGUCHIand Hiroshi NAKAMURA *

Department of AnalyticalChemistry, Faculty of Pharmaceutical Sciences, Science University of Tokyo,Ichigaya-funagawara-machi, Shinjuku, Tokyo 162-0826, Japan

A postcolumn phosphate-selective fluorescencedetection system was developed for the determination of phospholipids byhigh-performance liquid chromatography. Phospholipids were separated on asilica gel column and then on-line hydrolyzed by photolysis in the presenceof titanium dioxide under UV-irradiation (253.7 nm). The resultantorthophosphate was reacted with a molybdenum reagent to formphosphomolybdic acid, which was then reacted with the alkaline thiaminereagent. Finally, the fluorescence intensity of the resultant thiochromeconverted from thiamine by phosphomolybdic acid was measured by setting theexcitation wavelength at 380 nm and the emission wavelength at 442 nm. Thecalibration curves for phospholipids under the gradient elution programwere linear (correlation coefficient, 0.984 - 0.991) in the range 0.02 -0.5 mg ml^-1 (injection volume, 10 ml). When every 2 mg each offive phospholipids was repeatedly injected into this system, the relativestandard deviations were between 4.14 and 4.76% (n=5). (Keywords: High-performance liquid chromatography, postcolumnfluorescence detection, phospholipids, on-line hydrolysis, UV-irradiation,TiO₂, phosphomolybdic acid reaction, thiamine-thiochromereaction)

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