Solid Phase Extraction - Photochemical Fluorescence - High Performance Liquid Chromatographic Determination Of Folic Acid In Plasma

Folic acid is Vitamin Bc made up of 2-amino-4-hydroxyl-6-methylpterin, para-amino benzoic acid and glutamic acid, transforming into tetrahydro-folic acid in the human body, the ones that participated in formatting amino acid and nucleic acid. Because the human metabolic process is extremely complicated, it is particularly difficult for the analysis of the biological sample of folic acid, the ordinary analysis method is difficult to detetmine it. We choose the solid phase extration to deal with the biological sample of folic acid and use macroporous resins as static phase for the first time. Meanwhile, to studied the photochemistry behavior of the folic acid, we find the folic acid can produce the hypofluorescence, but it be changed into a strong photochemically induced fluorescence by ultraviolet radiation in the special medium. so the sensitivity of the analysis has been improved. To combine the solid phase extration, photochemically induced fluorescence and the high performance liquid chromatogram together, can improve the sensitivity and selectivity in further. Objectives To set up a new method to determine the concentration of folic acid in plasma that is sensitive and the selectivity is good. Methods After the biological sample of folic acid are purified by macroporous resins, put into the Na2HPO4-NaH2PO4 buffer (0.05mol·L^-1,pH₅.4) and irradiated by ultraviolet for 50 minutes. And then, let the sample flow through a C18 column of HPLC with methanol-Na2HPO4-NaH2PO4 buffer (0.05mol·L^-1,pH5.4) as the mobile phase(V/V27.5:72.5). The C18 column is used with flow rate at 0.8 ml·min^-1 and temperature at 25℃. The fluorescence detection is at 280nm (excitation wavelength) and 443nm (emission wavelength). Results The linear range is over 0.100¹00.0μg·ml^-1(r=0.9988). The extraction and method recoveries are higher than 80.2% and 79.4%. The coefficients of variation of within-day and between-day (n=5) are lower than 7.8%. The detectable limit is 10ng·ml^-1. Conclusions The method is accurate, sensitive, the selectivity is good, the recovery rate is high for folic acid and can be used in routine determination.