| Folic acid is one of important B-vitamins,and it is a carrier of one carbon unit in biochemical reaction in man.Folic acid deficiency will lead to not only megaloblastic anemia but also neural tube defects, tumor, cardiovascular diseases,and so on.So its importance in food is understood by more and more persons.In man, folic acid carries one carbon unit to form 5-methyltetrahydrofolic acids methylene tetrahydrofolic acid and other active forms ,then they become useful. 5-Methyltetrahydrofolic acid is the major folate in man.Measurement of folic acic in food and 5-methyltetrahydrofolic acid in man by reversed-phase high-performance liquid chromatography with fluorescent detection have very important significance.In this research,we developed a reversed-phase high-performance liquid chromatographic method with fluorescent detection for analysis of folic acid and 5-methyltetrahydrofolic acid ,then we used it to detect folic acid in food and 5-methyltetrahydrofolic acid in erythrocyte.We think that it provides condition for accurate determination of folic acid in food and study on erythrocyte 5-methyltetradrofolic acid in different people.l.Reverse-phase HPLC method with post-column derivatization for quantitation of folic acid in food and its applicationThe optimized HPLC procedure for determination of folic acid was chose.GOLD high-efficiency liquid chromatograph was used,a ODS CIS reverse-phase column was used protected with a guard column, and the volumn was enclosed in a heater maintained at 30癈. The mobile phase was a mixture of 50mM aqueous potassium phosphate buffer, PH 3.5, containing 12% acetonitrile, flow rate was Iml/min.The post-column derivatization reagent was potassium peroxodisulphate(K2S2Og),the optimized consistency was 0.3% and flow-rate was 0.5ml/min, temperature for derivatization was 60癈.The loop sizewas 20pi. The optimized excitation wavelength was 365nm and emission wavelength was 450nm. The sample was prepared with 60% perchloric acid, after centrifuging and filter the sample can be detected. The detection limits were 5ng/ml,linear range was 0.1 u g/ml~~3.2 u g/ml, correlation coefficient was 0.9999, recover rate was 98.07%,and variation coefficient was 2.31%.The method was suitable for determination of folic acid in food . 2.Reverse-phase HPLC method for quantitation of 5-methyltetrahydrofolic acid in erytnrocyte and its clinical applicationThe optimized HPLC procedure for determination of 5-methyltetrahydrofolic acid was chose.GOLD high-efficiency liquid chromatograph was used,a ODS CIS reverse-phase column was used protected with a guard column, and the column was enclosed in a heater maintained at 25 癈 .The mobile phase was a mixture of 50mM aqueous potassium phosphate buffer, PH 2.5, containing 8% acetonitrile, flow rate was Iml/min. The loop size was 20(Jl.The optimized excitation wavelength was 295nm and emission wavelength was 356nm. The fresh blood samples for HPLC were prepared by the addition of 0.8ml blood to 1.4ml ascorbic acid(10g/L) ,the mixture was incubated in a water-bath at 37癈 for 90min to allow the plasma carboxypeptidase to convert tne RBC folate polyglutamates to monoglutamates,next 0.2ml 60% perchloric acid was added.after centrifuging the sample can be detected.The detection limits were O.Olng/ml,linear range was lOn g/ml~100ng/ml, correlation coefficient was 0.9999, recover rate was 100.67% and variation coefficient was 3.85%.It can be used for determination of 5-methyltetrahydrofolic acid in erythrocyte.The RBC 5-methyltetrahydrofolic acid of health adults health senile and diabetes mellitus were 175.07 + 72.90 u g/L> 148.94?6.04 u g/L> 140.81 ?6.15 u g/L respectively.Conclusion:Reverse-phase HPLC method with post-column derivatization for quantitation of folic acid in food and reversed-phase HPLC method with fluorescent detection for analysis of 5-methyltetrahydrofolic acid are both simple > fast ^ accurate,and they have high sensitivity > recover rate and good repetition.so they have important value for application of determin...
|
PDF Full Text Request