Establishment Of A Highly Sensitive Assay For Folic Acid Based On Derivatization Technique

Folic acid is an essential micronutrient for humans and animals and plays an important role in single-carbon metabolism.Folate deficiency affects DNA synthesis,repair and methylation,which in turn has deleterious effects on maintaining genome stability and expression.Humans and animals cannot synthesize folic acid on their own and can only obtain it from their diet.Plant and animal foods are the main sources of dietary folic acid.Accurate determination of folate levels in different foods is an important prerequisite for implementing precise nutritional regulation by improving the composition of the diet.The low chemical stability of folic acid and the interconversion between different forms of folic acid hinder the precise analysis and affect the accuracy of its quantification results;in addition,the complex matrix in biological samples is an important factor affecting the quantification of folic acid.In this thesis,a method based on high performance liquid chromatography-tandem mass spectrometry(HPLC-MS)combined with stable isotope derivatization was developed to solve the above analytical problems.1.A high performance liquid chromatography-tandem mass spectrometry(HPLC-MS)method was developed for the determination of total folate and different forms of folate by using the "light" reagents sodium cyanoborohydride and12C-formaldehyde,and the "heavy" reagents sodium deuterated cyanoborohydride and 13C-formaldehyde as isotopic derivatization reagents,respectively.HPLC-MS)for the detection of total folate and different forms of folate,respectively.The optimum conditions for the derivatization of folate were determined: formic acid concentration of 1%,sodium cyanoborohydride concentration of 30 m M,formaldehyde concentration of 0.2 %,derivatization temperature of 30 ° C and derivatization time of 60 min,under which the highest conversion rate of folate was achieved.Meanwhile,the isotope internal standard dilution method was used for quantitative analysis,which can effectively overcome the matrix effect.2.Since folates are susceptible to temperature,p H and other factors,resulting in the interconversion between folates,a high performance liquid chromatography tandem mass spectrometry(LC-MS)method based on derivatization technique for the determination of different forms of folates was established;after that,various aspects such as linearity,sensitivity,MRM parameters,ion cleavage mode,limit of detection,limit of quantification and stability were investigated to verify the The method was validated in terms of linearity,sensitivity,MRM parameters,ion cleavage mode,limit of detection,limit of quantification and stability,and was able to accurately distinguish the two isomers 5-formyltetrahydrofolate and 10-formyltetrahydrofolate by secondary fragment ions and peak time.3.The established method for total folate detection was applied to compare the total folate content in folate-enriched eggs with that in ordinary eggs.150 μg/100 g of total folate was obtained in folate-enriched eggs and 100 μg/100 g in ordinary eggs.4.The established method for the determination of different forms of folic acid was applied to compare the differences in the contents of different forms of folic acid in tomatoes,corn and folic acid-rich eggs.Based on the results,it was found that tomatoes,corn and folic acid-rich eggs contained the highest amount of5-methyltetrahydrofolate,followed by 5,10-methylenetetrahydrofolate and5,10-submethyltetrahydrofolate,in which folic acid,dihydrofolate,tetrahydrofolate,5-formyltetrahydro folic acid,dihydrofolic acid,tetrahydrofolic acid,5-formyltetrahydrofolic acid and 10-formyltetrahydrofolic acid were found to be low.In this thesis,a method for the determination of total folate and different forms of folate was successfully established by stable isotope derivatization combined with high performance liquid chromatography-tandem mass spectrometry,and was successfully applied to actual biological samples such as folate-rich eggs,tomatoes and maize,which can be extended to the analysis of other different types of biological samples such as vegetables,fruits,meat,eggs and milk as well as cells and blood.This assay simplifies the sample processing steps and the results are accurate and rapid,which has a broad application prospect.