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Document Type

Thesis - University Access Only

Award Date


Degree Name

Master of Science (MS)


Nutrition, Food Science, and Hospitality

First Advisor

Padmanaban G. Krishnan


5-methyltetrahydrofolate (5 MeTHF) is the predominant natural form of folate in human blood and most foods. Therefore, it is of significance as an indicator of the natural folate content in foods and the folate status in humans. 5-methyltetrahydrofolate analysis was done in both human whole blood and food samples using reverse phase chromatography, an isocratic mobile phase of 6% acetonitrile: 94% 33 mM phosphate buffer (pH 2.3), a flow rate 1.0 ml/min, and fluorometric detection. Excitation and emission wavelengths were set at 290nm and 356nm, respectively. The analytical column used was a C18 column (250x4.6mm i.d., 4 µm particle size) protected by a guard column, and equipped with a 100 µl injection loop. Procedures for thermal extraction, enzymatic deconjugation, and clean up protocol using a strong anion exchange column (SAX) were optimized. Deconjugation of folate polyglutamates in red blood cells was done using 10 g/L of ascorbic acid, which made the pH of the final mixture favorable for endogenous plasma pteroylpolyglutamate hydrolase activity. A 90-minute incubation period at 37°C was sufficient for the conversion of red blood cell polyglutamate folates to monoglutamates. In food samples, a trienzyme extraction method consisting of α-amylase, protease, and rat plasma folate conjugase were used to totally liberate folate prior to quantification. Food sample extracts were incubated with a combined rat plasma folate conjugase and α-amylase for 4 hours at 37°C followed by protease treatment for I hour at 37°C. For food samples a second extraction was needed owing to complexity of sample matrices. In blood samples, a second extraction showed no gains in peak area. All samples extracts were purified using a strong anion exchange column (SAX) procedure prior to HPLC analysis. 5-methyltetrahydrofolate was eluted with 3 ml of 0.1 M sodium acetate containing 10% sodium chloride and 1 % ascorbic acid (pH 4.5). The SAX column proved effective for clean up of blood samples. The chromatograms of food samples after the SAX clean up showed peaks other than 5-methyltetrahydrofolate peak, although they generally did not interfere with the 5-methyltetrahydrofolate peak. The retention time for 5-methyltetrahydrofolate was approximately 18-22 minutes. Results for whole blood samples spiked with pure standard of 5-methyltetrahydrofolate yielded a mean recovery of 91.7± 5.1%. Food samples spiked in a similar fashion showed average recoveries of 103.7 ± 23.9%, 101.1 ± 4%, 131± 7.8%, 98.7± 10.6%, 107.9± 7 .5%, 100.1± 8.9% for Cheerios®, Kellogg's® Product 19 Kellogg's® Special K, milk powder, frozen mixed vegetables, and enriched macaroni, respectively. Both stock and working standard solutions were made using the buffer, which was used to elute 5-methyltetrahydrofolate from the SAX cartridge. A study of storage stability of working standard solutions indicated that there was no deterioration of the standard solutions up to 24 days of storage at -18°C.

Library of Congress Subject Headings

Folic acid
Blood -- Analysis
Food -- Analysis
High performance liquid chromatography




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