Document Type

Thesis - University Access Only

Award Date

2005

Degree Name

Master of Science (MS)

Department / School

Nutrition, Food Science, and Hospitality

Abstract

5-Methyltetrahyrofolate (5-MeTHF) was analyzed in food samples using reverse phase chromatography, a gradient mobile phase of 33 rnM phosphate buffer (pH 2.3) and acetonitrile, a flow rate 0.8ml/min, and fluorornetric detection. For natural folate analysis. procedures were optimized for thermal extraction, enzymatic deconjugation, and clean up protocol using a strong anion exchange columns (SAX) and affinity chromatography using folate bind protein (FBP) columns. SAX column clean up did not yield clearly resolved peaks for broccoli. However, affinity column clean up yielded well resolved peaks. While the peak areas were small, they were still quantifiable. The 5- MeTHF content in spinach assayed using affinity chromatography for sample clean-up was lower than the concentration determined using Solid phase extraction (SPE) columns (15.66±0.83 vs. 98.92±1.24 μg/100 g). Results from SAX column clean up were closer to LCMS values than that of FBP clean up values. 5-MeTHF in broccoli assayed using affinity chromatography for sample clean-up was lower than the concentration determined using SPE (6.25 ±0.3085 μg/100 g vs. 47.08±0.85 μg/100 g). No reasons are apparent as to why the two techniques yielded different values for 5-MeTHF in the same samples. Sample matrix effects must play a significant role as purified standards show comparable results between two methods when taken through the entire respective procedures. In the measurement of added folic acid in processes food, a recent multi-laboratory evaluation of HPLC folic acid methods in 21 ready to eat (RTE) cereal samples revealed considerable variability in folic acid data between laboratories. A closer look at the data showed that laboratories which employed simple aqueous extraction techniques showed good precision between blind duplicates and recovered folic acid values closest to values claimed in the food label. To replicate these findings in our laboratory, various sample extraction schemes (amylase & protease assisted extraction, non-enzyme pH 7.85 extraction, non-enzyme pH 11 extraction) and solid-phase strong anion exchange clean-up techniques (3 mL SPE vs 6mL SPE) were employed to analyze the pteroylglutamic acid (PGA) content of 21 RTE cereal samples. Mass spectroscopic stable isotope analysis of the samples was also done to reveal true enrichment levels. Unfortified flour samples spiked with PGA at a range of 1.25 to 10 μgig showed recovery of 89.6 to 97.8 % when measured by non-enzyme, high pH extraction and gradient HPLC. An alkaline pH of 11 and agitation using a stir-bar favored the complete dissolution of folic acid from sample matrices. The use of large capacity SPE devices (6mL capacity) ensured optimal retention of PGA on the SPE cartridges and recovery of added folic acid from most of the cereal matrices tested. Blind duplicates embedded within the 2lsamples yielded excellent precision as judged by coefficient of variation. Aqueous pH 11 extraction yielded PGA values for 15 samples that were statistically no different than those analyzed by LCMS. Some samples showed anomalously higher PGA values with alkaline pH 11 extraction that was not observed with extraction at pH 7.85. The use of a amylase and protease enzymes traditionally used in sample preparations did not provide advantages in the analysis of added folic acid in RTE cereals samples. An HPLC gradient system comprising IO to 30% acetonitrile in 33 mM phosphoric acid solution and a LiChrosorb 18 column achieved good separation of folic acid from other enrichment ingredients.

Library of Congress Subject Headings

Folic acid
Food -- Analysis
High performance liquid chromatography

Format

application/pdf

Number of Pages

107

Publisher

South Dakota State University

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