Approaches to folyl polyglutamate analysis

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Title: Approaches to folyl polyglutamate analysis
Author: Yang, Yingying
Other contributor: Helsingin yliopisto, Maatalous-metsätieteellinen tiedekunta, Elintarvike- ja ympäristötieteiden laitos
University of Helsinki, Faculty of Agriculture and Forestry, Department of Food and Environmental Sciences
Helsingfors universitet, Agrikultur- och forstvetenskapliga fakulteten, Institutionen för livsmedels- och miljövetenskaper
Publisher: Helsingfors universitet
Date: 2013
Language: eng
Thesis level: master's thesis
Discipline: Food Science
Food Science (Food Safety)
Food Science
Abstract: The literature review presented the effects of the polyglutamate chain on the biological and nutritional properties of folates and the main methods used for folate assays, with a special emphasis on the approaches to studying intact polyglutamates. A brief introduction regarding safety aspects of folate fortification was also given. The aim of this study was to develop a UPLC-FLR/PDA method for simultaneous determination of polyglutamyl folate vitamers. Chromatographic conditions were optimised for the resolution of polyglutamyl 5-methyltetrahydrofolates and major naturally-occurring monoglutamates. Method validation was conducted for both the UPLC method and affinity chromatography. Applicability of the validated method was evaluated on lupin flour, faba bean flour, and dry yeast, which were subjected to preparatory treatments with and without deconjugation. In addition, the effects of the sequential modification of preparatory treatments on the folate content and composition were investigated by using both the UPLC method and Lactobacillus rhamnosus assay. A desirable separation of target polyglutamates and monoglutamates was successfully achieved on the BEH C18 UPLC column within 11 minutes. The optimised UPLC method showed satisfactory selectivity, linearity, and sensitivity for the determination of methylated polyglutamates in the femtomole range and monoglutamates in the picogram range. Affinity chromatography showed satisfactory recoveries for polyglutamyl 5-methyltetrahydrofolates, but not for 5-formyl polyglutamates. In all three selected foods, 5-methyltetrahydrofolate was the dominant folate vitamer. Meanwhile, the analysis of undeconjugated samples showed that in the intact methylated folate pools, pentaglutamate predominated in legume flours and heptaglutamate in dry yeast. In addition, different sequences of enzyme and purification pretreatments were found to significantly affect both the total measurable folates and the folate profiles. Our standard preparatory procedures comprising simultaneous treatments with amylase and conjugase, then protease and affinity purification resulted in the greatest yield of total folates, but UPLC analysis indicated incomplete deconjugation. However, a modification in which deconjugation was conducted as the last step enhanced hydrolysis efficiency.
Subject: UPLC
folyl polyglutamate
folate food analysis

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