Water-soluble Vitamin Assays in Human Nutrition

Water-soluble Vitamin Assays in Human Nutrition

Water-soluble Vitamin Assays in Human Nutrition G.F.M. Ball SPRINGER-SCIENCE+BUSINESS MEDIA, B.V.

First edition 1994 1994 Springer Science+Business Media Dordrecht Originally published by Chapman & Hall in 1994 Softcover reprint of the hardcover 1st edition 1994 Typeset in Palatino 10/12 pt by Cambrian Typesetters, Frimley, Surrey ISBN 978-1-4613-5855-8 ISBN 978-1-4615-2061-0 (ebook) DOI 10.1007/978-1-4615-2061-0 Apart from any fair dealing for the purposes of research or private study, or criticism or review, as permitted under the UK Copyright Designs and Patents Act, 1988, this publication may not be reproduced, stored, or transmitted, in any form or by any means, without the prior permission in writing of the publishers, or in the case of reprographic reproduction only in accordance with the terms of the licences issued by the Copyright Licensing Agency in the UK, or in accordance with the terms of licences issued by the appropriate Reproduction Rights Organization outside the UK. Enquiries concerning reproduction outside the terms stated here should be sent to the publishers at the London address printed on this page. The publisher makes no representation, express or implied, with regard to the accuracy of the information contained in this book and cannot accept any legal responsibility or liability for any errors or omissions that may be made. A catalogue record for this book is available from the British Library (55? Printed on permanent acid-free text paper, manufactured in accordance with ANSI/NISO Z39.48-1992 and ANSI/NISO Z39.48-1984 (Permanence of Paper).

Contents Preface 1 Introduction 1.1 Definition and classification of vitamins 1.2 Biochemical role of the B-group vitamins 1.3 Dietary considerations 1.4 Biological activity and bioavailability References ix 1 1 2 3 7 8 2 Chemical and biological nature of the water-soluble vitamins 2.1 Thiamin 2.2 Riboflavin 2.3 Niacin 2.4 Vitamin B6 2.5 Pantothenic acid 2.6 Biotin 2.7 Folate 2.8 Vitamin B12 2.9 Vitamin C References 10 12 19 27 36 48 54 59 71 79 89 3 Analytical considerations 3.1 Analytical requirements 3.2 Scope of analytical techniques 3.3 The general analytical procedure 3.4 Method validation References 99 99 100 106 115 119

vi Contents 4 Extraction of the water-soluble vitamins 121 4.1 Objectives 121 4.2 Extraction procedures used for the individual vitamins 123 References 136 5 Physicochemical methods <excluding HPLC) for determining thiamin, riboflavin, niacin, vitamin B 6, pantothenic acid and vitamin C 142 5.1 Titrimetric methods for vitamin C 143 5.2 VoltammetricmethodsforvitaminC 149 5.3 Spectrophotometric determination of vitamin C 153 5.4 Colorimetric methods for niacin and vitamin C 155 5.5 Fluorimetric methods for thiamin, riboflavin, vitamin B6 and vitamin C 159 5.6 Continuous-flow analysis for the determination of thiamin, riboflavin, niacin and vitamin C 175 5.7 Gas chromatographic methods for thiamin, niacin, vitamin B 6, pantothenic acid and vitamin C 179 References 193 6 High-performance liquid chromatographic methods for the determination of thiamin, riboflavin, niacin, vitamin B 6, folate and vitamin C 203 6.1 General aspects of HPLC 203 6.2 Chromatographic modes 209 6.3 Detection systems 228 6.4 Applications of HPLC 232 References 305 7 Microbiological methods for the determination of the B-group vitamins 317 7.1 History 318 7.2 General principles 318 7.3 Generalassayprocedure 322 7.4 Developments in microbiological assays with respect to the individual vitamins 336 References 358 8 Biospecific methods for the determination of water-soluble vitamins 365 8.1 Introduction 366 8.2 The antigen-antibody relationship 366 8.3 Radioassay methods for the determination of biotin, folate and vitamin B12 367

Contents vii 8.4 Radioimmunoassay method for the determination of pantothenic acid 373 8.5 ELISA methods for the determination of pantothenic acid and vitamin B6 374 8.6 Non-isotopic protein-binding assay methods for the determination of biotin, folate and vitamin B12 379 8.7 Enzymatic methods for the determination of vitamin C 381 References 383 9 Appraisal of analytical techniques 387 9.1 Biological activity and bioavailability 387 9.2 Principal analyticaltechniques 388 9.3 Assay techniques for the individual vitamins 390 References 404 Index 410

Preface The demand for rapid, accurate and reproducible analytical methods for the quantitative determination of vitamins in foods is growing, due to an increased consumption of processed and convenience foods for which nutritional data are lacking. Such methods are also needed for the quality control of manufacturing processes and to investigate rates of vitamin loss during handling, processing and storage. Existing food compositional data for natural products are unreliable for certain vitamins (notably B6 and folate), owing to recently discovered problems with the assay methodology. In addition, new varieties of traditional food plants, including the cereal grains, have been introduced since the early analyses. There are a number of factors which complicate the assessment of a food commodity or diet in terms of its value in providing vitamins in a form that can be assimilated and utilized by the consumer. All natural foods are derived from animal or plant products and are therefore subject to biological variation in vitamin content. The variation is further compounded by losses incurred during processing, storage and cooking, especially for the more labile vitamins. Many of the B-group vitamins are present in coenzyme and other bound forms, often at extremely low levels, in animal and plant tissues, and these bound forms are absorbed and utilized by the body to varying extents. The dietary significance of a food or diet is complicated by interaction of the vitamins with other nutrients, and especially by alcohol, during intestinal absorption. Physiological factors such as age and state of health further complicate the evaluation. In the past, in vitro analytical methods have been developed to determine the total (potential) vitamin content of a food using the most rigorous extraction method commensurate with the stability of the vitamin. The data obtained have been used to compile food composition tables, which provide information about typical amounts of vitamin for

x Preface raw and processed foods as measured by microbiological or physicochemical assay, but they do not evaluate the nutritional value of the food in terms of biological vitamin activity. The determination of total vitamin content is useful when animal growth assays are being carried out on the same test material, as it allows the bioavailability of a nutrient to be determined as a ratio of standardized biological activity to the total nutrient content. On its own, however, the total vitamin content can sometimes grossly overestimate the biologically active vitamin content. There is an increasing trend nowadays toward the estimation of biologically active vitamin, rather than total vitamin content, using in vitro methods with selective extraction techniques. The purpose of this book is to survey current methodologies of determining the water-soluble vitamins in foods, with the theme of human nutrition very much in mind. This book has been published as a companion volume to an earlier book entitled Fat-Soluble Vitamin Assays in Food Analysis. A Comprehensive Review, G.F.M. Ball, Elsevier Applied Science, London and New York, 1988. As to the scope of this book, the introductory chapter establishes the nutritional theme and introduces the concepts of biological activity and bioavailability. A detailed discussion of animal bioassays is beyond the scope of this book, since such assays are the province of specialized laboratories equipped with the personnel and facilities for their evaluation. Chapter 2 sets out to acquaint the reader with a brief accoun't of the chemical and biological properties of thiamin, riboflavin, niacin, vitamin B6, pantothenic acid, biotin, folate, vitamin B12 and vitamin C. Emphasis is placed upon the vitamins' chemical structures, including the various bound forms, their stabilities and their occurrence in natural materials. Chapter 3 sets the stage for the subsequent analytical chapters by defining the analytical objectives and summarizing the scope of current analytical techniques in determining the vitamins and related vitamers. Chapter 4 is entirely devoted to the extraction of the vitamins from the food matrix. The chemistry of the extraction process and the quantitative efficiency have a great influence upon the accuracy of an assay method, yet extraction methods are the least studied and least characterized component of vitamin assays. Chapters 5-8 present the basic principles of the various physicochemical, microbiological and biospecific techniques encountered in vitamin analysis and discuss the applications of these techniques by reference to representative published methods. In the concluding chapter the various techniques are appraised by reference to comparative studies. Absorption and fluorescence spectra were kindly provided by A. C. Cowling. For the preparation of the manuscript, I am indebted to Ruth Harrisson, proprietor of Action Desk. G.F.M. Ball