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Home , B vitamins, Dihydrofolic acid, Folate, Multivitamin, Riboflavin, Thiamine

Double Fortification of with Folic Acid and

by

Angjalie Ruwanika Sangakkara

A thesis submitted in conformity with the requirements for the degree of Master of Applied Science Graduate Department of Chemical Engineering and Applied Chemistry University of Toronto

© Copyright by Angjalie Ruwanika Sangakkara 2011

Double Fortification of Salt with Folic Acid and Iodine

Angjalie Ruwanika Sangakkara

Master of Applied Science

Graduate Department of Chemical Engineering and Applied Chemistry University of Toronto

2011 Abstract

Salt iodization is widely available throughout the developing world. Incorporating other into the existing salt iodization process could prevent multiple deficiencies.

The thesis objective was to develop a stable formulation of salt dual fortified with folic acid and iodine, using a single solution that could be sprayed on. The micronutrients needed to be fully solubilized and stable in solution for at least one month. In the absence of an alkaline environment or , iodine losses occur most likely due to the oxidation of folic acid by iodate.

Optimal salt formulations were prepared by spraying a pH 9 carbonate-bicarbonate buffer solution containing folic acid and iodine dissolved at 0.35% (w/v) each. Acceptable retentions of > 90% were observed in refined salt after 6 months of storage at

45°C/60% relative humidity.

Further investigations into increasing the concentration of iodine and folic acid in the spray solution are recommended.

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Acknowledgments

I would like to express profound gratitude to my supervisor, Professor Levente L. Diosady, for his valuable support, encouragement, and useful suggestions during the course of my research.

His dedication to finding solutions for the alleviation of micronutrient deficiencies is remarkable, and I feel truly honoured that he welcomed me to be a small part of such a worthwhile cause.

I would like to thank The Micronutrient Initiative (MI) for generously supplying a sample of local salt from India, as well as for their financial support throughout this study.

I would also like to thank my friends and colleagues from the Engineering Group for their vital comments and suggestions throughout my research work.

Finally, I would like to thank my parents for their continual support and patience throughout my academic journey.

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Table of Contents

Abstract ...... ii

Acknowledgements...... iii

List of Tables ...... vi

List of Figures ...... vii

List of Appendices ...... ix

1 Introduction...... 1

2 Background ...... 3

2.1 Micronutrient Deficiencies ...... 3

2.1.1 Folic Acid...... 4

2.1.2 Iodine ...... 12

2.2 Prevention of Micronutrient Deficiencies...... 17

2.3 Fortification Strategy ...... 18

2.3.1 Requirements for Effective Programs ...... 18

2.3.2 Salt Iodization Technologies...... 20

2.3.3 Double Fortification of Salt with Folic Acid and Iodine ...... 22

2.3.4 Project Objectives ...... 23

3 Experimental Details...... 25

3.1 Materials ...... 25

3.2 Methods...... 26

3.2.1 Experimental Methods ...... 26

3.2.2 Analytical Methods...... 27

4 Results and Discussion...... 29

4.1 Selection of Final Formulations...... 29

4.1.1 Effect of pH on Micronutrient Stability in Buffered Solutions ...... 29

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4.1.2 Effect of Citrate on Micronutrient Stability in Spray Solutions ...... 33

4.2 Stabilities of Micronutrients in Final Formulations...... 34

4.2.1 Effect of Alkaline pH...... 34

4.2.2 Effect of Citrate...... 39

4.2.3 Effect of Micronutrients on Each Other...... 44

4.3 Assessment of Optimal Formulations...... 50

5 Conclusions...... 54

6 Recommendations...... 55

7 References...... 57

8 Nomenclature ...... 66

9 Appendices...... 68

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List of Tables

Table 2.1.1 Recommended daily intake (RDI) for in DFE (adapted from [43]) 9

Table 2.1.2 Recommended daily intake for iodine (adapted from [74]) 16

Table 2.3.1 Comparison of salt iodization methods (adapted from [91]) 22

Table 3.1.1 List of materials used in this study 25

Table 4.1.1 Components of the buffer solutions used 29

Table 4.1.2 Apparent first order degradation rate constants (k) estimated for the buffered solutions containing 0.35% (w/v), or 3500 mg/L, folic acid and 32 iodine when stored at 25ºC and 60% RH

Table 4.2.1 Buffered solution compositions at time 0 for final formulations 34

Table 4.2.2 Compositions at time 0 in 500g of salt sprayed with buffered solutions 35

Table 4.2.3 Apparent first order degradation rate constants (k) estimated for the 39 sprayed with the pH 9 spray solution, when stored at 45ºC and 60% RH

Table 4.2.4 Target citrate concentrations in solutions and salt at month 0 39

Table 4.2.5 Apparent first order degradation rate constants (k) estimated for the salt containing 45 ppm folic acid and iodine, and varying amounts of citrate, 43 when stored at 45ºC and 60% RH

Table 4.2.6 Apparent first order degradation rate constants (k) estimated for the solutions containing folic acid and/or iodine when stored at 25ºC and 60% 46 RH

Table 4.2.7 Apparent first order degradation rate constants (k) estimated for the salt 48 containing 45 ppm folic acid and iodine when stored at 45ºC and 60% RH

Table 4.3.1 SEM images of Canadian salt treated with different spray solutions 51

Table 4.3.2 Approximate FOB prices (US $/kg) of the constituents in the two best 52 formulations [108-110]

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List of Figures

Figure 2.1.1 Chemical structure of folic acid (adapted from [10]) 4

Figure 2.1.2 Chemical structure of folate (adapted from [11]) 4

Figure 2.1.3 Structures of common degradation products of folic acid (adapted from 6 [10])

Figure 4.1.1 The appearance of the buffered solutions after folic acid and iodine have been added at 0.35% (w/v) each (Note: the numbers indicate the pH of 30 each solution).

Figure 4.1.2 Folic acid retentions in the buffered solutions (that have iodine as well) during 5 months storage at room temperature in the dark (each bar 30 represents the mean of triplicates, and the error bars represent the relative standard deviations).

Figure 4.1.3 Iodine retentions in the buffered solutions (that have folic acid as well) during 5 months storage at room temperature in the dark (each bar 31 represents the mean of triplicates, and the error bars represent the relative standard deviations).

Figure 4.2.1 Comparing the colour of Canadian salt before (left) and after (right) double fortification with folic acid and iodine using a pH 9 spray 35 solution.

Figure 4.2.2 Folic acid retentions in refined dry salt during 6 months storage in the dark at 45°C/60% RH (each point represents the mean of triplicates, and 36 the error bars represent the relative standard deviations).

Figure 4.2.3 Iodine retentions in refined dry salt during 6 months storage in the dark at 45°C/60% RH (each point represents the mean of triplicates, and the 36 error bars represent the relative standard deviations).

Figure 4.2.4 Stability of micronutrients in impure, moist, coarse salt during 4 months storage in the dark at high temperature and humidity (each point 38 represents the mean of triplicates, and the error bars represent the relative standard deviations).

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Figure 4.2.5 Folic acid retentions in citrate-containing solutions (that have iodine as well) during 5 months storage in the dark at 25ºC/60% RH (each point 40 represents the mean of triplicates, and the error bars represent the relative standard deviations).

Figure 4.2.6 Iodine retentions in citrate-containing solutions (that have folic acid as well) during 5 months storage in the dark at 25ºC/60% RH (each point 41 represents the mean of triplicates, and the error bars represent the relative standard deviations).

Figure 4.2.7 Folic acid retentions in refined dry salt during 6 months storage in the dark at 45ºC/60% RH (each point represents the mean of triplicates, and 42 the error bars represent the relative standard deviations).

Figure 4.2.8 Iodine retentions in refined dry salt during 6 months storage in the dark at 45ºC/60% RH (each point represents the mean of triplicates, and the 42 error bars represent the relative standard deviations).

Figure 4.2.9 Folic acid (FA) retentions in aqueous solutions during 6 months storage in the dark at ambient conditions (the results are the mean values 44 obtained from four replicates, and the error bars represent the relative standard deviations).

Figure 4.2.10 Figure 4.2.10: Iodine retentions (I) in aqueous solutions during 6 months storage in the dark at ambient conditions (the results are the 45 mean values obtained from four replicates, and the error bars represent the relative standard deviations).

Figure 4.2.11 Figure 4.2.11: Folic acid retentions in refined dry salt during 6 months storage in the dark at 45ºC and 60% RH (the results are the mean values 47 obtained from four replicates, and the error bars represent the relative standard deviations).

Figure 4.2.12 Figure 4.2.12: Iodine retentions in refined dry salt during 6 months storage in the dark at 45ºC and 60% RH (the results are the mean values 48 obtained from four replicates, and the error bars represent the relative standard deviations).

viii

List of Appendices

Appendix 9.1 Detailed Analytical Methods 68

Appendix 9.2 Buffer Capacity Calculations 71

Appendix 9.3 Experimental Data 72

ix 1

1 Introduction

Deficiencies in micronutrients – and minerals needed in small quantities by the body for physical and mental development – are widespread in more than a third of the world’s population, contributing to approximately 7.3% of the global burden of disease [1]. The prevalence of micronutrient is much higher in developing regions of the world relative to developed countries, with young children and women of reproductive age being at a higher risk due to their greater need for vitamins and minerals. Important micronutrients with public health significance include A, , , iodine and folic acid.

Micronutrient intake can be increased by taking regular supplements or through dietary interventions that promote the regular consumption of micronutrient-rich like fruits, vegetables and products. However, these interventions are often inaccessible by those who need them the most, generally due to their high costs or unavailability. Fortification of staple foods and condiments increases their nutritional content and offers a simple, low-cost way of delivering micronutrients to a large number of people. When imposed on existing food patterns, it does not necessitate changes in the customary diet of the population and does not call for individual compliance. This makes food fortification a sustainable method of overcoming micronutrient malnutrition.

Salt is an excellent vehicle for food fortification that has been successful in eliminating disorders (IDD) such as irreversible mental retardation and goiter, in many parts of the world. It is universally consumed at a constant rate irrespective of economic status. It is centrally processed so that iodization can be carried out under controlled conditions. Furthermore, the equipment required for salt iodization is uncomplicated, easy to operate and maintain, and well established in many developing nations. With that said, it makes sense to incorporate other micronutrients into iodized salt to combat multiple deficiencies, ensuring that chemical interactions, organoleptic changes and incremental costs are kept to a minimal. One such example is the development of a double fortified salt (DFS) fortified with iodine and iron, which has shown simultaneous reductions in the prevalence of iron-deficiency and iodine deficiency disorders during field trials in India and Ghana [111]. The product was based on technologies developed by the Food Engineering Group at the University of Toronto, under the direction of Professor L. L. Diosady.

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More recently, folic acid has become a compound of interest since its deficiency is the predominant cause of neural tube defects (NTD) that prevent the proper development of the spine and brain, which occurs during the first 28 days after . Mandatory folic acid fortification of products by 1998 in the Americas was associated with significant reductions in NTD prevalence [2]. Now, more than fifty countries have implemented fortification of with folic acid globally; with most recently (effective October 2009) making it mandatory to add folic acid (via flour) and iodine (via iodized salt) to [3,4]. The inclusion of folic acid into the existing salt iodization practice seems a safe, cost-effective and sustainable option for simultaneously alleviating serious birth defects due to folic acid deficiency, and IDD including mental retardation and goiter, due to lack of iodine.

This project explored the feasibility of incorporating folic acid into the spray-mixing method of salt iodization that involves spraying a solution of potassium iodate (KIO3) onto salt at a uniform rate. This is the preferred method of salt iodization in developing nations where the salt is often unrefined and subject to adverse climatic conditions. Stability enhancing treatments were investigated to ultimately recommend a single spray solution containing both micronutrients fully dissolved and stabilized (for at least one month) that, when sprayed onto salt, keeps both folic acid and iodine stable for 6 months under hot and humid storage conditions typical of the target countries.

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2 Background 2.1 Micronutrient Deficiencies

Micronutrients are vitamins and minerals that are required by the body in very small amounts. Yet, their absence in the diet can have severe consequences on the body’s ability to produce hormones, and other substances that are essential for growth and development [5].

A significant proportion of the world's population suffers from, or is at risk of, deficiencies of micronutrients. Although the most severe problems of micronutrient malnutrition are found in developing nations, people in developed countries also suffer from various forms of these nutritional problems. These include, among others, progressive blindness due to deficiency; anemia due to ; gross skin lesions from ; goiter from iodine deficiency; and neural tube birth defects due to folic acid deficiency [6]. As a result, micronutrient deficiencies are now recognized as an important contributor to the global burden of disease, having a widespread impact on the physical and mental development, the general good health and the overall well being of all individuals and populations [7].

At the most basic level, deficiencies occur when especially poorer populations do not consume sufficient amounts of micronutrient-rich foods such as fruit, vegetables, animal products and fortified foods, usually because they are too expensive to buy or are locally unavailable. Micronutrient deficiencies can also easily develop during emergencies like natural disasters, in which food supplies are often destroyed; and infections break out that generally slow the appetite while increasing the need for micronutrients to help fight illness [8].

The groups most vulnerable to micronutrient deficiencies are young children and women of childbearing age, mainly because they have a relatively greater need for vitamins and minerals and are more susceptible to the harmful consequences of deficiencies. For instance, each year, approximately 18 million newborns suffer from brain damage due to iodine deficiency, and roughly 150,000 suffer from severe birth defects due to folic acid deficiency [9]. Both consequences can be prevented if the mother has a sufficient intake of these micronutrients during the early stages of pregnancy. Folic acid and iodine are therefore the focus of this study, which aims to present a solution for alleviating their deficiencies through a salt fortification approach. These micronutrients are discussed in greater detail in the following subsections.

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2.1.1 Folic Acid

Chemistry and Stability

Folic acid (pteroylglutamic acid) is the basic structural unit in the group of compounds denoted by the generic term folate. The chemical structure of this B-complex vitamin (C19H19N7O6, MW = 441.4) is shown in Figure 2.1.1. The molecule consists of a pteridine ring (2-amino-4-hydroxy- pteridine) linked by a methylene bridge to para-aminobenzoic acid (PABA), which in turn is coupled by an amide linkage to a single molecule of .

Figure 2.11: Chemical structure of folic acid (adapted from [10])

Natural in food are compounds with folic acid activity; typically their pteridine ring is reduced, substituted in the N5- and/or N10-position, and the compounds are usually polyglutamated, containing one to six additional glutamate molecules attached to the p- aminobenzoic group by peptide linkage. The main naturally occurring folates exist as tetrahydrofolate species (Figure 2.1.2), and over a hundred native folate are possible [11-12].

Figure 2.1.2: Chemical structure of folate (adapted from [11])

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Folic acid is the synthesized form that is used in supplements and for food fortification, and it is the most oxidized form of the vitamin. While natural folates rapidly lose activity in foods over periods of days or weeks, folic acid (e.g., in fortified foods) is almost completely stable for months or even years [13]. It is a yellow-orange, crystalline powder, almost tasteless, odourless and contains on average 8.0-8.5% [14]. It has no but undergoes significant degradation by 200°C resulting in an amorphous product above this temperature. The initial mechanism for its decomposition has been established. First, the glutamic acid component breaks away from the folic acid structure, followed by the decomposition of the and PABA in an overlapping mechanism [15]. Folic acid is practically insoluble in cold water (0.0016 mg/mL at 25˚C), sparingly soluble in boiling water (0.2 mg/mL), slightly soluble in , and butanol, and insoluble in acetone, chloroform benzene and diethyl ether. It is relatively soluble in acetic acid, phenol, , solutions of alkali hydroxides and carbonates.

Generally, folic acid is relatively low in acidic solutions and significantly higher in basic fluids. Solubility of folic acid at pH 7 has been shown to be 183-fold more than the solubility of folic acid at pH 1. The vitamin is moderately stable to heat, humidity and atmospheric , but will lose its activity upon exposure to sunlight, light, oxidizing or reducing agents, and acidic or alkaline environments. In addition, it interacts with several like and , causing the molecule to become inactive. However, its stability is greatly enhanced in the presence of ascorbate, reduced thiols and other [14].

Folic acid exhibits increased nutritional stability in alkaline conditions. The time required for 50% destruction of folic acid at pH 5 or above at room temperature, in buffered solutions, was over 700 hours. This sharply decreased to 64-24 hours when the pH dropped below 4 [16]. Pharmaceutical preparations of 1mg/mL folic acid dissolved in pH 6 or higher were stable for one year at room temperature. At the lower pH levels (3 to 4) the very small amount of folic acid that went into solution was unstable and formed p-aminobenzoylglutamic acid (pABG) due to acid reduction, but mixtures containing undissolved folic acid at concentrations exceeding its solubility exhibited good stability [16-22].

Treatment of folic acid with alkaline under aerobic conditions at room temperature in the presence of light oxidatively cleaves the molecule at the C9-N10 bond, yielding

6 pABG and pterin-6-carboxylic acid (Figure 2.1.3) [10]. Aerobic of folic acid under acid conditions gives pABG and 6-methylpterin.

Figure 2.1.3: Structures of common degradation products of folic acid (adapted from [10])

Folic acid was stable in aqueous solution at 100˚C for 10 h in a pH range 5.0-12.0 when protected from light. Below this pH, destruction occurs and the reaction was shown to be unimolecular with the decomposition velocity coefficient being directly proportional to the ion concentration. The thermal destruction of folic acid does not depend on the constituents of the buffer solutions, and follows first order kinetics. The temperature dependence of degradation follows the Arrhenius relationship [21-22].

Solutions of folic acid in water or 20% ethanol were rapidly decomposed with the splitting off of pABG when exposed to daylight in a test tube kept at a window for up to 24 hours, with the reaction proceeding most rapidly at pH 7 and below, and most slowly in alkaline (0.1N NaOH) solution. Likewise, aqueous solutions of folic acid irradiated at 250 nm with UV light at pH 2-10 degrade to give pterin-6-carboxylic acid and pABG under aerobic conditions, with maximum yields of the decomposition products obtained at 3–8 h depending upon the pH. Folic acid is photolyzed by an apparent first-order kinetics and the log k-pH profile shows a gradual decrease in rate in the pH range 2.0–10.0. The profile indicates the appearance of three steps that reflect the participation of different ionic species of folic acid (pKal = 2.3, pKa2 = 8.3) in the photolysis reaction. The rate of photodegradation varied from 0.1550×10− 3 min− l (pH 10.0) to 5.04×10− 3 min− l (pH 2.5) in the pH range studied [19-22].

Some studies hint that the degradation of folic acid by light occurs by a mechanism that involves dissolved oxygen. For instance, the removal of oxygen by dioxide reduces the rate of folic acid oxidation in the presence of light [22]. Folic acid does not undergo any chemical

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modification under irradiation at 350 nm in the absence of O2. In the presence of O2 however, it does undergo cleavage, yielding 6-formylpterin and (4-aminobenzoyl)glutamate as photoproducts [23].

Folic acid can also act as an antioxidant, since it was shown that folic acid can efficiently • • -• -• • • scavenge such free radicals as CCl3O2 , N3 , SO4 , Br2 , OH and O- ; and was able to reduce Fe3+ to Fe2+ in a ferric reducing antioxidant power (FRAP) assay. Folic acid is a better free scavenger at acidic and basic pH compared to neutral pH. This may be of biological relevance because the pH range of different human body fluids is known to vary widely. Furthermore, all folates contain an amide-like structure involving N3 and C4 atoms that resonates between two forms that differ in the position of the hydrogen atom on the aforementioned atoms. It has been suggested that this group is responsible for the antiradical activity of folic acid. Nevertheless, it still has a poor protective effect against free radicals and peroxidation compared to other folates, and therefore the synthetic form is not really thought to be relevant as an antioxidant [23-25].

Biological Role

Folic acid in vivo is reduced to 7,8- (DHF), in which one double bond of the pteridine ring system is reduced. DHF is subsequently reduced to 5,6,7,8- (THF), which is enzymatically converted into the plasma form of the vitamin, 5-methyl-5,6,7,8- tetrahydrofolic acid (5-MTHF), in which two double bonds of the pteridine ring are reduced. 5- MTHF functions as a that accepts and donates one- units (i.e. formyl, methyl, formate and hydroxymethyl units) in a variety of biosynthetic pathways critical to the of nucleic acids and amino acids. For instance, it is involved in the of a number of sites within DNA and RNA, the building blocks of cells. Methylation of DNA may be important in prevention [26]. Additionally, the synthesis of from requires 5-MTHF, as otherwise a buildup of plasma homocysteine occurs due to the decreased synthesis of methionine, which is a risk factor for cardiovascular and other chronic diseases [27-29].

A recent study from the Netherlands has estimated that approximately two-thirds of folate intake from a mixed unfortified diet is in the polyglutamyl form (conjugated to a polyglutamyl chain containing different numbers of glutamic acids depending on the type of food), derived

8 mainly from vegetables. These folates are unstable chemically as they are easily split between the C9 and N10 bond to yield a substituted pteridine and p-aminobenzoylglutamate, which have no biologic activity. Furthermore, the polyglutamyl chain in the natural folates is removed in the brush border of the mucosal cells by the folate conjugase, and folate monoglutamate is subsequently absorbed in the proximal . This process is apparently not complete, thereby reducing the of natural folates by as much as 25-50%. In contrast, synthetic folic acid appears to have a bioavailability of close to 100% [30-34].

Dietary Sources

Many pulses and vegetables like and are rich sources of dietary folate, with concentrations of up to 600 µg/100 g in some and chick peas, and around 200 µg/100 g in leafy green vegetables. A general rule is that the lower the water content in the vegetable, the higher the folate concentration. Folate concentrations in fruits and berries are usually one-tenth those of vegetables, ranging from a few micrograms to approximately 50 µg/100 g. and meat products, except (which is the storage organ and has 200-500µg/100 g), contain little folate, while and fish are moderate sources. Folate concentrations in are only around 5 µg/100 mL. Compressed baker’s has about 1000 µg/100 g, while egg is also high in folate with up to 90 µg/100 g [34]. However, most of these are rare or absent in the diets of the poor in developing countries.

Cooking and other food processing techniques can destroy 50-90% of a food’s folate through leakage and oxidative degradation, increasing losses with increasing severity of processing conditions in terms of heating temperature and time. This is in contrast to the stability of folic acid (from fortified foods and supplements), in which the pteridine ring is not reduced, making it very resistant to chemical oxidation [35].

Dietary Requirements

Although mammals have the ability to synthesize all of the component parts of folate, they lack the enzyme required for coupling the pteridine ring to PABA and as a result, cannot synthesize folate de novo. Therefore, with the exception being capable of incorporating some of the folate synthesized by intestinal flora, mammals need to obtain folate from dietary and supplemental sources [36-38].

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In 1992, the U.S. Public Health Service recommended that all women of childbearing age consume 400 µg of folate daily from food or supplements, in an effort to potentially reduce the number of NTD’s by 50% [39]. After fortification of flour with folic acid, some countries in the Americas like and Chile saw NTD reduction rates of 26-54% [40-42].

When the Food and Board of the US Institute of Medicine set the new dietary recommendation for folate (Table 2.1.1) in 1998, they introduced the dietary folate equivalent (DFE) to reflect the higher bioavailability of synthetic folic acid found in supplements and fortified foods, compared to natural food folates. The Food and Agriculture Organization /World Health Organization (FAO/WHO) expert group on human vitamin and requirements also agreed with the definition of this new unit, in which 1 µg of DFE = 1 µg of food folate = 0.5 µg of folic acid taken on an empty = 0.6 µg of folic acid with meals [44].

Table 2.1.1: Recommended daily intake (RDI) for folate in DFE (adapted from [43]) Life Stage Age RDA (µg/day) 0-6 months 65 Infant 7-12 months 80 1-3 years 150 Child 4-8 years 200 9-13 years 300 Adolescent 14-18 years 400 Adult 19 years and older 400 Pregnancy all ages 600 Breastfeeding all ages 500

No adverse effects have been associated with the consumption of excess folate from foods. However, the Food and Nutrition Board of the US Institute of Medicine further advisees that all adults limit their intake of folic acid (supplements and fortification) to 1000 µg daily as the tolerable upper limit.

Deficiency

Nutritional deficiency of folate is common in people consuming a limited diet. This is intensified by such conditions as and celiac disease, as well as chronic . tends to be more prevalent in populations that have a high intake of refined (which are low in folate) and a low intake of leafy greens and fruits (which are high in folate). Dietary surveys in India show that people eating predominantly cereal-based diets

10 only consume about 75µg folate per day; this is far below the daily requirement even for infants.

The combination of severe folate deficiency and to a lesser extent deficiency can result in nutritional , in which inadequate amounts of red and white cells and platelets are produced due to the general impairment of division. This is related to folate’s role in nucleic acid synthesis. Megaloblastic anemia from folic acid deficiency has been found in 2.5-5.0% of pregnant women in developed countries; considerably higher incidences have been observed in developing countries. Pregnant women are at higher risk of folate deficiency because of the increased need for folate during the second and third trimester, during which rapid fetal growth happens [44-49].

Numerous studies conducted over the past decade have confirmed that a woman's risk of a pregnancy being affected with a fetal NTD can be reduced substantially by taking folic acid periconceptionally [40-49]. Between days 21 and 27 post-conception, the neural tube closes to form what will eventually be the spinal cord and cranium; NTD’s are caused by the failure of closure of the neural tube. The two most common NTD’s diagnosed at birth are and . While the latter is not compatible with long-term survival, the former can be surgically corrected, but surgical repair of the lesion is not always associated with improvement in motor function. Going from a low to adequate intake of folate can reduce both the first occurrence as well as the recurrence in subsequent , of these birth defects by tenfold [48].

Food Fortification

Many studies support the efficacy of folic acid fortification prior to food processing. For instance, folic acid in (pH 7) had 75-92% retention after processing at 100°C for 2 hours, 68-85% retention after processing at 120°C for 20 minutes (a typical commercial thermal treatment for infant formula) and 40-55% retention after processing at 140°C for 15 minutes. In that study, folic acid had the highest stability in the iron-fortified liquid infant formula system. This was because oxygen partial pressure was the lowest (since ferrous iron oxidizes to ferric iron in the presence of water), and it is known that folic acid is degraded by oxidative mechanisms requiring the presence of molecular oxygen. Intermediate stability was noted in the ascorbate-fortified liquid infant formula system (ascorbate is oxidized to dehydroascorbate) and in the system containing both iron and ascorbate (iron was chelated by ascorbate, which in turn

11 lessens the oxygen consumption ability of each). The lowest stability was seen in the unfortified liquid infant formula system (oxygen partial pressure was the highest in this system). The degradation products were found to be pABG and pterin-6-carboxylic acid; and it was found that folic acid did not interact with the components of the tested infant formula formulation during thermal processing to form complexes that might affect its bioavailability [50]. Numerous other studies have demonstrated the acceptable stability of added folic acid in fortified and grains during typical storage and baking conditions, as well as in high moisture environments. Acceptable losses of added folic acid range from 10-20% during typical food preparation conditions [51-53].

Recent publications from the Americas show that mandatory folic acid fortification of cereal products are associated with a significant increase in population folate status and also with significant reductions in the incidences of NTD. For instance, folic acid fortification at 140 µg/100 g and 150µg/100g of flour respectively, became mandatory in 1998 in the US and Canada. These concentrations were selected on the assumption of a nearly uniform consumption of about 100 micrograms per day, depending upon the level of folic acid fortificant added to the flour. Recent studies show that the fortification of grain products and ready-to-eat breakfast cereals has clearly increased folate status in Canada and the US [54-55]. For example, a national health and nutrition examination survey comparison of pre-fortification data with post- fortification data in the US found that the impact of this measure has been an almost threefold increase in the mean folate concentration in women aged 15-44 years, and a 26% reduction in the incidence of neural tube defects [55]. Reductions of up to 54% in NTD rates after fortification were reported from similar studies done in Canada [55-60]. Likewise, starting January 2000, the Chilean Ministry of Health legislated the addition of 220µg/100g flour to reduce the risk of NTD, since the mean intake of bread (representing 90% of the total consumption of wheat flour in that country) is very high and consumption is widespread. In just over two years post-fortification, the NTD rate was reduced by 43% [61]. Since then, over 50 countries have begun adding folic acid to wheat flour, including Indonesia (200µg/100g), Mexico (200 µg/100 g) and Australia (200-300 µg/100 g) [1]. Two large countries, China and India, have begun small pilot programs to fortify wheat flour with folic acid, and the expansion of these programs would contribute enormously to prevention of folic acid-preventable NTD. No country in Europe has implemented mandatory folic acid fortification of flour yet, but it has been

12 recommended by the UK Food Safety Authority [1,58].

The consumption of folic acid in amounts normally found in fortified foods has not been associated with adverse health effects. However, there has been some concern that high folic acid intakes by individuals with undiagnosed could correct the megaloblastic anemia that is only a symptom of its deficiency (and indistinguishable from that associated with folate deficiency), without correcting the underlying vitamin B12 deficiency, potentially leaving individuals at risk of developing irreversible neurological damage. However, most cases of this sort of neurological progression in vitamin B12 deficiency have been seen at doses of folic acid of 5000 µg and above [62]. Many recent studies have also assured that no measurable harm has occurred from this phenomenon at the current levels of folic acid intake [45,53, 62]. Thus, 400 µg/day of folic acid from fortified food or supplements, in addition to dietary folate, is considered safe; especially because there is no evidence that it is possible to consume sufficient natural folate to pose a risk of , particularly in the poorer regions of the world.

2.1.2 Iodine

Chemistry and Stability

Iodine (symbol I; MW 126.9), an essential mineral, is a diatomic molecule at room temperature but sublimes when heated, giving off a violet vapor with a stinging odor similar to that of chlorine. Iodine forms compounds with many elements, but is the least reactive of the halogens. It dissolves readily in chloroform, carbon tetrachloride, or carbon disulfide to form purple solutions. Its poor solubility in water is improved by the presence of iodide ions that results in − the formation of I3 ions [63]. The primary source of iodine is marine flora, with micro- and macro- (e.g. phytoplankton, cyanobacteria and seaweed) releasing organic iodine gases and molecular iodine into the atmosphere. All of these gases are subject to dissociation in the atmosphere, primarily via photolysis, with I2 having the shortest lifetime and therefore being the dominant source of reactive iodine, particularly in coastal locations. After subsequent reactions, iodine gets deposited on the land where iodine is adsorbed onto the soil and vegetation (through rainfall or aerosol) that leads to eventual ingestion by and humans [64].

Iodine is normally added as the iodide or iodate of potassium in salt iodization practices [65]. (KI) in salt is not very stable and can easily be lost by oxidation to iodine under

13 harsh environmental conditions. For instance, when salt is damp to the touch, iodine losses occur though evaporation to the atmosphere due to migration and segregation of KI, which readily oxidizes and sublimes when wet. This cannot happen with potassium iodate (KIO3) because it is in its fully oxidized form [66-67]. Iodine losses are reduced when the salt iodized with KI is very pure (≥ 99.5%) and dry (moisture < 0.1%), and by the addition of stabilizers and drying agents. Upon exposure to sunlight, heat, excessive air currents and impurities, salt iodized with KI looses iodine. However, salt made slightly alkaline with bicarbonate does not lose iodine upon exposure to heat for several months [68]. Impure iodated salt can also lose most of the added iodine during extended storage at high temperature and humidity, due to the reduction of iodate [69]. Moisture naturally present in the salt or taken from the air by hygroscopic impurities can easily become the reaction medium for the reduction of added iodate. However, KIO3 was stabilized by carbonate in crude sea salt stored in fiber sacks for up to eight months at ambient temperatures and relative humidity, with only 3.5% of the added iodine being lost [68-70]. The iodide and iodate reactions discussed above are as follows:

- - 2I  I2 + 2e (Oxidation of iodide) 5+ - 2I + 10e  I2 (Reduction of iodate)

Overall, KIO3 is more suitable for underdeveloped areas that consume crude salts with minimal refinement and processing. On the other hand, KI is more suitable for high-grade, free-running salt that is refined and contains very little impurities, which is typical of developed nations like Canada and the US.

Biological Role

Iodine must be absorbed from either food or water, where it can be present in the iodide or iodate form. Iodate in food or water is converted to iodide, which circulates in the bloodstream and is rapidly and almost completely absorbed and transported to the gland for the synthesis into the thyroid hormones, triiodothyronine (T3) and thyroxine (T4). T3, the physiologically active thyroid hormone, binds to thyroid receptors in the nuclei of cells and regulate gene expression. T4, as the most abundant circulating thyroid hormone, is converted to T3 by enzymes in target tissues, and together they aid in the regulation of such physiologic processes as growth, development, metabolism and reproductive function [71].

In response to a hormone released by the brain known as thyrotropin-releasing hormone (TRH),

14 the secretes thyroid-stimulating hormone, TSH, which stimulates iodine trapping, thyroid hormone synthesis and release of T3 and T4 by the thyroid gland. When there is enough

T4 circulating, the sensitivity of the pituitary gland to TRH decreases and thus limits the secretion of TSH. However, when circulating T4 levels decrease, the pituitary excessively secretes TSH, which leads to increased iodine trapping. Iodine deficiency occurs in response to reduced blood levels of T4. The increased output of TSH by the pituitary gland leads to enlargement of the thyroid gland, also known as goiter [72].

Dietary Sources

The iodine content of most foods depends on the iodine content of the soil in which it was raised, with most foods providing 3 to 75 µg per serving [73]. Seafood is rich in iodine because marine animals are able to concentrate the iodine from seawater. Certain types of seaweed are also very rich in iodine. Iodine is also available through /multimineral supplements, usually as KI [74]. Generally, vegetarian and non-vegetarian diets that exclude iodized salt, fish and seaweed have been found to contain very little iodine [75-76].

Deficiency

Iodine deficiency is a major public health problem for populations throughout the world, but particularly for young children and pregnant women, and in some settings represents a significant threat to social and economic development. It is now accepted as the most common cause of preventable brain damage in the world. This is the primary motivation behind the current worldwide initiative to eliminate IDD [1]. Other growth and developmental abnormalities in the spectrum of IDD include hypothyroidism, goiter and cretinism, all of which result from inadequate thyroid hormone production from the lack of sufficient iodine. According to the WHO, IDD affects 740 million people throughout the world who are living in areas of iodine deficiency and at risk of its consequences. Mountainous regions like the Himalayas, the Andes and the Alps, and flooded river valleys like the Ganges, are among the most severely iodine- deficient areas in the world. Many other parts of Central Africa and Central Asia are also deficient [73-74].

Goiter, or thyroid enlargement, is one of the earliest and most visible signs of iodine deficiency, reflecting an attempt to adapt the thyroid to the increased need to produce thyroid hormones. The

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International Council for the Control of Iodine Deficiency Disorders (ICCIDD) and the WHO have recommended surveying schoolchildren for thyroid size as one of the most practical indicators of iodine deficiency, and many reports on iodine malnutrition are based primarily on surveys of thyroid size and urinary iodine [77]. Severe cases of deficiency result in hypothyroidism, of which the reversibility depends on an individual’s stage of development. It is most damaging to the developing brain, since the thyroid hormone is important for the maturation of the nerve cells in the central , which is most active before and shortly after birth. School children in iodine-deficient areas show poorer school performance and high incidences of learning disabilities than matched groups from iodine-sufficient areas. An analysis of 18 related studies concluded that iodine deficiency alone lowered mean IQ scores by 13.5 points [78]. One of the most devastating effects of fetal iodine deficiency is cretinism, also known as congenital hypothyroidism. It is characterized by mental retardation, along with varying degrees of , deaf mutism, and spasticity. Other consequences of iodine deficiency are impaired reproductive outcome, increased childhood mortality and economic stagnation [73-74].

Dietary Requirements

The RDI for iodine for different age groups have been calculated using several methods, including the measurement of iodine accumulation in the thyroid glands of individuals with normal thyroid function. All women of reproductive age require adequate iodine stores, especially because a significant degree of neurological development in the occurs during the early stages of conception. The Food and Nutrition Board of the US Institute of Medicine presented reevaluated RDI values in 2001 (Table 2.1.2), which are in agreement with those of the ICCIDD, the WHO and the United Nations Children's Fund (UNICEF) [74].

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Table 2.1.2: Recommended daily intake for iodine (adapted from [74]) Life Stage Age RDA (µg/day) 0-6 months 110 Infant 7-12 months 130 1-3 years 90 Child 4-8 years 90 9-13 years 120 Adolescent 14-18 years 150 Adult 19 years and older 150 Pregnancy all ages 220 Breastfeeding all ages 290

People whose diets contain large amounts of seaweed have been found to have iodine intakes of up to 80 milligrams per day. Excess iodine intake is commonly associated with elevated blood levels of TSH, hypothyroidism and goiter [74]. To minimize the risk of developing such conditions, the upper limit for iodine has been set at 1100 µg/day for individuals who are not being treated with iodine under medical supervision [73].

Fortification

There are two chemical forms of iodine that are suitable for use as food fortificants, namely, KI and KIO3. For historical reasons, countries in Europe and North America still use KI because of having salts of high purity and refinement, while most countries with tropical climates use KIO3 [1].

Iodine is added to a variety of foods, including bread, sweets, milk, and water. For instance, bread is used as a vehicle for iodine fortification in countries such as Russia and Tasmania, where bread is a [1]. Water fortification has also been successful in parts of Africa, Asia and Europe, although sources of drinking water are so ubiquitous that iodization becomes difficult to control [78-81].

Salt is the most widely used food vehicle for iodine fortification. Universal salt iodization (USI) is the strategy recommended by the WHO for the control of IDD. Because salt iodization is inexpensive and easy to implement, great strides in salt iodization programs have been made in a relatively short period of time. For instance, 34 countries have attained universal salt iodization, with at least 90 per cent of households consuming adequately iodized salt, while 60 countries have increased household consumption of adequately iodized salt by at least 20% during the past

17 decade [1]. Iodized salt is mandatory in Canada and used optionally by about 50% of the U.S. population [64]. Both countries iodize salt with KI at 76 mg iodine/kg salt. By 2009, 71.1% of the households in India were using salt iodized at the recommended level of 15 ppm (15 µg/g) or more [82]. Although there is no standard iodization value, salt is generally fortified at 25-100 mg iodine/kg salt, depending on the country.

2.2 Prevention of Micronutrient Deficiencies

There are a number of approaches to prevent micronutrient malnutrition that have been utilized in the past to deliver single and multiple micronutrients to target populations [3]. Of these, the more common interventions include dietary education/diversification, supplementation, and fortification.

Dietary education/diversification attempts to improve the availability, access and use of micronutrient-rich foods. Mass communication and education activities are promoted to motivate changes in behavior that increase consumption, production and feeding (especially in infants and young children) of nutritionally beneficial foods. There is some evidence that in more developed populations, such educational approaches can work well [83-85]. In more underdeveloped and impoverished populations, changes in food production and selection practices have shown improvements in micronutrient status. These include increasing dietary zinc availability through encouraging the use of animal source foods, and growing home gardens to include more vitamin A rich fruits and vegetables [1]. This approach has the advantage of being sustainable, since any changes in knowledge and practices can be carried forward to the subsequent generations. For it to properly work however, there should be no other serious constraining factors, such as insufficient access to the desired foods, lack of motivation by the target population group, and poor design and planning of the dietary education process because of economic constraints and other overwhelming priorities by policymakers. For instance, this method is not too well suited for the prevention of iron deficiency if there are religious constraints on increasing animal intake [83].

Supplementation refers to the addition of pharmaceutical preparations of —capsules, tablets, or syrups—to the diet. It was shown to be effective in correcting deficiencies of iron, vitamin A, iodine and zinc for groups in which there were severe health problems. It has the advantage of reaching segments of the population most at risk while not putting other groups of

18 the same population at risk of overdose. It can be integrated to the health care system, with the cost of worker training being lower than that for dietary modification. However, because of insufficient coverage, deficient individuals, especially in rural outskirts, can be missed. Other disadvantages that have been often encountered include poor compliance both by the target populations being given the supplements, and by policymakers due to lack of financial support [85].

Fortification involves the addition of one or more essential micronutrients to foods for the purpose of preventing or correcting their deficiencies in populations or subgroups. Since there is no or minimal behavior change required on the part of the consumers, a greater coverage of the population can be achieved. For instance, fortification of salt with iodine has been a major public health success for decades in reducing IDD worldwide. More recently in April 2011, government-run agencies dealing with food and nutrition programs in India made the use of salt dual fortified with iodine and iron mandatory in meals [87]. fortification of milk has also been mandatory in Canada since 1979 and has been almost universally practiced in the US since the 1940s [1]. Furthermore, iron-fortified cereal has been demonstrated to be one of the most effective food vehicles in combating iron deficiency anemia, and is still recommended by the WHO [1, 88]. Food fortification is relatively easier to implement than other strategies as it could often be incorporated into the existing food production and distribution system [86]. Its cost effectiveness has made this approach quite popular in developing nations. For example, in India, it costs only US$0.05/year per person to fortify salt with iodine [88], and in Chile, it costs US$ 0.16/year per woman to fortify flour with folic acid [61]. Although there are apparent benefits to food fortification, it must meet several criteria in order to be implemented as an intervention for micronutrient deficiencies. These requirements are discussed in the following section.

2.3 Fortification Strategy

2.3.1 Requirements for Effective Food Fortification Programs

A successful fortification program requires active and long-term participation by government, scientific bodies and the food industry. Fortification programs should only be implemented once documented evidence is presented indicating that there will indeed be a health benefit to increasing the presence of one or more micronutrients in the target food. Compiling

19 biochemical/chemical data, scanning dietary patterns to understand the composition of the usual diet of the target population, and obtaining detailed information on the existing dietary intakes of the desired micronutrients are some ways of accomplishing this. Although insufficient intake from food is a common risk factor for micronutrient deficiencies, other factors like the presence of infections and parasites (which can contribute to high rates of anemia) can also play a substantial role. Therefore, it is important to evaluate fortification against other strategies in terms of cost-effectiveness and ability to control the risk factors. In some instances, the best option is a combination of fortification and other interventions like school based deworming programs [1].

Once the need for intervention is established, the next steps include prioritizing the target population groups, deciding on the appropriate level of the selected fortificants, selecting a suitable carrier for the fortificants and identifying and understanding any constraints (e.g. safety, cost, technological) that may impact the amounts of nutrients that can be added to given foods [89]. Program monitoring and evaluation, along with communicating and marketing of the fortification programs should also be carried out in an on-going basis to see how the program has affected the intakes and nutritional status of the recipients.

When selecting the most appropriate form of the selected micronutrient to be added as the fortificant, some important considerations must be made:

• The fortificants must not cause unacceptable organoleptic changes (flavour, colour, texture or odour) in the fortified food, or segregate out from the food matrix.

• The fortificants should demonstrate acceptable stabilities in the fortified foods.

• The potential for any interactions that might occur between micronutrients in multiple- fortified systems, as well as interactions between the fortificant(s) and food vehicle that may interfere with the metabolic uptake of the fortificant, also needs be thoroughly assessed.

• The addition of extra additives such as binders, encapsulates and stabilizers to improve the retention of the fortificants should not require significant changes to existing technologies.

• The fortificant must be well absorbed from the food vehicle at the level of consumption

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compatible with a .

• The final cost of fortification must not decrease the affordability of the food or increase the competitiveness with the unfortified alternative.

Foods such as flour, wheat, cereals, dairy products, beverages, oils and various accessory foods such as salt, sauces and sugar are particularly well suited to fortification. They all share some or all of the following common characteristics of an ideal carrier [89]:

• The food is adequately consumed by a large portion of the population, on a regular basis.

• The food is centrally processed (this allows to better facilitate the implementation of quality control measures and effective monitoring and enforcement procedures)

• The nutrients can be added relatively easily using low-cost technology, and in such a way so as to ensure an even distribution within batches of the product.

• The food is used relatively soon after production and purchase, which improves vitamin retention and imparts minimal organoleptic changes, since only a small overage is required when fortifying.

Salt is an ideal food vehicle that has been favoured as a carrier for especially iodine, as well as other nutrients like iron, because of its consumption in fairly consistent amounts by all sectors of society at roughly constant levels throughout the year. Salt iodization has proven to be technologically feasible and cost-effective. Countries with effective salt double fortification programs have shown sustained reductions in the prevalence of IDD and iron deficiency anemia [89-90]. The following section describes the major technologies used in the addition of iodine to salt, since this project focuses on incorporating folic acid to the existing salt iodization technology.

2.3.2 Salt Iodization Technologies

Salt iodization began in 1922 in Switzerland and has been implemented in many countries as the major mechanism for eliminating iodine deficiency. Three technologies are widely used in the addition of iodine to salt. These are dry mixing, drip feed addition and spray mixing. Table 2.3.1 compares these methods.

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In the dry mixing process, a powdered form of the iodine compound is mixed with an anti-caking agent like calcium carbonate. This stock mixture is subsequently mixed with salt at a ratio of 1:10 to form a “premix”, which is introduced onto a conveyor at a constant rate. Salt is also introduced onto the conveyor and mixing occurs while the material is moving along the conveyer belt. This process is best suited for fine salt with a grain size of less than 2 mm. Dry mixing is not appealing for the unrefined coarse salt commonly used in developing countries because the powdered form of the iodine source will settle at the bottom of the packaging, due to having a finer particle size and being denser than salt. Nevertheless, it has been adopted in several countries of South and Central America [91].

In the drip feed addition method, salt moves along a conveyor while a solution of the iodine source is dripped at a constant rate onto the salt crystals. A capacity of five tons per hour is ideal for this system, which requires only a low pressure head to maintain the needed flow rate. This system is fairly simple and inexpensive. However, it is generally only suitable for coarse salt with particle sizes of up to 1 cm, having moisture contents less than 5%. Since fine salts have particle sizes typically less than 2 mm, the iodine solution is not dispersed uniformly [70]. This method is still used in some Asian countries like Indonesia [91].

In the spray mixing process, the salt moves inside a rotating drum or ribbon blender, while a finely atomized mist of a solution of the iodine compound (usually KI or KIO3) is sprayed onto the salt. The spray system disperses the iodine solution uniformly on the salt crystals as the salt is constantly mixed. This ensures better uniformity in mixing for all types of salt used, in comparison to the two above-mentioned methods. The concentration of solution and the spray rates are adjusted to obtain the required dosage of iodine in the salt, with KIO3 solutions typically ranging from 10-30 g/L. This method is being increasingly preferred in Asia, South America and Africa [91].

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Table 2.3.1: Comparison of salt iodization methods (adapted from [91])

Dry mixing Drip feed addition Spray mixing Iodization Technology Refined powder, dry √√√ √√ √√√ Unrefined powder, dry √√√ √√ √√√ Salt Unrefined powder, moist √√ √√ √√ Type Unrefined crystals, dry √ √√ √√ Unrefined crystals, moist √ √ √√ Cost (includes capital cost, operating cost High Low Medium and cost to consumer)

√ Poor √√Fair √√√ Good

Although the technology for salt iodization is well known, readily available and inexpensive, the best iodization technology for each country varies due to having different types of salt and environmental conditions. The target nations to which this project is intended to apply are the less developed countries, which rely on salts of uneven purity, moisture and refinement.

Therefore, crushing the salt and spray mixing by using a solution of KIO3 has become the preferred method of salt iodization in these regions. Furthermore, simple salt iodization can be easily achieved even at the village level using spray mixing, where a spray bottle can be used to manually spray a solution of the iodine source onto batches of salt, which are being stirred by hand [70]. Because of these reasons, the spray mixing approach was explored in this project as the method of transferring the micronutrients of interest onto salt.

2.3.3 Double Fortification of Salt with Folic Acid and Iodine

While great progress has been made on double fortifying salt with iodine and iron to successfully reduce multiple deficiencies, the addition of folic acid to salt is not widely addressed in the literature.

A recent study by the Food Engineering Group found that folic acid was generally stable when sprayed into unfortified, refined Canadian salt with retention values of over 90% after 9 months of storage at high temperature and relative humidity, without any apparent interaction with the salt. When sprayed onto already iodized Guatemalan salt and stored at the aforementioned conditions, over 80% remained after 9 months of storage. Iodine retention was found to be lower with ~60% of the original iodine remaining after 9 months, whereas retention was over 90% after

23 the same duration of time in salt unfortified with folic acid [92]. The excessive losses in iodine suggested that there might be interactions between iodine and folic acid. Therefore, it was recommended to incorporate folic acid as a separate premix to already iodized salt, since this resulted in improved folic acid stability and unaffected iodine retention. However, this may not be a viable option for small-scale producers in rural parts of developing countries, who use spray mixing as the prevalent method of salt production.

In order to facilitate spray mixing, there still was the need for a single spray solution that maintains folic acid and iodine at acceptable levels both in solution and once sprayed onto salt. This was the motivation for this project.

2.3.4 Project Objectives

The main goal of this study was to develop a formulation of salt dual fortified with folic acid and iodine (in the form of KIO3), in which high retention values (≥ 90%) for both micronutrients would be maintained for at least 6 months under high temperature and humidity storage conditions. The fortified salt needed to be prepared through spray mixing, since this is the preferred method of salt iodization in less developed countries.

Therefore, my goal was to prepare a spray solution that contains both micronutrients fully dissolved (for ease of spraying through a nozzle) and stable for at least 1 month, as salt iodization facilities usually replenish the spray solution every 2- 4 weeks. Once sprayed onto salt, the micronutrients must have acceptable stabilities for at least 6 months of storage. The effects of increasing the spray solution pH, or using an oxygen scavenging antioxidant in the spray solutions, were initially investigated to eventually establish the final formulations.

A secondary goal was to determine whether interactions occurred between the two micronutrients, both in solution and in salt. The losses of each micronutrient added by itself to the solutions and the salt, were compared to the losses observed when the micronutrients were added together.

Once the final formulations were established, salt fortified with 45 ppm (45 µgmicronutrient/gsalt) of both micronutrients was prepared based on the WHO/UNICEF/ICCIDD recommended level of salt iodization at the production sites of tropical and subtropical countries [93]. Since the previously mentioned study on Guatemalan salt saw a 20% reduction in folic acid when sprayed

24 onto iodized salt and stored at harsh environmental conditions for 9 months, it was estimated that in the worst case scenario, if the stability enhancing treatments were to have no effect on folic acid stability in salt, similar losses would be incurred in addition to further losses from other environmental factors during transport and storage in the target countries. It was hoped that there would be at least 30 ppm of both micronutrients remaining in the dual fortified salt samples after 6 months of storage under harsh environmental conditions. This was the target concentration recommended by The Micronutrient Initiative (MI), based on a per capita salt consumption of 10 g/day in the target countries, which would provide the salt consumer 300 µg/day of both micronutrients. This value is 75% of the RDA for folic acid, but the literature review suggested that most of natural folates become biologically inactive during food preparation steps. Therefore, the risk of overconsumption is unlikely, especially because people living in poor, remote parts of the world often do not have access to foods rich in folate. Likewise, iodine will be present in the salt at twice the RDA at the target level of fortification, but it has been shown in previous trials that ~50% of the iodine becomes lost during transport and storage in countries experiencing warm, humid climates [93]. Further losses can also be expected during the thermal processes of food preparation in the target countries, because salt is often added to the food while it is being cooked, rather than at the table. Thus ingesting excessive amounts of the two micronutrients from this fortified salt is extremely unlikely.

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3 Experimental Details 3.1 Materials

Table 3.1.1 summarizes the formulation ingredients and the analytical reagents used for micronutrient analysis.

Table 3.1.1: List of materials used in this study Chemical Supplier Description Non-iodized Salt Refined Canadian salt Sifto Canada Corp., Canada Clean & dry Coarse Orissa salt Micronutrient Initiative Impure & moist Micronutrients Folic acid Bulk Pharmaceuticals Inc., Canada USP grade

Potassium iodate (KIO3) Sigma-Aldrich Chemicals, Canada ACS reagent Antioxidant Trisodium citrate dihydrate Sigma-Aldrich Chemicals, Canada ACS reagent Buffering Agents

Citric acid (C6H8O7) Sigma-Aldrich Chemicals, Canada ACS grade Disodium Sigma-Aldrich Chemicals, Canada ACS grade (Na2HPO4)

Sodium carbonate (Na2CO3) Sigma-Aldrich Chemicals, Canada ACS grade

Sodium bicarbonate (NaHCO3) Sigma-Aldrich Chemicals, Canada ACS grade Analytical reagents Folic acid analysis Hydrochloric acid (HCl) EMD Chemicals Inc., USA R&D use; conc. 3-Aminophenol (3-AP) Sigma-Aldrich Chemicals, Canada ACS grade Sodium hydroxide (NaOH) Sigma-Aldrich Chemicals, Canada ACS grade

Sodium nitrite (NaNO2) Sigma-Aldrich Chemicals, Canada ACS grade

Sulfamic acid (H3NSO3) Sigma-Aldrich Chemicals, Canada ACS grade Zinc granules Sigma-Aldrich Chemicals, Canada Reagent grade Iodine analysis Potassium iodide (KI) Sigma-Aldrich Chemicals, Canada ACS grade

Sulphuric acid (H2SO4) EMD Chemicals Inc., USA R&D use; conc. ACS grade, 0.1 Sodium thiosulphate (Na S O ) BDH Inc., Canada 2 2 3 N solution 1% Starch indicator solution Caledon Laboratories Ltd., Canada ACS grade

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3.2 Methods

3.2.1 Experimental Methods

General Method of Spray Solution Preparation

The buffered spray solutions were prepared by dissolving folic acid and iodine at 0.35% (w/v) in the buffering agent (exact buffers used are discussed later). The citrate (antioxidant) containing spray solutions were prepared by dissolving both micronutrients at 1% (w/v) each, along with 0- 3% (w/v) sodium citrate, in distilled water.

In order to determine whether folic acid and iodine would interact, aqueous solutions of each micronutrient were prepared (as controls), as well as solutions containing both dissolved together. These solutions were prepared by dissolving folic acid and/or iodine in distilled water. Two concentrations of the micronutrients were chosen for the control solutions in order to correspond to those concentrations used in the buffered solutions (0.35% w/v) and the citrate solutions (1% w/v). The solutions were sprayed onto clean salt (reduces the effect of impurities) and the retention of folic acid and iodine in both solutions and salt were tested over time. The solutions were stored at ambient conditions, while the salt was stored at high temperature, to mimic the real temperatures likely encountered by the solutions (at the production facility) and salt (during transport), respectively. The actual loss of each micronutrient due to the interaction with the other was calculated by subtracting the loss in the control from the loss in the samples containing both micronutrients.

Potassium iodate (1 g KIO3 = 0.6 g I) was used as the iodine source in all preparations due its high stability in salt under harsh environmental conditions.

The constituents of each spray solution were added to a 100 mL volumetric flask, filled to the mark with either the buffering agent or distilled water, stoppered and mixed for 5 minutes by turning upside down and back. The contents were transferred to a 500 mL Erlenmeyer flask that was subsequently placed on a hot plate stirrer and the contents continuously stirred at high speed for 15 minutes using a stir bar. The solutions were prepared with minimal lighting and stored in tinted bottles in the dark at room temperature to avoid degradation of folic acid by light and heat.

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General Method of Salt Fortification

A bench-scale ribbon blender (Les Industries All-Inox Inc., Montreal) was used for the preparation of double fortified salt. It consists of a rotating shaft fitted with two helical ribbons that rotate in opposite directions inside a semicircular trough. To prepare each formulation, 500 g of non-iodized salt was placed in the blender and allowed to blend (with the blender operating at 22 rpm) for 3 minutes, in order to break any large clumps of salt. Next, a prefixed quantity of each spray solution was sprayed to the center of the blender using a hand-held spray bottle. Either 6.6 mL were sprayed from the solutions containing 0.35% (w/v) of the micronutrients, or 2.2 mL were sprayed from the solutions containing each micronutrient at 1% (w/v), to obtain a final concentration of 45ppm of each micronutrient in the salt at time 0. The batch was allowed to mix for 20 minutes. Afterwards, the blender was stopped, and the batch was discharged onto a baking tray and spread evenly for drying overnight in the dark at room temperature. Oven drying was avoided because folic acid degrades with heat, and studies in Thailand have found that over 20 ppm of iodine can be lost because of the drying process (>100°C) after spray mixing [94].

Each formulation was placed in Zip-LocTM polyethylene bags and stored in the dark at 45˚C and 60% relative humidity (RH), in a Model 307 Fisher Scientific Incubator, for up to 6 months.

3.2.2 Analytical Methods pH Analysis

A VWR Scientific Model 8000 pH meter was used to measure the pH of the spray solutions, and of 1% (w/v) solutions of the salt.

Moisture Analysis

The moisture content of the salt samples was measured gravimetrically. Five grams of salt was accurately measured and placed in a shallow aluminum dish, which was subsequently placed in a forced-air oven at 110oC for 24 hours. The following formula was used to calculate the moisture content:

Moisture content (%) = (Ww – Wd)/ Ww * 100, where Ww = Wet weight of the sample Wd = Dry weight of the sample

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Surface Morphology Assessment

The surface morphology of the salt pre- and post-fortification was assessed and photographed at 30X and 100X magnifications using a Model S-2500 Hitachi Scanning Electron Microscope. Salt grains were placed on a strip of carbon tape and coated with a gold sputter coater for 70 seconds. The grains were then magnified and photographed by the microscope.

Folic Acid Analysis

Folic acid in the spray solutions and fortified salt was determined by using the coupling reaction method (adapted and modified from [95-96]). Folic acid was first reductively cleaved in the presence of zinc and hydrochloric acid. Next, the product was diazotized; followed by coupling with 3-AP. Finally, the absorbance of the yellow-orange complex was measured at 460 nm using a Cary 50 Ultraviolet-Visible Spectrophotometer. The detailed procedure is presented in Appendix 9.1.1.

Iodine Analysis

The iodometric titration method (AOAC method 33.149) [97] was used for the determination of iodine in the spray solutions and fortified salt. The iodate was reduced to free iodine, which formed a deep blue colour complex with starch indicator. Titration with Na2S2O3 was carried out until the colour disappeared. The detailed procedure is presented in Appendix 9.1.2.

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4 Results and Discussion 4.1 Selection of Final Formulations

4.1.1 Effect of pH on Micronutrient Stability in Buffered Solutions

A series of buffered solutions from pH 2 to pH 10 were initially prepared to determine the effect of pH on the stability of folic acid and iodine. Common buffers were used, of which the components can be added to food based on good manufacturing practices, according to the standards for food additives outlined in the [98]. Citric acid-phosphate buffer was used for pH 2-8; carbonate-bicarbonate buffer was used for pH 10. Both micronutrients were simultaneously added to each solution, since the final salt formulations needed to be prepared by spraying a single solution. Folic acid and iodine were added at 0.35% (w/v) each, since this concentration would not shift the pH of the pH 10 solution significantly (buffer capacity calculations are presented in Appendix 9.2). Although the concentration could have been increased to at least 1% (w/v) – a more realistic concentration that is currently used in salt iodization – it was thought that a lower concentration would have the benefit of reducing the strength of the buffer components that will ultimately end up in the salt, if any of the solutions were to be chosen to be sprayed onto unfortified salt to prepare the final salt formulations. Adding a higher concentration of folic acid into the buffer solution requires a higher concentration of the buffer components, especially for the alkaline pH range, in which there needs to be a higher concentration of the base compared to the acid, in order for the hydrogen ions from folic acid to be taken up to prevent a pH decrease. The components of each buffer tested are presented in Table 4.1.1, while Figure 4.1.1 shows the physical appearance of the solutions.

Table 4.1.1: Components of the buffer solutions used Volume (mL) pH 0.1 M C6H8O7 0.2 M Na2HPO4 0.1 M Na2CO3 0.1 M NaHCO3 2 98 2 0 0 4 58.4 41.6 0 0 6 34 66 0 0 8 2.8 97.2 0 0 10 0 0 60 40

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Figure 4.1.1:The appearance of the buffered solutions after folic acid and iodine have been added at 0.35% (w/v) each (Note: the numbers indicate the pH of each solution).

While the potassium iodate was fully dissolved in all solutions, the improvement in the solubility of folic acid as the pH becomes less acidic can be clearly observed from the above figure. Figures 4.1.2 and 4.1.3 present the results from the initial 5-month stability studies.

Figure 4.1.2: Folic acid retentions in the buffered solutions (that have iodine as well) during 5 months storage at room temperature in the dark (each bar represents the mean of triplicates, and the error bars represent the relative standard deviations).

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Figure 4.1.3: Iodine retentions in the buffered solutions (that have folic acid as well) during 5 months storage at room temperature in the dark (each bar represents the mean of triplicates, and the error bars represent the relative standard deviations).

Based on the literature review, it was expected that both micronutrients would be more stable under alkaline conditions compared to storage in acidic or neutral conditions. In the latter conditions, the iodate would get reduced to iodine; and the folic acid likely degraded into pABG (and other compounds), since it has been reported that the decomposition of folic acid under a similar pH range has been accompanied by the liberation of an equivalent amount of aromatic amine [99].

Regression analysis using Excel showed that overall, the experimental data were consistent with first-order degradation kinetics. This was confirmed when the micronutrient retention data were plotted as ln (% retention) vs. the storage month, and a generally good correlation existed between the data and the linear regression lines (with the correlation coefficients ≥ 0.95 for all pH values). For folic acid, the differences in the first order degradation rate constants (k values) are not significant at a 95% confidence level for pH 2 to pH 4 solutions, and for pH 6 to pH 8 solutions. The results are presented in the table below, and clearly indicate that the rate of degradation in alkaline pH is much slower than that in acidic pH, especially for folic acid, where the degradation rate at pH 10 is almost ten times lower than the degradation rate at pH 2. Iodine

32 degradation is higher in the acidic range likely because of interaction with folic acid degradation products (that form faster in acidic environments [21]), which may cause the reduction of iodate, resulting in iodine loss. Although the overall reaction approximates first order kinetics, the detailed reaction mechanism could be of another order, especially for folic acid in the acidic range. This is because such common degradation compounds as pABG may further degrade folic acid by being yet another acidic catalyst that leads to the C9-N10 bond cleavage (the most common initial step in folic acid degradation [21]) of the molecule. Under the assumption that the reaction is likely acid catalyzed, the degradation rate constant is expected to increase as the pH decreases (since the [H+] concentration increases).

Table 4.1.2: Apparent first order degradation rate constants (k) estimated for the buffered solutions containing 0.35% (w/v), or 3500 mg/L, folic acid and iodine when stored at 25ºC and 60% RH Buffer solution pH k (month-1) Correlation coefficient, r Folic Acid 2 0.3207 ± 0.1049 0.97 4 0.1907 ± 0.0878 0.95 6 0.1058 ± 0.0469 0.95 8 0.0804 ± 0.0157 0.99 10 0.0266 ± 0.0090 0.97 Iodine 2 0.0449 ± 0.0150 0.97 4 0.0445 ± 0.0065 0.99 6 0.0315 ± 0.0128 0.96 8 0.0319 ± 0.0052 0.99 10 0.0284 ± 0.0057 0.99

From the above graphs, the pH 10 solution showed the highest retention values by the end of the 20-week study. It has been suggested that because the folic acid molecule exists as a predominantly anionic species, it becomes less susceptible to degradation due to existing as a mesomer stabilized anion; whereas the formation of a predominantly protonated species in the acidic mediums makes it highly susceptible to degradation [20]. It is interesting to note that there was almost a 10% loss of folic acid only after one month of storage in the pH 8 solution. This may be attributed to the fact that the pH 8 buffer had the greatest amount of 0.2 M disodium phosphate (Table 4.1.1), since it has been shown in literature that folic acid has a higher degree of instability in phosphate buffer at pH 6 and 8 compared to other buffers that have been tested at the same pH; but the mechanism is still unclear [17].

33

Therefore it was decided that the spray solutions for the final salt formulations would be prepared with only pH 9 and pH 10 carbonate-bicarbonate buffers containing folic acid and iodine dissolved together. It was decided to include pH 9 because if it were to show the same high stability of the micronutrients for at least one month, this would induce fewer production costs compared to preparing the pH 10 solution, as less buffering chemicals would be needed. The detailed results for the final formulations are presented in Section 4.2.1.

4.1.2 Effect of Citrate on Micronutrient Stability in Spray Solutions

Since folic acid has shown improved stability with antioxidants according to literature, the sodium salts (to maintain an alkaline pH) of two highly water soluble antioxidants – ascorbic acid and citric acid – were initially considered for preparing aqueous spray solutions containing the two micronutrients and an antioxidant.

Ascorbic acid is considered a multifunctional antioxidant since it can quench singlet oxygen (which is a that has been shown to commence the degradation of folic acid when there is headspace or dissolved oxygen), reduce free radicals and remove molecular oxygen when there are metal ions present. Citric acid is mainly considered as a metal chelator, but has been shown to inhibit active oxygen formation when added at different stages of oxygen reduction, although the mechanism of antioxidant action has not been clarified [101-102]. It would be useful especially for less refined salts containing metal ion impurities, often found in poorer areas of developing countries. In this study however, it was determined that ascorbate could not be used because a quick test confirmed that the iodine generated during the iodometric titration method used for iodine analysis would be reduced by the ascorbate. Although another iodine analytical method such as neutron activation analysis (which is not subject to interference from reducing or oxidizing agents) could have prevented the problem, it was thought that such analysis equipment would be more costly to implement in the salt iodization facilities of the developing countries, where the simple colourimetric titration method is predominantly used for iodine analysis.

Therefore, a test solution was prepared containing 1% (w/v) each of folic acid and iodine, along with sodium citrate at 3% (w/v), especially because it was found during the literature review that folic acid is stabilized in the presence of citrate. Such a high citrate concentration was used with the hope of achieving the maximum possible protective effect on folic acid in solution (and on

34 iodine once sprayed onto impure salt). The micronutrients were at 1% (w/v) so that a low volume of solution could be ultimately sprayed onto salt if this solution showed excellent retention of the micronutrients and was to be included in the final formulations. The solution was stored in a tinted glass bottle in the dark at room temperature for 3 months. The solution maintained a pH of 5.99 ± 0.03 throughout the study, and when compared to the pH 6 solutions discussed in the previous section, the retention of the micronutrients in the citrate-containing solution were higher and generally ≥ 99% by the end of the study. This suggests that folic acid degradation in solution occurs due to oxidative stress, likely caused by dissolved oxygen present in solution and in the headspace of the capped glass storage bottle. Although the iodine source used in this study is in its fully oxidized form, the slight improvement in iodine retention was likely because citrate prevented the formation of potential oxidative degradation compounds of folic acid that may otherwise react with the iodate. In order to utilize the apparent protective effect of citrate, solutions to be sprayed onto the final salt formulations were prepared to contain 0.1-3% (w/v) sodium citrate, and 1% (w/v) of both folic acid and iodine. It was hoped that a lower concentration of the antioxidant could provide the same level of micronutrient protection as the 3% (w/v) solution did, since this would reduce costs. The detailed results for the final formulations are presented in Section 4.2.2.

4.2 Stabilities of Micronutrients in Final Formulations

4.2.1 Effect of Alkaline pH

Tables 4.2.1 and 4.2.2 present the constituents at time 0 in the final solutions and salt, respectively. The control salt sample was prepared by spraying an aqueous solution of the two micronutrients dissolved at 0.35% (w/v) in water.

Table 4.2.1: Buffered solution compositions at time 0 for final formulations Solution pH Component 9 10 Buffering agent % Na2CO3 0.53 0.64 % NaHCO3 0.42 0.34 Micronutrient % Folic acid 0.35 0.35 % Iodine 0.35 0.35 Note: All percentages are w/v based.

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Table 4.2.2: Compositions at time 0 in 500g of salt sprayed with buffered solutions Salt Sprayed with Spray Solution pH: Component 9 10 Buffering agent in salt ppm Na2CO3 70 84 ppm NaHCO3 55 44 Micronutrient in salt ppm Folic acid 45 45 ppm Iodine 45 45 Note: 1 ppm = 1 µg/g

The pH 9 solution was identical in appearance to the pH 10 solution, while the control solution was not homogeneous and resembled the pH 6 solution due to the low solubility of folic acid in water (its pH was 5.93± 0.06 throughout the study). The stabilities of the micronutrients in the pH 9 solution (Appendix 9.3.1) were very similar to those in the pH 10 solution tested earlier, with retentions >99% after four weeks of storage. The apparent first order rate constants were 0.0278 ± 0.0111 month-1 for folic acid (r = 0.977) and 0.0247 ± 0.0113 month-1 for iodine (r = 0.969). These were quite close to those of the pH 10 solutions, with the differences in the k values being insignificant for folic acid at a 95% confidence interval. The appearance of the salt turns into a pale yellow upon spraying the solutions, as can be seen from Figure 4.2.1. However, this is not expected to be a turnoff for consumers, since the salt is uniformly yellow (due to micronutrients being able to adhere to the salt with the help of the moisture in the spray solution), instead of having dark patches that look like impurities, as is the case sometimes with some fortificants like unencapsulated iron that impart a dark colour similar to dirt. Besides this, the salt is often packaged in paper in the poorer regions of the target countries, so the colour would not be noticeable. The micronutrients’ stabilities in clean Canadian salt sprayed with pH 9 and pH 10 buffered solutions are presented in Figures 4.2.2-4.2.3.

Figure 4.2.1: Comparing the colour of Canadian salt before (left) and after (right) double fortification with folic acid and iodine using a pH 9 spray solution.

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Figure 4.2.2: Folic acid retentions in refined dry salt during 6 months storage in the dark at 45°C/60% RH (each point represents the mean of triplicates, and the error bars represent the relative standard deviations).

Figure 4.2.3: Iodine retentions in refined dry salt during 6 months storage in the dark at 45°C/60% RH (each point represents the mean of triplicates, and the error bars represent the relative standard deviations).

37

It can be seen from the above figures that spraying an alkaline solution of the two micronutrients onto salt greatly improves the retention of both folic acid and iodine, compared to the control that had no buffer, by slowing down the rate of degradation of folic acid and the reduction of the iodate. Although 1% (w/v) solutions of the salt were close to neutral pH throughout the study (the pH of the Canadian salt solutions fluctuated between 6.58 - 6.8 both pre- and post- fortification), the presence of the alkaline carbonate likely formed a protective shield, since otherwise acidic and neutral environments tend to cause faster degradation of folic acid. Furthermore, just as expected, the high temperature and humidity storage conditions caused high losses in folic acid, since it is known to be less stable under heat. Moisture may have been a factor that influenced the stability of especially iodine, since the addition of the spray solutions onto the unfortified salt initially increased the salt moisture from 0.05 ± 0.01% to 1.29 ± 0.03%. The salt was not oven dried due to the degradation of folic acid by heat, and drying in a vacuum oven set to 45°C overnight was not effective. After 6 months of storage however, the final moisture in the salts was 0.04 ± 0.01%. The reduction in moisture may have been a reason as to why the iodine losses occurred at a slower rate by the end of the study, since the high water solubility of KIO3 would have allowed it to easily leech to the bottom of the storage bag at the beginning of the study, when the salt was wet.

Since the micronutrient retentions in the clean salt were comparable for both pH 9 and pH 10, the pH 9 spray solution could be used for fortifying salts in tropical countries because, this would minimize ingredient costs compared to the pH 10 solution. Since the target countries consume

~10 g salt/day, the fortified salt will add 700 µg/day Na2CO3 and 550 µg/day NaHCO3 to the diet. These values are far below the LD50 values for humans for these two compounds: 2000 mg/kgbodyweight for Na2CO3; 4000 mg/kgbodyweight for NaHCO3 [103-104].

The pH 9 solution was therefore sprayed on a coarse, impure (grey-brown colour) and moist salt sample (moisture of unfortified salt was 4.94 ± 0.01%) from Orissa, an eastern state on the coastal belt of India that experiences temperatures as high as 46ºC. Air-drying for 48 hours both before and after spraying was inefficient at sufficiently reducing the moisture, with the final moisture being 5.98 ± 0.01% at month 0. The stabilities of the micronutrients were tested for only four months at 45ºC/60% RH due to the lack of enough salt for further micronutrient analysis. The results are presented in Figure 4.2.4.

38

Figure 4.2.4: Stability of micronutrients in impure, moist, coarse salt during 4 months storage in the dark at high temperature and humidity (each point represents the mean of triplicates, and the error bars represent the relative standard deviations).

It can be observed from the above figure that the buffered spray solution improved the stability of the micronutrients even in the less pure salt by maintaining about 90-95% of folic acid and iodine by the end of the month 4; this was about 10% more than the retentions observed in the control salts.

It was confirmed that the experimental data were consistent with first-order degradation kinetics for all salts sprayed with the pH 9 solution, when the micronutrient retention data were plotted as ln (% retention) vs. the storage month, and the correlation coefficients were > 0.96 (Table 4.2.3). The differences in the k values between spraying a pH 9 buffered solution versus a non-buffered (control) solution are significant at a 95% confidence level. Losses were generally much greater in the Orissa salt compared to the Canadian salt as expected, and the apparent first order rate constants were higher in the impure salt sprayed with both the control and the pH 9 solutions (the differences in the k values between the two salt types are significant at a 95% confidence interval for both micronutrients). This is likely due to a combination of high impurity levels (possibly and sulphur compounds that have been shown to lower iodine retention in iodated salt having a similar brown colour [67]) and the presence of moisture (moisture after 4th month of storage was 5.18± 0.01%) in the salt. Moisture allows for greater mobility of the reducing impurities, thus making it easier for those compounds to contact iodate and become oxidized.

39

The use of the pH 9 spray solution decreased the folic acid degradation rate to almost a third of the control, while the iodine degradation rate was reduced by almost half in the Orissa salt. This suggests that the presence of the pH 9 alkaline buffer in the salts of the tropical countries can retard iodate degradation caused by both the general salt impurities that can act as reducing agents to release iodine to the atmosphere, and the possible folic acid degradation compounds that can get oxidized by iodate.

Table 4.2.3: Apparent first order degradation rate constants (k) estimated for the salts sprayed with the pH 9 spray solution, when stored at 45ºC and 60% RH Canadian Salt Orissa Salt Solution Correlation Correlation k (month-1) k (month-1) coefficient, r coefficient, r Folic Acid Control 0.0300 ± 0.0059 0.985 0.0489 ± 0.0109 0.993 pH 9 0.0103 ± 0.0005 0.999 0.0165 ± 0.0051 0.987 Iodine Control 0.0332 ± 0.0106 0.962 0.0594 ± 0.0199 0.984 pH 9 0.0107 ± 0.0032 0.967 0.0279 ± 0.0054 0.995

4.2.2 Effect of Citrate

Sodium citrate was incorporated at different concentrations into the aqueous spray solutions containing 1% (w/v) each of folic acid and iodine to take advantage of its antioxidant properties that seem to stabilize folic acid. Table 4.2.4 presents the citrate levels in the final solutions and salt. The control sample was sprayed with an aqueous solution of folic acid and iodine at 1% (w/v), in the absence of citrate.

Table 4.2.4: Target citrate concentrations in solutions and salt at month 0 % Trisodium citrate dihydrate ppm (mg/L) in Spray ppm (µg/g) in Salt Solution 0.0 (Control) 0 0 0.1 1000 4.5 0.2 2000 8.8 0.5 5000 22 1 10000 45 2 20000 96.8 3 30000 132 Note: All percentages are w/v based

40

As can be seen from figures 4.2.5 and 4.2.6, in all of the solutions, at most ~3% of the micronutrients were lost after 5 months of storage at room temperature in the dark, even though the pH was mildly acidic at approximately 5.93 ± 0.08 (the appearance and consistency were similar to the pH 6 buffered solution in Figure 4.1.1 as well). This is in contrast to the lower retentions of the micronutrients in the pH 6 buffered solutions (Figures 4.1.2 and 4.1.3), suggesting that citrate may have removed at least a portion of the dissolved oxygen in the solutions, even at the 0.1% (w/v) citrate level, in which the ratio of citrate to micronutrients is 1:10. It is also possible for citrate to have removed any metals ions that could have catalyzed the oxidation. However, because the pH of the solutions was acidic, the folic acid was not fully dissolved. This is a disadvantage in an actual production setting, because extra equipment like mixers will be needed to thoroughly mix the contents of the spray solution storage container. It might also be difficult for the solution to pass through the spray nozzles.

Figure 4.2.5: Folic acid retentions in citrate-containing solutions (that have iodine as well) during 5 months storage in the dark at 25ºC/60% RH (each bar represents the mean of triplicates, and the error bars represent the relative standard deviations).

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Figure 4.2.6: Iodine retentions in citrate-containing solutions (that have folic acid as well) during 5 months storage in the dark at 25ºC/60% RH (each bar represents the mean of triplicates, and the error bars represent the relative standard deviations).

When the micronutrient retention data were plotted as ln (% retention) vs. the storage month, a generally good correlation existed between the data and the linear regression lines (with the correlation coefficients ≥ 0.95 for all concentrations of citrate tested), confirming that the degradation of the micronutrients followed apparent first order kinetics. The results are presented in Appendix 9.3.1, and clearly indicate that the rate of degradation of the micronutrients is much higher in the absence of citrate. Interestingly, concentrations higher than 1% (w/v) citrate do not have a significant impact on decreasing the rate of degradation of the micronutrients, suggesting that the protective effect of citrate can be maximized with 1:1 ratio of citrate to micronutrients. Nevertheless, even a ratio of 0.1:1 (citrate to micronutrients) can retain high levels of folic acid and iodine for over two months, which might make it economically attractive since the solution would not need to be replenished every month at the iodization facilities.

The stabilities of the micronutrients in refined Canadian salt containing both micronutrients with increasing amounts of citrate, were measured and presented in the figures below.

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Figure 4.2.7: Folic acid retentions in refined dry salt during 6 months storage in the dark at 45ºC/60% RH (each bar represents the mean of triplicates, and the error bars represent the relative standard deviations).

Figure 4.2.8: Iodine retentions in refined dry salt during 6 months storage in the dark at 45ºC/60% RH (each bar represents the mean of triplicates, and the error bars represent the relative standard deviations).

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Folic acid stability was enhanced by the presence of citrate ions in the salt at all concentrations, (Figure 4.2.7). This could be attributed to the antioxidant properties of citrate that may prevent the oxidative degradation of folic acid by reducing reactive oxygen species that may have entered the salt during preparation. Citrate concentrations of 45 ppm (i.e. a 1:1 ratio of citrate to folic acid) or greater can maintain the folic acid retention above 95% for at least 6 months of storage in high temperature conditions; this trend was also noted in the spray solutions. As can be seen from Figure 4.2.8, iodine retention was also positively affected by the presence of citrate. This is likely because the citrate slowed down the formation of some degradation product of folic acid that could otherwise interact with the iodate. In less purified salts that are common in the target developing nations, the presence of citrate can further aid to further maintain iodine stability by chelating unwanted metal ion impurities like iron, calcium and magnesium. A 1:1 ratio of iodine to citrate is also sufficient for achieving ~90% iodine retention in the salt after 6 months at the harsh temperature condition. The rates of degradation in salt for each micronutrient remain essentially unchanged as the citrate concentration increases above 45 ppm, as can be seen in the table below. A generally good correlation existed between the data and the linear regression lines when the ln of % retention was plotted against storage time (with the correlation coefficients > 0.98 for all concentrations of citrate tested), confirming that the degradation of the micronutrients followed apparent first order kinetics. The differences in the k values between the salt having citrate concentrations of 45 ppm and salt having citrate concentrations > 45 ppm, are not significant at a 95% confidence level especially for folic acid. As well, the differences in the k values for folic acid in all of the salts with citrate concentrations < 45 ppm are insignificant at a 95% confidence level, although the differences in k values are significant at a 95% confidence level between the salt having 45 ppm citrate versus salt with lower citrate concentrations.

Table 4.2.5: Apparent first order degradation rate constants (k) estimated for the salt containing 45 ppm folic acid and iodine, and varying amounts of citrate, when stored at 45ºC and 60% RH Citrate concentration (ppm) k (month-1) Correlation coefficient, r Folic Acid 0 0.0273 ± 0.0039 0.993 4.5 0.0229 ± 0.0046 0.987 8.8 0.0191 ± 0.0032 0.990 22 0.0184 ± 0.0027 0.989 45 0.0116 ± 0.0020 0.991 96.8 0.0114 ± 0.0020 0.987 132 0.0113 ± 0.0020 0.989

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Iodine 0 0.0284 ± 0.0065 0.981 4.5 0.0197 ± 0.0030 0.990 8.8 0.0209 ± 0.0041 0.984 22 0.0205 ± 0.0039 0.995 45 0.0174 ± 0.0130 0.991 96.8 0.0173 ± 0.0050 0.994 132 0.0173 ± 0.0017 0.995 Note: 1 ppm = 1 µg/g

4.2.3 Effect of Micronutrients on Each Other

The retentions of folic acid and iodine in aqueous solutions are presented in the following figures.

Figure 4.2.9: Folic acid (FA) retentions in aqueous solutions during 6 months storage in the dark at ambient conditions (the results are the mean values obtained from four replicates, and the error bars represent the relative standard deviations).

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Figure 4.2.10: Iodine retentions (I) in aqueous solutions during 6 months storage in the dark at ambient conditions (the results are the mean values obtained from four replicates, and the error bars represent the relative standard deviations).

It is clear from the above figures that the presence of one micronutrient reduces the retention of the other. By the 6th month of storage, the micronutrients retentions in the mixed solutions (folic acid and iodine together) had been reduced to about ≤90% of those in the solutions containing either folic acid or iodine alone. The % relative retentions of the micronutrients were generally higher at the 1% (w/v) level compared to the 0.35% (w/v) level. A molar comparison of the losses was carried out, and it was estimated that the molar ratio of iodate loss to folic acid loss was ~5.7:1. However, iodate (I at +5 ) needs to gain 5 electrons to be reduced to iodine (I at 0 oxidation state). Furthermore, in the presence of folic acid, the only plausible way for iodate losses to occur in the solution (assuming there are no other reducing impurities) is that folic acid or its degradation compounds are acting as the reducing agents in providing those - - electrons in the following five electron reduction: IO3 + 5e  ½ I2. Further investigation into the mechanism of interaction needs to be carried out, since the chemistry implies that the molar ratio of iodate to reducing agent should be 1:5, as is the case when there are ferrous iron impurities.

46

The degradation of both micronutrients seems to follow first order kinetics when the micronutrient retention data were plotted as ln (% retention) vs. the storage month, with a generally good correlation existing between the data and the linear regression lines (correlation coefficients were > 0.95 for both concentrations tested). The differences in the iodine k values in the solutions having both micronutrients at different concentrations (1% vs. 0.35%) are not significant at a 95% confidence interval; although they are significant at the 95% level when comparing solutions having just iodine to those having both micronutrients. For folic acid, the differences in all k values are significant at a 95% confidence level, indicating that both the folic acid concentration and the presence of iodine have an effect on degradation. The data are presented in Table 4.2.6.

Table 4.2.6: Apparent first order degradation rate constants (k) estimated for the solutions containing folic acid and/or iodine when stored at 25ºC and 60% RH Salt Sample k (month-1) Correlation coefficient, r Folic Acid FA only: 1% 0.0045 ± 0.0008 0.982 FA only: 0.35% 0.0090 ± 0.0024 0.977 FA + I: 1% 0.0216 ± 0.0046 0.982 FA + I: 0.35% 0.0335 ± 0.0046 0.992 Iodine I only: 1% 0.0016 ± 0.0059 0.974 I only: 0.35% 0.0045 ± 0.0096 0.951 I + FA: 1% 0.0330 ± 0.0195 0.997 I + FA: 0.35% 0.0437 ± 0.0212 0.992 Note: the salt was sprayed with the solution concentrations listed in the first column.

The pH of the solutions containing the micronutrients together was around pH 6 during this study, but it is thought that iodine degradation in the presence of folic acid was likely not due to a pH effect. This is because the iodine retention in the 0.35% (w/v) solution containing both micronutrients in Figure 4.2.10 is lower than the retention values of iodine at pH 6 in the buffered solution study (Figure 4.1.3). The rate of iodine degradation in the 0.35% (w/v) solution containing both micronutrients is also higher than the 0.0315 month-1 rate that was calculated for iodine in the pH 6 buffer at the same iodine concentration.

Unlike the solutions, which will be produced at the production facilities where the temperatures are not as extreme, the salt will likely encounter higher temperature conditions during transport and sale in the developing countries. Degradation of the micronutrients in salt samples stored at a

47 high temperature condition was therefore followed. Using linear regression, the degradation data were analyzed to determine the overall order and rate constant (k) for the degradation process of each micronutrient in the salt. A correlation coefficient > 0.9 in all of the cases suggested that the degradation of both micronutrients followed first order kinetics in salt. The differences in the iodine k values in the salt having both micronutrients at different concentrations (1% vs. 0.35%) are not significant at a 95% confidence interval; although the differences in the k values are significant at the 95% level when comparing salt having just iodine to those having both micronutrients. For folic acid, the differences in the k values are significant at a 95% confidence level when comparing the salts with and without iodine present, but insignificant at a 95% confidence level when comparing the salts with different concentrations of folic acid. The results are presented in the figures and table below.

Figure 4.2.11: Folic acid retentions in refined dry salt during 6 months storage in the dark at 45ºC and 60% RH (the results are the mean values obtained from four replicates, and the error bars represent the relative standard deviations).

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Figure 4.2.12: Iodine retentions in refined dry salt during 6 months storage in the dark at 45ºC and 60% RH (the results are the mean values obtained from four replicates, and the error bars represent the relative standard deviations).

Table 4.2.7: Apparent first order degradation rate constants (k) estimated for the salt containing 45 ppm folic acid and iodine when stored at 45ºC and 60% RH Salt Sample k (month-1) Correlation coefficient, r Folic Acid FA only: 1% 0.0084 ± 0.0017 0.977 FA only: 0.35% 0.0142 ± 0.0016 0.993 FA + I: 1% 0.0273 ± 0.0028 0.993 FA + I: 0.35% 0.0300 ± 0.0074 0.984 Iodine I only: 1% 0.0065 ± 0.0020 0.986 I only: 0.35% 0.0141 ± 0.0016 0.993 I + FA: 1% 0.0284 ± 0.0095 0.981 I + FA: 0.35% 0.0332 ± 0.0112 0.972 Note: the salt was sprayed with the solution concentrations listed in the first column.

While folic acid and iodine had similar degradation rates in salt containing only one micronutrient, the degradation of iodine was faster when the two compounds were added together. Moisture due to the spray solution addition onto the salt was likely not a significant factor, as much of the water was evaporated off due the hot storage condition by the end of the study. This suggests that the rapid degradation of iodine was caused by the presence of folic acid or other reducing impurity ions, especially because the estimated molar ratio of iodate loss to folic acid loss was ~5.7:1.

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Although a mechanism of interaction is unclear and not mentioned in literature, one possibility is that first folic acid oxidizes, which results in the C9-N10 bond cleavage, yielding pABG (a well- established oxidative degradation product of folic acid), pterine-6-carboxaldehyde and various other such biologically inactive pterin derivatives. These compounds were reported in aqueous folic acid solutions at similar pH values to that of the fortified salt in this study, although those solutions were subjected to photolysis [99]. Such a degradation compound (which can degrade further) might subsequently react with the iodate. For instance, a US patent claims it is possible to prepare -iodine complexes wherein the coordinate center is the iodine molecule (in - the form of the triiodide, I3 , ion) and the sites of complexing reaction are the carboxyl and amino groups of the amino acid [105]. Glutamic acid, one of the components of the folic acid and pABG molecules, is one such amino acid that the patent claims to form the complex with iodine by acid catalysis (e.g. H2SO4 and HCl) in water or organic . The ideal pH level for this reaction was reported to be between 2-5. Interestingly, the iodometric titration method used in this project for iodine analysis of the samples requires the use of H2SO4 to bring the pH to ~2.8, - and also forms the I3 ion for complexation with starch. Therefore, it is possible that the iodine in the sample gets used up during the analysis stage by forming a complex with the glutamic acid residue of folic acid (which may compete with the starch) that is present in the tested samples. However, the temperature required for the patented reaction was between 40°C-50°C; whereas the iodometric titration for each sample is carried out at room temperature.

Therefore, it is more likely that because iodate is a strong oxidizing agent, folic acid or its degradation compounds that have the pteridine ring in particular may be getting oxidized, since the other components of the folic acid molecule are highly stable. The glutamic acid component of folic acid is stable since carboxylic acids have the highest level of oxidation next to carbon dioxide; and the amide linkage (connecting the glutamic acid residue to the PABA portion of the molecule) is also quite stable due to amides being inert. Further studies will need to be carried out to characterize interactions between folic acid and iodine.

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4.3 Assessment of Optimal Formulations

Additional tests are required to verify the initial finding from this study suggesting that about 5.7 mols of iodate are used up by the presence of 1 mol of folic acid (or its degradation product(s)). Therefore, it is not yet possible to recommend preparing an aqueous solution of the two micronutrients in water – although it would be the simplest and least costly spray solution containing both micronutrients – that could be used to spray onto salt to have excess iodine, which then could be prepared in sufficient quantities at pilot-scale for efficacy and effectiveness tests.

Instead, either the pH 9 solution containing 0.35% (w/v) of each micronutrient, or the citrate solution containing 1% (w/v) each of citrate and the micronutrients, can be sprayed onto salt to retain excellent micronutrient stability during storage in harsh environmental conditions that are typical of the target countries. Furthermore, in both of these formulations, the salt crystals generally retained their cubic shape after being sprayed with different volumes of the spray solutions (6.6 mL of pH 9 solution or 2.2 mL of citrate solution for a 500 g salt batch), without causing the initially straight edges to be significantly tapered off after being wetted with the solutions. This can be seen from the images in Table 4.3.1 on the following page. Additionally, the SEM images in the table reveal that the presence of the extra moisture did not cause the salt to begin caking, even from the addition of a greater volume of the pH 9 solution (due to having almost a third of the concentration of the micronutrients compared to the citrate solution). The fortified salt crystals are fairly spread apart (100X) and retain the same size instead of forming large clumps (30X), making them indistinguishable from unfortified salt in terms of their texture. Although the refined Canadian salt was slightly coloured due to the addition of folic acid, salts that are sold for consumption in poorer regions are often already discoloured by impurities like iron. Accordingly, the presence of the spray solution will not significantly affect the physical attributes of the salt, and may even aid to lighten the colour of the impure salts. Furthermore, the pH of 1% solutions of the salt remained essentially neutral before and after fortification, suggesting that the extra additives would not have a significant effect on the taste of the salt.

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Table 4.3.1: SEM images of Canadian salt treated with different spray solutions Unfortified Sprayed with pH 9 solution Sprayed with citrate (6.6 mL/500 g) solution (2.2 mL/500 g) 30X Magnification

100X Magnification

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Regardless of the chosen spray solution, fortification should have a low impact on the price of salt to facilitate compliance. The following table lists the FOB prices of the different compounds in the two spray solutions discussed above.

Table 4.3.2: Approximate FOB prices (US $/kg) of the constituents in the two best formulations [108-110] pH 9 Spray Solution 1% (w/v) Citrate Spray Solution Folic acid: $2/kg Potassium iodate: $10/kg Sodium carbonate: $0.23/kg Trisodium citrate: $1.05/kg Sodium bicarbonate: $0.34/kg

While a detailed cost analysis is difficult to conduct at this time due to the wide range of variables that are involved, including investment, capital, packaging and transportation costs, it is generally agreed that the cost of iodization ranges from 2-7 US cents per kilogram, which is less than 5% of the retail price of salt in most countries. For instance, the salt cost in India is typically 20 US cents per kilogram or less [70]. Likewise, folic acid fortification (mostly flour) programs cost about 5 US cents per person annually [106].

When looking at the other (stability enhancing) components in the two formulations above, both formulations seem comparable, with the compounds needed for the pH 9 spray solution being almost US $0.50 cheaper than the citrate solution, but the latter solution requiring less material. However, the citrate solution had a thick consistency due to the folic acid not being fully dissolved, which might clog the spray nozzle during the spray mixing process of salt fortification. This was another reason for not recommending a simple solution of folic acid and excess iodine dissolved in water, as such a solution is also a mixture rather than a homogeneous solution, which would lead to extra maintenance costs due to having to constantly clean the spray nozzles. Because the recommended citrate solution from this study has 1% (w/v) each of citrate and the two micronutrients, the preparation of this solution also requires more of the micronutrients, compared to the pH 9 spray solution that has only 0.35% (w/v) of the micronutrients. This becomes a waste especially if quality assurance controls in the spray mixing facilities require the solution to be replenished once a month, even though the citrate solution can maintain essentially 100% retention for over a month. Although the citrate solution could be prepared at a 0.35% (w/v) level of all three compounds (citrate, folic acid and iodine) to obtain the 1:1 ratio of citrate:folic acid that is sufficient for high stability, the solution will still exist as a

53 mixture instead of a homogeneous solution. Furthermore, organic acid ions like citrate, ascorbate, malate and phytate have been found to have an inhibitory effect on the activity of the enzyme that converts natural folates in foods to the monoglutamate form for normal absorption in the small intestine [107]. Thus the presence of excess citrate in the double fortified salt may prevent consumers from obtaining the full amount of folic acid possible from natural foods like vegetables when consumed with the fortified salt.

Therefore, the salt prepared with the pH 9 spray solution seems a better option to recommend for pilot tests as the optimal formulation from this project. This simple technology uses two readily available stabilizers that are commonly used food additives, which can likely be purchased in bulk at prices even lower than those listed in Table 4.3.2 in the target countries. The spray solution is easily transferable to the current spray mixing method of salt iodization that is used by most developing countries – even at the village level.

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5 Conclusions

The main objective of this project was met by developing a stable formulation of double fortified salt containing folic acid and iodine, which is readily transferable to the existing salt iodization facilities in developing countries, without adding significant costs. The key findings of this study include the following:

1. A spray solution buffered to pH 9 (using a carbonate-bicarbonate buffer system) maintained folic acid and KIO3, both added at 0.35% (w/v) each, fully solubilized and stabilized for at least one month during storage at 25°C/60% RH. When the solution was sprayed onto salts of varying purity, moisture and refinement, acceptable retentions of over 90% of the initial levels of both micronutrients were achieved after 6 months of storage at 45°C/60% RH. This formulation is recommended for pilot-scale testing under extreme environmental conditions typical of developing countries.

2. The presence of citrate maintained the stability of the micronutrients at essentially 100% of the starting amount, in an aqueous preparation consisting of trisodium citrate, folic acid and iodine, each at the 1% (w/v) level. When sprayed onto salt, micronutrient retentions of over 95% of the initial were observed after 6 months of storage at 45°C/60% RH. This suggests that folic acid degradation in aqueous solution and salt occurs due to oxidative stress, which in turn causes iodine degradation possibly through iodate/iodine’s interaction with one or more folic acid degradation products. The addition of citrate likely protects the folic acid from oxidation.

3. A clear mechanism for the possible interaction of folic acid (or its degradation compounds, not all of which are known) with the iodine/iodate was not identified. However, the calculated molar ratio of iodate loss to folic acid loss was ~5.7:1, in the absence of any stability enhancing compounds.

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6 Recommendations

This study has demonstrated that double fortified salt prepared by spraying a homogeneous, stabilized solution of folic acid and iodine can successfully retain these micronutrients over a period of at least 6 months at high temperature conditions. The following investigations could build upon the knowledge gained in this project.

1. Conduct sensory evaluations to determine any differences in taste due to the presence of the micronutrients and stabilizers in the salt. If the organoleptic properties are deemed acceptable, conduct a detailed cost analysis of using the recommended pH 9 spray solution for pilot-scale testing, in order to estimate the cost per metric ton of fortified salt on the producers, as well as the annual cost per person. The production at pilot-scale, of sufficient quantities of salt fortified with the pH 9 spray solution, is recommended for efficacy and effectiveness tests at the extreme environmental conditions of developing countries.

2. Test the feasibility of preparing solutions of both micronutrients dissolved in the pH 9 buffer at higher concentrations (ideally 1-3% (w/v)), and subsequently spraying them onto salt, possibly with the simple addition of a mixture to stir any suspension. This also becomes useful for implementation to the existing iodization process that typically uses up to 4% (w/v) concentrations of KIO3 in the spray solutions to minimize the volume of solution added to salt. It may also be worthwhile to prepare the pH 9 spray solution with citrate incorporated with the micronutrients, and spray it onto salt, to test for any improvements in retention compared to using the alkaline treatment or the citrate treatment separately.

3. Study folic acid degradation kinetics in the presence of molecular oxygen and in its absence (-rich environment), at room temperature and high temperature, in solution and in salt that is kept in the dark, with and without citrate. If it is found that folic acid retention is higher in the presence of citrate in the absence of molecular oxygen, it can be concluded that the micronutrient degradation does indeed occur due to oxidative stress; and if so, other suitable and readily accessible compounds of higher antioxidant activity may be explored for inclusion into an aqueous spray solution containing iodine and folic acid. For instance, ascorbic acid may be useful if another iodine determination is used, since the compound acts as a vitamin and an oxygen scavenger.

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4. Test for well-established oxidative degradation products of folic acid such as pABG and pterin-6-carboxylic acid, in aqueous solutions of the two micronutrients and especially in the dual fortified salt, and correlate their formation with the iodine losses to determine whether the excess iodine losses in the presence of folic acid occurs due to an interaction with folic acid degradation products. If so, a possible mechanism of interaction could be identified. If not, the titrimetric method for determining iodine from iodate in the double fortified salt should be compared with other methods of iodine analysis that are not subject to interference. It may also be useful to prepare aqueous solutions with more iodine relative to folic acid (e.g. a ~5.7 to 1 iodate:folic acid molar ratio), to determine whether stability of iodine is improved in solution and once sprayed onto salt.

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[102] T. Triantis. “EVALUATION OF FOOD ANTIOXIDANT ACTIVITY BY PHOTOSTORAGE CHEMILUMINESCENCE.” National Research Centre for Physical Sciences. Athens. Internet: http://www.docstoc.com/docs/38754713/Evaluation-of-Food- Antioxidant-Activity-by-Photochemiluminescence, 2002 [January 04, 2011].

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8 Nomenclature

AOAC The Association of Official Analytical Chemists

DFE Dietary Folate Equivalent

DFS Double Fortified Salt

DHF Dihydrofolic acid

DNA Deoxyribonucleic Acid

FA Folic Acid

FAO The Food and Agriculture Organization

FOB Free On Board

FRAP Ferric Reducing Antioxidant Power

I Iodine

ICCIDD International Council for the Control of Iodine Deficiency Disorders

IDD Iodine Deficiency Disorders

IQ Intelligence Quotient

MI Micronutrient Initiative

MTHF Methyl-5,6,7,8-tetrahydrofolic acid

NTD Neural Tube Defects

PABA Para-aminobenzoic acid pABG p-aminobenzoylglutamic acid

RDI Recommended Daily Intake

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RH Relative Humidity

RNA Ribonucleic acid

SEM Scanning Electron Microscope

THF Tetrahydrofolic acid

TRH Thyrotropin-releasing hormone

TSH Thyroid-stimulating hormone

UNICEF United Nations Children's Fund

WHO World Health Organization

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9 Appendices 9.1 Detailed Analytical Methods

9.1.1 Folic Acid Determination

Reagent Preparation 1.) 5 N HCl: Dilute 431 mL of 11.6 N HCl to 500 mL of distilled water (dH2O). 2.) 0.1 N NaOH: Dissolve 4g of NaOH in dH2O in a 1L VF, and dilute to the mark with dH2O. 3.) 2% NaNO2: Dissolve 2g of NaNO2 in dH2O in a 100 mL volumetric flask (VF), and dilute to the mark with dH2O. 4.) 4% H3NSO3: Dissolve 4g of H3NSO3 in dH2O in a 100 mL VF, and dilute to the mark with dH2O. 5.) 1% 3-AP: Dissolve 2.5g of 3-AP in dH2O in a 250 mL VF, and dilute to the mark with dH2O. Prepare on the day of analysis; store in a dark place or cover with aluminum foil. 6.) 500ppm folic acid: Dissolve 0.05g of folic acid in 0.1N NaOH in a 100 mL VF, and dilute to the mark with the same reagent. Prepare on the day of analysis; store in a dark place or cover with aluminum foil.

Standardization 1.) Mix 2mL of the 500ppm folic acid with 1g of zinc granules and 10mL of 5N HCl in a 250 mL glass jar. Shake the solution vigorously for 20 minutes to allow for the reductive cleavage step to occur. 2.) Add 28 mL of dH2O to the jar to get the final volume to 40 mL. Shake vigorously, and wait 5 minutes to allow the zinc granules in the solution to settle to the bottom. Then aliquot 0, 1, 2, 3, 4, 5, and 6 mL (corresponding to 0-6 ppm of folic acid) of the diluted solution to labeled test tubes. 3.) Add 2mL of 5N HCl, 1mL of 2% NaNO2 and 1mL of 4% H3NSO3 to each of the tubes, mixing well and waiting 5 minutes between each addition to allow the diazotization step to go into completion. 4.) Add 5mL of 1% 3-AP to each test tube and mix well. Heat the tubes in a boiling water bath for 5-10 minutes to allow the coupling reaction step to go into completion. At this stage, the originally colourless solutions will turn into an orange-yellow complex. The intensity of the colour depends on the original folic acid content. 5.) Remove the test tubes from the water bath, cool to room temperature and add 3mL of 5N HCl to each tube. 6.) Dilute each solution to 25 mL with dH2O by adding the following volumes of distilled water to the labeled test tubes, and mix well.

Final ppm (mg/L) of folic acid mL dH2O to be added 0 13 1 12 2 11 3 10 4 9 5 8 6 7

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7.) Measure the absorbencies of the solutions in each of the test tubes by transferring each solution to a 10mm cuvette and using a UV/Vis Spectrophotometer at a 460 nm wavelength. The absorbance of the blank sample (0 ppm folic acid) is used to initially zero the instrument.

Sample Analysis

Spray Solutions 1.) Dilute 4mL of the spray solution of interest to 50 mL of dH2O, and mix 1mL of the diluted spray solution with 1g zinc granule and 10mL of 5N HCL in a 250 mL glass jar. Shake the solution vigorously for 20 minutes to allow for the reductive cleavage step to occur. 2.) Add 29 mL distilled water to the jar to get the final volume to 40 mL. Shake vigorously, and wait 5 minutes to allow the zinc granules in the solution to settle to the bottom. Aliquot 3-4 replicates of 2mL each to labeled test tubes. 3.) Repeat steps 3-5 from the Standardization procedure discussed above. The final volume in each test tube is 14 mL. Repeat step 7 from the Standardization procedure discussed above.

Salt Samples 1.) Place 20 g of the salt sample of interest in a 250 mL glass jar and add 8 mL 0.1N NaOH. Shake the solution vigorously for 10 minutes to extract the folic acid from the salt. 2.) Add 4g of zinc granules and 40mL of 5N HCl to the jar. Shake the solution vigorously for 30 minutes to allow for the reductive cleavage step to occur. 3.) Add 52 mL of distilled water to the jar to get the final volume to 100 mL. Shake vigorously, and wait 5 minutes to allow the zinc granules in the solution to settle to the bottom. Aliquot 3-4 replicates of 2mL each to labeled test tubes. 4.) Repeat steps 3-5 from the Standardization procedure discussed above. The final volume in each test tube is 14 mL. Repeat step 7 from the Standardization procedure discussed above.

Calculation of Folic Acid Content

Calibration Curve X-axis: concentration ( ppm = µg/mL) of folic acid in the standard solutions Y-axis: absorbencies of each standard solution at 460 nm

Folic Acid in Spray Solutions ppm (µg/mL) folic acid = {(absorbance at 460nm - intercept of calibration curve) / (slope of calibration curve)} * {14 mL / 2mL} * {40 mL / 1 mL} * {50 mL / 4 mL of the spray solution used}

Folic Acid in Salt Samples ppm (µg/gsalt) folic acid = {(absorbance at 460nm - intercept of calibration curve) / (slope of calibration curve)} * {14 mL / 2mL} * {100 mL / grams of salt used}

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9.1.2 Iodine Determination

Reagent Preparation 1.) 0.005 N Na2S2O3: Dilute 50 mL of 0.1 N Na2S2O3 to 1L of dH2O. 2.) 0.00125 N Na2S2O3: Dilute 12.5 mL of 0.1 N Na2S2O3 to 1L of dH2O. 3.) 0.2 N H2SO4: Dilute 1.4 mL of concentrated H2SO4 to 500 mL of dH2O. 4.) 0.05% KIO3: Dissolve 0.05g of KIO3 in dH2O in a 100 mL VF, and dilute to the mark with dH2O. 5.) 2% KI: Dissolve 10g of KI in dH2O in a 500 mL VF, and dilute to the mark with dH2O. Store in a dark place or cover with aluminum foil.

Standardization 1.) Mix 2 mL of the 0.05 % KIO3 with ~100 mL dH2O in a 500 mL Erlenmeyer flask. Repeat for 3-4 replicates. 2.) Add 2 mL each of 0.2 N H2SO4 and 2% KI solutions, mixing well after each addition. 3.) Stopper the flask and allow the yellow colour to develop (due to the liberation of iodine) for 10 minutes in a cool, dark place. 4.) Slowly titrate with either the 0.005 N Na2S2O3 solution (for the spray solutions) or the 0.00125 N solution (for the salt samples), until the yellow colour becomes a faint yellow. 5.) Add a few drops of the 1% starch solution to generate the blue/purple iodine-starch complex. 6.) Continue titrating until the blue colour disappears.

Sample Analysis

Spray Solutions 1.) Dilute 10 mL of the spray solution of interest to 100 mL of dH2O. Again dilute 10 mL of the initially diluted solution to 100 mL of dH2O. 2.) Mix 2 mL of the final solution from Step 1 with ~100 mL dH2O in a 500 mL Erlenmeyer flask. Repeat for 3-4 replicates. 3.) Repeat steps 2-6 from the Standardization procedure discussed above, using the 0.005 N Na2S2O3 solution as the titrant.

Salt Samples 1.) Add 10 g of the salt sample of interest to ~100 mL dH2O in a 500 mL Erlenmeyer flask. Repeat for 3-4 replicates. 2.) Repeat steps 2-6 from the Standardization procedure discussed above, using the 0.00125 N Na2S2O3 solution as the titrant.

Calculation of Iodine Content

Strength of Na2S2O3 Strength (µg I/ mL Na2S2O3) ={0.05%}*{59.5%}*{2 mL/ mL Na2S2O3 consumed} Iodine in Spray Solutions ppm (µg/mLsolution) iodine = {strength of Na2S2O3} * { mL Na2S2O3 consumed } / 2 mL of spray solution used Iodine in Salt Samples ppm (µg/gsalt) iodine = {strength of Na2S2O3} * { mL Na2S2O3 consumed } / grams of salt used

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9.2 Buffer Capacity Calculations

The buffer capacity corresponds to the amount of H+ or OH- ions that can be neutralized by the buffer. Therefore, the buffer capacity is related to the buffer concentration. If the addition of an acid or base causes the buffer pH to change by one pH unit above or below the pKa, the buffer loses so much buffer capacity that it no longer has any real buffer function.

- The pH 10 buffer solution was prepared by combining 60 mL of 0.1 M Na2CO3 (A ) and 40 mL of NaHCO3 (HA). Consequently, this 100 mL buffer solution now contains 0.06 M carbonate, and 0.04 M bicarbonate. Therefore, the maximum possible folic acid concentration must not exceed the base concentration, or 0.06 M, which translates into 2.6% (w/v) folic acid that can be added to the pH 10 buffer solution. However, this study used only 0.35% (w/v) folic acid as a precaution, since this amount equals to adding only ~0.008 M of folic acid to the buffer so that the concentration of carbonate ions is much higher in order to consume the H+ from folic acid.

- Similarly, the pH 9 buffer solution is prepared by combining 50 mL of 0.1 M Na2CO3 (A ) and

50 mL of NaHCO3 (HA). Consequently, this 100 mL buffer solution now contains 0.05 M carbonate, and 0.05 M bicarbonate. Therefore, the maximum possible folic acid concentration must not exceed the base concentration, or 0.05 M, which translates into 2.2% (w/v) folic acid that can be added to the pH 9 buffer solution. It was calculated that in order to have 1-3% (w/v) folic acid in a pH 9 spray solution, one requires a pH 9 buffer solution to be prepared with 2.1%

(w/v) M Na2CO3 and 1.7% (w/v) NaHCO3.

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9.3 Experimental Data

9.3.1 Relative Micronutrient Retentions in the Final Formulations

Optimal Formulation from Alkalinity Study: pH 9 spray solution

Micronutrient Month 1 Month 2 Month 3 Month 4 Month 5 Month 6 pH 9 solution containing 0.35% (w/v) each of folic acid and iodine (25°C/60% RH) % FA retained 99.02 95.67 91.48 90.54 N/A N/A ± %RSD ± 1.89 ± 3.21 ±5.49 ± 3.40 % I retained 99.05 97.49 93.00 91.23 N/A N/A ± %RSD ± 0.45 ± 4.05 ± 4.68 ± 2.38 Canadian salt containing 45 ppm each of folic acid and iodine (45°C/60% RH) % FA retained 99.00 97.98 97.03 96.11 95.02 94.09 ± %RSD ± 3.40 ± 1.90 ± 2.80 ± 1.20 ± 1.60 ± 2.30 % I retained 98.00 96.07 95.70 94.90 94.02 93.40 ± %RSD ± 1.20 ± 0.90 ± 1.90 ± 3.50 ± 2.40 ± 2.60 Orissa salt containing 45 ppm each of folic acid and iodine (45°C/60% RH) % FA retained 99.99 96.00 94.50 93.80 N/A N/A ± %RSD ± 2.20 ± 1.50 ± 2.00 ± 0.50 % I retained 96.70 93.70 91.40 89.50 N/A N/A ± %RSD ± 3.50 ± 2.40 ± 2.60 ± 2.10

Optimal Formulation from Citrate Study: Spray solution with 1% (w/v) each of citrate, folic acid and iodine

Micronutrient Month 1 Month 2 Month 3 Month 4 Month 5 Month 6 Solution containing 1% (w/v) each of sodium citrate, folic acid and iodine (25°C/60% RH) % FA retained 100 99.98 99.80 99.67 99.61 N/A ± %RSD ± 0.87 ± 0.11 ±0.90 ± 0.69 ± 0.22 % I retained 99.35 99.19 99.02 98.72 98.42 N/A ± %RSD ± 0.54 ± 0.56 ± 0.44 ± 0.02 ± 0.34 Canadian salt containing 45 ppm each of folic acid and iodine (45°C/60% RH) % FA retained 99.6 98.6 96.7 95.7 94.7 93.7 ± %RSD ± 4.30 ± 1.60 ± 0.02 ± 0.56 ± 1.70 ± 1.70 % I retained 99.7 98 95.4 94.1 91 89 ± %RSD ± 0.60 ± 1.45 ± 0.56 ± 1.80 ± 1.80 ± 1.80

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Apparent first order degradation rate constants (k) estimated for the citrate solutions containing 1% (w/v), or 10,000 mg/L of folic acid and iodine each, when stored at 25ºC and 60% RH Citrate Concentration (% w/v) k (month-1) Correlation coefficient, r Folic Acid 0 0.0216 0.972 0.1 0.0026 0.959 0.2 0.0023 0.955 0.5 0.0018 0.966 1 0.0009 0.950 2 0.0006 0.970 3 0.0003 0.957 Iodine 0 0.033 0.995 0.1 0.005 0.973 0.2 0.0045 0.963 0.5 0.0041 0.970 1 0.0029 0.975 2 0.0023 0.968 3 0.0022 0.963

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Relative Retentions (%) in Formulations from the Study on Micronutrient Interactions

These samples were used as the controls for the alkalinity study (the 0.35% solutions and their salts) and the citrate study (the 1% solutions and their salts) as well.

Spray Solutions

Solution Month 1 Month 2 Month 3 Month 4 Month 5 Month 6 Folic Acid FA only: 1% 99 98.86 98.45 98.02 97.74 97 FA only: 0.35% 98.45 97 96.81 95.98 95.56 94.08 FA + I: 1% 97 93.87 91.76 90.58 89.96 87 FA + I: 0.35% 95.56 92.28 88.89 85.49 83.67 82 Iodine I only: 1% 99.68 99.47 99.35 99.21 99.17 98.97 I only: 0.35% 99 98.31 97.89 97.54 97.37 97.20 I + FA: 1% 98 94.74 90.45 87.90 85.50 82.50 I + FA: 0.35% 96.35 92.23 88.30 86.70 80.76 76.34 Note: Relative standard deviations are ≤ ± 4.5%

Salt

Salt Sprayed with Listed Month 1 Month 2 Month 3 Month 4 Month 5 Month 6 Solutions Folic Acid FA only: 1% 98.78 98 96.45 96 95.83 94.98 FA only: 99.17 98.30 96.06 94.99 93.56 92.08 0.35% FA + I: 1% 98.48 95.4 92.1 89.4 87 86 FA + I: 0.35% 95.24 92.65 89.06 86.56 84.45 83.78 Iodine I only: 1% 98.95 98 97.71 97 96.68 95.92 I only: 0.35% 98.56 96.72 95 94 93 92 I + FA: 1% 96.39 92 89.49 87 85.89 84.38 I + FA: 0.35% 93.96 89.01 86.23 83.97 82.40 81.67 Note 1: Relative standard deviations are ≤ ± 4.5%