The Fortification of Salt with Iodine, Iron, and Folic Acid

Elisa June Teresa McGee

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

Elisa McGee

Master of Applied Science

Department of Chemical Engineering and Applied Chemistry University of Toronto

2012

Abstract

Micronutrient poor diets around the globe and in particular in the developing world cause deficiencies in iron and folic acid. This may be rectified by the incorporation of these micronutrients into currently running salt iodization processes. The objective of this project was to develop folic acid and iodine spray solutions to be ready for pilot scale testing and to investigate the stability of triple fortified salt containing iodine, folic acid and microencapsulated ferrous fumarate.

The optimal spray solutions were buffered to pH 9 with a carbonate/bicarbonate buffer to stabilize folic acid and contained 1%-2% w/v folic acid and 1%-3% w/v iodine (as KIO3). They remained in solution and retained ≥80% of both micronutrients after 5 months of storage at 25ºC and 45ºC. Double fortified salt produced using these spray solutions retained 100% of both folic acid and iodine over a 5 month period when stored at ambient conditions. Unfortunately triple fortified salt did not sufficiently retain the micronutrients due to excess moisture absorption and inadequate encapsulation of iron.

Acknowledgments

Firstly I would like to sincerely thank my supervisor Dr. Levente L. Diosady for giving me the opportunity to advance this project. I am very grateful for his guidance and support throughout my research.

I would like to thank The Micronutrient Initiative (MI) for their financial support. I would also like to thank Dan Mathers, the supervisor of the ANALEST analytical lab at the University of

Toronto, for his training and continued guidance pertaining to high performance liquid chromatography (HPLC). I would also like to thank him for the donation of solvents and HPLC columns.

Finally, I would like to thank the members of the Food Engineering Group for warmly welcoming me into the lab, always being willing to lend their assistance, and for being wonderful co-workers and friends. I would especially like to thank Angjalie Sangakkara, Lana

Kwan, and Dan Romita for their guidance on the salt fortification project and their encouragement to grow both professionally and personally.

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Table of Contents

Abstract ...... ii Acknowledgments ...... iii Table of Contents ...... iv List of Tables ...... vi List of Figures ...... vii List of Appendices ...... ix

1 Introduction...... 1 2 Background ...... 4 2.1 Nutrition Intervention Programs ...... 4 2.1.1 Focus on Micronutrient Malnutrition ...... 4 2.1.2 Impact on Developing Countries ...... 4 2.2 Salt Fortification Strategy Development ...... 5 2.3 Micronutrient Deficiencies ...... 6 2.3.1 Iodine ...... 6 2.3.2 Iron ...... 8 2.3.3 Folate ...... 10 2.4 Micronutrient Properties ...... 12 2.4.1 Iodine ...... 12 2.4.2 Iron ...... 13 2.4.3 Folate ...... 16 2.5 Analytical Methods to Quantify Folic Acid Based on p-ABGA ...... 18 2.6 Salt Fortification Technologies ...... 19 2.6.1 Iodine Salt Fortification ...... 19 2.6.2 Iron Salt Fortification ...... 21 2.6.3 Iodine and Iron Fortification ...... 21 2.6.4 Iodine and Folic Acid Fortification ...... 23 2.6.5 Multiple Fortification...... 24 2.7 Project Objectives ...... 24

3 Materials and Methods ...... 26 3.1 Materials ...... 26 3.2 Fortification Methods ...... 28 3.2.1 Buffered Spray Solution Preparation ...... 28 3.2.2 Spray Drying Microencapsulation ...... 30 3.2.3 Fortified Salt Preparation ...... 30 3.3 Analytical Methods...... 31 3.3.1 Iodine Stability Testing ...... 31 3.3.2 Iron Stability Testing ...... 31 3.3.3 Folic Acid Stability Testing ...... 31 3.3.4 pH Testing ...... 33 4 Results and Discussion ...... 34 4.1 Folic Acid Analytical Method Development...... 34 4.1.1 High-Performance Liquid Chromatography (HPLC) ...... 34 4.1.2 Spectrophotometry Based Coupling Method ...... 35 4.1.3 Effect of Folic Acid Degradation on the SBCM ...... 38 4.2 Triple Fortified Salt ...... 39 4.3 Double Fortified Salt ...... 44 4.3.1 Optimization of Spray Solution Formulations ...... 44 4.3.2 Stability of Spray Solutions ...... 46 4.3.3 Stability of Double Fortified Salt ...... 49 5 Conclusions ...... 52 6 Recommendations...... 54 8 Nomenclature ...... 64 9 Appendices ...... 65

List of Tables

Table 2.3.1: Appropriate Consumption of Iodine ...... 7 Table 2.3.2: Appropriate Consumption of Iron ...... 9 Table 2.3.3: Appropriate Consumption of Folate ...... 11 Table 2.4.1: Common Iron Fortificants ...... 14 Table 2.6.1: Comparison of Salt Iodization Methods ...... 20

Table 3.1.1: Materials Used in Salt Fortification ...... 26 Table 3.1.2: Materials Used in Folic Acid Analysis ...... 27 Table 3.1.3: Materials Used in Iodine and Iron Analysis ...... 28 Table 3.2.1: Resultant % RDA of Folic Acid in Spray Solutions ...... 29

Table 4.1.1: p-ABGA in Sangakkara’s Double Fortified Salt After 1 Year of Storage ...... 38 Table 4.1.2: p-ABGA in 1%-3% Spray Solutions After 5 Months of Storage ...... 39 Table 4.1.3: p-ABGA in Double Fortified Salt After 5 Months of Storage ...... 39 Table 4.2.1: Triple Fortified Salt Formulations ...... 40 Table 4.3.1: Ratio of Carbonate to Bicarbonate Required For pH 9 Solution ...... 45 Table 4.3.2: Final Spray Solution Formulations ...... 46 Table 4.3.3: pH Stability of Spray Solutions ...... 46 Table 4.3.4: Spray Solutions Used to Fortify Salt ...... 49 Table 4.3.5: Observed (Incorrect) Retention of Folic Acid in Spray Solutions (Time 0) ...... 50

Table 9.2.1: Working Solution Dilutions for Iron Calibration Curve ...... 67 Table 9.4.1: Spectrophotometry-Based Coupling Method Development ...... 94

List of Figures

Figure 2.4.1: Potassium Iodate Chemical Structure ...... 13 Figure 2.4.2: Ferrous Fumarate Chemical Structure ...... 15 Figure 2.4.3: Folic Acid Chemical Structure ...... 16 Figure 2.6.1: Spray Drying Process ...... 23

Figure 3.3.1: Reaction #1 Reductive Cleavage of Folic Acid ...... 32 Figure 3.3.2: Reaction #2 p-ABGA Diazotization ...... 33 Figure 3.3.3: 3-Aminophenol Coupling Reaction ...... 33

Figure 4.2.2: Retention of Iodine in Triple Fortified Salt After 1 Year of Storage ...... 42 Figure 4.2.3: Retention of Folic Acid in Triple Fortified Salt After 1 of Year of Storage ...... 43 Figure 4.3.1: 0.1 M Carbonate/Bicarbonate Spray Solution Selection...... 44 Figure 4.3.2: 0.2 M Carbonate/Bicarbonate Spray Solution Selection...... 45 Figure 4.3.4: Retention of Folic Acid in Spray Solutions Stored at 25ºC ...... 47 Figure 4.3.5: Retention of Folic Acid in Spray Solutions Stored at 45ºC ...... 47 Figure 4.3.6: Retention of Iodine in Spray Solutions Stored at 25ºC ...... 48 Figure 4.3.7: Retention of Iodine in Spray Solutions Stored at 45ºC ...... 48 Figure 4.3.8: 3% Iodine/3% Folic Acid Spray Solutions After 2 Months Storage ...... 49 Figure 4.4.9: Retention of Folic Acid in Double Fortified Salt ...... 51 Figure 4.4.10: Retention of Iodine in Double Fortified Salt...... 51

Figure 9.2.1: Sample Calibration Curve ...... 68 Figure 9.3.1: p-ABGA HPLC Calibration Curve Using Area (7 replicates) ...... 74 Figure 9.3.2: p-ABGA HPLC Calibration Curve Using Height (7 replicates) ...... 74 Figure 9.3.3: Folic Acid HPLC Calibration Curve Using Area (8 replicates) ...... 74 Figure 9.3.4: Folic Acid HPLC Calibration Curve Using Height (8 replicates) ...... 75 Figure 9.3.7: Linear Range Determination of Folic Acid Using Area (3 Replicates) ...... 76 Figure 9.3.8: Linear Range Determination of Folic Acid Using Height (3 Replicates) ...... 76 Figure 9.3.9: Linear Range Determination of p-ABGA Using Area (4 Replicates) ...... 76 Figure 9.3.10: Linear Range Determination of p-ABGA Using Height (4 Replicates) ...... 77 Figure 9.3.15: Folic Acid Addition to Fortified Salt Measured Using Peak Height ...... 77 vii

Figure 9.3.16: HPLC Salt Peak from Sodium Chloride Solution ...... 78 Figure 9.3.17: HPLC Salt and Folic Acid Peaks from a Mixture ...... 78 Figure 9.3.18: Salt Peak Area ...... 78 Figure 9.3.19: Salt Peak Height ...... 78 Figure 9.3.20: Salt Skew of Folic Acid HPLC Calibration Curve using Area ...... 79 Figure 9.3.21: Salt Skew of Folic Acid HPLC Calibration Curve using Height ...... 79 Figure 9.3.22: Folic Acid Reading After HPLC Extraction Method 1 ...... 80 Figure 9.3.23: Folic Acid Reading After HPLC Extraction Method 2 (No Filter) ...... 80 Figure 9.3.24: Folic Acid Reading After HPLC Extraction Method 2 (5 μm Filter) ...... 81 Figure 9.3.25: Folic Acid Readings’ Dependence on Filtration ...... 81 Figure 9.3.26: p-ABGA Chromatograph ...... 82 Figure 9.3.27: p-ABGA and Folic Acid Chromatograph ...... 82 Figure 9.3.28: Folic Acid in Double Fortified Salt Stored for 1 Year (25ºC or 45ºC) ...... 82 Figure 9.3.29: pH of HPLC Salt Sample Solutions ...... 83 Figure 9.4.1: Sample Calibration Curve of Original SBCM Procedure ...... 86 Figure 9.4.2: Standard Folic Acid Solutions Tested With and Without Reaction 1 ...... 87 Figure 9.4.3: Sample Calibration Curve of Revised SBCM Procedure ...... 90 Figure 9.4.4: 5 N HCl Causes Higher Absorbance Readings than Stock Concentration HCl .... 90 Figure 9.4.5: Folic Acid Detected in Spray Solutions Using Different HCl Concentrations ...... 91 Figure 9.4.6: Absorbance Measurements of Salt Using Different HCl Concentrations ...... 91 Figure 9.4.7: Salt (50 ppm Folic Acid) Calibrated Using Different Concentrations of HCl ...... 92 Figure 9.4.8: Change in Dilution Cause Rectification of Inaccuracies Due to 3-AP ...... 92 Figure 9.4.9: Effect of Iron on Folic Acid Readings Using Original Procedure (30 ppm FA) ... 93 Figure 9.4.10: Rectification of Iron Effects Through Folic Acid Extraction ...... 93 Figure 9.4.11: Standard Additions of Folic Acid to Different Salts (Revised Method) ...... 94 Figure 9.5.1: Spray Solutions After 2 Months Storage at 25ºC ...... 95 Figure 9.5.2: Spray Solutions After 2 Months Storage at 45ºC ...... 95

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List of Appendices

Appendix 9.1: Analytical Determination of Iodine ...... 65 9.1.1 Solution Preparation ...... 65 9.1.2 Standardization ...... 65 9.1.3 Spray Solution Analysis ...... 65 9.1.4 Salt Sample Analysis ...... 66 9.1.5 Calculation of Iodine Content ...... 66 Appendix 9.2: Analytical Determination of Iron ...... 67 9.2.1 Solution Preparation ...... 67 9.2.2 Calibration Curve Preparation ...... 67 9.2.3 Salt Sample Analysis ...... 68 9.2.4 Calculation of Iron Content ...... 69 Appendix 9.3: Analytical Determination of Folic Acid by HPLC ...... 70 9.3.1 Detailed Sample Preparation Methods ...... 70 9.3.2 Detailed HPLC with UV Detection Methods ...... 72 9.3.3 Calculation of Folic Acid Content ...... 73 9.3.4 HPLC Successes ...... 74 9.3.5 HPLC Underperformances ...... 77 Appendix 9.4: Analytical Determination of Folic Acid by SBCM ...... 84 9.4.1 Solution Preparation ...... 84 9.4.2 Original Procedure ...... 84 9.4.3 Additional Round for the Determination of p-ABGA ...... 86 9.4.4 Revised Procedure ...... 87 9.4.5 The Effect of HCl Concentration on Folic Acid Readings ...... 90 9.4.6 The Effect of 3-Aminophenol (3-AP) on Folic Acid Readings ...... 92 9.4.7 The Effect of Iron on Folic Acid Readings ...... 93 9.4.8 Accurate Standard Additions ...... 94 9.4.9: Summary of SBCM Revisions ...... 94 Appendix 9.5: Spray Solutions After 2 Months of Storage ...... 95

1 Introduction

Malnutrition is the world’s most severe health problem [1]. The major contributing factor is micronutrient malnutrition – deficiencies in nutrients needed in small amounts (i.e. vitamins and minerals) [2]. Micronutrient deficiency directly affects more than 2 billion people worldwide [3]. The most extensive problems arise in developing countries where people consume micronutrient-poor cereal and tuber diets [4] [5]. This type of diet does not provide a sufficient amount of iodine, iron, folic acid, vitamin A, or zinc [5]. These micronutrients are the focus of many aid programs including those of the Micronutrient Initiative (MI) [6].

The fortification of salt with micronutrients is a well established technique for ensuring proper nutrition. The first micronutrient to be incorporated into salt was iodine in Switzerland back in 1921 [7]. The State of Michigan in the United States quickly followed in 1924 and soon the majority of developed nations were using iodized salt [8]. In the 1980s the significance of moderate iodine deficiency became known, causing UNICEF to call for salt to be fortified with iodine (iodized) worldwide [7]. In the year 2000, two thirds of the developing world’s salt was being iodized [7]. The success of iodized salt prompted the use of salt as a carrier for other micronutrients as well.

The Food Engineering Group at the University of Toronto and MI worked together to find an appropriate formulation for salt double fortified with iron and iodine. Microencapsulation is the strategy employed by the Food Engineering Group to create a physical barrier between adsorbed water, iron, and iodine on the salt surface where they were found to cause degradation [9]. The first strategy employed was to microencapsulate iodine [10]. This strategy was effective in preventing iodine loss in extreme conditions of storage, retaining iron in a bioavailable form, and reducing micronutrient deficiencies [10]. Then double fortified salt where iron was incorporated as ferrous fumarate microencapsulated using fluidized bed agglomeration with a variety of binders, a colour coating agent, and a soy stearine lipid coating was developed [11]. It was demonstrated that microencapsulated iron can protect both the stability of iodine and iron, and thus there was no need to encapsulate both micronutrients [11]. Because ferrous fumarate is coloured it is more important to encapsulate it (simultaneously masking its colour) instead of iodine. One issue was that the salt with microencapsulated ferrous fumarate was found to cause sensory changes when put into food and found to segregate in coarse salt [12]. Therefore a new

2 process based on extrusion agglomeration with glassy polymer coating was developed [13]. It was determined that encapsulated ferrous fumarate is the best choice for iron source in double fortified salt because of its high bioavailability, mild taste, acceptable colour due to colour masking, high iodine retention achieved in double fortified salt, and its competitive cost [13]. The extrusion agglomeration and glassy polymer coating formulation resulted in iron microcapsules with improved particle surface properties and higher density which was thought to reduce the potential of iron loss during food preparation and segregation during transport [12] [13]. It also reduced the capital and operating cost of the process [14]. However, the sizes of these microcapsules were similar to refined salt crystals and therefore were still segregating in coarse salt.

The most recently developed method for the double fortification of salt with iron and iodine, by Romita et al. (2011) at the University of Toronto (Food Engineering Group), uses a microencapsulated ferrous fumarate premix that is created using spray dry microencapsulation [15]. This strategy is targeted towards coarse iodized salt where matching the particle size to avoid segregation is impractical [15]. The particles produced through spray dry microencapsulation are too small to be visually detected (<20μm) and are small enough to adhere to the surface of salt crystals [15]. It was concluded that double fortified salt using this new premix was stable and bioavailable [15]. Spherical particles that were uniform in size were obtained with the use of hydroxypropyl methylcellulose (HPMC) as the coating material and sodium fumarate as the excipient [15]. Titanium dioxide was successfully used as a colour masking agent rendering the premix visually undetectable in salt [15].

Another area of research for the double fortification of salt pursued by the Food Engineering Group was to fortify it with iodine and folic acid. Li et al. (2011) found that formulations where folic acid was encapsulated though an extrusion-based process were stable in iodized salt and the poorest results occurred when adding folic acid by aqueous solution [16]. A spray solution was desired however because it would cause the least change to current salt iodization plant processes. Sangakkara (2011) developed two stable formulations of double fortified salt that use a single solution to spray both micronutrients onto the salt [17]. One used the effects of pH and the other used citrate, a metal chelator, to keep folic acid stable. The formulation controlling pH was much more cost effective [17]. Some issues with the work include that the analytical

3 method used by Sangakkara to test the stability of folic acid did not distinguish between folic acid and its common degradation product para-aminobenzoyl glutamic acid (p-ABGA) and the concentrations of the micronutrients in the spray solutions were not as high as those used commercially (1%-3% w/v).

In order to reach those in most need, a fortified salt formulation must not cause a large increase in cost and must be suitable for fortifying coarse unrefined salt. Therefore, the objective of this project was to develop a process for double and triple fortifying salt using existing equipment and technologies employed commercially in developing countries. This required further development of the folic acid and iodine spray solution established by Sangakkara (2011) and an investigation of the stability of triple fortified salt using iron microcapsules developed by Romita (2011) [17] [15] [18].

2 Background 2.1 Nutrition Intervention Programs 2.1.1 Focus on Micronutrient Malnutrition Nutrition intervention programs, such as MI, that focus on developing countries have been active since the 1930s. However, their focus on micronutrient (i.e. vitamin and mineral) deficiency is relatively new. Until the 1960s it was thought that protein deficiency was the main nutritional problem in the developing world [2]. However, in 1959 Derrick B. Jelliffe coined the term protein-calorie deficiency pulling the emphasis off of solely protein deficiency [19]. In the 1970s a shift of focus from protein malnutrition to calorie (energy) malnutrition, caused by an insufficient quantity of food, took place [2]. In 1977 the National Research Council in the United States began a world food study which concluded that the quality of food, particularly micronutrient content, was far more influential with respect to nutritional status in developing countries than quantity of food [2].

Aid programs began to shift towards the prevention of micronutrient deficiencies in the 1980s [2]. The first two strategies employed were food fortification (the deliberate increase of micronutrient content in a food or condiment) and supplementation (the administration of relatively large doses of micronutrients usually in the form of pills, capsules, or syrups) to boost iodine and vitamin A intake respectively [4] [2]. Further interventions include: the supplementation of zinc, iron, and folic acid; fortification of foods with single and multiple micronutrients; home-based multiple micronutrient supplements (to be added to food within the home); and dietary diversification (the increase in amount and variety of micronutrient-rich foods consumed) [6].

2.1.2 Impact on Developing Countries Micronutrient interventions have been proven very successful. There has been a 23% reduction in mortality rates for children under the age of 5 and a 70% reduction in childhood blindness where vitamin A supplementation programs have been established [6]. There has been a 6% reduction in child mortality and a 27% reduction in diarrhoea incidence in children due to zinc supplementation [6]. Iodine fortification of salt has caused an average 13-point increase in IQ [6]. Programmes focused on iron deficiency, such as supplementation to women of childbearing

5 age and food fortification, have caused a 20% reduction in maternal mortality [6]. There has also been a 50% reduction in severe neural tube birth defects in women receiving adequate amounts of folate through supplementation and/or fortification of food [6].

Despite the efforts and successes of aid programs, micronutrient deficiencies continue to have a substantial negative impact on human life and wellbeing. Deaths of 1.1 million children under the age of 5 per year are caused by vitamin A and zinc deficiencies [6]. 350,000 children become blind due to vitamin A deficiency [6]. 18 million babies are born each year mentally impaired because of maternal iodine deficiency [6]. Deaths of 136,000 women and children per year are due to iron deficiency, this includes one fifth of the world’s maternal mortalities [6]. Iron deficiency also causes 600,000 stillbirths or deaths of babies within 7 days of birth per year [6]. 150,000 babies are born with severe birth defects, often leading to death or paralysis, due to folate deficiencies [6]. Also, more than half of the children with vitamin and mineral deficiencies are harbouring more than one deficiency [7]. Therefore, micronutrient intervention must be developed further to reach those currently not being assisted adequately.

2.2 Salt Fortification Strategy Development There are several technical reasons why large groups of people are not being reached by micronutrient programs. Firstly, programs focused on dietary diversification require behavioural change (regarding types of foods consumed), public education on nutrition, resources (financial and natural) for producing micronutrient-rich foods, and financial resources for purchasing higher quality foods [4]. Also, these programs take the longest to implement and thus must be supplemented by other programs while they are being established [4]. Secondly, supplementation programs were noted by program managers to suffer from lack of supplies and poor compliance [4]. This is caused in part by the high expense of the packaged supplements, insufficient nutrition education, and difficulty in procurement of the supplements (i.e. distance, cost, time constraints) [4]. Finally, food fortification requires members of the target group to eat the fortified food in sufficient regular amounts, the financial resources for the purchase of the foods, the resources for foods to be processed, and an effective distribution channel [4]. Although food fortification does have some difficulties, they are the easiest to overcome because food fortification does not require active consumer participation and is the most cost effective [4].

The effectiveness of a food fortification program in reaching those in need is dependent on the food that is chosen to be fortified (known as the food vehicle or carrier). To ensure that the food is eaten in sufficient regular amounts, staple foods or condiments are generally chosen. Foods that have been chosen as food vehicles include: salt, wheat flour, rice, edible oil, sugar, fish sauce, and soy sauce [6]. The poorest of a population often have lower purchasing power so they often rely on staple foods grown themselves or grown locally and thus are not processed [4]. The selection of a condiment instead of a staple food provides a solution to this issue. Effective and established distribution channels are necessary for reaching many people. The most extensive of these has been created for salt. Some advantages of using salt include that it is consumed in constant daily amounts (regardless of economic status), is generally purchased, is often manufactured in just a few large plants, and has proven efficacy in the developing world (iodized salt found in 70% of developing world households in 2009) [20] [7] [6]. Therefore, given the appropriate technology, salt is the most ideal food vehicle to reach the largest population

2.3 Micronutrient Deficiencies Micronutrients are nutrients needed in small amounts (i.e. vitamins and minerals). The most common cause of micronutrient deficiency is a lack of micronutrient intake which often occurs in developing countries [4] [5]. Iodine, iron, and folate deficiencies are common and have large devastating effects. Iodine deficiency causes 18 million babies per year to be born mentally impaired [6]. Every year iron deficiency causes 136 thousand deaths of women and children as well as 600 thousand stillbirths or deaths of babies less than 1 week old [6]. Folic acid deficiency causes 150 thousand babies to be born with severe birth defects [6]. In the following subsections these micronutrient deficiencies will be discussed in more depth.

2.3.1 Iodine Iodine from the diet is absorbed throughout gastrointestinal tract and is cleared from circulation by the thyroid and kidneys [5] [21]. It is very bioavailable, especially in the form of iodide or iodate salts, with more than 90% being absorbed [21]. The thyroid uses iodine to synthesize thyroid hormones whereas the kidneys allow iodine to be excreted with urine [5]. The major thyroid hormone secreted by the thyroid gland is thyroxine (T4) which is taken up by cells and

7 converted into triiodothyronine (T3) [21] [5]. T3 is most important for developmental and metabolic processes [5].

Iodine deficiency is the most common cause of mental impairment [4] [22]. Severe iodine deficiency of a fetus causes cretinism which is a severe form of fetal neurological damage characterized by mental retardation, stumped growth, and deaf mutism [21]. Thyroid stimulating hormone (TSH) released by the pituitary gland increases when circulating thyroid hormones are low (when persistent this is called hypothyroidism). TSH stimulates the thyroid to uptake more iodine and send more hormones out to the blood [21]. Goitre (a swelling of the thyroid gland) is caused by an overstimulation of the thyroid gland by TSH due to hypothyroidism. In later stages goitre can cause thyroid follicular cancer [21]. Further consequences of iodine deficiency include decreased fertility rate, miscarriages, stillbirths, and congenital abnormalities [5] [4]. Pregnant women, lactating women, women of reproductive age, and those under 3 years of age are considered at the highest risk [5]. To avoid iodine deficiency, an appropriate amount of iodine should be consumed usually defined in terms of recommended dietary allowance (RDA) or adequate intake (AI) (Table 2.3.1).

Table 2.3.1: Appropriate Consumption of Iodine (adapted from [23], Health Canada) Life Phase/Gender Age RDA/AI* Tolerable Upper (μg/day) Intake Level (μg/day) Infant 0-6 months 110 no data 7-12 months 130 no data Child 1-3 years 90 200 4-8 years 90 300 9-13 years 120 600 Adult 14-18 years 150 900 19+ years 150 1100 Pregnancy ≤18 years 220 900 19-50 years 220 1100 Lactation ≤18 years 220 900 19-50 years 220 1100 *Bold is used to indicate AI instead of RDA.

Acquiring iodine through natural food alone is difficult so interventions have been made. Marine food sources such as seaweed and coral reef fish are high in iodine [5]. However, other food sources are dependent on iodine content in the soil [5]. Unfortunately, much of the soil on

Earth has insufficient iodine due to leaching by glaciation and repeated flooding [5]. The areas most affected by iodine deficiency are South-East Asia, the Western Pacific, Africa, the Eastern Mediterranean, and Eastern/Western Europe [4]. Supplementation with iodized oil is one strategy used to combat iodine deficiency [5]. Another is food fortification which has been practiced for more than 90 years [7]. Foods noted to have been fortified with iodine include: salt, tea, water, fish paste, bread, soya sauce, dairy, and poultry [24] [5]. The most widespread and most mature technology is salt iodization [4]. Salt iodization has caused an average 13-point increase in IQ in developing countries where fortification was introduced [6].

2.3.2 Iron Iron is absorbed into the body in the upper portion of the small intestine by two separate pathways [21]. One pathway is for non-heme iron from vegetable and dairy sources and the other is for heme iron acquired though consumption of meat [21]. Once absorbed, approximately 60% is found in hemoglobin of red blood cells (erythrocytes); 25% is stored in the liver as ferritin and hemosiderin (mobilized and transported as protein transferrin); and 15% is located in myoglobin of muscle tissue as well as enzymes [21] [5]. Hemoglobin is used to transport oxygen from the lungs to tissues [21]. Myoglobin is used to store oxygen and increase the diffusion rate of oxygen from blood to the mitochondria of muscle cells [21]. Iron is used in cytochrome enzymes that act as electron carriers aiding in energy acquisition by aerobic respiration [21]. Other functions of iron-containing enzymes include the synthesis of steroid hormones and bile acids; control of some neurotransmitters; and the detoxification of substances in the liver [5].

Iron deficiency is the most common and widespread nutritional disorder in the world, affecting an estimated 40% of the population [4]. Insufficient intake of iron is responsible for 50% of anemia, characterized by a severely low amount of erythrocytes in the blood [4]. Iron deficiency anemia is associated with impaired physical capacity, developmental delay, cognitive impairment, increased maternal mortality, premature delivery, low birth weight, increased infant mortality, and others [21]. This results in the death of 136,000 women and children annually [6]. Iron deficiency also causes 600,000 stillbirths or neonatal deaths (within 7 days of birth) per year [6]. Iron deficiency decreases physical capacity due to slowing of oxidative metabolism in muscles [5]. It also lowers defence mechanisms of the body to fight infection because the

9 production and action of immune T lymphocytes is iron dependent [5]. Also, iron deficiency causes a decreased absorption of iodine and vitamin A [4]. Those at most risk are infants, children, adolescents, and women of childbearing age (especially those who are pregnant) [5]. Adequate levels of iron consumption in order to avoid deficiency are given in Table 2.3.2.

Table 2.3.2: Appropriate Consumption of Iron* (adapted from [23], Health Canada) Life Phase/Gender Age RDA/AI** Tolerable Upper (mg/day) Intake Level (mg/day) Infant 0-6 months 0.27 40 7-12 months 6.9 40 Child 1-3 years 7 40 4-8 years 10 40 9-13 years 8 40 Adult (Male) 14-18 years 11 45 19-50 years 8 45 Adult (Female) 14-18 years 15 45 19-50 years 18 45 Adult 51+ years 8 45 Pregnancy ≤50 years 27 45 Lactation ≤18 years 10 45 18-50 years 9 45 *Requirement for iron is 1.8 times higher for vegetarians due to lower bioavailability of iron in vegetarian diets. **Bold is used to indicate AI instead of RDA.

There are many dietary sources of iron including heme iron from hemoglobin and myoglobin in meat (beef, poultry, and fish) and non-heme iron present in legumes, fruits, and vegetables [5]. Absorption rate of heme iron is very high and only calcium is known to inhibit it. However, absorption of non-heme iron is heavily dependent on in promoters (e.g. ascorbic acid) and inhibitors. Inhibitors include calcium, inositol phosphates (bran, oats, rice, etc.), iron-binding phenolic compounds (tea, coffee, cocoa, etc.), and some vegetable proteins (e.g. soy protein) [5]. Cereal and tuber diets in developing countries lack iron, especially heme-iron, and often there is a need for iron supplementation and iron fortification of foods [5]. Supplementation involves giving iron tablets to high risk groups such as pregnant women and children [5]. Many foods have been fortified with iron including salt, soy sauce, chocolate/cocoa, wheat flour, maize flour, cereal based complimentary foods (for infants weaning off milk), fish sauce, soy sauce, milk (dry and fluid), sugar, soft drinks, and breakfast cereals [21]. These programs have

10 been successful and caused a 20% reduction in maternal mortality in developing countries which adopted them [6].

2.3.3 Folate Folate is absorbed through the intestinal mucosal cells [5]. Folic acid, a synthetic folate, is converted to 5-methyltetrahydrofolate (5-MTHF) within mucosal cells and released into the blood [5]. If there is too much folic acid it may be absorbed into the blood unconverted [5]. Body cells will later absorb it and convert it into an active form [5]. Natural folates are conjugated to a polyglutamate chain that must be removed in order to be absorbed and converted into 5-MTHF [5]. Because of this, natural folates are 25%-50% less bioavailable than folic acid [5]. Within the body, folate assists in biosynthesis reactions by passing one-carbon groups from one molecule to another [5]. It is particularly important for DNA synthesis and the methylation cycle [5]. In the methylation cycle the enzyme methionine synthase requires both folate and vitamin B12 in order to be formed [5].

A decrease in DNA production leads to reduced cell division which is most obvious in cells that divide rapidly (e.g. erythrocytes, immune cells, platelets, and those that line the gut) [5]. In adults folic acid deficiency can lead to anemia, increased susceptibility to infection, decrease in blood coagulation, and decrease in absorption of nutrients [5]. Because of an impaired methylation cycle, blood homo-cystein levels may be elevated (due to homo-cysteine not being re-methylated) [5]. Elevated levels of homo-cysteine have been linked to cardiovascular disease [5]. The methylation cycle is also connected to the methylation of myelin basic protein (MBP), which is a component of myelin [5]. Myelin is the material responsible for nerve cell insulation. If there is a severe prolonged deficiency in folic acid the nerves will not conduct electrical signals efficiently which leads to ataxia (lack of muscle movement coordination), paralysis, and even death [5]. However, other signs of folate deficiency would be noticed earlier. These symptoms are more often observed due to a deficiency in vitamin B12 because it is also required in the methylation cycle [5]. The most prominent effect of folate deficiency is neural tube birth defects (NTDs) [5]. During the 21st to 27th days post-conception the neural plate closes to form a neural tube which will later form the spinal cord and cranium of the foetus [5]. With a folic acid deficiency, the likelihood of improper closure is greatly increased [5]. Two common NTDs are spina bifida (which causes lower body paralysis), and anencephaly (usually leads to death

11 within hours of birth) [5]. There has also been an indication that adequate folic acid intake may decrease the risk of colorectal cancer and cognitive impairment [5] [4]. The group at most risk are pregnant and lactating women because of the risk of NTDs and also the increased need for folic acid during the rapid growth of the fetus (2nd and 3rd trimester) and during lactation [5]. Because neural tube closure occurs before many women become aware of their pregnancy, all women of childbearing age are to be targeted [5]. To prevent folate deficiency the levels to be consumed are given in Table 2.3.3.

Table 2.3.3: Appropriate Consumption of Folate in DFE* (adapted from [23], Health Canada) Life Phase/Gender Age RDA/AI** Tolerable Upper (μg/day DFE) Intake Level (μg/day DFE) Infant 0-6 months 65 no data 7-12 months 80 no data Child 1-3 years 150 300 4-8 years 200 400 9-13 years 300 600 Adult 14-18 years 400 800 19+ years 400 1000 Pregnancy ≤18 years 600 800 19-50 years 600 1000 Lactation ≤18 years 500 800 19-50 years 500 1000 *DFE (Dietary Folate Equivalent) = 1 μg food folate = 0.6 μg folic acid (fortified food/supplement with food) = 0.5 μg folic acid (supplement with no food) **Bold is used to indicate AI instead of RDA.

Folate is found in high concentrations naturally in liver and fresh green vegetables [5]. It is also found in certain legumes and fruits [4]. However, because natural folates easily degrade and the poorer population consumes food low in folate, intervention is needed. The interventions that have been used are food fortification and selected supplementation for women of childbearing age [4]. Folic acid has been added to cereal fortification programs. Wheat flour and breakfast cereals have been fortified with folic acid [4]. In 1998 it became mandatory in the United States to fortify grain products. This led to a 26% reduction in NTDs [4]. Now more than 30 countries fortify their flour with folic acid and often together with iron [4].

2.4 Micronutrient Properties 2.4.1 Iodine 2.4.1.1 Choice of Fortificant - - There are two main forms of iodine used in fortification: iodide (I ) and iodate (IO3 ). They are usually added to foods as potassium salts but can sometimes be added as calcium or sodium salts [4]. Potassium iodide (KI) was the first fortificant used followed by potassium iodate

(KIO3) [4]. Potassium iodate is less water soluble, more resistant to oxidation, and more resistant to evaporation [4]. Therefore it is more stable and is preferred over potassium iodide, especially in hot and humid climates [4]. Iodine loss due to oxidation is intensified due to humidity (moisture), elevated temperature, sunlight, and salt impurities (e.g. magnesium chloride) [4] [25]. Countries in Europe and North America continue to use potassium iodide but most tropical regions use potassium iodate [4]. Potassium iodate was used by Sangakkara (2011) in the development of spray solutions for use in India which were further developed in this project [17].

2.4.1.2 Properties of Potassium Iodate Potassium iodate is an ionic compound made up of a potassium cation and iodate polyatomic anion (see Figure 2.4.1). It takes the form of white odourless crystals or a crystalline powder [26]. Potassium iodate is heat stable (melting with partial decomposition at 560ºC) and soluble in water (9.16 g KIO3/100 g H2O at 25ºC and 32.2 g KIO3/100 g H2O at 100ºC) [26] [27]. Its method of decomposition is reduction followed by sublimation [26].

- + - 2IO3 (aq) + 12H + 10e  I2(s) + 6H2O Eo = +1.195 V (1 atm, 25ºC) [28] o I2(s)  I2(g) ΔH = 62.438 kJ/mol (1 bar, 25ºC) [28]

It is used as an oxidizing agent in analytical chemistry and as a maturing agent in dough conditioning because it promotes disulfide bond formation in gluten [26] [27].

- O + K I

O O Potassium Cation Iodate Anion

Figure 2.4.1: Potassium Iodate Chemical Structure

2.4.2 Iron 2.4.2.1 Choice of Fortificant There are many iron compounds used as fortificants in food fortification [4]. They are split into three general categories: water soluble, sparingly soluble in water (dilute acid soluble), and water insoluble (sparingly soluble in dilute acid) [4]. Each category requires some trade-offs. Generally, higher water solubility is tied to higher bioavailability, lower stability, and more severe undesired organoleptic properties [4]. Encapsulation of iron and the use of stabilizers can improve stability and reduce the development of undesirable organoleptic properties [29]. Also, the bioavailability of less water soluble iron may be improved by increasing particle surface area through comminution, and by the use of absorption promoters (e.g. sodium acid sulfate (SAS) and ascorbate) [29]. Cost is also an important factor to consider. A list of commonly used iron compounds and important properties are given in Table 2.4.1.

Table 2.4.1: Common Iron Fortificants (adapted from [4]) Compound Relative Bioavailability (%) Relative cost (per mg iron) (%) Water Soluble Ferrous sulfate, 7H2O 100 100 Ferrous sulfate, dried 100 100 Ferrous gluconate 89 670 Ferrous lactate 67 750 Ferrous bisglycinate >100 1,760 Ferric ammonium citrate 51 440 Sodium iron EDTA >100 1,670 Poorly water soluble, soluble in dilute acid Ferrous fumarate 100 220 Ferrous succinate 92 970 Ferric saccharate 74 810 Water insoluble, poorly soluble in dilute acid Ferric orthophosphate (FOP) 25-32 400 Ferric pyrophosphate (FPP) 21-74 470 Elemental Iron: H-reduced 13-148* 50 Atomized 24 40 CO-reduced 12-32 <100 Electrolytic 75 80 Carbonyl 5-20 220 *Experimental data only for a greatly reduced particle size

The least expensive and most bioavailable options for salt fortification are ferrous sulfate and ferrous fumarate. Unfortunately their stability is relatively low and our group found that stabilizers were ineffective at retaining iodine in salts fortified with these iron salts at elevated temperatures and humidity typical of tropical countries [9]. Zimmerman et al. (2004) used a water insoluble iron source with a reduced particle size (micronized FPP) [30]. However, our group found that the increased surface area of the smaller particles lead to more contact between the iron and iodine causing degradation of iodine. The iodine retention was unacceptable in course unrefined salt stored at elevated temperature and humidity [12] [13]. The retention was even less than that of non-encapsulated ferrous fumarate which is more reactive due to its higher water solubility [13]. Physical separation of the micronutrients by microencapsulation was developed by our group to prevent this degradation and to mask undesirable colour. Ferrous fumarate and ferrous sulfate were used but ferrous sulfate developed an unacceptably strong

15 flavour [9]. Therefore, microencapsulated ferrous fumarate was chosen by our group to be the fortificant [9].

2.4.2.2 Properties of Ferrous Fumarate Ferrous fumarate is an iron salt composed of iron in the 2+ oxidation state and fumarate which is derived from an unsaturated dicarboxylic acid (see Figure 2.4.2) [26]. Ferrous fumarate takes the form of an odourless and almost tasteless reddish-orange to reddish-brown powder [26]. It is slightly soluble in water and is soluble in dilute hydrochloric acid [26]. It is practically insoluble in organic solvents [26]. In salt fortification iron is always retained but it may be oxidized from ferrous iron (Fe(II) or Fe2+) to ferric iron (Fe(III) or Fe3+). Ferric iron is less bioavailable than ferrous iron because it is less soluble (soluble only in strong complex formation) [31].

2+ 3+ - Fe  Fe + e Eo = -0.771 V (1atm, 25ºC) [28]

When ferrous salts (including ferrous fumarate) are dissolved in a strong base they are converted into ferrous hydroxide (Fe(OH)2) (dirty-green colour) [32] [33]. Ferrous hydroxide is chemically unstable in the presence of oxygen and readily oxidises to ferric oxide (Fe2O3•nH2O) (red colour) [32] [26].

- 2+ Fe O - O

O Ferrous Ion Fumarate

Figure 2.4.2: Ferrous Fumarate Chemical Structure

All ferrous salts are susceptible to the potential redox reaction with iodate. The reaction is energetically favorable and results in the loss of iodine through sublimation and the reduction of ferrous iron to ferric iron.

- + 2+ 3+ 2IO3 (aq) + 12H + 10Fe  I2(s) + 10Fe + 6H2O Eo = 0.421V (1atm, 25ºC) [28]

2.4.3 Folate Folic acid, a synthetic folate, is the only folate fortificant used in food fortification [5]. Folic acid is an organic molecule made up of three basic parts: pteridine ring, para-aminobenzoyl group, and glutamic acid (see Figure 2.4.3). Natural folates consist of a variety of reduced folate polyglutamates. The polyglutamate chain is conjugated to the pteridine ring lowering the bioavailability of the folate, as mentioned in section 2.3.3 [5]. Also, natural folates are more susceptible to oxidative cleavage of the C9-N10 bond because their pteridine rings are reduced [5].

Folic acid appears as yellowish-orange thin long platelets or powder [26]. For spray solutions and extraction purposes it is important to be aware of folic acid solubility. It is insoluble in acetone, chloroform, ether, and benzene [26]. It is only very slightly soluble in water (0.0016 mg FA/mL H2O at 25ºC and 0.01 mg FA/mL H2O at 100ºC) [26]. It is slightly soluble in methanol and significantly less soluble in ethanol and butanol [26]. Finally, it is soluble in alkali solutions, hot diluted hydrochloric acid, and sulphuric acid [26].

O OH O C9-N10 bond OH OH NH N O N NH

H N N N 2 (6-methylpetrin) pteridine ring para-aminobenzoyl glutamic acid

Figure 2.4.3: Folic Acid Chemical Structure

The four main modes of folic acid degradation studied have been due to thermal degradation, gamma radiation, reduction, and oxidation. Solid folic acid is heat stable degrading between 148ºC-262ºC [34]. It melts and decomposes rapidly starting with the loss of glutamic acid, then two overlapping reactions beginning with the loss of pterin and then p-aminobenzoic acid [34]

[35]. Gamma-radiolysis of folic acid in aqueous solution was found to yield a mixture of pteroic acid, glutamic acid, 6-methylpteridine, p-aminobenzoic acid, and γ-aminobutyric acid [36]. Because the salt should not be exposed to temperatures as high as 148ºC or gama-irradiation these modes of degradation are not of concern. The most expected modes of degradation are reduction and oxidation.

Both reduction and oxidation of folic acid results in the cleavage of the C9-N10 bond yielding p- ABGA as a product [37]. The reduction of folic acid in aqueous solution is pH dependent. In acidic pH folic acid undergoes a series of reduction reactions (chemical, electrochemical, or catalytic) one of which involves a cleavage reaction which liberates p-ABGA and a pterin [38] [39] [40]. In acidic anaerobic conditions an electrochemical reductive cleavage reaction occurs in solution below pH 4 [41]. The reductive cleavage of folic acid has been achieved by the use of ascorbic acid, zinc, and ferrous ions in acidic solutions [42] [39] [43] [37] [44]. In a basic solution only one reaction takes place with no cleavage. It yields 5,6-dihydrofolic acid which is still a bioavailable form of folate [38]. Therefore, alkaline conditions should be used for fortification especially where ferrous ions are present.

Oxidation of folic acid occurs in the presence of light (photooxidation). Most experiments reported in the literature irradiated folic acid with UV-A light (most at 350 nm). Folic acid is photostable in acidic and alkaline solution in the absence of oxygen [45] [46]. Oxidative cleavage of folic acid in an acid solution yields p-ABGA and a pterin (6-formylpterin which is transformed into 6-carboxypterin with further oxidation) [46] [47] [45] [48]. In basic solution the same pathway occurs but an additional one has been seen where a product is formed with a higher molecular weight than folic acid possibly due to an increase in oxygen atoms and decrease in hydrogen atoms [47]. Photooxidation is an autocatalytic reaction with 6- formylpterin acting as a catalyst for further oxidation of folic acid [46] [49]. Other photosensitizers studied include other B-vitamins (riboflavin and thiamine) and other unconjugated pterins (e.g. 6-methylpterin) [50] [51] [46] [52]. In studies with riboflavin and thiamine it was found that they catalyze a folic acid degradation reaction even in the dark, yielding p-ABGA [52]. Degradation of folic acid in the presence of riboflavin and light was seen to slow in the absence of oxygen [53]. However antioxidants were shown to stop the reaction [53]. In salt fortification light may increase the rate of degradation of folic acid and,

18 because it is an autocatalytic reaction, the rate may increase with time. Also the addition of other B-vitamins should be avoided.

Oxidation of folic acid to p-ABGA can also occur with oxidizing agents such as potassium permanganate under mildly alkaline conditions [44] [37]. Alkaline oxidation of folic acid using sodium N-bromo-p-toluene sulfonamide as the oxidant has been reported [54]. This reaction can be catalyzed by metal ions (Ru(III), Os(VIII), Pd(II), and Pt(I)) [54]. It was predicted that this would also occur with other metal oxidants and oxygen as an oxidant [54]. Therefore metal oxidant impurities in unrefined salt may catalyze degradation and exposure to oxygen may increase degradation rate. Despite potassium iodate being an oxidizing agent there have been no reports of it oxidizing folic acid and therefore should not cause an issue.

Folic acid has also been found to exhibit free radical scavenging behaviour and thus is a possible - - - antioxidant [55]. It has been seen to scavenge CCl3O2•, N3•, SO4• , Br2• , •OH, and O• very efficiently at pH 6.8 to 12.8 [55]. It was also found that folic acid is a less efficient free radical scavenger in neutral pH [56]. Free radical scavenging activity increases from pH 2 to a maximum at pH 3.5. Then it decreases from pH 3.5 to pH 6 and increases again to at least pH 10 [56]. The hydroxyl radical has been shown to cause cleavage of folic acid resulting in p-ABGA [57]. Therefore, in basic pH, folic acid will scavenge free radicals. Thus if the formulation or exposure to light causes the production of many free radicals an antioxidant may be used to scavenge them before folic acid.

2.5 Analytical Methods to Quantify Folic Acid Based on p-ABGA Several methods have been developed for the quantification of folic acid involving the degradation of folic acid into p-ABGA (an aromatic amine) followed by a diazotization and coupling reaction leading to a coloured product. These methods are not specific to p-ABGA but would effectively measure any aromatic amine, including p-aminobenzoic acid and p- aminobenzoylglycine.

The first method was developed by Hutchings et al. (1974) where 5.0 N hydrochloric acid and zinc are used for the reductive cleavage of folic acid resulting in p-ABGA and a pteridine [40].

The aromatic amine content is measured before and after reduction, the difference is assumed to be due to folic acid [40]. During the procedure folic acid in non-reduced samples should be kept in the dark so as to prevent photodegradation. Any compound that will give rise to an aromatic amine on reduction will develop a colour and interfere with the analysis [40]. It was later found that ferrous ions and ascorbic acid interfere with this procedure causing reductive cleavage of folic acid in the samples that were not to be reduced [44]. This resulted in lower readings of folic acid. Because of this complication another method was put forward where oxidative cleavage was employed using potassium permanganate in a slightly basic solution [44]. However it was found that p-ABGA is not stable under these conditions (pH 9) [58]. This also indicates that p-ABGA may not appear as a degradation product of folic acid in alkaline conditions due to its instability.

The most recent advancement in these methods was the use of novel coupling reagents after reductive cleavage using zinc and hydrochloric acid. The coupling agent of choice had been the Bratton-Marshall reagent (N-(1-naphthyl)ethylenediamine dihydrochloride) [43] [39]. Nagaraja et al. (2002) suggested the use of iminodibenzyl, 3-aminophenol (3-AP), or sodium molybdate- pyrocatechol as the coupling agent [43] [39]. The method using 3-AP was adapted by Sangakkara (2011) who conducted folic acid stability tests on aqueous solutions and double fortified salt [17]. However, instead of taking the difference between reduced and non-reduced samples, only reduced samples were tested [17]. This means that the results do not distinguish between folic acid and p-ABGA which may be present in the sample due to folic acid degradation. In order to move the project forward it is important to determine the accuracy of the stability tests for which salt and solutions are stable [17].

2.6 Salt Fortification Technologies 2.6.1 Iodine Salt Fortification Salt iodization is a well matured technology that began in Switzerland in 1921 [7]. The majority of developed nations quickly followed [8]. In the 1980s UNICEF called for worldwide salt iodization and in the year 2000 two thirds of the developing world’s salt was being iodized [7].

Iodine is typically added to salt after refinement and drying however these technologies are not globally available [4]. There are two main categories of salt fortification technologies: dry and wet [4]. In the dry method potassium iodide or potassium iodate powder is sprinkled over dry salt followed by intense mixing to distribute the powder [4]. Salt with small homogeneous crystals is required for proper distribution of powder throughout the salt [4]. For wet fortification a solution of potassium iodate is either dripped or sprayed onto salt passing by on a conveyor belt or corkscrew [4] [59]. The method chosen is dependent on salt refinement, salt moisture content, and cost (see Table 2.6.1).

Table 2.6.1: Comparison of Salt Iodization Methods (adapted from [59]) Method Dry Wet (Drip) Wet (Spray) Salt type Refined dry powder 3 2 3 Unrefined dry powder 3 2 3 Unrefined moist powder 2 2 2 Unrefined dry crystals 1 2 2 Unrefined moist crystals 1 1 2 Cost Culmination of capital cost, 1 3 2 operating cost, and cost to consumer. 1=poor/highest cost, 2=fair/in between cost, 3=good/lowest cost

The wet methods are more cost effective than the dry method. The spray method is applicable for the widest range of salt types and thus is the most desirable method for use in developing countries. Because spray fortification of salt is advantageous, salt iodization plants currently in developing countries have spray equipment in place. Thus the addition of more micronutrients to spray solutions would be an effective way to fortify because the only cost would be of the micronutrient itself. Sangakkara (2011) investigated spray solutions containing iodine and folic acid for salt fortification purposes [17].

Both continuous processes and batch processes are used for spray fortification [29] [59]. The continuous process is the most widely used because it offers cost effective large scale production [29] [59]. However, the batch process is necessary for small-scale manufacturers in developing countries (e.g. India, Bangladesh, and Vietnam) [59]. In the continuous process salt is crushed from crystal form into a coarse powder in a roller mill and fed into a feed hopper through a sieve [29]. The salt is discharged from the hopper via a shaft which regulates the salt’s

21 flow onto an inclined conveyor belt [29]. On the conveyor belt the salt enters a spray chamber and is sprayed with an atomized aqueous solution which contains 1%-3% iodine [29] [59]. The salt then falls onto a screw conveyor which mixes it before it is discharged to outlets for packaging [29]. A more simplified process is now preferred where the conveyor belt has been replaced with an inclined screw conveyor [59]. In the batch process salt is fed into a ribbon blender and sprayed with an iodine solution by overhead nozzles by a hand pump or compressor [29].

2.6.2 Iron Salt Fortification In the 1970s researchers began investigating the fortification of salt with iron. The strategy quickly turned to the addition of stabilizers (e.g. sodium hexametaphosphate (SHMP)) and absorption promoters (e.g. sodium acid sulfate (SAS)) due to difficulty with absorption of insoluble stable iron sources and unstable well absorbed soluble iron sources [60] [61] [62] [29]. Colour development was a major factor causing the failure of a few attempts [61] [62]. The first success was from Rao et al. (1975) who fortified salt using ferric orthophosphate (FOP) and SAS [63]. Improvements to the fortified salt soon came with the use of a spray-mixing process for double fortification similar to the one established for salt iodization using ferrous sulfate, SHMP, and SAS (Suwanik et al. in 1978); a more inexpensive formulation using ferrous sulfate, orthophosphoric acid (a stabilizer), and SAS (Rao (1978)); an improved iron absorption using ferrous sulfate and SHMP (S. Ranganathan (1992)); and also a dry mixing procedure using ferrous glycine sulfate (three times more bioavailable that ferrous sulfate) was developed (S. Ranganathan et al. (1996)) [64] [65] [66] [67]. These salt formulations were unsuccessful when double fortification was attempted with iodine and iron.

2.6.3 Iodine and Iron Fortification As a method to remedy iodine and iron deficiency simultaneously, double fortified salt was investigated. The first strategy employed was the use of stabilizers, absorption promoters, and different iron sources. Some successes include Suwanik (1978) using ferrous sulfate, SHMP, and sodium acid sulfate; Mannar et al. (1989) using ferrous fumarate and potassium iodide without stabilizers; and Rao (1994), who found adding iodine to his past formulation (using FOP and SAS) was unsuccessful, used ferrous sulfate and SHMP [29] [64] [68].

The Food Engineering Group in collaboration with MI investigated double fortified salt. Methods where iron is added with stabilizers and absorption promoters were seen to be unacceptable when added to salt with high moisture content and stored in a warmer temperature environment (typical of targeted developing countries) [9]. Encapsulation was necessary in order to prevent interaction between iodine and iron [9]. Fluidized bed agglomeration followed by lipid coating was the first iron encapsulation strategy developed for double fortified salt and was tested on a large scale [11]. This method was effective at preventing the interaction between iron and iodine but the iron capsules segregated during food preparation due to their low density [11] [12]. As an improvement to the process, extrusion agglomeration followed by polymer coating was investigated [13]. This method formed denser particles, was more cost effective, and formed a more uniform coat (thus higher stability) [13]. Both fluidized bed agglomeration with lipid coating and extrusion agglomeration with polymer coating produce particles which match to those of refined salt [11] [13].

In unrefined salt, crystals are much larger which would cause the premix to segregate if it were to remain the size of small refined salt crystals. If the particle sizes were increased to match those of the unrefined salt they would be organoleptically obvious (visually, by taste, and by texture) plus cause dosing issues. Therefore a spray dry encapsulation method was developed by Romita (2011) where premix particles <20μm in diameter were produced. These small particles attach to the surface of salt crystals by electrostatic attraction when dry or adhere due their glutinous nature in the presence of moisture [15] [18].

A schematic of the spray drying process used is presented in Figure 2.6.1. A drying gas is fed through an electric heater and then through an atomizing nozzle along with a solution, suspension, emulsion, or colloid that is to be dried. The two fluids enter the drying cylinder where the droplets are dried and entrained in the gas. Particles that are not dried or are too heavy for entrainment are collected in a container at the bottom of this cylinder. The gas and entrained particles move to the cyclone where the particles are separated and travel downward into a collection vessel and gas moves to a filter. The filter is used to remove any residual solids from the gas. Then the gas moves out through the aspirator which is used to create suction (thus gas flow) through the system.

① Atomizing nozzle ② Electric heater ③ Drying cylinder ④ Cyclone ⑤ Filter ⑥ Aspirator

Figure 2.6.1: Spray Drying Process (adapted from BUCHI product information pamphlet [69])

2.6.4 Iodine and Folic Acid Fortification Another area of research the Food Engineering Group has explored is the double fortification of salt with iodine and folic acid. Li et al. (2011) compared folic acid fortification though iodized salt and vitamin A-fortified sugar [16]. Formulations where folic acid was encapsulated though an extrusion-based process were stable in iodized salt [16]. The lowest retention of folic acid occurred when folic acid was added by aqueous solution [16]. An ideal process for salt fortification with iodine and folic acid would cause the least change to current salt iodization processes so as to lower capital and production costs. Thus an aqueous solution would be advantageous if folic acid could be stabilized. Sangakkara (2011) developed two stable formulations of double fortified salt that use a single aqueous solution to spray iodine and folic acid onto the salt [17]. The least expensive used the effects of pH while the other used citrate as an antioxidant to keep folic acid stable [17]. The least expensive formulation was composed of a pH 9 carbonate-bicarbonate buffer system, 0.35% w/v folic acid, and 0.35% w/v potassium iodate [17]. Retentions of both micronutrients was >90% after 6 months of storage at 45ºC and 60% relative humidity [17]. Unfortunately, the analytical method used by Sangakkara to test the stability of folic acid did not distinguish between folic acid and its common degradation product

24 p-ABGA and the concentrations of the iodine in the spray solutions were not as high as used commercially (1%-3% w/v).

2.6.5 Multiple Fortification To stave off multiple micronutrient deficiencies, there have been attempts to fortify salt with three or more micronutrients. A variety of microencapsulation techniques have been investigated for salt triple fortified with iron, iodine and vitamin A [70] [71] [72]. There have also been attempts to fortify salt with up to 10 micronutrients at once including iron; iodine; and vitamins A, B1, B2, B3, B5, B6, B9 (folic acid), and B12 [73] [74]. These studies indicate that multiple fortification of salt is a plausible method for reducing micronutrient deficiency because the micronutrients were seen to remain stable, remain bioavailable, and not cause organoleptic changes.

2.7 Project Objectives The main objective of this project was to develop a process for double and triple fortifying salt using existing equipment and technologies used commercially in developing countries. The double fortified salt was to contain iodine (KIO3) and folic acid whereas the triple fortified salt was to contain iron (ferrous fumarate) in addition. Spray fortification is the method most common for salt iodization in developing countries. Thus the further development of the folic acid and iodine spray solution established by Sangakkara (2011) was required [17]. Also the iron microcapsules developed by Romita (2011) were compatible with course unrefined salt common in developing countries [15] [18]. Thus they were used in the production of triple fortified salt. In order to complete this task a series of sub-objectives were fulfilled.

1) The first sub-objective was to develop a reliable analytical method for stability testing folic acid in spray solutions, double fortified salt, and triple fortified salt. The method used by Sangakkara (2011) for spray solutions and double fortified salt did not distinguish between folic acid and its degradation product p-ABGA [17]. Also, an analytical method for the determination of folic acid in triple fortified salt had not been developed. The methods chosen for investigation were high-performance liquid chromatography (HPLC) and spectrophotometry-based coupling (SBCM) because of their time efficiency and low cost.

2) The second sub-objective was to validate the work of Sangakkara (2011) who found folic acid to be stable in spray solutions and double fortified salt [17]. It was required to ensure that the stability of folic acid found in the salt was valid and not skewed by the production of p-ABGA. This validation would affect the direction of further spray solution development.

3) The third sub-objective was to produce spray solutions containing the commercially used concentration of 1%-3% w/v iodine and 1%-3% w/v folic acid. The spray solutions must not form a precipitate and micronutrients within them must remain stable for 2 months. Precipitate formation would cause settling within the process equipment. This goal was set because it is safely longer than the 2-4 week regular solution replacement used in salt iodization facilities. They must also be used to fortify salt that would be stable for 5 months at 50 ppm iodine. The concentration of 50 ppm iodine was based on the highest recommended level for salt iodization from WHO/UNICEF/ICCIDD which was for warm temperature and high moisture conditions typical of tropical countries [75]. This amount is assumed to degrade due to transportation and storage in the harsh conditions. The target concentration to reach the consumer recommended by MI, based on a per capita salt consumption of 10 g/day, was 30 ppm for iodine and folic acid. The higher 50 ppm amount was also chosen because interactions between the micronutrients are more likely to be evident when they are present in higher concentrations.

4) Finally, the fourth sub-objective was to produce a triple fortified salt and investigate its stability after 1 year. Iodine and folic acid were to be sprayed onto the salt and iron was to be added as spray dry encapsulated ferrous fumarate. To investigate the effect of each component on the others in the system, different combinations of the micronutrients were added to salt as well as non-encapsulated or encapsulated iron. Because the behaviour of the micronutrients were unknown the salt was fortified with the MI recommended 30 ppm of iodine and folic acid. The concentration of iron added was 1000 ppm, the concentration added by Romita (2011) [18]. Assuming 10 g/day consumption of salt, the amount of iron consumed would be 10 mg/day which is 56% of the RDA of an adult female. The rest would be supplemented by diet. This is well below the 45 mg tolerable upper intake level.

3 Materials and Methods 3.1 Materials Tables 3.1.1-3.1.3 summarize the materials used in this project.

Table 3.1.1: Materials Used in Salt Fortification Purpose Material Supplier Grade/Description Non-iodized salt Refined Canadian Sifto Canada Corp. Fine grain, clean, Salt dry Micronutrients Folic acid Bulk Pharmaceuticals USP grade Inc. Potassium iodate Sigma-Aldrich ACS reagent grade Chemicals Ferrous fumarate Dr. Paul – Lohmann Food-grade Chemicals, Germany (mean diameter ~10μm) Spray Solution Buffer Sodium carbonate, T. J. Baker Chemical Reagent grade anhydrous Co. (99.9%) Sodium bicarbonate Caledon Laboratory ACS Reagent grade Chemicals Microencapsulation Maltodextrin Cerestar, Indianapolis C*Dry MD Inc. DE=7 Hydroxypropyl Dow Chemicals Co., Hydroxypropyl methylcellulose USA. methylcellulose (HPMC) (HPMC E15)

Table 3.1.2: Materials Used in Folic Acid Analysis Purpose Material Supplier Grade/Description Spectrophotometry- Hydrochloric acid Caledon Laboratory ACS reagent grade based Coupling Chemicals Method 3-aminophenol Alfa Aesar (A For use in research Johnson Matthey and development Company) 98+% Sodium hydroxide Caledon Laboratories ACS reagent grade Ltd. Sodium nitrite Caledon Laboratories ACS reagent grade Ltd. Sulfamic acid Sigma-Aldrich ACS reagent grade Chemicals Zinc granules Caledon Laboratories ACS reagent grade Ltd. High Performance Methanol Caledon Laboratories HPLC grade Liquid Ltd. Chromatography 20 mM hexane ANALEST For use in HPLC sulfonic acid and experiments run by 0.1% phosphoric acid, ANALEST pH 2.2 Citric acid Sigma-Aldrich ACS reagent grade Chemicals Sodium phosphate, T. J. Baker Chemical Reagent grade dibasic Co. (99.7%)

Table 3.1.3: Materials Used in Iodine and Iron Analysis Purpose Material Supplier Grade/Description Iodine Analysis Potassium iodide Caledon ACS reagent grade Laboratories Ltd. Potassium iodate Sigma-Aldrich ACS reagent grade Chemicals (99.5%) Sulfuric acid EMD ACS grade Sodium thiosulfate BHD ACS grade solution, 0.1 N Starch indicator, 1.0% LabChem Inc. For laboratory and manufacturing use only Iron Analysis Potassium hydrogen Sigma-Aldrich ACS acidimetric phthalate Chemicals standard grade (99.95- 100.05%) Hydroxylamine Sigma-Aldrich ACS reagent grade hydrochloride, 98% Chemicals 1,10-phenanthroline Sigma-Aldrich ACS grade monohydrate Chemicals Sulfuric acid EMD ACS grade Ferrous ammonium sulfate Caledon ACS reagent grade hexahydrate Laboratories Ltd.

3.2 Fortification Methods 3.2.1 Buffered Spray Solution Preparation Sangakkara (2011) found that solutions of folic acid and iodine in a solution buffered to pH 9 using a 0.1 M sodium carbonate/bicarbonate buffer remained stable at high temperatures [17]. Sangakkara used solutions of 0.35% w/v folic acid and iodine so as to not affect the pH of the buffer [17]. However, a solution of 1%-3% w/v of iodine is what is applied in practice so as to add less moisture and use less solution when fortifying salt. 1%-3% w/v folic acid is also added to the spray solutions. This amount is required because the target concentration recommended by MI, based on a per capita salt consumption of 10 g/day, was 30 ppm of both iodine and folic acid. This means that the population is supplied with 300 μg/day of each micronutrient. This is double the RDA of iodine for an adult but still much below the tolerable upper intake level of 1100 μg/day and is therefore safe. This amount is 125% of the RDA of folic acid for an adult (RDA = 400 μg/day DFE = 240 μg/day folic acid from fortified food). It is much less than the 1000 μg/day DFE tolerable upper intake level but because folic acid is present in the natural diet a lower concentration is required. A target of about 30% of the RDA may be appropriate for

29 folic acid. Therefore spray solutions were investigated with lower concentrations of folic acid then iodine. The concentrations of micronutrients used in the spray solutions and the resultant % RDAs of folic acid are given in Table 3.2.1. Also, because the concentration of folic acid in the spray solutions was higher, the strength of buffer and ratio of bicarbonate to carbonate to reach pH 9 in solutions had to be determined.

Table 3.2.1: Resultant % RDA of Folic Acid in Spray Solutions % w/v Iodine % w/v Folic Acid % RDA Folic Acid (assuming 30 ppm I on salt) 1 1 125 2 2 125 3 3 125 2 1 62 3 1 42

In order to determine these amounts a range of folic acid solutions were made with buffer strengths of 0.1 M (the same as Sangakkara (2011)) and 0.2 M varying the carbonate to bicarbonate ratio. Because folic acid will alter the pH of the spray solutions much more than potassium iodate, these trials were only done with added folic acid. Stock solutions of carbonate and bicarbonate were made at 0.1 M and 0.2 M concentration. For 0.1 M folic acid solutions, different volumetric ratios of the 0.1 M carbonate and the 0.1 M bicarbonate solutions were combined. For 0.2 M folic acid solutions the same thing was done with the 0.2 M carbonate and bicarbonate solutions. Solutions of 1%, 2%, and 3% w/v folic acid were made in solutions of 11 incremental steps from 100% v/v carbonate to 100% v/v bicarbonate at both 0.1 M and 0.2 M buffer strengths. More detail on the procedure used can be found in Appendix 9.5.

These optimal formulations were used in making buffered spray solutions. In 500 mL glass bottles covered in aluminum foil, folic acid and potassium iodate were measured and added so that they would be 1%-3% of the final solution, then carbonate and bicarbonate solutions were added in the correct ratio adding to 500 mL. These solutions were split into two batches, one stored at 25ºC and the other at 45ºC in a Model 307 Fisher Scientific Incubator. They were all stored in the absence of light.

3.2.2 Spray Drying Microencapsulation Microcapsules used were prepared by Dan Romita as described in Romita (2011) [18]. They contained 9% w/w iron (as ferrous fumarate) coated using 80% w/w Dextrin (DE7) and 20% w/w HPMC (E15).

3.2.3 Fortified Salt Preparation Salt was fortified in a bench-scale ribbon blender from Les Industries All-Inox Inc. (Montreal). Salt was added to the ribbon blender (250-1000 g) and blended for 2 minutes to break down any large salt clusters. Then folic acid and/or iodine were added as a solution via spray bottle. Iron was added in powder form either as non-encapsulated ferrous fumarate or as spray dried microcapsules. The salt was mixed for 15 minutes in the ribbon blender and then was taken out and put on sheets to dry overnight. The salt was stored in Zip-LocTM polyethylene bags in the dark at 25ºC.

Double fortified salt was fortified to 50 ppm iodine and folic acid. This is on the highest recommended level for salt iodization from WHO/UNICEF/ICCIDD which was for warm temperature and high moisture conditions typical of tropical countries [75]. This amount is assumed to degrade due to transportation and storage in the harsh conditions. This high amount was also chosen because interactions between the micronutrients are more likely to be evident when they are present in higher concentrations. The target concentration recommended by MI, based on a per capita salt consumption of 10 g/day, was 30 ppm. With equal amounts of each micronutrient being added to salt this provides 300 μg/day of each. This is double the RDA of iodine for an adult but still much below the tolerable upper intake level of 1100 μg/day and will therefore be safe. This is 125% of the RDA for an adult for folic acid. Since the diets tend to be low in folic acid and that the addition level is well below the 1000 μg/day DFE (600 μg/day folic acid from fortified food) tolerable upper intake level it should be safe. Also, spray solutions with less folic acid than iodine were investigated.

Triple fortified salt was fortified with the MI recommended 30 ppm of iodine and folic acid. The concentration of iron added was 1000 ppm. This was the concentration added by Romita (2011) [18]. Assuming 10 g/day consumption of salt, the amount of iron consumed would be 10 mg/day which is 56% of the RDA of an adult female. The rest would be supplemented by diet.

This is also well below the 45 mg tolerable upper intake level. To investigate the effect of each component on the others in the system, different combinations of the micronutrients including non-encapsulated or encapsulated iron were added to salt.

3.3 Analytical Methods 3.3.1 Iodine Stability Testing Method 33.149 by the Association of Official Analytical Chemists (AOAC) was used for iodine quantification in salt and spray solutions [76]. In this method iodate is reduced to iodine (I2) and titrated with sodium thiosulfate using a starch indicator [76]. Four replicates were used for each sample. The detailed procedure is outlined in Appendix 9.1.

3.3.2 Iron Stability Testing The total iron and ferrous iron (Fe(II)) content was determined by the complexation of ferrous iron with 1,10-phenanthroline followed by spectrophotometry at 512nm. To measure total iron, a reducing agent (hydroxylamine hydrochloride) was added to convert any ferric iron (Fe(III)) into ferrous iron before it was complexed with 1,10-phenanthroline. This method was first developed by Harvey et al. (1955) and adapted by Oshinowo et al. (2004) [11] [77]. The method is described in greater detail in Appendix 9.2.

3.3.3 Folic Acid Stability Testing Two methods were used for folic acid stability testing in salt and spray solutions: high performance liquid chromatography (HPLC) with UV detection and spectrophotometry-based coupling (SBCM).

3.3.3.1 High Performance Liquid Chromatography (HPLC) with UV Detection Two slightly different HPLC programs were developed due to the fouling of one of the columns. The two columns used were C18 columns with the following specifications: CSC-Intersil 150A/ODS2 5 μm pore size, 100 mm length, 4.6 mm diameter; and Waters Symmetry Column, 5μm pore size, 4.6mm diameter, 150 mm length. The mobile phase used was an isocratic mixture of 70-75% v/v 20 mM hexane sulfonic acid (HSA) adjusted to pH 2.2 with 0.1% phosphoric acid and 25-30% v/v methanol. 20 μL samples were fed into the HPLC system by a

Perkin Elmer Series 200 auto sampler. They were pumped through the HPLC system at a temperature of 40ºC and a flow rate of 0.7 mL/min or 1.0 mL/min using a Perkin Elmer series 410 LC pump. Once eluted from the column they were fed through a UV-Vis detector (SPD- 10A SHIMADZU) and the absorbance was measured at 280 nm. The results were interpreted using TurboChrom, a component of Total Chrom Software. More detail of the method used can be found in Appendix 9.3 sections 9.3.1 to 9.3.3.

3.3.3.2 Spectrophotometry-Based Coupling Method (SBCM) This method was adapted from Hutchings et al. (1974) and Nagaraja et al. (2002) [40] [43]. Folic acid is reductively cleaved in hydrochloric acid by zinc. The product, p-ABGA, is diazotized and then coupled with 3-AP. The coupled substance is yellow-orange coloured with maximum absorbance at 460 nm. Figures 3.3.1-3.3.3 show the reactions that take place in the SBCM. A Cary 50 UV Spectrophotometer was used to measure the absorbance which was correlated to folic acid concentration. Four replicates were used. A more detailed description of the procedure is given in Appendix 9.4.

O OH

H OH O O N HCl H2N OH O Zn N OH O H N N NH O H N N N 2 HO

folic acid p-aminobenzoylglutamic acid (p-ABGA)

Figure 3.3.1: Reaction #1 Reductive Cleavage of Folic Acid

O O O O H N OH + 2 N N OH N NaNO 2 N H H + H O O HO HO

p-ABGA intermediate product

Figure 3.3.2: Reaction #2 p-ABGA Diazotization

O O N + N NH N N OH 2 HCl N H2N O O HO H + N H OH O OH HO

HO O 3-aminophenol (3-AP) intermediate product orange/yellow compound

Figure 3.3.3: 3-Aminophenol Coupling Reaction

3.3.4 pH Testing A VWR Scientific Model 8000 pH meter was used to measure the pH of spray solutions and dissolved salt samples.

4 Results and Discussion 4.1 Folic Acid Analytical Method Development High-performance liquid chromatography with UV detection and spectrophotometry-based coupling were investigated as potential methods to quantify folic acid in triple fortified salt. These methods were selected because they are less expensive and more time efficient than other methods such as microbial assays and biospecific methods.

4.1.1 High-Performance Liquid Chromatography (HPLC) The high performance liquid chromatography (HPLC) method was successful in a few ways. Calibration curves of folic acid and p-ABGA were very linear with R2 values of 0.9999 for both folic acid and p-ABGA when area and over 0.9980 when peak height was used (see Appendix 9.3, section 9.3.4.1). Folic acid’s linear range extended above 100 ppm whereas p-ABGA was linear to only 15 ppm (see Appendix 9.3, section 9.3.4.3). Folic acid was successfully separated from p-ABGA using both the CSC-Intersil and Waters Symmetry C18 columns (see Appendix 9.3, section 9.3.4.2). When standard amounts of folic acid were added to fortified salt, it resulted in the recovery of the correct amount calculated when peak height was used for quantitation (see Appendix 9.3, section 9.3.4.4).

However there were some points of concern. When samples were made by dissolving salt a peak appeared right at the beginning of the HPLC scan (see Appendix 9.3 section 9.3.1.2 for salt sample preparation; and see Appendix 9.3, section 9.3.5.1 for salt peaks). This peak increased in size with an increase in salt concentration and would appear even if pure salt was tested, even though salt should not absorb light in the measured wavelength range (see Appendix 9.3, section 9.3.5.1). This may indicate the degradation of the column by high salt concentrations. Measured folic acid values were a bit low in the presence of salt (see Appendix 9.3, section 9.3.5.2). Salt may have been pulling folic acid that is in its ionized state through the column faster. In order to rectify this issue, extraction was attempted using water and methanol (see Appendix 9.3, section 9.3.1.2). Extraction is also required when salt fortified with ferrous fumarate is tested because the ferrous fumarate can foul the column. Unfortunately, extraction was not entirely successful and some folic acid was not extracted (see Appendix 9.3 section 9.3.5.3). When a filter was used very little folic acid was found in sample solutions (see Appendix 9.3 section 9.3.5.3).

Furthermore, even when folic acid was extracted, iron had a tendency to precipitate out of solution causing fouling of the column. Another issue is that p-ABGA would result in two peaks or one malformed peak possibly due to degradation. The sum of the areas under the two peaks gave reasonable results. Also a small peak would appear at the beginning of the chromatogram, which indicates degradation or ionization of p-ABGA (see Appendix 9.3, section 9.3.5.4). Another interesting observation was that folic acid results were much too high in salt fortified with pH 8-10 buffered solutions and stored for 1 year (see Appendix 9.3, section 9.3.5.5). I originally thought that the pH of the spray solutions could be significantly affecting the pH of the sample solutions resulting in higher readings. However the measured pH of the sample solutions were all very close (see Appendix 9.3, section 9.3.5.5) so this could not be the cause. This raised issues that must be rectified before HPLC with UV detection is used to measure folic acid in triple fortified salt.

4.1.2 Spectrophotometry Based Coupling Method The spectrophotometry based coupling method had to be improved in several ways. Firstly, the method used by Sangakkara (2011) did not distinguish between p-ABGA and folic acid [17]. Therefore the difference must be taken between reduced and non-reduced samples (which omit reaction 1) (see Appendix 9.4, section 9.4.3). This was done by omitting the addition of zinc to the first reaction. It was important to test if p-ABGA was still produced since folic acid in acidic solution is known to degrade. Only minimal degradation was found and no trend was seen between absorbance and folic acid concentration when omitting the first reaction (see Appendix 9.4, section 9.4.3). The average amount measured correlates to -7 ± 7 ppm of folic acid on salt and is therefore insignificant. However, a deviation of 7 ppm would result in >20% error when measuring salt that contains ≤50 ppm folic acid.

Large standard deviations were obtained when measuring the folic acid and p-ABGA content of salt (>100%). An example of this can be seen in Figure 4.1.1 where p-ABGA content was measured in a few salt formulations. Absorbance from salt samples was very low due to dilution during the reductive cleavage reaction and subsequent quenching. The calibration curve tested solutions from 0 ppm to 6 ppm folic acid however a 50 ppm salt sample would be diluted to a 1.4 ppm solution. The absorbance of this solution was approximately 0.075 AU (see Appendix 9.4, section 9.4.2, Figure 9.4.1). This low absorbance value would be imprecise. An

36 extraction procedure was implemented, for reasons to be explained below, and the quenching of the reductive cleavage reaction was achieved with separation of the liquid from zinc instead of the addition of water which diluted the sample. The amount of HCl added to this reaction was also decreased from 40 mL to 11 mL. This resulted in final solutions concentration of 2.9 ppm based on 50 ppm salt (~0.15 AU) (see Appendix 9.4, section 9.4.4, Figure 9.4.3). It also resulted in standard deviations of 3.6% ± 2.5% on average when testing salt fortified with 50 ppm folic acid. This is a maximum error of 3 ppm (6% error) which is acceptable.

The concentration of HCl was also initially increased to ensure that the reaction would go to completion, since a smaller volume was being used. However this caused a degradation of p- ABGA when it was used to create calibration curves or test spray solutions (see Appendix 9.4, section 9.4.5, Figure 9.4.4). This degradation of p-ABGA into a non-diazotizable form (thus not detected by this method) was reported earlier by Hutchings et al. (1974) [40]. Spray solutions containing more than 1% iodine were found to degrade p-ABGA more quickly than solutions containing 0%-1% iodine, such as those used in folic acid calibration curves. Thus spray solutions with 1% iodine tested using stock HCl solution gave the correct value when the stock HCl solution calibration curve was used, but spray solutions containing 2% or 3% iodine measured incorrectly low amounts of folic acid (see Appendix 9.4, section 9.4.5, Figure 9.4.5). The increase in iodine content resulting in a decrease of folic acid detected indicates that iodine contributes to the degradation of p-ABGA into a non-diazotizable form. When 5 N HCl was used, all solutions tested gave the expected results for folate. Also, p-ABGA did not degrade when using concentrated HCl on salt samples (see Appendix 9.4, section 9.4.5, Figure 9.4.6). Therefore, salt samples tested using this method with a concentrated HCl calibration curve, resulted in higher than expected values (>140% retention). Once 5 N HCl was used and the results were recalibrated, they were accurate (see Appendix 9.4, section 9.4.5, Figure 9.4.7).

The coupling reagent 3-AP is coloured, therefore standards and samples must dilute the 3-AP to the same volume (see Appendix 9.4.6). In the original method the standard solutions were diluted to 25 mL whereas the sample solutions were only diluted to 14 mL. This resulted in inaccurately high readings where salt not fortified with folic acid would read at approximately 10 ppm because of the increased concentration of 3-AP in the solution. In the revised method both standards and samples were diluted to 12 mL.

Extraction was necessary when iron was present. Ferrous fumarate reacts with the zinc metal making iron metal and zinc chloride (which goes into solution). The iron metal quickly reacts with the hydrochloric acid producing ferrous chloride and hydrogen gas. Ferrous chloride is a yellow colour and interferes with the absorbance, making it appear as if more folic acid was present (see Appendix 9.4, section 9.4.7). Since zinc is used for reducing iron, it is not used in reducing the folic acid. Sodium hydroxide and methanol were used in an attempt to dissolve folic acid while leaving ferrous fumarate behind. Methanol alone did not dissolve all of the folic acid resulting in readings that were too low. Sodium hydroxide alone reacted with ferrous fumarate making ferrous hydroxide (green colour) which did not easily separate by centrifugation. Thus the same problems arose from iron in the samples. A 50/50 v/v solution of methanol and 0.1 N sodium hydroxide was then used and was successful. Extraction had to be done twice because although folic acid does dissolve in basic aqueous solutions and methanol, salt saturates the solution before all the folic acid can dissolve. When one extraction is done folic acid is centrifuged out. So a second extraction is necessary to dissolve the folic acid before centrifugation. Improved results can be found in Appendix 9.4 (section 9.4.7). Standard amounts of folic acid were added to fortified salt and tested. The additions tested accurately indicating that the revised method is acceptable (see Appendix 9.4, section 9.4.8). See Appendix 9.4, section 9.4.9, for a summary of the development of the SBCM for use in triple fortified salt.

The final test procedure involved standardization with solutions of 0-31.25 ppm folic acid in 50/50 v/v (methanol)/(0.1N NaOH) added into the first reductive cleavage reaction. Spray solutions were diluted into this range using 50/50 v/v methanol/NaOH. Folic acid in salt samples were extracted by adding 50/50 v/v methanol/NaOH to a test tube of salt, shaking it, transferring the suspension located above the salt to a centrifuge tube, adding more 50/50 v/v methanol/NaOH so that the folic acid dissolves, and then centrifuging it. From this point forward the rest of the procedure is identical for all samples. 14 mL of each of these solutions is added to a jar with 11 mL of 5 N HCl and 2 g of zinc granules. The zinc granules were not added to samples that were to not be reduced (test for p-ABGA). The reductive cleavage reaction is undergone in the dark while jars are mixed at regular intervals. Then 4 replicates of 2 mL aliquots are put into test tubes. 2 mL of 5 N HCl and 1 mL of NaNO2 are added to the test tubes and mixed in order for the diazotization reaction to take place. 1 mL of sulfamic acid is

38 then added to each test tube to clear any residual amines. 5 mL of 1% 3-AP was then added to each test tube and then placed in a boiling water bath. The coupling reaction was then allowed to go to completion. Absorbance of the solutions was measured at 460 nm in a 10 mm cuvette. See Appendix 9.4 for more detail on the original and revised SBCM.

4.1.3 Effect of Folic Acid Degradation on the SBCM The SBCM does not distinguish between folic acid and p-ABGA unless the difference is taken between the sample when reduced and not reduced. Unfortunately, Sangakkara (2011) only tested reduced samples [17]. However if p-ABGA is not present in the samples only testing reduced samples is necessary. It was determined that p-ABGA is only slightly produced, if at all, in spray solutions or in double fortified salt (folic acid and iodine). Salt fortified by Angjalie Sangakkara was tested for p-ABGA after 1 year of storage. This salt was fortified using spray solutions adjusted to various pH values [17]. The results can be seen in Table 4.1.1.

Table 4.1.1: p-ABGA in Sangakkara’s Double Fortified Salt After 1 Year of Storage Spray Solution pH p-ABGA as % Initial FA Content (ppm p-ABGA/ppm Initial FA * 100%) 2.21 48 ± 43 3.03 4 ± 3 4.07 9 ± 7 5.98 24 ± 37 7.02 58 ± 63 7.76 122 ± 176 9.27 2 ± 8

The test is not precise because it was conducted before SBCM optimization where the concentration of the test solutions was increased. Before optimization slight contamination would cause large positive fluctuations in concentrations detected (even above 100%). These fluctuations are represented in the standard deviations of the readings. Thus the samples are accurate within their standard deviations, however some are quite imprecise. The samples that indicate high conversion to p-ABGA are accompanied by large standard deviations indicating contamination is responsible for these large amounts. In samples with small standard deviations (<50% FA) there was very little gain of p-ABGA. Therefore there was little to no significant p- ABGA in the samples.

The p-ABGA content was also tested after 5 months of storage in the 1%-3% w/v iodine and folic acid spray solutions and in salt double fortified using those solutions. The results also indicate little or no increase in p-ABGA (see Tables 4.1.2-4.1.3). At most only ~10% of the folic acid degraded into p-ABGA and remained in this form, which is acceptable.

Table 4.1.2: p-ABGA in 1%-3% Spray Solutions After 5 Months of Storage Formulation p-ABGA in Solutions Stored p-ABGA in Solutions Stored at 25ºC at 45ºC (% Initial FA Content) (% Initial FA Content) 1% FA, 1% I, 0.1 M Buffer 7 ± 7 4 ± 1 1% FA, 2% I, 0.1 M Buffer 10 ± 3 5 ± 4 1% FA, 3% I, 0.1 M Buffer 9 ± 8 3 ± 3 1% FA, 1% I, 0.2 M Buffer 11 ± 5 4 ± 2 1% FA, 2% I, 0.2 M Buffer 4 ± 1 6 ± 3 1% FA, 3% I, 0.2 M Buffer 4 ± 2 6 ± 2 2% FA, 2% I, 0.2 M Buffer 5 ± 2 5 ± 2 3% FA, 3% I, 0.2 M Buffer 3 ± 1 9 ± 1

Table 4.1.3: p-ABGA in Double Fortified Salt After 5 Months of Storage Spray Solution Formulation Used to Fortify Salt p-ABGA in Salt (% Initial FA Content) 1% FA, 1% I, 0.1 M Buffer 9 ± 8 1% FA, 1% I, 0.2 M Buffer 2 ± 4 2% FA, 2% I, 0.2 M Buffer 7 ± 4 3% FA, 3% I, 0.2 M Buffer -1 ± 2

The reason for so little p-ABGA is likely due to its instability within the samples. It was reported earlier that p-ABGA is unstable in pH 9 solution and will degrade into non-diazotizable products that will not be detected by the SBCM by Maruyamaa et al. (1978) [58]. It is unlikely that folic acid degradation is following a pathway that does not produce p-ABGA since both oxidation and reduction of folic acid result in the cleavage of the C9-N10 bond in folic acid resulting in the production of p-ABGA [37]. Since the salt and spray solutions contained negligible p-ABGA, folic acid may be measured directly from reduced samples. Thus, the results of Sangakkara (2011) are valid.

4.2 Triple Fortified Salt Analytical method development and triple salt fortification tests were performed in parallel so that the analytical methods could be tested on salt that contained iron. Due to time constraints triple fortified salt was not prepared after the analytical method was finalized and stable spray

40 solutions were developed. Therefore the salt made for analytical method development was fortified by spraying a mixture of dissolved 0.1% w/v iodine (I) and/or suspended 0.1% w/v folic acid (FA) in water onto the salt. The concentration of these mixtures was the same as the initial solution used in making folic acid calibration curves. This concentration also made the number of sprays required easily determinable (1 gnutrient/mL * 1 mL/spray = 1 gnutrient/spray). Different variations of the fortified salt were made. 500 g of salt was put into a ribbon blender. 30 ppm of folic acid and/or iodine was added to salt by spraying 15 mL of solution into the ribbon blender. This corresponds to the addition of 2.9% moisture (% moisture = (weight of water/ (weight of salt + weight of water)) * 100) which is relatively high. However, 2.4% moisture is typical of inexpensive commercial coarse salt [18]. Iron was added to the ribbon blender as a solid, spray dry microencapsulated, premix of ferrous fumarate (nFe) or as non- encapsulated ferrous fumarate (Fe) so that the salt contained 1000 ppm of iron. Below is a list of salt samples that were made through this process:

Table 4.2.1: Triple Fortified Salt Formulations Salt Sample # Iodine (I) (ppm) Folic Acid (FA) Iron (Fe) (ppm) Encapsulated (ppm) Iron (nFe) (ppm) 1 0 30 0 0 2 30 30 0 0 3 0 0 1000 0 4 0 0 0 1000 5 0 30 1000 0 6 0 30 0 1000 7 30 30 1000 0 8 30 30 0 1000

These salt formulations were used in developing the analytical method for folic acid quantification. They were also stored for 1 year in Zip-LocTM polyethylene bags at ambient conditions in a dark place. The salt samples were tested 1 year after production. It was confirmed that encapsulated iron retained its ferrous form better than non-encapsulated iron. The retention of ferric iron in triple fortified salt with encapsulated iron was 80% ± 1% (Figure 4.2.1). There was no significant change in ferrous iron retention due to folic acid or iodine in the salt. The high concentration of iron relative to iodine or folic acid in the salt may account for this.

100 90 80 70 60 50 Fe(II) 40 nFe(II)

%Retention Fe(II) 30 20 10 0 Fe/nFe FA, Fe/nFe FA, I, Fe/nFe Salt Formulations

Figure 4.2.1: Retention of Ferrous Iron in Triple Fortified Salt After 1 Year of Storage

Contrastingly, iodine retention was greatly affected by iron. Without iron, iodine remained very stable in the salt (100% retention ± 3%). With the addition of encapsulated iron only a small fraction was retained after 1 year of storage (11% ± 1%). When ferrous fumarate was added without encapsulation, iodine was no longer measurable after 1 year of storage (Figure 4.2.2). - The poor iodine retention is mostly due to the ferrous iron and iodate redox reaction (2IO3 (aq) + + 2+ 3+ 12H + 10Fe  I2(s) + 10Fe + 6H2O). The retention of iodine in these capsules was much lower than that reported by Romita (2011) where after six months more than 80% of iodine was retained when salt was stored at high humidity [18]. Romita added only 2.4% moisture whereas I added 2.9% moisture by spraying a solution of iodine and/or folic acid onto the salt. The difference of retention is likely due to a loss of capsule integrity though capsule solubilisation allowing the ferrous fumarate to contact potassium iodate. A way to avoid this is to use more concentrated spray solutions. In industry spray solution concentrations are between 1%-3% w/v which would add only 0.1%-0.3% moisture. As iron is more soluble in acidic environments, using an alkaline buffer solution may increase iodine retention. Less iron is able to come in contact with iodine and react if it is in solid form.

120

100

%Retention I 40

0 FA, I FA, I, nFe FA, I, Fe Salt Formulations

Figure 4.2.2: Retention of Iodine in Triple Fortified Salt After 1 Year of Storage

Folic acid retention in salt was about 100% in salt fortified with just folic acid (105% ± 6%) or with folic acid and iodine (101% ± 6%). Iron seemed to have an effect on folic acid decreasing the retention by >20% in all cases. Although not statistically significant, iodine seems to protect folic acid from iron. Iron encapsulation protected folic acid to some extent. In all formulations folic acid retention was better than 50%. In the salt fortified with encapsulated iron and spray solution containing iron and iodine, the retention was 76% ± 7% after 1 year. The decreased retention of folic acid in salt fortified with iron may be due to the catalytic effect of iron on the oxidation of folic acid, similar to the mechanisms of metal catalyses discussed in section 2.4.3.

120 110 100 90 80 70 60 50

%Retention FA 40 30 20 10 0 FA FA, I FA, Fe FA, nFe FA, I, Fe FA, I, nFe Salt Formulations

Figure 4.2.3: Retention of Folic Acid in Triple Fortified Salt After 1 of Year of Storage

The interaction of iron and folic acid may be decreased by better encapsulation of ferrous fumarate or protection of the coat by limiting moisture. The addition of an overage of folic acid may be used to make up for the observed 24% loss. Since iodine was seen to protect folic acid from degradation, adding more iodine than folic acid would be advantageous. If equal amounts of iodine and folic acid are added to the salt at the MI recommended amount (30 ppm), 125% of the RDA for folic acid and 200% of the RDA for iodine are reached when the expected daily intake (10 g/day) is consumed. Since folate may be found in the natural diet, adding less of it (about 30% RDA), may be more acceptable. Iodine is not found in the natural diet and has a high tolerable upper intake level (733% RDA for adults). Therefore 200% RDA of iodine is safe and remains effective for those who consume less salt than estimated.

Therefore, triple fortification of salt seems feasible if moisture content is controlled or capsules were reformulated to be more resilient in high moisture conditions. Lowering moisture may be accomplished by using higher concentration spray solutions (1%-3% w/v of micronutrients). Also spray solutions buffered to pH 9, the optimal pH determined by Sangakkara (2011), may protect iodine and folic acid from ferrous fumarate because of its lower solubility in basic solutions [17].

4.3 Double Fortified Salt 4.3.1 Optimization of Spray Solution Formulations Angjalie Sangakkara (2011) developed a folic acid and iodine spray solution using a 0.1 M carbonate/bicarbonate buffered solution [17]. She found that pH 9 was optimal for the retention of folic acid. However the solutions used were at 0.35% w/v folic acid and iodine. Since industry uses 1% to 3% solutions of iodine, it is advantageous to use these same concentrations in these spray solutions. Folic acid is a weak acid and thus will lower the pH of basic solutions. Sangakkara chose 0.35% w/v folic acid so that it would not cause too large a pH reduction. In this project first a 0.1 M buffer carbonate/bicarbonate buffer was attempted. Solutions of 0.1 M sodium carbonate and 0.1M sodium bicarbonate were made. They were added together in a range of volumetric ratios from 100% v/v carbonate to 100% v/v bicarbonate. Folic acid was added to these solutions at a concentration of 1% to 3% w/v. For further method details see Appendix 9.5. Only one solution reached pH 9 and was suitable (see Figure 4.3.1). This solution was 1% w/v folic acid, 80% v/v 0.1 M carbonate, and 20% v/v 0.1 M bicarbonate. Because of this a 0.2 M strength buffer was attempted using the same methodology (see Figure 4.3.2).

10 9.5 9

8.5

8 pH 1% Folic Acid 7.5 2% Folic Acid 7 3% Folic Acid 6.5 6 0 20 40 60 80 100 Volume % Carbonate

0.1 M Buffer

Figure 4.3.1: 0.1 M Carbonate/Bicarbonate Spray Solution Selection

10.5 10 9.5 9

8.5

pH 8 1% Folic Acid 7.5 2% Folic Acid 7 3% Folic Acid 6.5 6 0 20 40 60 80 100 Volume % Carbonate 0.2 M Buffer

Figure 4.3.2: 0.2 M Carbonate/Bicarbonate Spray Solution Selection

The suitable ratios to attain pH 9 with different strength buffers and folic acid concentrations are outlined in Table 4.3.1. Solutions of 0.1 M buffer strength and 2% to 3% folic acid did not reach pH 9 and thus no solutions were acceptable (see Figure 4.3.1). The 0.2 M buffer with 3% folic acid did not reach pH 9 but was close (pH 8.84) when 100% of the carbonate solution was used (see Figure 4.3.2).

Table 4.3.1: Ratio of Carbonate to Bicarbonate Required For pH 9 Solution Buffer Strength (M) % w/v Folic Acid % v/v Carbonate % v/v Bicarbonate 0.1 1 80 20 0.1 2 N/A N/A 0.1 3 N/A N/A 0.2 1 50 50 0.2 2 80 20 0.2 3 100* 0* *Did not reach pH 9, however was close at pH 8.84

The final spray solutions were produced using the carbonate to bicarbonate ratios determined to raise the pH to 9 in the folic acid solutions mentioned previously. Table 4.3.2 lists the final spray solution formulations their initial pH. Solutions were made with less than or equal amounts of folic acid to iodine. As mentioned previously, the target concentration recommended by MI, based on a per capita salt consumption of 10 g/day, was 30 ppm of both iodine and folic acid. This is double the RDA of iodine for an adult but still much below the tolerable upper

46 intake level of 1100 μg/day and is therefore safe. It is 125% of the RDA of folic acid for an adult but because folic acid is present in the natural diet a lower concentration (providing ~ 30% RDA) may be beneficial.

Table 4.3.2: Final Spray Solution Formulations Buffer Strength (M) % w/v Folic Acid % w/v Iodine Initial pH (Month 0) 0.1 1 1 9.0 0.1 1 2 9.0 0.1 1 3 9.0 0.2 1 1 9.1 0.2 1 2 9.1 0.2 1 3 9.0 0.2 2 2 9.0 0.2 3 3 8.7

4.3.2 Stability of Spray Solutions Spray solutions must remain stable for 2-3 months as this is the maximum time they would be in use in a plant. I monitored the pH, as it is tied to the stability of folic acid. As can be seen in Table 4.3.3 the pH was stable for a 2 month period.

Table 4.3.3: pH Stability of Spray Solutions Formulation Month 0 Month 1 Month 2 Month 1 Month 2 pH pH (25ºC) pH (25ºC) pH (45ºC) pH (45ºC) 1% FA, 1% I, 0.1 M Buffer 9.0 8.9 8.9 9.0 9.1 1% FA, 2% I, 0.1 M Buffer 9.0 9.0 9.0 9.0 9.1 1% FA, 3% I, 0.1 M Buffer 9.0 9.0 9.0 9.0 9.0 1% FA, 1% I, 0.2 M Buffer 9.1 9.1 9.1 9.1 9.1 1% FA, 2% I, 0.2 M Buffer 9.1 9.1 9.1 9.0 9.1 1% FA, 3% I, 0.2 M Buffer 9.0 9.0 9.0 9.0 9.1 2% FA, 2% I, 0.2 M Buffer 9.0 9.0 9.0 9.0 9.1 3% FA, 3% I, 0.2 M Buffer 8.7 8.7 8.7 8.8 8.9

The folic acid and iodine in the spray solutions were stable over a 5 month period (Figures 4.3.4-4.3.7). However, the 3% folic acid spray solution formed a precipitate when stored at 45ºC for 2 months (see Figure 4.3.8). For images of all spray solutions at month 2 see Appendix 9.5, Figures 9.5.1-9.5.2. By 5 months the 2% and 3% folic acid solutions stored at both 45ºC and 25ºC contained precipitates. The spray solution is generally replaced every month however to ensure stability a two month goal was set. Therefore the 3% folic acid solution is unsuitable. The 1%-3% iodine solutions caused no issues; therefore the formulation may be adjusted to

47 provide different ratios of iodine to folic acid (range from 42.7%-125% RDA of folic acid) with the addition of 0.1%-0.3% moisture.

180 160

140

120 100 Month 0 80

60 Month 2 %Retention FA 40 Month 4 20 Month 5 0 1% FA, 1% FA, 1% FA, 1% FA, 1% FA, 1% FA, 2% FA, 3% FA, 1% I, 2% I, 3% I, 1% I, 2% I, 3% I, 2% I, 3% I, 0.1M 0.1M 0.1M 0.2M 0.2M 0.2M 0.2M 0.2M Formulations

Figure 4.3.4: Retention of Folic Acid in Spray Solutions Stored at 25ºC

160 140

120 100

80 Month 0

60 Month 1 %Retention FA 40 Month 4 20 Month 5 0 1% FA, 1% FA, 1% FA, 1% FA, 1% FA, 1% FA, 2% FA, 3% FA, 1% I, 2% I, 3% I, 1% I, 2% I, 3% I, 2% I, 3% I, 0.1M 0.1M 0.1M 0.2M 0.2M 0.2M 0.2M 0.2M Formulations

Figure 4.3.5: Retention of Folic Acid in Spray Solutions Stored at 45ºC

Retentions of folic acid measured to be higher than 100% were due to an error that occurred during the first reaction prior to the samples being split into replicates. In some of these samples HCl used in the first reaction of the SBCM caused degradation of p-ABGA, the reaction

48 product. Samples left in the HCl for a few minutes shorter time had elevated readings because this degradation occurred to a lesser extent than in the calibration curve solutions.

140

120

100

80 Month 0 60 Month 1 %Retention I 40 Month 2 20 Month 5 0 1% FA, 1% FA, 1% FA, 1% FA, 1% FA, 1% FA, 2% FA, 3% FA, 1% I, 2% I, 3% I, 1% I, 2% I, 3% I, 2% I, 3% I, 0.1M 0.1M 0.1M 0.2M 0.2M 0.2M 0.2M 0.2M Formulations

Figure 4.3.6: Retention of Iodine in Spray Solutions Stored at 25ºC

140

120

100

Figure 4.3.7: Retention of Iodine in Spray Solutions Stored at 45ºC

Storage Temperature: 25ºC Left & 45ºC Right Figure 4.3.8: 3% Iodine/3% Folic Acid Spray Solutions After 2 Months Storage

Folic acid is stable in basic solution because reduction and oxidation are the main modes of degradation at temperatures below 148ºC. The reduction of folic acid is pH dependant and the reductive cleavage reaction only occurs in acidic conditions. In basic pH it is reduced to 5,6- dihyrofolic acid which is a bioavailable form of folate [38]. Oxidization of folic acid was found to occur in the presence of light so samples were stored in the dark until analyzed. Folic acid in basic solution has also been shown to oxidize in the presence of oxidizers. Although potassium iodate is an oxidizing agent, degradation in its presence was not seen and has not been reported in literature.

4.3.3 Stability of Double Fortified Salt Salt was fortified using the following spray solutions:

Table 4.3.4: Spray Solutions Used to Fortify Salt Salt # Carbonate/Bicarbonate Buffer (M) Folic Acid (% w/v) Iodine (% w/v) 1 0.1 1 1 2 0.2 1 1 3 0.2 2 2 4 0.2 3 3

Salt was not fortified with spray solutions containing more iodine than folic acid. Analytical error due to the use of stock HCl caused incorrectly low readings of folic acid in solutions, especially those with >1% w/v iodine. Therefore I believed folic acid did not remain stable during solution production when iodine was present in >1% w/v concentrations. The observed

50 retentions of folic acid in the spray solutions immediately after they were produced are given in Table 4.3.2. Solutions with an observed retentions ≥80% were selected for use in salt fortification. However, now that the analytical method has been rectified it is known that the spray solutions with higher iodine to folic acid ratios were stable as well. The error due to stock solution HCl is discussed further in section 4.1.2. Because these results were due to an analytical error and folic acid was found to be stable in the presence of iodine, salt may be fortified using 1% w/v folic acid and 2%-3% iodine as well.

Table 4.3.5: Observed (Incorrect) Retention of Folic Acid in Spray Solutions (Time 0) Formulation Initial % Retention of Folic Acid Observed in Spray Solutions 1% FA, 1% I, 0.1 M Buffer 98 ± 3 1% FA, 2% I, 0.1 M Buffer 69 ± 6 1% FA, 3% I, 0.1 M Buffer 47 ± 1 1% FA, 1% I, 0.2 M Buffer 93 ± 3 1% FA, 2% I, 0.2 M Buffer 64 ± 3 1% FA, 3% I, 0.2 M Buffer 43 ± 2 2% FA, 2% I, 0.2 M Buffer 80 ± 2 3% FA, 3% I, 0.2 M Buffer 88 ± 2

Salt was fortified with 50 ppm of each micronutrient by altering the amount of each solution sprayed onto the salt. Therefore the higher the micronutrient concentration, the less water was added to the salt during fortification. The salt was stored at ambient conditions in Zip-LocTM polyethylene bags. Initial folic acid tests resulted in inaccurately high readings (see section 4.1.2 for further description). Therefore, time was spent on resolving the analytical method issue. It was determined that recalibration could be used to rectify the inaccurate results from the salt. After the analytical method was corrected, time constraints prevented the production of double fortified salt stored at high temperature and humidity.

All salts fortified with these spray solutions were stable over the 5 month test period. There was no significant loss of folic acid or iodine over five months in all formulations tested (see Figures 4.4.9-4.4.10). Clearly, the slight increase in moisture due to the addition of the micronutrients did not affect the stability. Solid folic acid is very stable [52] Therefore, once the salt was dry folic acid content was expected to remain stable.

140

120

100

80 Month 0

60 Month 1

%Retention FA Month 3 40 Month 5 20

0 1%FA, 1%I, 0.1M 1%FA, 1%I, 0.2M 2%FA, 2%I, 0.2M 3%FA, 3%I, 0.2M Spray Solution Formulations Used to Fortify Salt

Figure 4.4.9: Retention of Folic Acid in Double Fortified Salt

120

100

80 Month 0 60 Month 1 Month 2 %Retention I 40 Month 4

20 Month 5

0 1% FA, 1% I, 0.1M 1% FA, 1% I, 0.2M 2% FA, 2% I, 0.2M 3% FA, 3% I, 0.2M Spray Solution Formulations Used to Fortify Salt

Figure 4.4.10: Retention of Iodine in Double Fortified Salt

5 Conclusions

The objective of this project was to develop a process for double and triple fortifying salt using existing equipment and technologies used commercially in developing countries. Double fortified salt was to contain iodine (potassium iodate) and folic acid. Triple fortified salt was to contain these and iron (ferrous fumarate). Spray fortification is the method most common for salt iodization in developing countries and iron microcapsules developed by Romita (2011) were compatible with coarse unrefined salt common in developing countries [15] [18]. Thus spray solutions for iodine and folic acid application was developed followed by an investigation of triple fortified salt using iron microcapsules. The main findings of this study are the following:

1. Spray solutions containing 1%-3% w/v folic acid and 1%-3% w/v iodine (as KIO3) buffered to pH 9 using a carbonate-bicarbonate buffer were developed. These solutions have iodine content similar to what is used commercially. The solution containing 3% w/v folic acid formed a precipitate after 2 months of storage at 45ºC and is not acceptable. Iodine was stable in solutions at concentrations up to 3%. Folic acid and iodine retention was ≥80% after 5 months for all formulations. Salt fortified with these spray solutions retained both folic acid and iodine 100% over a 5 month period when stored at ambient conditions. Therefore this system is ready to be tested on larger scales.

2. A reliable analytical method for stability testing folic acid in spray solutions, double fortified salt, and triple fortified salt was developed. The method is based on that of Hutchings et al. (1974) and Nagaraja et al. (2002). In this method folic acid undergoes a series of three reactions leading to a coloured product which is measured using spectrophotometry [40] [43]. Modifications include folic acid extraction, decreasing sample dilution, and testing non-reduced samples. This method distinguishes between folic acid and its degradation product p-ABGA.

3. The work of Sangakkara (2011) was validated though demonstration that p-ABGA is only slightly produced, if at all, in spray solutions or in double fortified salt. Sangakkara found folic acid to be stable in spray solutions and double fortified salt using analytical methods that did not distinguish between folic acid and p-ABGA [17]. Salt fortified by Sangakkara was tested for p-ABGA after 1 year of storage resulting in little to no significant production.

Double fortified salt and spray solutions I produced were tested for p-ABGA after 5 months of storage and observed conversion into p-ABGA was in an acceptable range of ~10%.

4. Triple fortified salt using folic acid and iodine (KIO3) sprayed onto salt from a water mixture, microcapsules produced by Romita of ferrous fumarate (spray dried with coating of 80% w/w dextrin and 20% w/w HPMC), and 2.9% moisture was not stable when stored for 1 year in ambient conditions. The iron encapsulated system developed by Romita (2011) is not effective at high moisture levels [18]. The capsules degrade allowing the iron to react with iodine (~10% retention) and to some extent folic acid (~75% retention). Therefore a better encapsulant is needed.

6 Recommendations

1. Test the processing of double fortified salt using 1% w/v folic acid and 3% w/v iodine (as

KIO3) on a pilot scale, and determine the stability of the double fortified salt under local conditions.

2. Further develop the coating system used for iron addition to ensure its stability under the moisture and pH conditions required for folic acid and iodine addition.

7 References

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8 Nomenclature

3-AP 3-Aminophenol 5-MTHF Methyl-5,6,7,8-tetrahydrofolic acid ACS American Chemical Society AI Adequate Intake ANALEST Analytical Lab for Environmental Science Research and Training (University of Toronto) AOAC The Association of Official Analytical Chemists AUFS Absorbance Units Full Scale DFE Dietary Folate Equivalent DNA Deoxyribonucleic Acid

Eo Standard Redox Potential o ΔH Standard Enthalpy Difference FA Folic Acid Fe Iron FOP Ferric Orthophosphate CFPP Ferric Pyrophosphate HCl Hydrochloric Acid HPLC High Performance Liquid Chromatography I Iodine IQ Intelligence Quotient MI Micronutrient Initiative nFe Encapsulated Iron NTD Neural Tube Birth Defects p-ABGA para-Aminobenzoyl Glutamic Acid RDA Recommended Dietary Allowance RO Reverse Osmosis SAS Sodium Acid Sulfate SBCM Spectrophotometry-based Coupling Method SHMP Sodium Hexametaphosphate UNICEF United Nations Children’s Fund UV/Vis Ultraviolet Light/Visual Light

9 Appendices Appendix 9.1: Analytical Determination of Iodine 9.1.1 Solution Preparation

1) 0.005 N Na2S2O3: 50 mL of 0.1 N Na2S2O3 was diluted to 1 L with RO water.

2) 0.00125 N Na2S2O3: 12.5 mL of 0.1 N Na2S2O3 was diluted to 1 L with RO water.

3) 2 N H2SO4: 800 mL of RO water was added to a 1 L volumetric flask. 56 mL of

concentrated H2SO4 was then added and diluted to the line with RO water.

4) 0.2 N H2SO4: 200 mL of RO water was added to a 250 L volumetric flask. 1.4 mL of

concentrated H2SO4 was then added and diluted to the line with RO water.

5) 0.05% KIO3: 1.0000 g of KIO3 was dissolved in 100 mL of RO water. 5 mL of this solution was diluted to 100 mL. 6) 2% KI: 10 g of KI was dissolved and diluted to 500 mL with RO water.

9.1.2 Standardization

1) 2 mL of the 0.05% KIO3 was diluted with approximately 100 mL of RO water in a 500 mL Erlenmeyer flask. This was repeated for each of 4 replicates.

2) 2 mL of the 0.2 N H2SO4 and 2% KI solutions were added to the flasks and mixed well. 3) Yellow colour was allowed to develop for 10 minutes in a dark place.

4) Solutions were titrated with 0.005 N Na2S2O3, to standardize for spray solutions, or the

0.00125 N Na2S2O3, to standardize for the salt samples. 5) Once yellow colour was almost gone a few drops of 1% starch indicator were added. 6) The solution was titrated until the blue colour disappeared. For iron solutions titration continued until the purple colour became stable (no longer getting lighter).

9.1.3 Spray Solution Analysis 1) 5 mL of the spray solutions were diluted to 100 mL with RO water. 2) 2 mL of these solutions were added to approximately 100 mL of RO water in a 500 mL Erlenmeyer flask. This was repeated for 4 replicates.

3) Steps 2-6 from the 9.1.2 Standardization procedure were repeated using 0.005 N Na2S2O3 as the titrant.

9.1.4 Salt Sample Analysis 1) 5 g of the salt sample samples were added to approximately 100 L of RO water in a 500 mL Erlenmeyer flask. This was repeated for 4 replicates.

2) Steps 2-6 from the 9.1.2 Standardization procedure were repeated using 0.00125 N Na2S2O3 as the titrant.

9.1.5 Calculation of Iodine Content

Mass KIO3 per volume of Na2S2O3 (Strength):

Strength (μgI/mLNa2S2O3)

= ((0.05/100)g/mL* 1,000,000μg/g * 2mL* 0.593 gI/gKIO3) / XmLNa2S2O3 X = volume of titrant used

Concentration I in spray solutions ([I]solution) (ppm):

[I]solution (μgI/mLsolution) = Strength (μgI/mLNa2S2O3) * XmLNa2S2O3 / 2mLSolution

Concentration I in salt ([I]salt) (ppm):

[I]salt (μgI//gsalt) = Strength (μgI/mLNa2S2O3) * XmLNa2S2O3 / 5gsalt

Appendix 9.2: Analytical Determination of Iron 9.2.1 Solution Preparation 1) Reacting Solution (0.3% 1,10-Phenanthroline Monohydrate): 3 g of 1,10-phenanthroline monohydrate powder was added to a 1 L Erlenmeyer flask with 500 mL of RO water and a magnetic stir bar. The flask was heated on low and stirred on a hot plate for 10 minutes (until all crystals dissolved). The solution was allowed to cool to room temperature then transferred to a 1 L volumetric flask where it was diluted to the mark with RO water. 2) Buffer Solution (4.085% Potassium Hydrogen Phthalate): 40.85 g of potassium hydrogen phthalate powder was added to a 1 L Erlenmeyer flask with 500 mL of RO water and a magnetic stir bar. The flask was heated and stirred for 10 minutes (until all crystals dissolved). The solution was allowed to cool to room temperature then transferred to a 1 L volumetric flask were it was diluted to the mark with RO water. 3) Reducing Solution (10% Hydroxylamine Hydrochloride): 10 g of hydroxylamine hydrochloride was dissolved in approximately 30 mL of RO water in a 100 mL volumetric flask then diluted to the mark with RO water. 4) Stock Solution (1000ppm Iron): 7.0213 g of ACS grade ferrous ammonium sulfate hexahydrate was dissolved in approximately 200 mL of RO water which contained 3 mL of concentrated sulphuric acid. Solution was diluted to 1 L with RO water in a volumetric flask. 5) Working Solution (100 ppm iron): 10 mL of the 1000 ppm iron stock solution was diluted to 100 mL with RO water in a volumetric flask.

9.2.2 Calibration Curve Preparation 1) Standard iron(II) solutions were prepared by adding the following amounts of working solution to 25 mL volumetric flasks (Table 9.2.1). Four replicates were used.

Table 9.2.1: Working Solution Dilutions for Iron Calibration Curve Standard Solution Concentration (ppm) Volume of Working Solution (mL) 0 0.0 2 0.5 4 1.0 6 1.5 8 2.0 10 2.5

2) 5 mL of buffer solution followed by 10 mL of reacting solution was added to each flask. 3) The flasks were then filled to the mark with RO water. 4) The absorbance of each solution was measured at 512 nm by UV/Vis spectrophotometry and the concentration was plotted against absorbance data. A sample calibration curve is shown below in Figure 9.2.1.

2.5

1.5 1 y = 0.2034x 0.5 Absorbance R² = 0.9998 0 -0.5 0 2 4 6 8 10 12 Concentration of Fe(II) (ppm)

*Standard deviations too small to be visible. Figure 9.2.1: Sample Calibration Curve

9.2.3 Salt Sample Analysis 1) Approximately 10 g of fortified salt (1000 ppm iron) was put in a 125mL Erlenmeyer flask and the exact weight was recorded. Four replicates for each sample. 2) Approximately 40 mL of RO water was added to each flask with a few boiling stones and 1 mL of concentrated sulfuric acid. 3) The Erlenmeyer flasks were heated until the solution boiled and kept on low heat for 10 minutes (until particles dissolve). 4) The solutions were allowed to cool to room temperature and transferred into 100mL volumetric flasks and diluted to the mark with RO water. 5) 1 mL of the sample solution from the 100 mL flask was pipetted into a 25 mL volumetric flask. 6) 5 mL of buffer solution and 10 mL of reaction solution were added to the 25mL volumetric flasks and then filled to the mark with RO water. These were used to test ferrous iron content. 7) Step 5 was repeated followed by the addition of 1mL of reducing solution and then step 6 was repeated. These were used to test total iron content. 8) The absorbance of each solution was found at 512 nm using UV/Vis spectrophotometry.

9.2.4 Calculation of Iron Content 2+ Ferrous iron (Fe step 6 solutions) and Total iron (FeT step 7 solutions) content:

Concentration in 25mL Flask ([Fe]F):

[Fe]F (μg/mL) = Absorbance/Slope of Calibration Curve

Concentration in Salt ([Fe]salt (ppm)):

[Fe]salt (μg/gsalt) = [Fe]F (μg/mL) * (25 * 100)mL / Mass of Salt Used (g) Ferric iron (Fe3+) content: 3+ Concentration in Salt ([Fe ]salt (ppm)): 3+ 2+ [Fe ]salt (μg/gsalt) = [FeT]salt (μg/gsalt) - [Fe ]salt (μg/gsalt)

Appendix 9.3: Analytical Determination of Folic Acid by HPLC 9.3.1 Detailed Sample Preparation Methods 9.3.1.1 Solution Preparation Methods 1) 5 N HCl: 200 mL of RO water was added to a 500 mL volumetric flask. 215.5mL of concentrated HCl was added to the flask and RO water was added to the mark. 2) 0.180 M Citrate/Phosphate Buffer (pH 6.94): 3.391g of citric acid was put into a 1 L

volumetric flask. 23.384 g of sodium phosphate dibasic (Na2HPO4) was also added to the volumetric flask. RO water was added up to the mark and the chemicals were dissolved and mixed. 3) 0.018 M Citrate/Phosphate Buffer: 100 mL of the 0.180 M citrate/phosphate buffer was diluted to 1 L using RO water in a volumetric flask. 4) 20 mM Hexane Sulfonic Acid and 0.1% Phosphoric Acid, pH 2.2: Prepared by ANALEST. 20 mM solution of hexane sulfonic acid was made using sodium hexane sulfonic acid in HPLC grade water. The pH was then adjusted to 2.2 with the addition of 0.1% phosphoric acid. 5) 1000 ppm Folic Acid: 0.1 g of folic acid was diluted to 100 mL using RO water or 0.018 M citrate/phosphate buffer. 6) 500 ppm Folic Acid: 0.05 g of folic acid was diluted to 100 mL using RO water or 0.018 M citrate/phosphate buffer. 7) 25 ppm p-ABGA: 2-4 mL of 1000 ppm or 500 ppm folic acid was added to a jar. 1-4 g of zinc and 10 mL-40 mL of 5 N HCl was added. The reacting mixtures were kept out of bright light and mixed every five minutes for 20-30 minutes. RO water and/or methanol were added to quench the reaction and dilute samples to 25 ppm. HPLC was able to determine if the reaction had gone to completion. 8) Folic Acid/p-ABGA Mixture: Dilutions of the 1000 ppm folic acid solution and 25 ppm p- ABGA solution were mixed together. 9) Methanol (MeOH)/Water (25/75 v/v): 250 mL of methanol was measured and diluted to 1000 mL using RO water in a volumetric flask. For other ratios the corresponding amounts of methanol were diluted to 1000 mL with RO water. These solutions were used to make calibration curves when attempting to extract folic acid from salt using methanol and water.

9.3.1.2 Calibration Curve Preparation Solutions of 25 ppm p-ABGA and 1000 ppm folic acid were diluted to specific concentrations using water, methanol, and/or 0.018 M citrate/phosphate buffer. They were then run through HPLC with UV detection (280 nm). Peak height and area were plotted against the known concentrations to create calibration curves. Peak area was chosen to calculate concentrations in samples because it had smaller standard deviations than peak height.

9.3.1.2 Salt Sample Preparation Method 1: Dissolution of Salt 1 g of salt was dissolved in 3mL of diluted solvent (0.018 M citric acid/phosphate buffer) or water. The difference in density between the calibration solutions which did not contain salt and the sample salt solutions was required because HPLC injects sample at a constant volume. Thus the calibration curve solutions would be less dense than the salt sample solutions, so although the concentration in mg/L is correct, it is incorrect to convert to ppm without taking into account the change in density.

Calibration Curve Solution Density = 1.02 g/mL ± 0.02 Salt Sample Solution Density = 1.21 g/mL ± 0.02

Densities measured by weighing specific volumes of solutions.

Method 2: Extraction from Salt Extraction Method 1: 10 g of salt was put into a jar and 8 mL of MeOH was added. This was mixed well for 5 minutes. 2 mL of the MeOH was then removed and put into a vial. 12 mL of RO water was then added.

Extraction Method 2: 10 g of salt was put into a test tube and 9 mL of MeOH was added. The vortex was used to stir the test tube every 30 seconds for 10 minutes. 1.5 mL of the MeOH was then removed and put into a vial. 3.5 mL of RO water was then added. This may or may not be followed by filtration through a 5 μm filter to remove iron.

9.3.2 Detailed HPLC with UV Detection Methods The methods used are based on a method outlined in the Symmetry® Columns Application Notebook [78, p. 181]. This was selected during past undergraduate work because it uses a simple C18 column, a simple isocratic mobile phase, and materials (mobile phase and column) were donated for use by ANALEST [79].

In both methods used, the conversion to absorbance units (AU) from the readout is as follows: Conversion to AU: readout: height (μV) or area (μV*s) height (AU) = (readout (μV) / 1,000,000 μV/V) * 0.1 AU/V area (AU*s) = (readout (μV*s) / 1,000,000 μV/V) * 0.1 AU/V

9.3.2.1 HPLC/UV Method 1 Column: CSC-Intersil 150A/ODS2, 5μm (4.6 mm, 100 mm) Mobile Phase: (A) 20 mM hexane sulfonic acid and 0.1% phosphoric acid, pH 2.2 (B) Methanol Gradient: Isocratic (A/B  75/25) Temperature: 40ºC Flow Rate: 0.7 mL/min Detection UV: 280 nm Injection Volume: 20 μL Range: 0.1 AUFS

9.3.2.2 HPLC/UV Method 2 Column: Waters Symmetry Column, 5 μm (4.6 mm, 150 mm) Mobile Phase: (A) 20mM hexane sulfonic acid and 0.1% phosphoric acid, pH 2.2 (B) Methanol Gradient: Isocratic (A/B  70/30) Temperature: 40ºC Flow Rate: 1.0 mL/min Detection UV: 280 nm Injection Volume: 20 μL Range: 0.1 AUFS

Conversion to AU: readout: height (μV) or area (μV*s) height (AU) = (readout (μV) / 1,000,000 μV/V) * 0.1 AU/V area (AU*s) = (readout (μV*s) / 1,000,000 μV/V) * 0.1 AU/V

9.3.3 Calculation of Folic Acid Content

[FAsampleH] = Concentration of Folic Acid in Sample Solution using Peak Height

[FAsampleA] = Concentration of Folic Acid in Sample Solution using Peak Area

[FAsample] = Concentration of Folic Acid in Sample Solution

[FAsalt] = Concentration of Folic Acid in Extract

[FAsalt] = Concentration of Folic Acid on Salt

[FAsampleH] (mg/L) = (Peak Height – Intercept of Height Calibration Curve)/Slope of Height Calibration Curve

[FAsampleA] (mg/L) = (Peak Area – Intercept of Area Calibration Curve)/Slope of Area Calibration Curve

9.3.3.1 Dilution Method

[FAsalt] (ppm) = [FAsample] * Volume of Sample (mL) / Mass of Salt (kg) Volume of Sample = Density of Sample * Mass of Sample

9.3.3.2 Extraction Method

[FAextract] (mg/L) = [FAsample] * Volume of Sample (mL) / Volume of Extract (mL)

[FAsalt] (ppm) = [FAextract] * Volume of MeOH Added to Salt (mL) / Mass of Salt (kg)

9.3.4 HPLC Successes 9.3.4.1 Calibration Curves

250000

200000

150000

V*s) μ 100000 y = 18208x - 1559.3 Area( 50000 R² = 0.9999 0 0 2 4 6 8 10 12 Concentration (mg/L) Figure 9.3.1: p-ABGA HPLC Calibration Curve Using Area (7 replicates)

25000

20000

V) μ 15000 10000

Height Height ( y = 1864.3x - 83.135 5000 R² = 0.9997 0 0 2 4 6 8 10 12 Concentration (mg/L) Figure 9.3.2: p-ABGA HPLC Calibration Curve Using Height (7 replicates)

The p-ABGA calibration curves using both area and height have similar standard deviations relative to the measurements. However the R2 value for area is slightly closer to 1.

1400000 1200000 1000000

V*s) 800000 μ 600000

Area ( Area 400000 y = 80774x - 2905.3 200000 R² = 0.9999 0 0 5 10 15 20 Concentration (mg/L) Figure 9.3.3: Folic Acid HPLC Calibration Curve Using Area (8 replicates)

80000

60000

V) μ 40000

y = 4501.1x - 604.24 Height ( 20000 R² = 0.998 0 0 5 10 15 20 Concentration (mg/L)

Figure 9.3.4: Folic Acid HPLC Calibration Curve Using Height (8 replicates)

The folic acid calibration curve using height has much larger standard deviations relative to the measured values than the calibration curve using area. Its R2 is also further from 1. Therefore the calibration curve using height will result in less precise sample concentrations.

9.3.4.2 Separation of Folic Acid and p-ABGA Separation of folic acid from p-ABGA was attained using both HPLC methods outlined in sections 9.3.2.1 (Method 1) and 9.3.2.2 (Method 2).

Figure 9.3.5: HPLC Separation – HPLC/UV Method 1 (p-ABGA 15ppm & Folic Acid 5ppm)

Figure 9.3.6: HPLC Separation – HPLC/UV Method 2 (p-ABGA 10ppm & Folic Acid 10ppm)

9.3.4.3 Linear Range The linear range for folic acid was determined to be higher than 100 ppm. The linear range for p-ABGA was found to be much lower at about 15 ppm. This can be seen in Figures 9.3.7- 9.3.10.

10000000

8000000

V*s) 6000000 μ 4000000

Area( y = 81957x + 10914 2000000 R² = 0.9999 0 0 50 100 150 Folic Acid Concentration (ppm)

Figure 9.3.7: Linear Range Determination of Folic Acid Using Area (3 Replicates)

500000

400000

μ 300000 200000

Height Height ( y = 3763x - 1024.7 100000 R² = 0.9999 0 0 50 100 150 Folic Acid Concentration (ppm) Figure 9.3.8: Linear Range Determination of Folic Acid Using Height (3 Replicates)

500000

400000

300000

V*s) 200000 μ

100000 Area( 0 0 5 10 15 20 25 30 Concentration (ppm) Figure 9.3.9: Linear Range Determination of p-ABGA Using Area (4 Replicates)

50000

40000

30000

μ 20000

10000 Height Height ( 0 0 5 10 15 20 25 30 Concentration (ppm) Figure 9.3.10: Linear Range Determination of p-ABGA Using Height (4 Replicates)

9.3.4.4 Adding Standard Amounts of Folic Acid The amount added (or expected amount) of folic acid was very close to the amount measured using peak height as can be seen in Figure 9.3.15.

4 Expected Measured 3

1 Folic Folic Concentration Acid Difference (ppm) 0 Salt #1 Salt #8

Salt #1: Spray Solution pH 2.21; 30ppm Folic Acid/ 30ppm Iodine  1 year storage Salt #8: Spray Solution pH 10.04; 30ppm Folic Acid/ 30ppm Iodine  1 year storage Figure 9.3.15: Folic Acid Addition to Fortified Salt Measured Using Peak Height

9.3.5 HPLC Underperformances 9.3.5.1 HPLC Salt Peaks When salt was run through HPLC with UV detection at 280 nm it caused an absorbance at the beginning of the readout (see Figures 9.3.16-9.3.17). This peak increased in size with an increase in salt concentration (see Figures 9.3.18-9.3.19).

Figure 9.3.16: HPLC Salt Peak from Sodium Chloride Solution

Figure 9.3.17: HPLC Salt and Folic Acid Peaks from a Mixture

500000 400000

300000 V*sec) μ 200000 y = 15124x + 13559 Area( 100000 R² = 0.9848 0 0 5 10 15 20 25 30 Salt Concentration (% Weight)

Figure 9.3.18: Salt Peak Area

60000

50000 V)

μ 40000 30000 20000 Height Height ( y = 1855.3x + 3698.1 10000 R² = 0.9291 0 0 5 10 15 20 25 30 Salt Concentration (% Weight)

Figure 9.3.19: Salt Peak Height

9.3.5.2 Folic Acid Reading is Salt Content Dependant

1400000 y = 80774x - 2905.3 1200000 R² = 0.9999 1000000

800000 y = 56381x - 224873

V*sec) 600000

μ R² = 0.9911 400000

Area( No Salt 200000 With Salt 0 0 2 4 6 8 10 12 14 16 Concentration (ppm)

Figure 9.3.20: Salt Skew of Folic Acid HPLC Calibration Curve using Area

80000 70000 y = 4501.1x - 604.24 R² = 0.998 60000

50000 V)

μ y = 3575.1x - 11907 40000 R² = 0.995 30000

Height Height ( 20000 No Salt 10000 With Salt 0 0 2 4 6 8 10 12 14 16 Concentration (ppm)

Figure 9.3.21: Salt Skew of Folic Acid HPLC Calibration Curve using Height

9.3.5.3 Low Readings for Folic Acid Extracted from Salt Extraction was carried out according to methods outlined in section 9.3.1.2.

Measured 15 Concentration (Area) 10 Measured Measured(ppm) Concentration

5 (Height) Concentration ofFolic Acid

0 10 20 30 Concentration of Folic Acid Added to Salt (ppm)

Figure 9.3.22: Folic Acid Reading After HPLC Extraction Method 1

25 Measured 20 Concentration (Area) 15 Measured

10 Concentration Measured(ppm)

5 (Height) Concentration of Folic Folic of ConcentrationAcid 0 10 20 30 -5 Concentration of Folic Acid Added to Salt (ppm) Figure 9.3.23: Folic Acid Reading After HPLC Extraction Method 2 (No Filter)

Using the extraction method 1 was not effective in extracting the folic acid. Extraction method 2 was a bit better but always came out about 2.5 ppm short. When iron was added to the salt additional filtration was required also. Graphs showing the outcome of that are below.

70000

60000

50000

V*s) 40000 μ

30000 Area( 20000

10000

0 FA & I FA & Fe FA & nFe FA, I, Fe FA, I, nFe Salt Formulations

Figure 9.3.24: Folic Acid Reading After HPLC Extraction Method 2 (5 μm Filter)

If the amounts are calculated using a calibration curve they are all negative because the intercept is higher than what was read for these samples.

1000000 900000 800000

700000

600000 V*s) μ 500000 filtered 400000 Area( 300000 not filtered 200000 100000 0 0 20 40 60 80 100 120 Concentration FA Added

Figure 9.3.25: Folic Acid Readings’ Dependence on Filtration

When filtered, folic acid does not entirely pass through the filter. The larger the amount of folic acid in the solution the larger the amount that passes through the filter because more is in solution. However this reaches a plateau as the solution reaches full capacity.

9.3.5.4 Peaks of p-ABGA

Figure 9.3.26: p-ABGA Chromatograph

Figure 9.3.27: p-ABGA and Folic Acid Chromatograph

9.3.5.5 Folic Acid Detection Erroneously High in Double Fortified Salt Folic acid results were much too high in salt fortified with pH 8-10 buffered solutions and stored for 1 year (see Figure 9.3.28).

140

120

100

80 Area 25C Height 25C 60 Area 45C Height 45C 40

Concentration(ppm) 20

0 2.21 3.03 4.07 5.98 7.02 7.76 9.27 10.04 pH of Spray Solution Figure 9.3.28: Folic Acid in Double Fortified Salt Stored for 1 Year (25ºC or 45ºC)

The readings should all be less than 30 ppm as this was the amount the salt was fortified with initially. It was thought that the pH of the spray solution was influencing the pH of the sample solutions. Therefore their pH was measured against blank salt.

7 6 5 4 3 2 pH of Salt of pH Salt HPLC Sample 1 0 Blank 2.21 3.03 4.07 5.98 7.02 7.76 9.27 10.04 pH of Spray Solution

Figure 9.3.29: pH of HPLC Salt Sample Solutions

The pH of the salt sample solutions was between pH 5.5 and 6 (Figure 9.3.29). This is only a small variation so it is unlikely to be the factor contributing to the high readings of salts fortified with spray solutions of pH 8-10.

Appendix 9.4: Analytical Determination of Folic Acid by SBCM 9.4.1 Solution Preparation 1) 5 N HCl: 200 mL of RO water was added to a 500 mL volumetric flask. 215.5 mL of concentrated HCl was added to the flask and RO water was added to the mark.

2) 2% NaNO2: 10 g of NaNO2 was dissolved in 500 mL of RO water. 3) 4% Sulfamic Acid: 20 g of sulfamic acid was dissolved in 500 mL of RO water. 4) 1% 3-Aminophenol (3-AP): 2.5 g of 3-AP was dissolved in 250 mL of RO water. It was made each day of testing and kept in the dark. 5) 500 ppm Folic Acid: 0.05 g of folic acid was suspended in 100 mL of water. 6) 1000 ppm Folic Acid: 0.1 g of folic acid was suspended in 100 mL of water. 7) 0.1 N NaOH: 4 g of NaOH pellets was dissolved in 1000 mL of water. 8) 50/50 v/v Methanol/NaOH: Equal volumes of methanol and 0.1 N NaOH were mixed together.

9.4.2 Original Procedure 9.4.2.1 Standardization 1) 2 mL of the 500 ppm folic acid was mixed with 1 g of zinc granules and 10 mL of 5 N HCl in a 250 mL glass jar. It was shaken every 5 minutes for 30 minutes to allow for the reductive cleavage reaction to go to completion. 2) 28 mL of RO water was added to the jar to get the final volume to 40 mL. The jar was shaken and zinc granules were allowed to settle to the bottom. 3) 0, 1, 2, 3, 4, 5, and 6 mL (corresponding to 0-6 ppm of folic acid) were dispensed into labeled test tubes.

4) 2 mL of 5 N HCl and 1 mL of 2% NaNO2 were added to each of the test tubes. The test tubes were mixed with a vortex and left to sit for 5 minutes to allow the diazotization step to go into completion. 5) 4% sulfamic acid was added to each of the tubes. The test tubes were mixed with a vortex and left to sit for 5 minutes. This is to remove the excess nitrite. 6) 5 mL of 1% 3-AP was added to each test tube and mixed with a vortex. The test tubes were heated in a boiling water bath for 10 minutes to allow the coupling reaction to go to completion. At this stage, the originally colourless solutions turned orange-yellow.

7) The test tubes were removed from the water bath, cooled to room temperature, and 3 mL of 5 N HCl was added to each tube. 8) Each solution was diluted to 25 mL with RO water and mixed with the vortex. 9) The absorbance of each solution was measured at 460 nm in a 10 mm cuvette using a UV/Vis spectrophotometer. The absorbance of the blank sample was used to initially zero the instrument.

9.4.2.2 Sample Analysis Spray Solutions: 1) 4 mL of the spray solution of interest was diluted to 50 mL with RO water. 2) 1 mL of the diluted spray solution was mixed with 1 g zinc granule and 10 mL of 5 N HCl in a 250 mL glass jar. It was shaken every 5 minutes for 30 minutes to allow for the reductive cleavage reaction to go to completion. 3) 29 mL of RO water was added to the jar to get the final volume to 40 mL. The jar was shaken and zinc granules were allowed to settle to the bottom. 4) 2 mL of the jar’s contents (4 replicates) was distributed into labeled test tubes. 5) Steps 3-9 from the 9.4.2.1 Standardization procedure were repeated. The final volume in each test tube was 14 mL.

Salt Samples: 1) 20 g of the salt sample of interest was placed in a 250 mL glass jar and 8 mL of 0.1 N NaOH was added. The mixture was shook for 10 minutes to extract the folic acid from the salt. 2) 4 g of zinc granules and 40 mL of 5 N HCl were added to the jar. The solution was shaken every 5 minutes for 30 minutes to allow for the reductive cleavage reaction to go to completion. 3) 52 mL of RO water was added to the jar (final volume = 100mL). The jar was shaken and zinc granules were allowed to settle to the bottom. 4) 2 mL of the jar’s contents (4 replicates) was distributed into labeled test tubes. 5) Steps 3-9 from the 9.4.2.1 Standardization procedure were repeated. The final volume in each test tube was 14 mL.

9.4.2.3 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 460 nm - 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 460 nm - intercept of calibration curve) / (slope of calibration curve)} * {14 mL / 2 mL} * {100 mL / grams of salt used}

0.25

0.2 0.15 0.1 0.05 y = 0.0328x + 0.0112

Absorbance(AU) R² = 0.9982 0 0 1 2 3 4 5 6 7 Folic Acid Concentration (ppm)

Figure 9.4.1: Sample Calibration Curve of Original SBCM Procedure

9.4.3 Additional Round for the Determination of p-ABGA All procedures are repeated omitting the addition of zinc to the initial reaction. The difference in concentration between these samples and those prepared with the use of zinc can be used to quantify folic acid.

0.25 y = 0.031x + 0.0199 0.2

R² = 0.9991

0.15

0.1 Reactions 1,2,3 Reactions 2,3

0.05 Absorbance(AU)

0 0 1 2 3 4 5 6 7 -0.05 Concentration (ppm)

Figure 9.4.2: Standard Folic Acid Solutions Tested With and Without Reaction 1

Figure 9.4.2 indicates that p-ABGA is not created when folic acid is tested without the addition of zinc, thus skipping the first reaction, when 5 N HCl is used. Therefore this method may be used to determine the p-ABGA content of samples.

9.4.4 Revised Procedure 9.4.4.1 Standardization 1) 25 mL of the 1000 ppm folic acid solution was diluted to 100 mL using 50/50 v/v methanol/NaOH. 2) 25 mL of that solution is taken and diluted to 100 mL using 50/50 v/v methanol/NaOH. 3) 0, 2.5, 5, 7.5, 10, and 12.5mL were taken from the solution above and diluted to 50 mL with 50/50 v/v methanol/NaOH. 4) 7 mL of each solution was added into a glass jar along with 7 mL of 50/50 v/v methanol/NaOH. 5) 11 mL of 5 N HCl were added. Then 2 g of zinc granules were added and the glass jars were put in the dark to react while being mixed every 5 minutes for 30 minutes. 6) 2 mL of the solutions were transferred into labelled test tubes (4 replicates).

7) 2 mL of 5 N HCl and 1 mL of NaNO2 were added to the test tubes. The test tubes were then agitated with a vortex and allowed to sit for 5 minutes. 8) 1mL of sulfamic acid was then added to each test tube. The test tubes were then agitated with a vortex and allowed to sit for 5 minutes. 9) 5 mL of 1% 3-AP was added to each test tube and mixed with a vortex. The test tubes were

heated in a boiling water bath for 10 minutes to allow the coupling reaction to go to completion. At this stage, the originally colourless solutions turned orange-yellow. 10) The absorbance of each solution was measured at 460 nm in a 10 mm cuvette using a UV/Vis spectrophotometer. The absorbance of the blank sample was used to initially zero the instrument. 11) The test tubes were removed from the water bath, cooled to room temperature, and 1 mL of 5 N HCl was added to each tube. 12) The absorbance of each solution was measured at 460 nm in a 10 mm cuvette using a UV/Vis spectrophotometer. The absorbance of the blank sample was used to initially zero the instrument.

9.4.4.2 Sample Analysis Spray Solutions: 1) Dilute all solutions to 1% folic acid with RO water. Take 0.5 mL of each 1% spray solution (or diluted solution) and dilute to 100 mL using 50/50 v/v methanol/NaOH. 2) Steps 4-12 were repeated from the 2.4.4.1 Standardization procedure.

Salt Samples: 1) 20 g of the salt sample of interest was placed in a test tube and 16 mL of 50/50 v/v methanol/NaOH was added. The tube was shook for 5 minutes to extract the folic acid from the salt. 2) 8 mL was removed and put into a centrifuge tube. 8 mL of 50/50 v/v methanol/NaOH was added. The tube was shook for 5 minutes to dissolve the folic acid. 3) The centrifuge tube was centrifuged for 2 minutes. 4) 14 mL was removed and put into a glass jar. 5) Steps 5-12 were repeated from the 2.4.4.1 Standardization procedure.

9.4.4.3 Standard Addition 1) 25 mL of 1000 ppm folic acid solution was diluted to 100 mL using 50/50 v/v methanol/NaOH solution. 2) 2.5 mL and 5 mL of that solution were diluted to 50 mL with 50/50 v/v methanol/NaOH solution.

3) 16 mL of the 0 mL, 2.5 mL, and 5 mL folic acid solutions were added to 20 g of salt. They were shaken for 5 minutes. 4) 8 mL of each solution was removed and put into a centrifuge tube. 8 mL of the 50/50 v/v methanol/NaOH solution were added. The tube was shaken for 5 minutes and centrifuged for 2 minutes. 5) 14 mL of the centrifuged solutions were put into a jar. 11 mL of 5 N HCl and 2 mg of zinc were added to the jar. The jar was mixed in dark place every 5 minutes for 30 minutes. 6) Steps 6-12 from the 2.4.4.1 Standardization procedure were followed.

9.4.4.4 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 460 nm - intercept of calibration curve) / (slope of calibration curve)} * {12 mL / 2 mL} * {25 mL / 7 mL} * {100 mL / 0.5 mL spray solution (or diluted spray solution)}

2% solution  multiply result by 2 3% solution  multiply result by 3

Folic Acid in Salt Samples: ppm (µg/gsalt) folic acid = {(absorbance at 460nm - intercept of calibration curve) / (slope of calibration curve)} * {12 mL / 2 mL} * {25 mL / 14 mL} * {16 mL / 8 mL} * {16 mL / 20 gsalt}

0.2

0.15

0.1 y = 0.0452x + 0.0195 0.05 R² = 0.9935

Absorbance(AU) 0 0 0.5 1 1.5 2 2.5 3 3.5 Concentration in Salt (ppm)

Figure 9.4.3: Sample Calibration Curve of Revised SBCM Procedure

9.4.5 The Effect of HCl Concentration on Folic Acid Readings

0.18 0.16 y = 0.0026x + 0.0195

0.14 R² = 0.9935

0.12 y = 0.0016x + 0.0227 0.1 R² = 0.9972 0.08

0.06 Absorbance(AU) 0.04 Stock Conc. HCl 0.02 5N HCl 0 0 10 20 30 40 50 60 Concentration in Salt (ppm)

Figure 9.4.4: 5 N HCl Causes Higher Absorbance Readings than Stock Concentration HCl

The reason for this change in absorbance when a more concentrated HCl solution is used is likely due to the instability of p-ABGA in the more concentrated acid. It therefore degrades into a non- diazotizable form and is not detected by the method. This has been found to happen when using this method by others in literature as well [40].

3.5 3 2.5

2 1.5 1 Stock Conc. HCl

Acid (% Acid wt) 0.5 5N HCl 0 1% FA, 1% FA, 1% FA, 1% FA, 1% FA, 1% FA, 2% FA, 3% FA,

MeasuredConcentration ofFolic 1% I, 2% I, 3% I, 1% I, 2% I, 3% I, 2% I, 3% I, 0.1M 0.1M 0.1M 0.2M 0.2M 0.2M 0.2M 0.2M Spray Solution Formulations

Figure 9.4.5: Folic Acid Detected in Spray Solutions Using Different HCl Concentrations

0.25

0.2

0.15 Stock Conc. HCl 0.1

5N HCl Absorbance(AU) 0.05

0 1%FA, 1%I, 0.1M 1%FA, 1%I, 0.2M 2%FA, 2%I, 0.2M 3%FA, 3%I, 0.2M Spray Solutions on Salt

Figure 9.4.6: Absorbance Measurements of Salt Using Different HCl Concentrations

80 70 60 50 40 Stock Conc. HCl Calibration 30 5N HCl Calibration 20

10 Folic Folic Concentration Acid (ppm) 0 1%F, 1%I, 0.1M 1%F, 1%I, 0.2M 2%F, 2%I, 0.2M 3%F, 3%I, 0.2M Spray Solution Formulation

Figure 9.4.7: Salt (50 ppm Folic Acid) Calibrated Using Different Concentrations of HCl

Salt calibrated using a 5 N calibration curve resulted in accurate results of approximately 50 ppm.

9.4.6 The Effect of 3-Aminophenol (3-AP) on Folic Acid Readings

16 14 12 10 8

(ppm) 6 4 2 0 Concentration ofFolic in Acid Salt -2 Blank Salt (Original I Salt (Original Method) 3-AP Alone (Original I Salt (Revised Method) Method) Method) Types of Salt Samples and Method Used

Figure 9.4.8: Change in Dilution Cause Rectification of Inaccuracies Due to 3-AP

The original method did not dilute calibration curve solutions and sample solutions the same amount. The colour of 3-AP was leading to inaccurate readings. The revised method diluted the calibration curve solutions the same amount as the sample solutions.

9.4.7 The Effect of Iron on Folic Acid Readings

90 80 70 60 50 40 30 20 10

0 Folic Folic Concentration Acid Salt in (ppm) Fe nFe Folic Acid, Fe Folic Acid, nFe Folic Acid, Fe, I Folic Acid, nFe, I Salt Samples

Figure 9.4.9: Effect of Iron on Folic Acid Readings Using Original Procedure (30 ppm FA)

When the original procedure was used to measure folic acid in fortified salt much higher results were found.

(ppm) Original Procedure 20 Revised Procedure

10 Folic Folic Concentration Acid Salt in 0 Fe nFe Salt Sample Fortificants

Figure 9.4.10: Rectification of Iron Effects Through Folic Acid Extraction

9.4.8 Accurate Standard Additions

15 30ppm FA, I, Fe 30ppm FA, I, nFe 10 0ppm FA, nFe

Acid Added Acid Salt to (ppm) 5

MeasuredConcnetration ofFolic 0 10 20 Concentration of Folic Acid Added to Salt (ppm)

Figure 9.4.11: Standard Additions of Folic Acid to Different Salts (Revised Method)

The standard addition of folic acid was accurate and successful when the revised method was used (see Figure 9.4.11).

9.4.9: Summary of SBCM Revisions Table 9.4.1: Spectrophotometry-Based Coupling Method Development Issue Solution Before Correction After Correction Imprecise Increased test solution >100% 3.6% ± 2.5% concentration standard deviation standard deviation Low folic acid Reduced ~50% of folic acid ~100% of folic acid detection in >1% w/v concentration of HCl detected detected iodine spray solutions from stock High folic acid concentration to 5 N ~140% of folic acid detection in salt detected Folic acid detection in Diluted 3-AP the 11 ± 3 ppm folic acid -0.4 ± 0.4 ppm folic unfortified salt same amount in detected in blank salt acid detected in blank calibration curve and salt test samples. High folic acid Extracted folic acid Fe  49 ± 6 ppm Fe  3 ± 1 ppm folic detection in presence from salt twice folic acid detected acid detected of iron nFe  33 ± 1 ppm nFe  4 ± 1 ppm folic acid detected folic acid detected

Appendix 9.5: Spray Solutions After 2 Months of Storage The spray solutions after 2 months of storage are shown below:

Figure 9.5.1: Spray Solutions After 2 Months Storage at 25ºC

Figure 9.5.2: Spray Solutions After 2 Months Storage at 45ºC

The 3% w/v folic acid and iodine solution stored at 45ºC formed a precipitate and appears cloudy whereas all of the other mixtures remained in solution.