Triple Fortification of Salt with Vitamin A, Self-

Emulsifying Drug Delivery System, Iron, and Iodine

by

Lana Kwan

A thesis submitted in conformity with the requirements for the Degree of Master of

Applied Science

Graduate Department of Chemical Engineering and Applied Chemistry

University of Toronto

© Copyright by Lana Kwan 2012

ii

Triple Fortification of Salt with Vitamin A, Self-Emulsifying Drug

Delivery System, Iron, and Iodine

Lana Kwan

Master of Applied Science

Graduate Department of Chemical Engineering and Applied Chemistry

University of Toronto

2012

Abstract

Triple fortification of salt with vitamin A, iron, and iodine has been investigated in the

past to reduce micronutrient deficiencies in the developing world.

The objective is to develop integrated nutrient delivery technology by

microencapsulating a self-emulsifying drug delivery system (SEDDS) made of

surfactants and a bioactive compound, retinyl palmitate. The SEDDS is used to enhance

absorption of the vitamin A through food systems and to achieve targeted release of the

active ingredient.

Encapsulating vitamin A was difficult when using the spray dryer and the enteric coating,

Aquacoat®. Losses of the micronutrient after a three month storage period ranged from

50-99% at both 25°C/20% RH and 45°C/60% RH. The result of a matrix encapsulation

and poor coating formation contributed to the high losses.

Further investigation of coating systems with the aim of stabilizing all three samples for a

six month storage period such as using other encapsulating methods is recommended.

iii

Acknowledgements

I wish to extend my gratitude to my supervisor Professor Levente Diosady, who

provided me with great support and guidance, as well as an invaluable learning

experience. Thank you for the opportunity to extend my research abilities and stretch

myself to limits that I never knew I could go.

In addition, I would like to thank everyone in the Food Engineering Group,

especially Dan Romita, Kristen Palynchuk, Angjalie Sangakkara, Elisa McGee, Olive Li,

and Bih-King Chen.

Finally, I would like to give a special thank you to my family and friends for their

continual love and support.

iv

Table of Contents 1.0 Introduction ................................................................................................................... 1

2.0 Background ................................................................................................................... 5

2.1 Micronutrient Deficiencies ........................................................................................ 5

2.1.1 Vitamin A Deficiency (VAD) ............................................................................ 5

2.1.2 Iron Deficiency Anemia ..................................................................................... 5

2.1.3 Iodine Deficiency Disorder (IDD) ...................................................................... 5

2.1.4 Micronutrient Intake ........................................................................................... 6

2.2 Chemistry of Vitamin A, Iron, and Iodine ................................................................ 7

2.2.1 Vitamin A Chemistry .......................................................................................... 7

2.2.2 Iron Chemistry .................................................................................................. 10

2.2.3 Iodine Chemistry .............................................................................................. 12

2.3 Nutritional Aspects and Fortification Programs of Vitamin A, Iodine, and Iron.... 12

2.3.1 Salt as the Food Vehicle ................................................................................... 12

2.4 Self-Emulsifying Drug Delivery Systems (SEDDS) .............................................. 14

2.4.1 Background ....................................................................................................... 14

2.4.2 Types ................................................................................................................ 15

2.4.3 Structure............................................................................................................ 16

2.4.4 Composition...................................................................................................... 18

2.4.5 Uses .................................................................................................................. 19

2.5 Microencapsulation ................................................................................................. 21

2.5.1 Spray Drying..................................................................................................... 21

2.5.2 Droplet Formation ............................................................................................ 22

2.5.3 Defects in Coatings ........................................................................................... 24

2.5.4 Enteric Coating ................................................................................................. 25

3.0 Materials and Methods ................................................................................................ 27

3.1 Materials .................................................................................................................. 27

3.2 Equipment .............................................................................................................. 27

3.2.1 Spray Drying Conditions .................................................................................. 27

3.2.2 Particle Imaging ................................................................................................ 28

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3.2.3 Salt Blending .................................................................................................... 28

3.3 Experimental Set-up ................................................................................................ 28

3.3.1 Triple Fortification of Salt and Stability Test ................................................... 28

3.3.2 Formulation Conditions .................................................................................... 28

3.4 Analytical Methods ................................................................................................. 29

3.4.1 Moisture Determination .................................................................................... 29

3.4.2 Vitamin A Analysis .......................................................................................... 29

3.4.3 Iodine Analysis ................................................................................................. 29

3.4.4 Iron Analysis..................................................................................................... 29

4.0 Results and Discussion ............................................................................................... 30

4.1 Formulation and Process ......................................................................................... 30

4.2 Capsule Evaluation .................................................................................................. 32

4.3 Storage Evaluation .................................................................................................. 34

4.3.1 Vitamin A Stability ........................................................................................... 34

4.3.2 Iodine Stability ................................................................................................. 42

4.4 Plasticizers ............................................................................................................... 49

5.0 Conclusions ................................................................................................................. 52

6.0 Recommendations ....................................................................................................... 53

7.0 References ................................................................................................................... 54

8.0 Nomenclature .............................................................................................................. 60

9.0 Appendices .................................................................................................................. 61

9.1 Solution Process ...................................................................................................... 61

9.2 Preliminary Trial Runs ............................................................................................ 62

9.3 Final Formulas......................................................................................................... 63

9.4 Calibration Curves................................................................................................... 64

9.5 Analytical Methods ................................................................................................. 65

9.5.1 Vitamin A- Method based on Bessey (Bessey, 1946) ...................................... 65

9.5.2 Iron – Method based on Harvey Smart and E. Amis (Smart, 1955) ................. 65

9.5.3 Iodine- Iodometric titration based on the AOAC official method 33.3149

(AOAC, 1984) ........................................................................................................... 66

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List of Tables

Table 1 Recommended dietary allowances (RDA) of vitamin A for different ages/group

(Data from the National Academy of Sciences, 1989) ..................................................... 10

Table 2 Recommended dietary allowance (RDA) of iron at different ages and groups

(Data from the National Academy of Sciences, 1989) ..................................................... 11

Table 3 Experimental formulation and preliminary formulation results .......................... 31

Table 4 Moisture Content of Salt and Samples A, D, and E ............................................ 31

Table 5 Plasticizer Effect on Tg of Aquacoat®

................................................................. 50

Table 6 Spray drying trial runs ......................................................................................... 62

Table 7 Spray drying conditions with sample A ............................................................... 62

Table 8 Formulation of samples A, B, D, and E ............................................................... 63

Table 9 Microencapsulated SEDDS formulation design .................................................. 63

vii

List of Figures

Figure 1 Structural formulae of vitamin A forms and β-Carotene (adapted from [43]) ..... 8

Figure 2 Characterization of O/W (top) and W/O (bottom) microemulsions (© R.

Nagarajan, the use is by permission of the copyright holder)........................................... 15

Figure 3 Pseudoternary phase diagram of oil, water and surfactant displaying

microemulsion regions (© S. Talegaonkar Ast. Professor, Dept. of Pharmaceutics,

Faculty of Pharmacy, Jamia Hamdard New Delhi-62, the use is by permission of the

copyright holder) ............................................................................................................... 16

Figure 4 Diagram of the Buchi mini 290 spray dryer (© Buchi, the use is by permission

of the copyright holder) .................................................................................................... 22

Figure 5 Pure liquid droplet drying balance (© D. Romita, the use is by permission of the

copyright holder) ............................................................................................................... 23

Figure 6 Mass balance on a drying droplet (© D. Romita, the use is by permission of the

copyright holder) ............................................................................................................... 24

Figure 7 Schematic of the different dried particle morphologies that can result when

drying droplets containing dissolved or suspended solids (© M. Kraft, the use is by

permission of the copyright holder) .................................................................................. 25

Figure 8 Structure of cellulose acetate phthalate .............................................................. 30

Figure 9 SEM Images from top left to right (a) Sample A at 2000x (b) Sample D at 2000x

(c) Sample E at 2000x magnification (d) Sample A at 6000x (e) Sample D at 6000x (f)

Sample E at 6000x magnification ..................................................................................... 33

Figure 10 Vitamin A in sample A over 3 months of storage at 25°C ............................... 35

Figure 11 Vitamin A in sample A over 3 months of storage at 45°C ............................... 36

Figure 12 Vitamin A in sample D over 3 months of storage at 25°C ............................... 37

Figure 13 Vitamin A in sample D over 3 months of storage at 45°C ............................... 38

Figure 14 Vitamin A in sample E over 3 months of storage at 25°C ............................... 39

Figure 15 Vitamin A in sample E over 3 months of storage at 45°C ............................... 40

Figure 17 Iodine content in Sample A over 3 months of storage at 45°C ........................ 44

Figure 18 Iodine content in Sample D over 3 months of storage at 25°C ........................ 45

Figure 19 Iodine content in Sample D over 3 months of storage at 45°C ........................ 46

Figure 20 Iodine content in Sample E over 3 months of storage at 25°C ......................... 47

Figure 21 Iodine content in Sample E over 3 months of storage at 45°C ......................... 48

Figure 22 SEM Images from top left to right (a) 20% TEC 700x magnification (b) 20%

TEC 6000x magnification (c) 30% TEC at 700x magnification (d) 30% TEC at 6000x

magnification .................................................................................................................... 50

Figure 23 Process of making up sample solutions ............................................................ 61

Figure 24 Calibration curve of SEDDS with retinyl palmitate and the comparison of pure

retinyl palmitate and Aquacoat® calibration curves.......................................................... 64

Figure 25 Calibration curve of absorption vs. concentration of iron ................................ 64

1

1.0 Introduction

Nutrient deficiency disorder is due to the insufficient intake of food and/or certain

nutrients (WHO, 2011). With nutrient deficiency, individuals may develop serious health

problems. This is especially true in children because interference with growth and

development can lead to infection or chronic diseases. Micronutrient deficiency is the

lack of essential vitamins and trace minerals (Black, 2003). According to the World

Health Organization, one out of three people in developing countries are affected by

vitamin and mineral deficiencies. The inadequate intake of essential vitamins has caused

clinical manifestations in humans and is recognized as an important contributor to

worldwide illness, disability, and death.

Vitamin A is vital for the functions of vision, growth, reproduction, skin and epithelial

integrity, and the immune system. Lack of vitamin A can result in visual or ocular

malfunctions, as well as reduced immune responsiveness. In developing countries, a

significant percentage of children under the age of five have vitamin A deficiencies, and

as a result, approximately 1 million children die each year (MI, 2011). Cases of vitamin

A deficiency (VDA) in children lead to xerophthalmia, diarrhea, measles, malaria, and

other infections. 10% of the estimated 5 million xerophthalmia cases among pre-

schoolers are considered potentially blinding. Unfortunately, vitamin A deficiency is the

leading cause of preventable pediatric blindness in the developing world (Semba, 2008).

Not only are preschool-aged children at high risks, but reproductive-aged women are as

well. There is an estimated 7 million affected in low-income countries. In pregnant

women, deficiencies of the micronutrient can lead to maternal morbidity and mortality.

Iron deficiency is a serious issue, categorized as one of the top ten most serious health

problems in the modern world. It affects 2 billion people and iron deficiency anemia

(IDA) is responsible for a fifth of early neonatal mortality (Black, 2003). Those with IDA

have inadequate intake and/or impaired absorption or transport at the cellular level. Thus,

IDA leads to physiological losses that are related to chronological or reproductive age. It

2

is also the cause of chronic blood loss secondary to disease (Clark, 2008). The outcomes

of IDA include immune dysfunction, GI disturbances, and neurocognitive impairment.

Iodine deficiency is the single greatest cause of preventable developmental delays in the

world. Globally, an estimated 38 million infants are born without the protection that

iodine provides for brain growth, thus leading to 18 million who are mentally impaired.

Even though successful programs for making iodized salt available in iodine-deficient

areas have been implemented, such as the universal salt iodization (USI) program, 30%

of households worldwide still do not consume adequately iodized salt (MI, 2011), and

thus iodine deficiency disorders (IDD) still affects 0.2% of the world (Black, 2003).

Currently, the main solutions for micronutrient deficiencies in the developing world are

supplementation and fortification due to their cost-effectiveness and ease of delivery.

Supplementation is beneficial in that it can target larger doses of micronutrients to

specific individuals and it has a rapid impact. However, it has the disadvantages of not

providing all the necessary nutrients, neglecting individuals in non-targeted groups, and

low compliance (Allen, 2003). Supplementation programs are viewed as ―top-down‖

approaches, which divert the deficiency problems from food-based approaches.

Food fortification programs have the advantage of increasing the intake of multiple

micronutrients simultaneously in a cheap and sustainable method. However, the problem

associated with food fortification is that many of those individuals, who require fortified

products the most, cannot afford them. Also, there are adverse effects associated with the

sensory qualities of foods, nutrient-nutrient interactions, and poor bioavailability (Allen,

2003).

Because salt is universally consumed, it has the potential to deliver micronutrients to

those in developing countries. Several methods to produce salt include natural

evaporation by the sun and wind for sea salt, natural evaporation of underground or saline

lake brine for solar salt, and thermal evaporation of brine from solution of rock salt for

vacuum salt (Aquaron, 2000). There have been successful salt fortification programs in

3

the past, and it has been used as the vehicle for iodine since the early 1900‘s. Salt

consumptions per capita in different countries vary from 3 to 20 g per day (Mannar,

1995) based on climate, culinary habits, and occupation; however, salt consumption

within any societal group tends to be uniform and independent of social status. Because

iodized salt has many advantages in delivering micronutrients (access to large

populations, ease of fortification, and low cost) it was hypothesized that triple fortifying

salt with vitamin A, iron, and iodine would have the same advantages.

The research objectives of this thesis program are (1) to test the concepts of delivering

fat-soluble bioactive compounds in systems that can survive the harsh acidic conditions

of the stomach, (2) to deliver the fat-soluble bioactive compound through an O/W

microemulsion in order to enhance absorption of the compound of interest, and (3) to

look into the chemical interaction between the fat-soluble bioactive compound in the

microemulsion with the additional compounds in the surrounding system. The fat-

soluble bioactive compound selected in this case was retinyl palmitate, the surrounding

compounds were iodine and iron, and the food carrier was salt. Even though research on

triple fortification of salt has been conducted in the past (Raileanu, 2002; Liang Tay,

2002; Zimmermann, 2004), the use of self-emulsifying drug delivery systems to contain

the bioactive compound has not been investigated until now.

There are difficulties due to chemical interactions that degrade the micronutrients.

Vitamin A is susceptible to oxidation and isomerization, thus resulting in a loss of

nutritional efficacy. In addition, vitamin A is sensitive to light, moisture, and acidity.

Retinoids in foods are usually found in the all-trans form but the presence of iodine or

iron can lead to the isomerization of the vitamin A to cis-isomer form, resulting in some

activity loss (Loveday, 2008).

Similarly to vitamin A, iodine is susceptible to oxidation and high temperatures. In

addition, iodine in either iodate or iodide form can be easily reduced or oxidized to the

elemental iodine (I2) which then sublimes or evaporates readily into the atmosphere.

4

Iron is added in the ferrous (Fe 2+

) form, which can be easily oxidized to the ferric (Fe 3+

)

form, which is less bioavailable. When in contact with iodate, ferrous iron can react with

the iodine in a redox reaction that leads to the reduction of iodate to the molecular iodine

(I2), which readily sublimes.

5

2.0 Background

2.1 Micronutrient Deficiencies

Micronutrients are essential vitamins and trace minerals, and the deficiency of any

essential micronutrient can lead to increased rates of diseases and in case of severe

deficiencies, death. Mild to moderate deficiencies have a great impact on human health

by reducing immune and non-immune defenses and by compromising normal physiology

or development (Black, 2003). There are three micronutrient deficiencies that are

particularly important: vitamin A, iron, and iodine.

2.1.1 Vitamin A Deficiency (VAD)

Vitamin A deficiency (VAD) is common in developing countries, in particular, those in

Africa and Southeast Asia. Young children and pregnant women in low-income countries

are the most affected. An estimated 250 million preschool children are vitamin A

deficient. VAD causes preventable blindness in children, night blindness in pregnant

women and can increase the risk of disease and death (WHO, 2011).

2.1.2 Iron Deficiency Anemia

Iron deficiency is the most widespread and common nutritional disorder. Not only is it

prevalent in developing countries, but it also affects industrialized countries as well. Over

2 billion people are anemic and an estimated 40% of preschool children are anemic.

Malaria, HIV/AIDS, and hookworm infestation are just some of the critical factors that

can lead to the high prevalence of anemia in some areas (WHO, 2011). Overall, anemia

contributes to 20% of all maternal deaths.

2.1.3 Iodine Deficiency Disorder (IDD)

Iodine deficiency is rated as the most prevalent cause of impaired cognitive development

in children; however, it is easily preventable (WHO, 2011). Iodine deficiency disorder

6

(IDD) has been linked to cretinism and fetal wastage (Black, 2003). The important

consequences of IDD are mental and physical retardation, impaired reproductive

outcome, and goiter. IDD is caused because of the inadequate production of the thyroid

hormones thyroxine (T4) and triiodothyronine (T3) (Dunn, 1996).

2.1.4 Micronutrient Intake

Micronutrient deficiencies are counteracted through three principal interventions:

supplementation, dietary improvements, and food fortification.

Supplementation is the administration of pills, capsules or injections containing one or

more micronutrients. Supplementation is preferred when large doses of micronutrients

are needed by specific individuals. Even though it has a rapid impact, supplementation is

a ―top-down‖ approach. It does not supply all necessary nutrients or meet non-targeted

groups, and compliance is low because supplementation is required to be taken frequently

and for extended periods of time (Allen, 2003).

Improving nutritional qualities of food has the benefit of taking many nutrients

simultaneously, even those that are not provided in either supplementation or fortification

programs (Allen, 2003). However, similarly to food fortification, the disadvantage of

dietary improvement may be that the poor population cannot purchase quality foods,

therefore, they cannot benefit from dietary improvements.

Food fortification is the addition of micronutrients to foods during processing. It can

increase the intake of multiple micronutrients simultaneously and it provides a cost-

effective and sustainable supply of these nutrients (Allen, 2003). The disadvantage to

food fortification is that the poorer and undernourished population will more likely

produce their own food and not purchase fortified products. In terms of technology,

fortified foods may have problems in producing undesirable sensory qualities and may

have nutrient-nutrient interactions and poor bioavailability. However, it is known to be

the most rapid, economical, flexible, and socially acceptable method.

7

There are several criteria for selecting the food to fortify: food must be consumed by the

vast majority of the target population and at relatively constant level on a daily basis;

food must have minimal regional variation; food must be unrelated to socio-economic

status; food must have minimal variation between individuals‘ consumption and low

potential for excessive in-take; and food must be low in cost. In addition, fortified foods

must have desirable sensory qualities and bioavailability. Overall, it must appeal to the

population and accomplish its goal in delivering the micronutrient.

2.2 Chemistry of Vitamin A, Iron, and Iodine

2.2.1 Vitamin A Chemistry

Vitamin A can be found in foods, either as preformed vitamin A in animal products or as

provitamin A, carotenoids, in plant products. Preformed vitamin A products can be found

in dairy products, such as whole milk and cheese, or fish, such as tuna and sardines.

However, carotenoids are synthesized by plants in nature, so they are found naturally in

many fruits and vegetables (Simpson, 2011). β-carotene, one of the most abundant

carotenoids, shows the greatest provitamin A activity.

Vitamin A can be found in several forms in nature, including retinol, retinal, retinoic

acid, retinyl ester, and synthetic analogues. Provitamin A carotenoids are a group of

compounds that are precursors of vitamin A (Simpson, 2011). However, only about 10%

of the approximately 600 carotenoids that exist can be converted to vitamin A.

8

Figure 1 Structural formulae of vitamin A forms and β-Carotene (adapted from [43])

There is an interrelationship between retinol, retinal, and retinoic acid and their biological

roles. Retinol can be oxidized to either the aldehyde, retinal, which is necessary for

vision, or retinoic acid, which is a major metabolite of retinol. Oxidation of retinol is a

reversible reaction, since retinal can then be converted by the body to retinol. The

importance of retinol is immense, as retinol is required for growth and reproduction.

Retinoic acid can be substituted for retinol in growth promotion, but it cannot be

substituted completely for retinol in maintaining the reproductive function (Pawson,

1981).

Vitamin A degrades easily during storage due to its sensitivity to light, oxygen, heat and

humidity. This is because the conjugated double bond system of retinoids and carotenoids

in the electron-dense region is attractive to electron-deficient species, also known as

9

radicals. Thus, retinoid degradation shows the characteristics of free-radical reactions

(Loveday, 2008). In ultraviolet radiation, vitamin A undergoes photo-dimerization,

photo-isomerization and photo-oxidation (Mousseron-Canet, 1971). In addition, its

degradation is strongly affected by heavy metal ions and water (Bondi and Sklan, 1984).

With trace metals present, vitamin A oxidation accelerates (Bauernfeind, 1980). Thus,

when being handled, vitamin A must be kept in dark containers under an inert

atmosphere. Contact with ingredients such as acids and peroxides can lead to a rapid

degradation of the vitamin (Albertini, 2010), and so processing and storing of foods can

lead to partial losses of vitamin A activity.

Retinoids have a double bond in the polyene chain which can undergo cis-trans

isomerization (Loveday, 2008). Endogenous retinoids in foods are mostly in the all-trans

form (Brinkmann, 1995), which has maximal vitamin A activity (Loveday, 2008).

Vitamin A activity is expressed in International Units (IU) and one IU is equivalent to the

activity of 0.300 µg of all-trans retinol and 0.550 µg of all-trans retinyl palmitate (Otten,

2006). The daily vitamin A intake should not exceed 3000 µg or 10,000 IU/day of retinol

to prevent overdosing.

The recommended dietary allowances for vitamin A increases with age and it ranges

from 375 µg RE/day for infants to 1300 µg RE/day for lactating women, where RE is

micrograms of retinol equivalents. Table 1 displays the recommended vitamin A intakes.

10

Table 1 Recommended dietary allowances (RDA) of vitamin A for different ages/group (Data from

the National Academy of Sciences, 1989)

Age /Group Recommended vitamin A intake (µg

RE/day)

Infant (0-1 yrs) 375

Children (1-3 yrs) 400

Children (4-6 yrs) 500

Children (7-10 yrs) 700

Children (11-19 yrs) 1000

Adult (men) 1000

Adult (women) 800

Pregnant women 800

Lactating women (1st 6 months) 1300

Lactating women (2nd

6 months) 1200

2.2.2 Iron Chemistry

Iron is a transition metal element and the second most abundant metal in the earth‘s crust.

The element exists in either ferrous (Fe2+

) or ferric (Fe3+

) oxidation states (British

Nutrition Foundation, 1995). Iron has a vital role in energy metabolism of living cells.

Iron can also take part in redox processes. It is known to bind to oxygen either on its own

or as a part of a complex (British Nutrition Foundation, 1995).

There are two major forms of iron that exists in foods: heme iron, which is mostly found

in meat as part of hemoglobin or myoglobin, and non-heme iron, which is found in

cereals, vegetables, and other foods. Each type of iron is absorbed differently and with

different degrees of efficiency. In fact, approximately 20-30% of heme iron is absorbed

and most of the absorbed iron is incorporated into red blood cells, as opposed to

approximately 1-3% of non-heme iron from vegetables.

The bioavailability of iron is influenced by its form and quantity. Depending on the form

of iron, there is a positive relationship between the iron dose and percentage of

absorption. Unfortunately, acute iron poisoning can occur if there is an overload of iron

in the intestinal mucosal homeostasis (Aisen, 1977). The average lethal dose of iron is

200-250 mg/kg of body weight but there have been cases of death occurring after the

ingestion of 40 mg/kg of body weight (National Research Council, 1979). However,

there is a stronger emphasis on iron deficiency, especially in children and women,

11

because of the widespread iron deficiency anemia in these populations. Iron is most

bioavailable in the ferrous, Fe2+

, form (Diosady, 2002). Ferrous iron is converted to the

ferric form, which is less bioavailable, by oxidation, as shown.

Fe2+

(green) Fe3+

(orange/red) + e-

Iron can undergo oxidation in alkaline conditions or in the presence of oxidizing agents.

High humidity can speed up the process. In addition, iron has many interactions with

other micronutrients that may oxidize it to Fe3+

. Calcium and nickel can increase the

uptake of iron, while the uptake can be impaired by riboflavin, vitamin A, and copper

deficiencies (British Nutrient Foundation, 1995).

The recommended dietary allowances (RDA) of iron depend on age, sex, physiological

status, and iron bioavailability. The range for RDA of iron is 10-30 mg/day, as shown in

Table 2.

Table 2 Recommended dietary allowance (RDA) of iron at different ages and groups (Data from the

National Academy of Sciences, 1989)

Age /Group Recommended iron intake (µg RE/day)

Infant (0-6 months) 6

Infant (6-12 months) 10

Children (1-10 yrs) 10

Children male (11-18 yrs) 12

Children female (11-18 yrs) 15

Adult (men) 10

Adult (women 19-50 yrs) 15

Elderly women (50+ yrs) 10

Pregnant women 30

Lactating women (1st 6 months) 15

Lactating women (2nd

6 months) 15

12

2.2.3 Iodine Chemistry

Iodine is an essential element: the human body requires ~150-200 µg of iodine every day.

The only role for iodine in vertebrates is being part of the thyroid hormones (Dunn,

1996). The thyroid contains 70-80% of the total iodine content (15-20 mg). Therefore, if

a person does not consume a sufficient amount of iodine, the thyroid glands may enlarge,

leading to abnormal swelling in the neck, known as goiter.

Potassium iodate (KIO3) is chosen to iodize salt because it is stable and already in its

fully oxidized form, compared to potassium iodide (KI), which is easily lost through

oxidation. Iodide and iodate reactions are shown below.

2I- I2 + 2e

-

2I5+

+ 10e- I2

Ferrous compounds can react with iodate, which can result in losses of iodine and the

oxidation of the ferrous iron to the ferric form (Diosady, 2002).

2 I5+

+ 10 Fe2+ I2 + 10 Fe

3+

2.3 Nutritional Aspects and Fortification Programs of Vitamin A,

Iodine, and Iron

2.3.1 Salt as the Food Vehicle

Choosing a food vehicle that is easily accessible and commonly consumed by the

targeted population is a challenge. In order to achieve successful fortification programs,

the food vehicle must be based on the dietary habits of the targeted population without

any risk of excess consumption, which greatly reduces the vehicle choices. In addition,

the food vehicle must remain stable under extreme conditions, be relatively low in cost,

and must retain its appearance and taste after fortification.

13

Salt is a prospective vehicle for micronutrients because it is universally and uniformly

consumed (Diosady, 2002). In addition, fortification of salt programs to combat IDD and

IDA have been implemented for the past decade and therefore have made salt technically

and economically feasible. It has been shown that iron fortified salt is the simplest and

cheapest method of controlling IDA (Ranganathan, 1992), costing a few cents per annum

per person for a few milligrams or micrograms of the micronutrient per day (Diosady,

2002). Worldwide, salt is consumed in the range of 3 to 20 g per day, depending on the

environment and culinary habits of the population (Mannar, 1995). Thus, it is the most

suitable vehicle for fortification of all three micronutrients: iodine, iron, and vitamin A

for the population in developing countries.

In the past, several formulas of salt fortified with micronutrients have been conducted,

including iron by Naransinga Rao. His work consisted of an iron compound in

combination with a stabilizing agent or an absorption promoter. Salt fortified with ferrous

sulphate (3200 ppm) added to sodium acid phosphate or othophosphoric acid (3200 ppm)

and sodium acid sulphate (5000 ppm) had good bioavailability and stability during

storage (Rao, 1972). Research conducted by Venkatesh Mannar suggested that using the

combination of ferrous fumarate and KIO3 would create a more stable formulation that

would improve the organoleptic properties and stability of the nutrients in salt (Mannar,

1989). Overall, both Rao and Mannar‘s work indicated that salt was a worthy vehicle for

micronutrients.

Triple fortification of salt with iron, iodine, and vitamin A is relatively new. Over the past

decade, only a handful of studies have been conducted. Potassium iodide, ferrous

fumarate, and vitamin A Durarome® encapsulated with soy steraine and encapsulated by

a rotating pan was found to be stable (95% retention of iodine and 75% retention of

vitamin A) after three months of storage (Tay, 2002). Iron, iodine, and vitamin A were

successfully microencapsulated using hydrogenated palm oil with a spray cooling

technique. Only 12-15% loss of vitamin A occurred over a six month storage period

(Zimmermann, 2004). Thus, it has been proven that stable triple-fortified salt is

technically feasible.

14

2.4 Self-Emulsifying Drug Delivery Systems (SEDDS)

Microemulsions are systems created when two immiscible liquids are brought into a

single phase such that they are macroscopically homogeneous but microscopically

heterogeneous (Paul, 2001). They are optically clear, thermodynamically stable, and

usually have low viscosity. Additionally, they can have ultralow interfacial tension, large

interfacial area and capacity to solubilize both aqueous and oil-soluble compounds (Paul,

2001).

The main differences between microemulsions and emulsions are thermodynamic

stability and particle size: 10-200 nm in size for the former and 1-20 µm for the latter,.

The stability of microemulsions is influenced by ionic strength, additives, temperature,

and pressure. Microemulsions have ultra low levels of interfacial tension (10-2

– 10-3

mN/m). The lowering of IFT is done through a gain in system entropy ΔS due to the

creation of a large number of sub-micron sized droplets. An increase in ΔS leads to a

decrease in free energy change for microemulsion formation (ΔGm<0), which makes it

thermodynamically stable.

Microemulsions form in the intestine during digestion and absorption of fat. Many foods

have natural microemulsions. However, the use of microemulsions for food production

has not been exploited in the food industry. There are few reports on microemulsion use

in the food industry. Such reports include incorporating food ingredients within

microemulsions and using cereal and edible lipid systems to form microemulsions (Paul,

2001). An important use of microemulsions in food is the provision of improved

antioxidation effectiveness because of the possibility of a synergistic effect between

hydrophilic and lipophilic antioxidants (Paul, 2001).

2.4.1 Background

Self-emulsifying drug delivery systems, or SEDDS, belong to lipid-based formulations

(Tang, 2008) and are isotropic mixtures of oil, co/surfactants, and/or co/solvents that

15

emulsify under gentle agitation, supposedly to mimic conditions in the gastro-intestinal

tract. The main idea behind SEDDS is that it can form fine oil-in-water (O/W)

microemulsions followed by a dilution in an aqueous phase (Patel, 2009). It has been

shown that microemulsions are more easily absorbed than oil droplets. Microemulsions

do not form normally; however, if all the components of a microemulsion system are

delivered without the aqueous phase, the microemulsion will form in situ in an aqueous

environment in the stomach and the small intestine. In fact, the agitation required for the

self-emulsification comes from the stomach and intestinal motility (Talegaonkar, 2008),

making self-emulsifying drug delivery systems quite beneficial to use.

2.4.2 Types

Microemulsions can be either a droplet or a non-droplet type. The droplet type of

microemulsions are either with spherical oil droplets dispersed in a continuous medium

of water known as oil-in-water microemulsions (O/W, Winsor Type I) or with spherical

water droplets dispersed in a continuous medium of oil known as water-in-oil

microemulsions (W/O, Winsor Type II), in which both are shown in Figure 2. The

droplet type can then be further categorized as either single- or two-phase system, in

which the microemulsion phase coexists with an excess dispersed phase, an upper phase

of excess oil for O/W and a lower phase of excess water in W/O microemulsion

(Nagarajan, 2000).

Figure 2 Characterization of O/W (top) and W/O (bottom) microemulsions (© R. Nagarajan, the use

is by permission of the copyright holder)

16

Non-droplet type microemulsions, also known as middle-phase microemulsions, are part

of a three-phase system in which the microemulsion phase is in the middle coexisting

with an upper phase of excess oil and a lower phase of excess water. A possible structure

of the middle-phase microemulsion can be attributed to the randomly distributed oil and

water micro-domains and bicontinuity in both oil and water domains (Nagarajan, 2000),

which is known as a bicontinous microemulsion (Winsor Type III or IV). In Figure 3, a

pseudoternary phase diagram depicts the different microemulsion zones, which can be

separated into W/O and O/W microemulsions by the composition of whether it has

excess oil or excess water.

Figure 3 Pseudoternary phase diagram of oil, water and surfactant displaying microemulsion regions

(© S. Talegaonkar Ast. Professor, Dept. of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard

New Delhi-62, the use is by permission of the copyright holder)

2.4.3 Structure

The structure of both O/W and W/O droplet type microemulsions consist of a core region

surrounded by an interfacial film. The interfacial film does not include the head groups of

the surfactant and the alcohol; instead, they belong to the micelle core. The core is free of

water molecules and is thus considered hydrophobic.

Given that each droplet contains gk molecules of kind k (k= S for surfactant, A for

alcohol, O for oil, and W for water), among with gkl molecules are present in the

interfacial layer and the remaining ones are present in the core containing mainly the

17

dispersed phase. The radii Ro and Rw as shown in Figure 2 provide the boundaries of the

interfacial layer. The volume of the aggregate Vg can be related to the radii Ro and Rw by

the following equation for the water-in oil droplets:

Vg =

For the oil-in-water droplets, the equation becomes:

Where vSP and vAP are the volumes of the polar headgroups of the surfactant and alcohol

molecules, respectively (Nagarajan, 2000).

The volume VI of the interfacial layer per surfactant molecule is given by:

|

|

Where vST, vAT and vO are the volumes of the surfactant, alcohol, and oil tails,

respectively (Nagarajan, 2000).

The surface area of the droplet in contact with the water, per surfactant molecule, is given

by

Combining the surface area of the droplet per surfactant molecule equation with the

volume of the interfacial layer per surfactant molecule results in:

(

| |

)

Simplifying the equation further leads to the thickness of the interfacial layer

| |

The composition average of the surfactant tail length ls and the alcohol tail length la leads

to the equation

| | (

)

18

Structurally, oil entrapped pockets are found inside the oil/water boundary and make up

the dispersed phase. The oil is stabilized by surfactants and co-surfactants. The size and

shape of the oil molecules in relation to the hydrophobic region determine the relative

abundance and oil entrapment in the surfactant layer. On the other hand, the surfactant

molecules self-associate to form micelles in the bulk phase. Both the dispersed phase and

bulk phase coexist in equilibrium (Narang, 2007).

2.4.4 Composition

The main components of SEDDS are oil and surfactants. Both long and medium-chain

triglycerides have been used as the oil component because of their ability to dissolve

large amounts of hydrophobic drugs and their efficient self-emulsification. Additional

advantages include higher fluidity and better solubility properties than short-chain

triglycerides. (Patel, 2009).

Surfactants are organic compounds that lower the surface tension between oil and water.

They are amphiphilic (Pletnev, 2001) and reduce the surface tension of water by

adsorbing at the liquid-liquid interface. In choosing surfactants for SEDDS, those with a

relatively high hydrophilic and lipophilic balance (HLB) are used most often (Patel,

2009). The surfactant concentration in self-emulsifying formulations required to form and

maintain an emulsion state in the GI tract ranges from 30%-60% w/w of the formulation

(Patel, 2009).

Co-surfactants are amphiphilic molecules that accumulate with the surfactant at the

interfacial layer (Narang, 2007). Usually a high HLB surfactant is chosen, so a low HLB

co-surfactant is used in conjunction because it can modify the overall HLB of the system.

Co-surfactants are not able to form self-associated structures (Narang, 2007).

There are some systems that use co-solvents which act as co-surfactants to increase drug

solubility by co-solvency and to stabilize the dispersed phase (Narang, 2007).

Additionally, co-solvents reduce the dielectric constant of water, thus making it more

hydrophobic. They also increase the amount of molecularly dispersed surfactant in the

19

aqueous phase (Narang, 2007). However, in literature, there have been other systems

which do not use co-solvents. Self-emulsification depends on the oil/surfactant ratio,

surfactant concentration and the temperature at which self-emulsification occurs. Even

with excipients added to SEDDS, the efficiency of self-emulsification depends on the

physicochemical compatibility of the drug system (Tang, 2008).

Linker molecules are amphiphiles that segregate near the oil/water interface (Sabatini,

2003). Adding polar oil additives to microemulsions helps improve the solubilization in

surfactant-oil-water microemulsions due to the lipophilic linker effect. The improvements

were caused by long-chain alcohols (above C8) and their low ethoxylation derivatives.

Lipophilic linkers were found to enhance oil solubilization without directly participating

in the interfacial interactions (Salager, 1998). From past research, it has been shown that

lipophilic linkers are segregated in the vicinity of the interfacial layer due to its polarity

(Salager, 1998). Because the predominant orientation of linkers is more likely to be

perpendicular to the interface, it would prefer the oil molecules in one or several layers

next to the interface, leading to an increase in the indirect interactions between the

surfactant and bulk oil (Salager, 1998). Hydrophilic linkers, on the other hand, are

surfactant-like molecules with six to nine carbons per head group. They co-adsorb with

the surfactant to increase surfactant-water interactions (Yuan, 2007). Thus, linker based

microemulsions with lecithin have solubilization capacity for a wide range of oils

(Acosta, 2005).

2.4.5 Uses

Of new drug candidates, 40% have been found to have low solubility in water, which

leads to poor bioavailability, high intrasubject/intersubject variability, and lack of dose

proportionality (Tang, 2008). SEDDS have been used to improve the solubility and

bioavailability of poorly soluble and lipophilic drugs by allowing the drug to remain in

solution in the gut, thereby avoiding the dissolution step, which limits the absorption rate

of hydrophobic drugs (Tang, 2008). SEDDS allow for fine oil droplets, which would pass

rapidly through the GI tract, minimizing irritation that frequently is encountered during

extended contact between bulk drug substances and the gut wall (Tang, 2007).

20

The advantages of SEDDS are that they are physically stable and easy to manufacture.

Emulsions, on the other hand, are sensitive, metastable dispersed forms. SEDDS provide

a large interfacial area for partitioning of the drug between oil and water (Tang, 2007).

They are known to be more consistent in temporal profiles of drug absorption and

provide protection of drugs from the hostile environment in the gut (Kumar, 2010). They

can also enhance bioavailability of a drug, which allows for the reduction in doses. They

provide control of the delivery profiles, reduce the variability including food effects, and

can have high drug payloads. Finally, they can be found in either liquid or solid dosage

forms (Kumar, 2010).

From past research on self-emulsifying drug delivery systems, it has been viewed that

lipids can enhance bioavailability through many potential mechanisms, including:

1. Reduction in the gastric transit allows for an increase in the time available for

dissolution in the small intestine (Porter, 2001 & Tang, 2007).

2. With the presence of lipids in the GI tract, the secretion of bile salts (BS) and

endogenous biliary lipids such as phospholipids (PL) and cholesterol (CH)

increases, leading to a BS/PL/CH intestinal mixed micelles and thus an increased

solubilization capacity of the GI tract (Porter, 2001 & Tang, 2007).

3. Highly lipophilic drugs can enhance the extent of lymphatic transport and

bioavailability directly or indirectly via a reduction in first-pass metabolism

(Porter, 2001 & Tang, 2007).

4. Specific lipids and surfactants can attenuate the activity of intestinal efflux

transporters, which can lead to changes in the biochemical barrier function of the

GI tract (Porter, 2001 & Tang, 2007).

5. Also, different combinations of lipids, lipid digestion products and surfactants can

have permeability-enhancing properties, which can then lead to changes in the

physical barrier functions of the GI tract (Porter, 2001 & Tang, 2007).

21

2.5 Microencapsulation

Microencapsulation is a process in which a core material is completely surrounded by an

outer wall or coat. Protection of the interior material (core) and control of the flow of

materials (permeation) across the membrane are the two main functions of

microencapsulation (Fanger, 1974). Coating materials range from natural and modified

polysaccharides to synthetic polymers. The coat provides protection of the core from the

surrounding environment, including heat and moisture. Encapsulation has been used

extensively in the food industry for items such as flavouring agents, acids, bases, artificial

sweeteners, colourants, and preservatives (Gibbs, 1999), for protection, masking

undesirable odours and taste, and controlling the release of active ingredients.

2.5.1 Spray Drying

There are several encapsulating techniques that have been widely used in the food and

pharmaceutical industries, including pan-coating, extrusion coating, and fluidized bed

coating. However, the most widely employed technique for microencapsulation of heat-

sensitive products is spray drying. Spray drying allows for rapid evaporation of the

solvent from the droplets to form reliably produced capsules less than 20 µm in size for

the encapsulation of products, such as volatile flavours (Madene, 2006), oils (Jafari,

2008), drugs (Takeuchi, 1992), and microorganisms (Lian, 2003). Spray drying is

efficient in drying and is relatively inexpensive.

The spray dryer generally operates with a co-current air and product stream, which is an

advantage because the dried product is only in contact with the coolest air. The high rates

of moisture evaporation enable the temperature of the dried product to be lower than that

of the air leaving the drying chamber. There are four main stages in the spray drying

process. The first is atomization of the feed into a spray, the second stage is the spray air

contact, after which drying of the spray occurs, and finally the dried product is separated

from the drying gas. A diagram of the spray drying process is shown in Figure 4.

22

Figure 4 Diagram of the Buchi mini 290 spray dryer (© Buchi, the use is by permission of the

copyright holder)

When the mixture is fed into the spray dryer, it is atomized with the nozzle, liquid is

evaporated by the hot air contacting the atomized material (Gibbs, 1999), and the final

product is then collected in the collection chamber at the bottom of the cylinder.

2.5.2 Droplet Formation

Both heat and mass transfer processes are responsible for the droplet drying process. In

general, droplets are entrained in warm air after they are formed. The heat from the warm

air is then transferred to the droplets while water vapour from the surface of the droplets

transfers along a concentration gradient to the bulk air (Romita, 2011).

In terms of the gas phase, the convection heat transfer constant is usually between 2-25

W/((m2)(K)) (Incropera, 2002).

23

Figure 5 Pure liquid droplet drying balance (© D. Romita, the use is by permission of the copyright

holder)

For spray drying, both the Nusselt and the Sherwood number are equal to 2 when the

droplets are moving at the same speed as the surrounding gas. To determine the heat and

mass transfer constants, the following formulas are used:

Where r is the radius of the droplet, α is the thermal diffusivity through the film layer and

Dv is the diffusivity of water vapour in air (~0.305 cm2/s at 60°C). The concentration and

temperature differences are then calculated using the log means because the gradients are

non-linear (Masters, 1991).

For the mass balance on a single droplet, the diagram is shown in Figure 6 and the

following formula is used to determine drying rate prior to shell formation after

incorporating the effects of Stefan Flow:

(

)

(

)

24

Figure 6 Mass balance on a drying droplet (© D. Romita, the use is by permission of the copyright

holder)

After the surface of the droplets is saturated, a crust or skin is formed. This is thickened

through Ostwald ripening over time during which the droplet continues to dry

(Handscomb, 2010). To model the effects of droplet drying after initial precipitation, two

groups of models are used: the semi-empirical approach using the Characteristic Drying

Curves (CDCs) and the empirical approach which uses the Reaction Engineering

Approach (REA) that includes crust formation and droplet shrinkage (Romita, 2011).

2.5.3 Defects in Coatings

Several defects in coatings of particles were observed, even though the technology for the

application of coating has advanced significantly over the past years.

In terms of the morphology of drying droplets, there are several drying routes that can

occur. A dried particle begins with the appearance of a surface shell. Three distinct

categories of droplets have been noted by Walton and Mumford‘s work: crystalline, skin-

forming, and agglomerate. The differences in the morphology following shell formation

in each category depend on the drying air temperatures and drying rate. At low

temperatures, drying does not always result in the formation of solid dried particles.

Some droplets dry like a porous solid medium with moisture menisci receding into the

25

droplet (Walter & Mumford, 1999). Likewise, aerated feeds, bubbles or voids can occur

if the droplet can become super-saturated with any dissolved air as a result of increasing

solute concentration or entrained air pockets can coalesce and expand during drying to

produce hollow particles, as shown in Figure 7 (Handscomb, 2010).

Figure 7 Schematic of the different dried particle morphologies that can result when drying droplets

containing dissolved or suspended solids (© M. Kraft, the use is by permission of the copyright

holder)

2.5.4 Enteric Coating

Enteric coating refers to the release of drugs in the small intestine. The main difference

between enteric coating and microencapsulation is that the enteric coating is the physical

barrier that is applied to a bioactive compound to provide protection from its

surroundings, while microencapsulation is the process that applies the protective coatings

to capsules. Thus, microcapsules are small spheres with a uniform wall surrounding it.

26

Most common enteric coatings use pH-dependent polymers that contain carboxylic

groups, which remain un-ionized in the acidic environment of the stomach until they

reach the basic conditions of the small intestine, where they then ionize (Liu, 2009). Most

enteric coating is made up of cellulose esters, polyvinyl derivatives, or

polymethacrylates. Some coating mediums, such as enteric polymers, are soluble in

organic solvents. There are concerns about the use of organic solvents, due to their

toxicity, flammability, and explosiveness. Thus, aqueous-based enteric coating

formulations were developed. Aqueous-based enteric coating has its own disadvantages,

such as microbial contamination and instability of the dispersions (Rafati, 2006).

27

3.0 Materials and Methods

3.1 Materials

Liquid retinyl palmitate was received from Sigma-Aldrich, Toronto, Canada. Glyceryl

monooleate was generously provided by Dr. Paul Lohmann Chemicals, Germany, and

Dermofeel G6CY was provided by Kinetik, Hazlet, NJ. Lecithin was obtained from

Fisher Scientific, Toronto, Canada, and Aquacoat® was received by FMC BioPolymer,

Philadelphia, PA. The food-grade ferrous fumarate (mean diameter ~10 µm) was donated

by Dr. Paul Lohmann Chemicals, Germany, and maltodextrin (C*Dry MD DE=7) was

given by Cerestar, Indianapolis, IN. Potassium iodate was received from Sigma-Aldrich,

Toronto, Canada.

All analytical grade reagents used in analyses were purchased from Sigma-Aldrich,

Toronto, Canada. Food grade, un-iodized salt was obtained from Toronto Salt Chemical

Co., Toronto, ON.

3.2 Equipment

3.2.1 Spray Drying Conditions

All samples were sprayed dried using a Buchi B290 Mini-Spray Dryer. Determining the

settings of temperature and flowrate were based on product yields of spray dried 60 mL

samples of SEDDS (1%-25% w/v) in dissolved Aquacoat® (12-16% v/v). Parameters

were adjusted to determine the optimal yield. The inlet temperature was found to be 80°C

and the feed flowrate was set to 0.15 L/h. All feed solutions were agitated while being fed

to the spray drying.

28

3.2.2 Particle Imaging

The encapsulated particles and salt were imaged using a Hitachi S-2500 Scanning

Electron Microscope (SEM) after sputter-coating with gold. The SEM settings were:

accelerating voltage of 15 kV, a working distance of 10 mm, and a tilt angle of 15°.

3.2.3 Salt Blending

Salt was blended with KIO3 with the use of the Leroy Somer LStronics Blender at a

setting of 22 rpm.

3.3 Experimental Set-up

3.3.1 Triple Fortification of Salt and Stability Test

Un-iodized salt samples were sprayed with KIO3 solution at a target of 30 ppm while

blending using the bench-scale ribbon blender for 20 minutes at 22 rpm and for an

additional 20 minutes after spraying. The iodized salt samples were then air-dried for 2

hours at 20°C. The SEDDS capsules were blended in at varied target concentrations of

encapsulated material, depending on the formulation, for 15 minutes at 22 rpm. The iron

capsules were then blended in at a target concentration of 1000 ppm for an additional 15

minutes at 22 rpm.

Salt samples were stored in two conditions for three months: 25C (~20% RH) in a

closed box and 45C (60% RH) in an environmental chamber. Samples were analyzed

monthly for vitamin A and iodine.

3.3.2 Formulation Conditions

Within each sample, there were four possible conditions for each temperature, i.e. 25°C

and 45°C. The conditions consisted of pure capsules, capsules in un-iodized salt, capsules

in 30 ppm iodized salt, and capsules in 30 ppm iodized salt with 1000 ppm encapsulated

iron.

29

3.4 Analytical Methods

3.4.1 Moisture Determination

Moisture content of salt was determined gravimetrically by weighing 5 g of un-iodized

salt in an aluminum tray before and after heating at 110°C for 24 hours in a forced air

oven.

3.4.2 Vitamin A Analysis

Preformed vitamin A analysis was determined using spectrophotometry. The Perkin

Elmer Fluorescence LS-50B Luminescence Spectrophotometer was used for vitamin A

analysis. This method is modified from Bessey (Bessey, 1946) in which retinyl palmitate

can be determined from UV spectrophotometry at approximately 325 nm.

Tetrahydrofuran was used as the solvent that could dissolve Aquacoat®. The coating was

then filtered out with the use of 0.45 µm PTFE membrane filters. Four replicates were

taken for each sample.

3.4.3 Iodine Analysis

Iodine content was determined using iodometric titration based on the AOAC official

method 33.3149 (AOAC, 1984). This method reduces iodate to I2 and is titrated using

sodium thiosulphate with starch as the indicator. Four replicates were taken for each

sample.

3.4.4 Iron Analysis

Iron content was determined using spectrophotometry of iron (II) and total iron. The Cary

50 UV-Visible Spectrophotometer was used for iron analysis. The method is based on

Harvey Smart and E. Amis (Smart, 1955).

30

4.0 Results and Discussion

4.1 Formulation and Process

The spray drying conditions of the SEDDS were chosen based on preliminary trials done

with sample A, a formula of 20:1 (ratio of coating to SEDDS by mass). This sample was

chosen because of its high product yield (see Table 3). Results are included in the

appendices.

The formulation of the SEDDS was generously donated by M.A.Sc. graduate Jackie Chu

in Dr. Edgar Acosta‘s lab. The SEDDS formulation consisted of 20% liquid retinyl

palmitate and 80% surfactants: 37.1% glyceryl monooleate 22.3% lecithin, and 20.6%

dermofeel G6CY.

Aquacoat® was chosen to be the enteric coating used in each formula based on trial runs

done on several other enteric coatings. However, most of the enteric coatings were unable

to form spherical particles. The list of enteric coatings investigated is in the appendix. It

is an aqueous dispersion of cellulose acetate phthalate (CAP) with its structure shown in

Figure 8. CAP is a cellulosic derivative and is more permeable than acrylic polymers

because it is hydrophilic and has less dense molecular arrangements (Williams, 2001). In

addition, CAP has been used extensively in the past and has been determined to provide

adequate acid resistance on capsules at sufficient coating levels (Williams, 2001).

Figure 8 Structure of cellulose acetate phthalate

31

The ratio of coating to SEDDS was varied and labeled as shown in

Table 3. After spray drying an average of four trials for each sample, average product

yield, percentage of SEDDS, and percentage of vitamin A were calculated. It was noted

that as the ratio of coating to SEDDS decreased, the product yield decreased. Based on

the calculated percentage of SEDDS left in the resulting product, a possible explanation

could be that the solid contents were lost in spray drying. The ratios 10:1 (Sample B) and

2:1 (Sample C) of coating to SEDDS produced the lowest amount of SEDDS, and thus

vitamin A (0.107% and 0.175%), in the final product. Therefore, these ratios were

discarded in the final experimental formulations.

Table 3 Experimental formulation and preliminary formulation results

Sample Label Ratio of Coating

to SEDDS

Product Yield

(%)

SEDDS (%) Vitamin A (%)

A 20:1 75 2.43 0.486

B 10:1 55.7 0.534 0.107

C 2:1 51.5 0.873 0.175

D 1:1 10.9 8.76 1.75

E 1:2 14.6 44.1 8.82

Formulations for samples A, D, and E are included in the appendices. Samples will be

referred to as sample A, sample D, and sample E. Please refer to

Table 3 for sample information. The table shows the ratio of coating to SEDDS by mass,

the product yield based on the percent of solids recovered after spray drying, and the

percent of SEDDS (w/v) and vitamin A (w/v) in each sample.

The moisture of blank salt, prepared 30 ppm iodine salt, and the spray dried particles of

samples A, D, and E were measured.

Table 4 Moisture Content of Salt and Samples A, D, and E

Sample Moisture (%)

Blank Salt 0.039

30 ppm Iodine Salt 0.080

Sample A 4.47

Sample D 5.30

Sample E 8.56

32

Spray drying a fat-soluble micronutrient is difficult because of the many mechanisms of

product loss. There are three main areas where the product cannot be collected: the spray

cylinder, the cyclone, and the filter. Throughout the spray drying process, the oily

solution was found to be layering the cylinder walls, which created a film for dried

particles to stick to.

Without the right spray drying conditions, fat-soluble products have a difficult time

drying fully because the coating cannot coalesce on time, which leads to fat-soluble

residues being stuck on the side of the chamber.

4.2 Capsule Evaluation

The capsules were evaluated based on SEDDS formation and on physical images. It was

found that the capsules formed a SEDDS after dissolving in a base (pH 9) of ammonium

hydroxide. The SEDDS were visually inspected and were found to be optically clear.

SEM images were captured for samples A, D, and E, as shown in Figure 9. As the ratio of

coating to SEDDS decreased, it can be seen that the product did not dry properly. Sample

E had approximately twice the amount of moisture (8.56%) left in the final product

compared to sample A (4.47%), as shown in Table 4. Another reason may be that because

there is a higher amount of SEDDS to water in sample E, it is assumed that the

surfactants did not diffuse properly in water, thus leaving oily particles. Sample E in

Figures 9c and 9f have agglomerated into one large mass. According to previous

research, using high temperatures to coat particles with CAP led to poorly coalesced film

structure. Reducing the temperature led to improvements in film formation but a higher

tendency for agglomeration (Williams, 2001).

33

Figure 9 SEM Images from top left to right (a) Sample A at 2000x (b) Sample D at 2000x (c) Sample

E at 2000x magnification (d) Sample A at 6000x (e) Sample D at 6000x (f) Sample E at 6000x

magnification

Particle formation is based on many parameters, including bulk temperature, material

concentration, initial droplet surface area and rheology of the droplet. If the conditions

are unfavorable, such that the particle formation pathway is disturbed and the coating

cannot form a skin or shell, then it can lead to shriveled, collapsed, or inflated particles

(Handscomb, 2010). Samples A and D were both able to form solid spherical particles,

such as the particle on the right in Figure 9e, indicating that the particle underwent ‗dry

shell‘ formation, as described by Handscomb. However, there is evidence that some of

the particles may have also undergone ‗well shell‘ formation, in which the particles

slightly collapsed and then re-inflated, such as the particle on the left in Figure 9e.

Based on pharmaceutically relevant polymer studies, there have been poor film

formations of aqueous CAP using the spray method (Obara, 1995). Rapid drying

conditions prevented CAP latex particles from coalescing. Instead, sufficient moisture

34

content with a high spray rate and slow post-drying allowed for the CAP film to form.

The CAP film performance may also be influenced by many factors, including moisture

content and coating temperature. With high coating temperatures, spray dried polymer

particles may have poorly coalesced film structures. It has been shown that using a

reduced coating temperature improved film formation but increased the tendency of

agglomeration (Williams, 2001). In general, spray drying oxidative-sensitive products

inevitably will lead to instantaneous oxidation, especially with the use of a large volume

of hot air. Rapidly drying the surface of capsules can lead to the potential production of

blow holes (Wiley, 2007), also known as blistered particles (Handscomb, 2010), leaving

a possibility of micronutrients exposed to the surface, which can oxidize easily. One

solution would be to fortify liquids with excess volatiles to allow for rounding off during

drying.

4.3 Storage Evaluation

4.3.1 Vitamin A Stability

Vitamin A content was monitored over three months in 25°C and 45°C environmental

conditions and the results are shown in Figures 10-15.

35

d Figure 10 Vitamin A in sample A over 3 months of storage at 25°C

36

d Figure 11 Vitamin A in sample A over 3 months of storage at 45°C

37

d Figure 12 Vitamin A in sample D over 3 months of storage at 25°C

38

d Figure 13 Vitamin A in sample D over 3 months of storage at 45°C

39

Figure 14 Vitamin A in sample E over 3 months of storage at 25°C

40

d Figure 15 Vitamin A in sample E over 3 months of storage at 45°C

41

When exposed to oxidizing agents and free radicals, retinyl palmitate isomerizes to the

cis-form, producing a high amount of 13-cis and 9-cis. In addition, vitamin A can

undergo fragmentation at high temperatures (Belitz, 2009), which can then lead to

degradation. Vitamin A can undergo cis-trans isomerization with the presence of acid,

leading to the rearrangement of the double bonds and dehydration (Schwieter, 1967).

Isomerization leads to the lower potency cis isomers and is directly promoted by light

containing wavelengths of less than 500 nm. In addition, heat applied to of all-trans

vitamin A can result in the formation of cis isomers and eventual equilibrium between the

two isomers. Because the analytical method did not differentiate between cis and trans

isomers, it cannot be determined if the resulting vitamin A are cis isomers after storage.

Sample A capsules stored at room temperature had a vitamin A loss of 53.6%, compared

to sample D‘s loss of 58%. For products stored at 45°C, sample A had a 61.2% loss and

sample D had a 74.4% loss. Capsules in non-iodized salt at 25°C resulted in losses 54.2%

and 58.7% for sample A and sample D, respectively. In 45°C, sample A had a 65.1% and

sample D had a 75.1% loss.

Sample E suffered a loss of 99% of vitamin A for both storage environments. This was

expected as seen from Figure 9. From such conditions, the vitamin A degraded entirely

within the first month of storage due to its exposure to the surrounding environment,

including oxygen and high heat.

Sample A‘s double fortified salt (DFS) at 25°C had a large loss of vitamin A with 81.9%,

and sample D had a 73% loss. At 45°C, sample A had a 80.7% loss and sample D

resulted in a loss of 89.0%. This is not surprising since potassium iodate is an oxidizing

agent when in contact with vitamin A, which itself is a reducing agent that is susceptible

to oxidation. It was noted that there was a lower amount of vitamin A loss for sample A

in 45°C than at 25°C. The results could be attributed to human error during vitamin A

analysis and sampling error in sampling the solid salt.

42

Triple fortified salt (TFS) had the highest loss of vitamin A for all samples. At 25°C

sample A had a 85.2% loss and sample D had a 76.2% loss. At 45°C, sample A had a

91.1% loss and sample D suffered vitamin A losses of 90.4%. In the presence of

oxidizing agents, such as iron, moisture, and light, vitamin A losses can be high

(Zimmermann, 2004). A slightly higher percentage of vitamin A loss in triple fortified

salt than double fortified salt was observed, which may indicate that the encapsulated iron

may have been exposed and thus expedited vitamin A‘s degradation.

4.3.2 Iodine Stability

The iodine content was monitored for the 3 months of storage and the loss for each

sample are shown in Figures 16-21.

43

d Figure 16 Iodine content in Sample A over 3 months of storage at 25°C

44

d Figure 17 Iodine content in Sample A over 3 months of storage at 45°C

45

d Figure 18 Iodine content in Sample D over 3 months of storage at 25°C

46

d Figure 19 Iodine content in Sample D over 3 months of storage at 45°C

47

d Figure 20 Iodine content in Sample E over 3 months of storage at 25°C

48

d Figure 21 Iodine content in Sample E over 3 months of storage at 45°C

49

From the results, iodized salt with encapsulated iron particles suffered a higher iodine

loss over the three months. Also, there was a higher loss of iodine from the control

samples stored at 45°C (23.1%) than at 25°C (9.44%). Based on the results of the

controls, it appears that iodine underwent oxidative reactions caused by the encapsulated

iron particles. In DFS, there exists some interaction between vitamin A and iodine,

leading to the reduction of iodine. As stated before, because KIO3 is an oxidizing agent

and vitamin A is a reducing agent, the incompatibility between KIO3 and vitamin A

resulted in the loss of both micronutrients.

Over the storage period, sample A stored at room temperature had 31.5% loss of iodine in

DFS and 37.5% in TFS. When sample A was stored at 45°C, DFS had a 48.3% loss and

51% loss of iodine for TFS. For both DFS and TFS, the increase in iodine loss was

greater than 10% when stored at a higher temperature. This was expected, as the heat

would accelerate the iodine formation.

Sample D had a higher iodine loss under both environmental conditions. DFS stored at

25°C lost 54.5%, and TFS stored at 25°C lost 57.4% of the added iodine. DFS stored at

45°C resulted in a 69.4% loss of iodine and TFS stored at 45°C resulted in 68.8% iodine

loss. The similarities between DFS and TFS results could be attributed to human error

during iodine analysis. With TFS, iron capsules gave a reddish tinge to the solution and

when starch was added, it was difficult to tell the exact endpoint.

Sample E had the lowest iodine losses for both DFS and TFS under the two

environmental conditions. DFS at 25°C had a 22.9% loss and TFS at 25°C had a 23.7%

loss of iodine. For 45°C, DFS had a 37.9% loss and TFS had a 47.6% loss. Once again, a

higher loss of iodine at a higher temperature was expected.

4.4 Plasticizers

Plasticizers are used with CAP to provide flexibility to the polymeric material by

softening and swelling the polymer to help overcome its resistance to deformation, thus

decreasing the glass transition temperature of CAP (67-68C). One plasticizer used often

50

with Aquacoat® is triethyl citrate (TEC). From Williams‘s work, plasticizers were shown

to have a significant role of curing CAP coated beads. Table 5 indicates the effect of

plasticizer levels and Tg. For TEC, a minimum level of 15% was required to obtain acid

resistance at a coating temperature of 46°C. Samples of 20% and 30% plasticizer levels

were spray dried for sample A and SEM images are shown in

Figure 22.

Table 5 Plasticizer Effect on Tg of Aquacoat

®

Plasticizers Plasticizer level (as %

of latex solids)

Tg of Aquacoat®

Triethyl citrate 0 40

Triethyl citrate 20 36

Triethyl citrate 30 33

Figure 22 SEM Images from top left to right (a) 20% TEC 700x magnification (b) 20% TEC 6000x

magnification (c) 30% TEC at 700x magnification (d) 30% TEC at 6000x magnification

At a 20% level of TEC, the particles were found to have undergone ‗dry shell‘

morphology and were more spherical than those particles with a 30% TEC level. From

these trial results, it was hypothesized that TEC could provide a positive effect in helping

51

CAP films coalesce, supporting past research that moisture and plasticizers are both

necessary during a heat-humidity curing process. However, it was noticed that at the 30%

level, there were noticeably more collapsed particles than from samples A, D, or E. From

past research, a higher plasticizer level can lead to a higher percentage of plasticizer loss.

This can then lead to an increased evaporation rate of the plasticizer, which then leads to

a higher amount of collapsed particles (Williams, 2001). Thus, for the optimization of the

coated SEDDS particles, it is recommended to use 20% levels of TEC.

Overall, the research encountered many difficulties. Not only was it challenging to spray

dry vitamin A, but it was also difficult to incorporate all three micronutrients into one

food vehicle and maintain a stable environment. However, the knowledge CAP film

performance is influenced by many factors, including moisture content and coating

temperature; that adjusting for proper coat formation is difficult with other variables

involved; and that the use of plasticizers and stabilizers can be vital in the physical

properties of encapsulated particles, was acquired. In addition, the encapsulation method

of SEDDS had led to the formation of a matrix encapsulation. Because the load was high

compared to the encapsulant, a large amount of the SEDDS was exposed on the surface

of the capsules. The matrix encapsulation would have then resulted in high losses of the

unprotected vitamin A. Future steps include testing other coatings, plasticizers and

stabilizers to help improve the performances of particle formation. In addition, using

other encapsulating equipments can broaden the scope of the research.

Even though the capsules did not satisfy all the research objectives, the work conducted

here is still able to further the development of stable micronutrient capsules so that the

delivery of fat-soluble bioactive compound through an O/W microemulsion can survive

harsh acidic conditions of the stomach and can enhance absorption of the compound of

interest.

52

5.0 Conclusions

1. The creation of a stable self-emulsifying drug delivery system was achieved and

vitamin A was successfully incorporated into an O/W microemulsion.

2. The vitamin A SEDDS was encapsulated with Aquacoat® using a spray dryer;

however, the encapsulation method was found to be difficult as there were several

problems associated with spray drying the SEDDS. Some of the problems

included large losses of solid particles throughout the process and poor coat

formation due to high heat and low drying time.

3. The formulation for sample E (ratio of coating to SEDDS is 1:2) led to high

agglomeration and poor solid formation. The formula was too oily for the spray

dryer to properly evaporate all the moisture and dry the particles. Thus, it was not

a viable formula to work with.

4. The encapsulated SEDDS particles in both sample A (coating to SEDDS ratio of

20:1) and sample D (coating to SEDDS ratio of 1:1) were influenced by iodine in

DFS and both iodine and iron in TFS, which led to the vitamin A degrading over

a three month period.

5. Out of the three formulations, sample A had the best retention of vitamin A and

iodine, while sample E was not able to encapsulate vitamin A properly and thus

had large losses of the micronutrient.

6. The capsules suffered large losses of vitamin A due to both the matrix

encapsulation, which exposes the SEDDS, and to insufficient softness/film

forming ability of the encapsulant, which led to the requirement of a plasticizer.

53

6.0 Recommendations

1. Testing other enteric coatings and incorporating plasticizers would help to

improve the quality and effectiveness of a coat formation.

2. In addition to the coating, reformulating solutions using different ratios of

materials could improve vitamin A retention.

3. Reaching the Tg (67-68C) of cellulose acetate phthalate during spray drying

could help improve coat formation leading to a reduction in vitamin A‘s oxidation

and free radical sensitivity.

4. Determining product losses by weighing out the cylinder and cyclone before and

after processing, and by creating a bag filter, could help to improve estimation of

product yields.

5. Using other encapsulating methods, such as a capsule filling and extrusion, could

improve vitamin A retention.

6. Determining the isomeric composition of retinoids in the added retinyl palmitate

by saponification could help to improve the accurate nutritional estimates of the

particles.

54

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60

8.0 Nomenclature

AOAC The Association of Official Analytical Chemists

CAP Cellulose acetate phthalate

DFS Double fortified salt

Fe Chemical symbol for iron

I Chemical symbol of iodine

IDA Iron Deficiency Anemia

IDD Iodine Deficiency Disorder

IFT Interfacial tension

KI Potassium iodide

KIO3 Potassium iodate

MI Micronutrient Initiative

ppm Parts per million

PTFE Polytetrafluoroethylene

RDA Recommended Daily Allowance

RH Relative humidity

rpm Revolutions per minute

SEDDS Self-emulsifying drug delivery system

SEM Scanning electron microscope

TEC Triethyl citrate

TFS Triple fortified salt

Tg Glass transition temperature

WHO World Health Organization

°C Degrees Celsius

61

9.0 Appendices

9.1 Solution Process

Figure 23 Process of making up sample solutions

62

9.2 Preliminary Trial Runs Table 6 Spray drying trial runs

Sample Water

(mL)

Coating

(mL)

NH4OH

(mL)

SEDDS

(g)

Yield (%) %

SEDDS

A 50 10 0.8 0.5 75 2.43

B 50 10 0.8 1.0 55.7 0.534

C 50 10 0.8 1.5 51.5 1.43

D 50 10 0.8 2.5 40.8 2.84

E 50 10 0.8 5.0 24.6 0.873

F 50 10 0.8 3.75 18.6 4.12

G 50 10 0.8 10 10.8 8.76

H 50 10 0.8 20 4.5 44.1

Table 7 Spray drying conditions with sample A

Temperature (˚C)

80 90 100 110 120

Pu

mp

Sp

ee

d

(%)

5 55.31% 52.44% 53.81% 54.28% 52.34%

10 50.12% 52.22% 49.81% 47.37% 50.45%

15 48.09% 51.73% 51.36% 48.80% 51.48%

20 46.91% 46.13% 46.03% 48.16% 49.93%

63

9.3 Final Formulas

Table 8 Formulation of samples A, B, D, and E

Sample

Water

(mL)

Coating

(mL)

SEDDS

(g)

NH40H

(mL) Total

A 4303.65 860.73 43.036 68.86 5276.27

B 17136.02 3427.20 342.72 274.18 21180.12

C 10652.26 2130.45 1065.23 170.44 14018.38

D 1474.37 294.87 294.87 23.59 2087.71

E 124.44 24.89 49.77 1.99 201.09

Several factors were incorporated such that the possibility of variability was narrowed

down. Choosing enteric coatings were based on product yield and particle formation.

Samples using different enteric coatings, as shown in Table 9, were made with a 10%

(w/v) SEDDS. These samples were then sprayed dried and SEM images were analyzed

for acceptability. From such results, it was determined that Aquacoat®

was the only

enteric coating that had a successful yield and particle formation. Therefore, varying the

formulations would be based on changing the ratio of SEDDS to coating while using the

successful coating.

Table 9 Microencapsulated SEDDS formulation design

Encapsulating Material Nominal SEDDS content % (w/v)

Aquacoat®

10

Eudragit®

FS30D 10

Eudragit®

L30 D-55 10

Eudragit®

L100 D55 10

Opadry®

Enteric 95 10

Opadry®

Enteric 95 10

Acryl-EZE® 93A 10

Sureteric 10

Ratio of Coating to SEDDS Sample Label

20:1 A

10:1 B

2:1 C

1:1 D

1:2 E

64

9.4 Calibration Curves

Based on the calibration curve of the SEDDS as shown in Figure 24, it was determined

that the surfactants in the SEDDS and Aquacoat® did not have any adverse effects on the

retinyl palmitate spectrophotometry reading at a wavelength of 325 nm. Each calibration

curve did not intersect the SEDDS curve.

d Figure 24 Calibration curve of SEDDS with retinyl palmitate and the comparison of pure retinyl

palmitate and Aquacoat® calibration curves

d Figure 25 Calibration curve of absorption vs. concentration of iron

y = 0.0591x R² = 0.9985

0.00

0.20

0.40

0.60

0.80

1.00

0 5 10 15 20

Ab

sorb

ance

Concentration (IU/mL)

Absorbance vs. Concentration Solvent: THF

Wavelength: 325 nm

Pure Retinyl Palmitate

SEDDS with RetinylPalmitate

SEDDS without RetinylPalmitate

Pure Aquacoat

y = 0.2039x + 0.0551 R² = 0.9996

0

0.5

1

1.5

2

2.5

0 2 4 6 8 10 12

Absorp

tio

n

Concentration (ppm)

65

9.5 Analytical Methods

9.5.1 Vitamin A- Method based on Bessey (Bessey, 1946)

Reagent Preparation

1.) HPLC Grade THF

Sample Analysis

1.) 50 mg of retinyl palmitate dissolved in 50 mL volumetric flask of THF

2.) Transfer to an Erlenmeyer flask and mix on a stir plate for 10 minutes

3.) Filter with 0.45 µm PTF filter

4.) Using the spectrophotometer, read at 325 nm

9.5.2 Iron – Method based on Harvey Smart and E. Amis (Smart, 1955)

Reagent Preparation

1.) Reacting solution (1,10-phenanthroline)

2.) Buffer solution

3) Reducing solution

Standardization

1.) Stock solution is 1000 ppm of iron. Dilute 7.0213g of ACS grade ferrous ammonium

sulphatehexahydrate in 200 mL distilled H20 containing 3 mL of concentrated sulphuric

acid. Dilute to 1 L volumetric flask.

2.) Working solution is 100 ppm of iron. Pipette 10 mL of the 1000 ppm stock solution

into a 100 mL volumetric flask and fill flask to mark with distilled H20.

3.) After adding specific amounts of working solutions to 25 mL volumetric flask, add 5

MI buffer solutions to each flask followed by 10 mL of the reacting solution.

Sample Analysis

1.) Weigh out sample into 125 mL Erlenmeyer flasks and record weight. Repeat 3-4

samples.

2.) Add approximately 40 mL of distilled H2O to each flask and 1 mL of concentrated

sulphuric acid.

3.) Heat Erlenmeyer flasks until the solution boils and keep on low heat for 10 minutes

until the iron particles completely disintegrate.

4.) Cool the solutions to room temperature and transfer to 100 mL volumetric flask.

5.) Pipette 1 mL of the sample solution from the 100 mL flask into a 25 mL volumetric

flask.

6.) Add 5 mL of buffer solution followed by 10 mL of reacting solution to each 25 mL

volumetric flask

7.) Fill the volumetric flask with distilled H20 to the mark.

8.) For total iron content, repeat step 5 and add 1mL of reducing solution to each flask

before continuing to step 6.

9.) Use the spectrophotometer to read the absorbance of the sample for both ferrous and

total iron content.

66

Calculation of Iron Content

Ferrous iron content

Fe2+

=Cf*25*100 mL/W (where Fe2+

(ppm) is ferrous iron content, Cf (µg/mL) is the

concentration of ferrous iron calculated from the calibration curve, and W(g) is the

weight of sample replicate).

Total iron content

Fe = Ct * 25 * 100 mL/W (where Fe2+

(ppm) is ferrous iron content, Ct (µg/mL) is the

concentration of total iron calculated from the calibration curve, and W(g) is the weight

of sample replicate).

9.5.3 Iodine- Iodometric titration based on the AOAC official method 33.3149 (AOAC,

1984)

Reagent Preparation

1.) 0.005 N Na2S2O3: Dilute 50 mL of 0.1 N Na2S2O3to 1 L of distilled H2O.

2.) 0.00125 N Na2S2O3: Dilute 12.5 mL of 0.1 N Na2S2O3 to 1 L of distilled H2O.

3.) 0.2 N H2SO4: Dilute 1.4 mL of concentrated H2SO4 to 500 mL of distilled H2O.

4.) 0.05% KIO3: Dissolve 0.05 g of KIO3 in distilled H2O in a 100 mL volumetric flask

and dilute to the mark with distilled H2O.

5.) 2% KI: Dissolve 10g of KI in distilled H2O in a 500 mL volumetric flask and dilute to

the mark with distilled H2O. Store in a dark place or cover with aluminum foil.

6.) Fill all standard flasks with distilled H2Oto the mark.

Standardization

1.) Mix 2 mL of the 0.05 % KIO3 with ~100 mL distilled H2O in a 500 mL Erlenmeyer

flask. Repeat for 3-4 replicates.

2.) Add 2 mL each of 0.2 N H2SO4 and 2% KI solutions, mixing well after each addition.

3.) Stopper the flask and allow the yellow colour to develop (due to the liberation of

iodine) for 10 minutes in a cool, dark place.

4.) Slowly titrate with either the 0.005 N Na2S2O3 solution (for the spray solutions) or the

0.00125 N solution (for the salt samples), until the yellow colour becomes a faint yellow.

5.) Add a few drops of the 1% starch solution to generate the blue/purple iodine-starch

complex.

6.) Continue titrating until the blue colour disappears.

Sample Analysis

Salt Samples

1.) Add 10 g of the salt sample of interest to ~100 mL distilled H2O in a 500 mL

Erlenmeyer flask. Repeat for 3-4 replicates.

2.) Repeat steps 2-6 from the standardization procedure discussed above, using the

0.00125 N Na2S2O3solution as the titrant.

Calculation of Iodine Content

Strength ofNa2S2O3

Strength (μg I/ mL Na2S2O3) ={0.05%}*{59.5%}*{2 mL/ mL Na2S2O3consumed}

67

Iodine in Salt Samples

ppm (μg/g salt) iodine = {strength of Na2S2O3} * { mL Na2S2O3consumed } / grams of

salt used.