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The Fortification of Salt with Iodine, Iron, and Folic Acid
by
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
© Copyright by Elisa June Teresa McGee 2012
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The Fortification of Salt with Iodine, Iron, and Folic Acid
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.
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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.
iv
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
v
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
vi
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
vii
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
viii
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
ix
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
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].
4
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].
6
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*
(μg/day)
Tolerable Upper
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
8
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**
(mg/day)
Tolerable Upper
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 27
th 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**
(μg/day DFE)
Tolerable Upper
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].
12
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]
I2(s) I2(g) ΔHo = 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].
13
I
O O
O-
K+
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.
14
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].
Fe2+
Fe3+
+ 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].
Fe2+ O
-
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.
16
2IO3-(aq) + 12H
+ + 10Fe
2+ I2(s) + 10Fe
3+ + 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].
OOH
O
OH
NH2 N
NN
N
OH
NH
NH
O
(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]
C9-N10 bond
17
[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].
19
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].
20
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,
operating cost, and cost to
consumer.
1 3 2
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].
22
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.
23
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.
25
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.
26
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
Salt
Sifto Canada Corp. Fine grain, clean,
dry
Micronutrients Folic acid Bulk Pharmaceuticals
Inc.
USP grade
Potassium iodate Sigma-Aldrich
Chemicals
ACS reagent grade
Ferrous fumarate Dr. Paul – Lohmann
Chemicals, Germany
Food-grade
(mean diameter
~10μm)
Spray Solution Buffer Sodium carbonate,
anhydrous
T. J. Baker Chemical
Co.
Reagent grade
(99.9%)
Sodium bicarbonate Caledon Laboratory
Chemicals
ACS Reagent grade
Microencapsulation Maltodextrin Cerestar, Indianapolis
Inc.
C*Dry MD
DE=7
Hydroxypropyl
methylcellulose
(HPMC)
Dow Chemicals Co.,
USA.
Hydroxypropyl
methylcellulose
(HPMC E15)
27
Table 3.1.2: Materials Used in Folic Acid Analysis
Purpose Material Supplier Grade/Description
Spectrophotometry-
based Coupling
Method
Hydrochloric acid Caledon Laboratory
Chemicals
ACS reagent grade
3-aminophenol Alfa Aesar (A
Johnson Matthey
Company)
For use in research
and development
98+%
Sodium hydroxide Caledon Laboratories
Ltd.
ACS reagent grade
Sodium nitrite Caledon Laboratories
Ltd.
ACS reagent grade
Sulfamic acid Sigma-Aldrich
Chemicals
ACS reagent grade
Zinc granules Caledon Laboratories
Ltd.
ACS reagent grade
High Performance
Liquid
Chromatography
Methanol Caledon Laboratories
Ltd.
HPLC grade
20 mM hexane
sulfonic acid and
0.1% phosphoric acid,
pH 2.2
ANALEST For use in HPLC
experiments run by
ANALEST
Citric acid Sigma-Aldrich
Chemicals
ACS reagent grade
Sodium phosphate,
dibasic
T. J. Baker Chemical
Co.
Reagent grade
(99.7%)
28
Table 3.1.3: Materials Used in Iodine and Iron Analysis
Purpose Material Supplier Grade/Description
Iodine Analysis Potassium iodide Caledon
Laboratories Ltd.
ACS reagent grade
Potassium iodate Sigma-Aldrich
Chemicals
ACS reagent grade
(99.5%)
Sulfuric acid EMD ACS grade
Sodium thiosulfate
solution, 0.1 N
BHD ACS grade
Starch indicator, 1.0% LabChem Inc. For laboratory and
manufacturing use
only
Iron Analysis Potassium hydrogen
phthalate
Sigma-Aldrich
Chemicals
ACS acidimetric
standard grade (99.95-
100.05%)
Hydroxylamine
hydrochloride, 98%
Sigma-Aldrich
Chemicals
ACS reagent grade
1,10-phenanthroline
monohydrate
Sigma-Aldrich
Chemicals
ACS grade
Sulfuric acid EMD ACS grade
Ferrous ammonium sulfate
hexahydrate
Caledon
Laboratories Ltd.
ACS reagent grade
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.
30
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.
31
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
32
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.
N
NNH2
OH
N
N
NH
O
NH
O OH
O
OHClH
Zn
NH2
O
N
H
O
OH
O
OH
folic acid p-aminobenzoylglutamic acid (p-ABGA)
Figure 3.3.1: Reaction #1 Reductive Cleavage of Folic Acid
33
NaNO2
H+
NH2
O
N
H
O
OH
O
OH
N N+
O
N
H
O
OH
O
OH
p-ABGA intermediate product
Figure 3.3.2: Reaction #2 p-ABGA Diazotization
OH
NH2
+
OH
NH2
NN
O
NH
O
OH
OOH
ClHN N
+
O
N
H
O
OH
O
OH
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.
34
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).
35
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.
37
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.
39
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
at 25ºC
(% Initial FA Content)
p-ABGA in Solutions Stored
at 45ºC
(% 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)
(ppm)
Iron (Fe) (ppm) Encapsulated
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.
41
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) +
12H+ + 10Fe
2+ I2(s) + 10Fe
3+ + 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.
0
10
20
30
40
50
60
70
80
90
100
Fe/nFe FA, Fe/nFe FA, I, Fe/nFe
% R
ete
nti
on
Fe
(II)
Salt Formulations
Fe(II)
nFe(II)
42
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.
0
20
40
60
80
100
120
FA, I FA, I, nFe FA, I, Fe
% R
ete
nti
on
I
Salt Formulations
43
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].
0
10
20
30
40
50
60
70
80
90
100
110
120
FA FA, I FA, Fe FA, nFe FA, I, Fe FA, I, nFe
% R
ete
nti
on
FA
Salt Formulations
44
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).
Figure 4.3.1: 0.1 M Carbonate/Bicarbonate Spray Solution Selection
6
6.5
7
7.5
8
8.5
9
9.5
10
0 20 40 60 80 100
pH
Volume % Carbonate 0.1 M Buffer
1% Folic Acid
2% Folic Acid
3% Folic Acid
45
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
6
6.5
7
7.5
8
8.5
9
9.5
10
10.5
0 20 40 60 80 100
pH
Volume % Carbonate 0.2 M Buffer
1% Folic Acid
2% Folic Acid
3% Folic Acid
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
pH
Month 1
pH (25ºC)
Month 2
pH (25ºC)
Month 1
pH (45ºC)
Month 2
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.
Figure 4.3.4: Retention of Folic Acid in Spray Solutions Stored at 25ºC
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
0
20
40
60
80
100
120
140
160
180
1% FA,1% I,0.1M
1% FA,2% I,0.1M
1% FA,3% I,0.1M
1% FA,1% I,0.2M
1% FA,2% I,0.2M
1% FA,3% I,0.2M
2% FA,2% I,0.2M
3% FA,3% I,0.2M
% R
ete
nti
on
FA
Formulations
Month 0
Month 2
Month 4
Month 5
0
20
40
60
80
100
120
140
160
1% FA,1% I,0.1M
1% FA,2% I,0.1M
1% FA,3% I,0.1M
1% FA,1% I,0.2M
1% FA,2% I,0.2M
1% FA,3% I,0.2M
2% FA,2% I,0.2M
3% FA,3% I,0.2M
% R
ete
nti
on
FA
Formulations
Month 0
Month 1
Month 4
Month 5
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.
Figure 4.3.6: Retention of Iodine in Spray Solutions Stored at 25ºC
Figure 4.3.7: Retention of Iodine in Spray Solutions Stored at 45ºC
0
20
40
60
80
100
120
140
1% FA,1% I,0.1M
1% FA,2% I,0.1M
1% FA,3% I,0.1M
1% FA,1% I,0.2M
1% FA,2% I,0.2M
1% FA,3% I,0.2M
2% FA,2% I,0.2M
3% FA,3% I,0.2M
% R
ete
nti
on
I
Formulations
Month 0
Month 1
Month 2
Month 5
0
20
40
60
80
100
120
140
1% FA,1% I,0.1M
1% FA,2% I,0.1M
1% FA,3% I,0.1M
1% FA,1% I,0.2M
1% FA,2% I,0.2M
1% FA,3% I,0.2M
2% FA,2% I,0.2M
3% FA,3% I,0.2M
% R
ete
nti
on
I
Formulations
Month 0
Month 1
Month 2
Month 5
49
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.
51
Figure 4.4.9: Retention of Folic Acid in Double Fortified Salt
Figure 4.4.10: Retention of Iodine in Double Fortified Salt
0
20
40
60
80
100
120
140
1%FA, 1%I, 0.1M 1%FA, 1%I, 0.2M 2%FA, 2%I, 0.2M 3%FA, 3%I, 0.2M
% R
ete
nti
on
FA
Spray Solution Formulations Used to Fortify Salt
Month 0
Month 1
Month 3
Month 5
0
20
40
60
80
100
120
1% FA, 1% I, 0.1M 1% FA, 1% I, 0.2M 2% FA, 2% I, 0.2M 3% FA, 3% I, 0.2M
% R
ete
nti
on
I
Spray Solution Formulations Used to Fortify Salt
Month 0
Month 1
Month 2
Month 4
Month 5
52
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.
53
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.
54
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.
55
7 References
[1] A. Lakshman, "Nutrition, health and the role of micronutrients," Micronutrient Initiative,
22-24 March 2010. [Online]. Available:
http://www.sph.emory.edu/wheatflour/IndiaTOT10/1Nutrition%20Health%20and%20ther
oleofmicronutrients_Anand%20Lakshman.pdf. [Accessed 7 March 2012].
[2] L. H. Allen, "Interventions for micronutrient deficiency control in developing countries:
past, present and future," The Journal of Nutrition, vol. 133, no. 11, p. 3875S–3878S, 1
November 2003.
[3] World Health Organization, World Food Programme and United Nations Children's Fund,
"Preventing and controlling micronutrient deficiencies in populations affected by an
emergency; multiple vitamin and mineral supplements for pregnant and lactating women,
and for children aged 6 to 59 months," 2007. [Online]. Available:
http://www.who.int/nutrition/publications/WHO_WFP_UNICEFstatement.pdf. [Accessed
12 March 2012].
[4] World Health Organization and Food & Agricultural Organization of the United Nations,
"Guidelines on food fortification with micronutrients," 2006. [Online]. Available:
http://whqlibdoc.who.int/publications/2006/9241594012. [Accessed 7 March 2012].
[5] World Health Organization and Food & Agriculture Organization of the United Nations,
"Human Vitamin and Mineral Requirements: Report of a joint FAO/WHO expert
consultation Bangkok Thailand," 2002. [Online]. Available:
http://www.fao.org/DOCREP/004/Y2809E/Y2809E00.htm. [Accessed 12 March 2012].
[6] Flour Fortification Initiative, GAIN, Micronutrient Initiative, USAID, The World Bank
and UNICEF, "Investing in the future; a united call to action on vitamin and mineral
deficiencies; global report 2009," 2009. [Online]. Available:
http://www.unitedcalltoaction.org/documents/Investing_in_the_future.pdf. [Accessed 6
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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
ΔHo 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
65
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.
66
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
67
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
68
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.
*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.
y = 0.2034x R² = 0.9998
-0.5
0
0.5
1
1.5
2
2.5
0 2 4 6 8 10 12
Ab
sorb
ance
Concentration of Fe(II) (ppm)
69
9.2.4 Calculation of Iron Content
Ferrous iron (Fe2+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:
Concentration in Salt ([Fe3+
]salt (ppm)):
[Fe3+
]salt (μg/gsalt) = [FeT]salt (μg/gsalt) - [Fe2+
]salt (μg/gsalt)
70
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.
71
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.
72
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
73
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)
74
9.3.4 HPLC Successes
9.3.4.1 Calibration Curves
Figure 9.3.1: p-ABGA HPLC Calibration Curve Using Area (7 replicates)
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.
Figure 9.3.3: Folic Acid HPLC Calibration Curve Using Area (8 replicates)
y = 18208x - 1559.3 R² = 0.9999
0
50000
100000
150000
200000
250000
0 2 4 6 8 10 12
Are
a (μ
V*s
)
Concentration (mg/L)
y = 1864.3x - 83.135 R² = 0.9997
0
5000
10000
15000
20000
25000
0 2 4 6 8 10 12
He
igh
t (μ
V)
Concentration (mg/L)
y = 80774x - 2905.3 R² = 0.9999
0
200000
400000
600000
800000
1000000
1200000
1400000
0 5 10 15 20
Are
a (μ
V*s
)
Concentration (mg/L)
75
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)
y = 4501.1x - 604.24 R² = 0.998
0
20000
40000
60000
80000
0 5 10 15 20
He
igh
t (μ
V)
Concentration (mg/L)
76
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.
Figure 9.3.7: Linear Range Determination of Folic Acid Using Area (3 Replicates)
Figure 9.3.8: Linear Range Determination of Folic Acid Using Height (3 Replicates)
Figure 9.3.9: Linear Range Determination of p-ABGA Using Area (4 Replicates)
y = 81957x + 10914 R² = 0.9999
0
2000000
4000000
6000000
8000000
10000000
0 50 100 150
Are
a (μ
V*s
)
Folic Acid Concentration (ppm)
y = 3763x - 1024.7 R² = 0.9999
0
100000
200000
300000
400000
500000
0 50 100 150
He
igh
t (μ
V)
Folic Acid Concentration (ppm)
0
100000
200000
300000
400000
500000
0 5 10 15 20 25 30
Are
a (μ
V*s
)
Concentration (ppm)
77
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.
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).
0
10000
20000
30000
40000
50000
0 5 10 15 20 25 30
He
igh
t (μ
V)
Concentration (ppm)
0
1
2
3
4
5
6
7
8
Salt #1 Salt #8
Folic
Aci
d C
on
cen
trat
ion
Dif
fere
nce
(p
pm
)
Expected
Measured
78
Figure 9.3.16: HPLC Salt Peak from Sodium Chloride Solution
Figure 9.3.17: HPLC Salt and Folic Acid Peaks from a Mixture
Figure 9.3.18: Salt Peak Area
Figure 9.3.19: Salt Peak Height
y = 15124x + 13559 R² = 0.9848
0
100000
200000
300000
400000
500000
0 5 10 15 20 25 30
Are
a (μ
V*s
ec)
Salt Concentration (% Weight)
y = 1855.3x + 3698.1 R² = 0.9291
0
10000
20000
30000
40000
50000
60000
0 5 10 15 20 25 30
He
igh
t (μ
V)
Salt Concentration (% Weight)
79
9.3.5.2 Folic Acid Reading is Salt Content Dependant
Figure 9.3.20: Salt Skew of Folic Acid HPLC Calibration Curve using Area
Figure 9.3.21: Salt Skew of Folic Acid HPLC Calibration Curve using Height
y = 80774x - 2905.3 R² = 0.9999
y = 56381x - 224873 R² = 0.9911
0
200000
400000
600000
800000
1000000
1200000
1400000
0 2 4 6 8 10 12 14 16
Are
a (μ
V*s
ec)
Concentration (ppm)
No Salt
With Salt
y = 4501.1x - 604.24 R² = 0.998
y = 3575.1x - 11907 R² = 0.995
0
10000
20000
30000
40000
50000
60000
70000
80000
0 2 4 6 8 10 12 14 16
He
igh
t (μ
V)
Concentration (ppm)
No Salt
With Salt
80
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.
Figure 9.3.22: Folic Acid Reading After HPLC Extraction Method 1
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.
0
5
10
15
20
25
10 20 30
Co
nce
ntr
atio
n o
f Fo
lic A
cid
M
eas
ure
d (
pp
m)
Concentration of Folic Acid Added to Salt (ppm)
MeasuredConcentration(Area)
MeasuredConcentration(Height)
-5
0
5
10
15
20
25
30
35
10 20 30
Co
nce
ntr
atio
n o
f Fo
lic A
cid
M
eas
ure
d (
pp
m)
Concentration of Folic Acid Added to Salt (ppm)
MeasuredConcentration(Area)
MeasuredConcentration(Height)
81
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.
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.
0
10000
20000
30000
40000
50000
60000
70000
FA & I FA & Fe FA & nFe FA, I, Fe FA, I, nFe
Are
a (μ
V*s
)
Salt Formulations
0
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
0 20 40 60 80 100 120
Are
a (μ
V*s
)
Concentration FA Added
filtered
not filtered
82
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).
Figure 9.3.28: Folic Acid in Double Fortified Salt Stored for 1 Year (25ºC or 45ºC)
0
20
40
60
80
100
120
140
2.21 3.03 4.07 5.98 7.02 7.76 9.27 10.04
Co
nce
ntr
atio
n (
pp
m)
pH of Spray Solution
Area 25CHeight 25CArea 45CHeight 45C
83
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.
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.
0
1
2
3
4
5
6
7
8
Blank 2.21 3.03 4.07 5.98 7.02 7.76 9.27 10.04
pH
of
Salt
HP
LC S
amp
le
pH of Spray Solution
84
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.
85
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.
86
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}
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.
y = 0.0328x + 0.0112 R² = 0.9982
0
0.05
0.1
0.15
0.2
0.25
0 1 2 3 4 5 6 7
Ab
sorb
ance
(A
U)
Folic Acid Concentration (ppm)
87
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
y = 0.031x + 0.0199 R² = 0.9991
-0.05
0
0.05
0.1
0.15
0.2
0.25
0 1 2 3 4 5 6 7
Ab
sorb
ance
(A
U)
Concentration (ppm)
Reactions 1,2,3
Reactions 2,3
88
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.
89
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}
90
Figure 9.4.3: Sample Calibration Curve of Revised SBCM Procedure
9.4.5 The Effect of HCl Concentration on Folic Acid Readings
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].
y = 0.0452x + 0.0195 R² = 0.9935
0
0.05
0.1
0.15
0.2
0 0.5 1 1.5 2 2.5 3 3.5
Ab
sorb
ance
(A
U)
Concentration in Salt (ppm)
y = 0.0016x + 0.0227 R² = 0.9972
y = 0.0026x + 0.0195 R² = 0.9935
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
0 10 20 30 40 50 60
Ab
sorb
ance
(A
U)
Concentration in Salt (ppm)
Stock Conc. HCl
5N HCl
91
Figure 9.4.5: Folic Acid Detected in Spray Solutions Using Different HCl Concentrations
Figure 9.4.6: Absorbance Measurements of Salt Using Different HCl Concentrations
0
0.5
1
1.5
2
2.5
3
3.5
1% FA,1% I,0.1M
1% FA,2% I,0.1M
1% FA,3% I,0.1M
1% FA,1% I,0.2M
1% FA,2% I,0.2M
1% FA,3% I,0.2M
2% FA,2% I,0.2M
3% FA,3% I,0.2M
Me
asu
red
Co
nce
ntr
atio
n o
f Fo
lic
Aci
d (
% w
t)
Spray Solution Formulations
Stock Conc. HCl
5N HCl
0
0.05
0.1
0.15
0.2
0.25
1%FA, 1%I, 0.1M 1%FA, 1%I, 0.2M 2%FA, 2%I, 0.2M 3%FA, 3%I, 0.2M
Ab
sorb
ance
(A
U)
Spray Solutions on Salt
Stock Conc. HCl
5N HCl
92
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
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.
0
10
20
30
40
50
60
70
80
90
1%F, 1%I, 0.1M 1%F, 1%I, 0.2M 2%F, 2%I, 0.2M 3%F, 3%I, 0.2M
Folic
Aci
d C
on
cen
trat
ion
(p
pm
)
Spray Solution Formulation
Stock Conc. HCl Calibration
5N HCl Calibration
-2
0
2
4
6
8
10
12
14
16
Blank Salt (OriginalMethod)
I Salt (Original Method) 3-AP Alone (OriginalMethod)
I Salt (Revised Method)Co
nce
ntr
atio
n o
f Fo
lic A
cid
in S
alt
(pp
m)
Types of Salt Samples and Method Used
93
9.4.7 The Effect of Iron on Folic Acid Readings
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.
Figure 9.4.10: Rectification of Iron Effects Through Folic Acid Extraction
0
10
20
30
40
50
60
70
80
90
Fe nFe Folic Acid, Fe Folic Acid, nFe Folic Acid, Fe, I Folic Acid, nFe, I
Folic
Aci
d C
on
cen
trat
ion
in S
alt
(pp
m)
Salt Samples
0
10
20
30
40
50
60
Fe nFe
Folic
Aci
d C
on
cen
trat
ion
in S
alt
(pp
m)
Salt Sample Fortificants
Original Procedure
Revised Procedure
94
9.4.8 Accurate Standard Additions
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
concentration
>100%
standard deviation
3.6% ± 2.5%
standard deviation
Low folic acid
detection in >1% w/v
iodine spray solutions
Reduced
concentration of HCl
from stock
concentration to 5 N
~50% of folic acid
detected
~100% of folic acid
detected
High folic acid
detection in salt
~140% of folic acid
detected
Folic acid detection in
unfortified salt
Diluted 3-AP the
same amount in
calibration curve and
test samples.
11 ± 3 ppm folic acid
detected in blank salt
-0.4 ± 0.4 ppm folic
acid detected in blank
salt
High folic acid
detection in presence
of iron
Extracted folic acid
from salt twice
Fe 49 ± 6 ppm
folic acid detected
nFe 33 ± 1 ppm
folic acid detected
Fe 3 ± 1 ppm folic
acid detected
nFe 4 ± 1 ppm
folic acid detected
0
5
10
15
20
25
30
10 20
Me
asu
red
Co
ncn
etr
atio
n o
f Fo
lic
Aci
d A
dd
ed
to
Sal
t (p
pm
)
Concentration of Folic Acid Added to Salt (ppm)
30ppm FA, I, Fe
30ppm FA, I, nFe
0ppm FA, nFe
95
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.
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