“Training Manual”

Regional Training on Laboratory and

Quality Control/Quality Assurance Procedures
for Universal Salt Iodisation Programme

1st to 11th November, 2009

Venue: Centre for Community Medicine,

All India Institute of Medical Sciences,
New Delhi, India

Organised by:
WHO-SEARO and ICCIDD

1
 Social Process Model for Health Care Programmes (The Wheel)

The Social Process model (“The Wheel”) below describes the iterative cycle of IDD
elimination. The key lies in the sustainability of IDD elimination, not just its short-term
elimination.

2
 Contents
S No. Topic Page No.
1 Iodine and Thyroid Hormone 4
2 Iodine Deficiency – Mechanism 10
3 Spectrum of IDD & its Consequences 12
4 IDD control: Why and how? 14
5 Laboratory Procedure for Iodine Estimation of Salt 21
6 Laboratory Procedure for Urinary Iodine Estimation 31
7 Laboratory Quality Assurance For salt iodine 42
content estimation
8 Pictorial representation of salt iodine estimation 51

3
1. Iodine and Thyroid Hormone

1.1) Role of Iodine

Iodine is one of the essential micronutrients required for the normal mental and physical well
being of human beings. The healthy human adult body contains 15 – 20 mg of Iodine of
which 50 – 80 percent is in the thyroid gland, which weighs only 15 – 25 gm. The
requirement of iodine is 150 micrograms per person per day. This works out to an amount
less than a tea spoonful over a life span of 70 years. The tiny quantity of iodine is required
everyday y the thyroid gland for adequate production of the hormone thyroxine. It requires
four atoms of Iodine to make one molecule of thyroxine. It is against this backdrop that a
Joint consultation of the WHO, UNICEF and the International Council for Control of Iodine
Deficiency Disorder (ICCIDD) made recommendation of optimal iodine intake in the various
age groups (Table 2).

1.2) Thyroid Gland: Anatomical Consideration

Thyroid tissue is present in all vertebras. In mammals, the thyroid originates from an
evagination of the floor of the pharynx, and a thyroglossal duct marking the path of the
thyroid from the tongue to the neck sometimes persist in adult. The butterfly shaped gland is
situated in the lower part of the front of the neck. The two lobes of the human thyroid are
connected by a bridge of tissue, the thyroid isthmus, and there is sometimes a pyramidal lobe
arising from the isthmus in front of larynx. The gland is well vascularised, and the thyroid has
one of the highest rates of blood flow per gram of tissue of any organ in the body.

The thyroid is made up of multiple acini (follicles). Each spherical follicle is surrounded by a
single layer of cells and filled with pink staining proteinaceous material called colloid. When
the gland is inactive, the colloid is abundant, the follicles are large, and the cells lining them
are flat. When the gland is active, the follicles are small, the cells are cuboid or columnar, and
the edge of the colloid is scalloped, forming many small “re-absorption lacunae”.

1.3) Iodine Metabolism & Thyroid Hormone Synthesis (Fig 1)

A. Iodide (I⁻) is rapidly absorbed through the gut. The normal intake is 100-150
microgram per day. Iodine is then excreted by the Kidney. The level of excretion

4
 correlates well with the level of the intake. Thus, urinary iodine can be used to
assess the level of iodine intake.
B. Concentration of iodine (I⁻): The thyroid, along with several other epithelial tissues
including mammary gland, chorion, salivary gland, and stomach, is able to
concentrate I⁻ against a strong electro-chemical gradient. This is an energy-
dependent process and is linked to the ATPase-dependent Na+ - k+ pump. The
intrinsic plasma membrane transport protein, Na+/I- Symporter (NIS) couples the
inward “downhill” translocation of Na+ to the inward “uphill” translocation of I-.
The driving force for the process is the inwardly directed Na+ gradient generated by
the Na+/K+ adenosine triphosphatase (ATPase)
The I⁻ transporter is inhibited by two classes of molecules. The first group consists
of perchlorate (CIO4⁻), perrhenate (Reo4⁻), and pertechnetate (TcO4⁻), all anions
with a similar partial specific volume to I⁻. These anions compete with I⁻ for its
carrier and are concentrated by thyroid. A radioisotope of TcO4⁻ is commonly used
to study iodide transport but is not concentrated by the thyroid.
C. Oxidation of I⁻: The thyroid is the only tissue that can oxidize I⁻ to a higher valence
state, an obligatory step in I⁻ organification and thyroid hormone biosynthesis. This
step in I⁻ organification and thyroid hormone biosynthesis. This step involves a
heme-containing peroxidise and occur at the luminal surface of the follicular cell.
Thyroperoxidase, a tetrameric protein with a molecular mass of 60 KDa, requires
hydrogen peroxide as an oxidizing agent. H2O2 is produced by an NADPH-
dependent enzyme resembling cytochrome C reductase. A number of compounds
inhibit I⁻ oxidation and therefore its subsequent incorporation into MIT and DIT.
The most important of these clinically are the thiourea drugs. They are known as
antithyroid drugs because of their ability to inhibit thyroid hormone biosynthesis at
this step.
D. Iodination of Tyrosine: Oxidized I⁻ reacts with the tyrosyl residues in thyroglobulin
in a reaction that probably also involves thyroperoxidase. The 3 position of the
aromatic ring is iodinated first and then the position 5 to form MIT and DIT,
respectively. Once iodination occurs, the iodine does not readily leave the thyroid.
E. Coupling of Iodotyrosyls: the coupling of two DIT molecules to form T4 or of an
MIT and DIT to form T3 occurs within the thyroglobulin molecule. A separate
coupling enzyme has not been found, and since this, is an oxidative process, it is

5
assumed that the thyroperoxidase catalyzes this reaction by stimulating free radical
formation of iodotyrosine.
This hypothesis is supported by the observation that the same drugs which inhibit I⁻
oxidation also inhibit coupling. The formed thyroid hormones remain as integral
parts of Tg until the latter is degraded, as described above. Tg hydrolysis is
stimulated by TSH, but is inhibited by I⁻, this latter effect is occasionally exploited
by using potassium iodide to treat hyperthyroidism.

Fig 1: Iodine metabolism in Thyroid follicles.

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1.4) Regulation of Thyroid Hormone Synthesis

The regulation of thyroid hormones is a complex process involving not only the thyroid, but
the pituitary, the brain, and the peripheral tissues. Thyroid secretion is under the control of
the pituitary gland through the thyroid stimulating hormone (TSH). TSH is a glycoprotein
with a molecular weight of approximately 28,000. It has two units: the ‘X subunit’ has
virtually the same structure as other pituitary hormones, while the β subunit is specific for
TSH, but essentially the same across the different animal species.

The action of TSH occurs through the binding of β subunit to specific receptor sites in the
membranes of the thyroid cells. The ensuing sequence of events involves the activation of
denylate cyclise which in turn activates the phosphorylation of various proteins and enzymes
within each cell including the nucleus, where one major effect is on gene structures for RNA
replication and protein synthesis.

TSH activates all stages of iodine metabolism in the thyroid from trapping to the secretion of
T4 and T3, I⁻ transport is accelerated, there is an increase in the organic binding of iodide in
the tyrosine molecules, there is an increase in the coupling rate of MIT and DIT to form T4
and T3, and then pinocytosis of colloid by the thyroid cell leads to the release of T4 and T3
into the circulation.

The control of TSH secretion is by a “feed back” mechanism related closely to the level of
T4 in the blood, as the blood T4 falls, the pituitary TSH secretion rises to increase thyroid
activity and the output of T4 into the circulation, and so maintain the necessary level of
circulating hormone. If this is not possible, due for example to severe iodine deficiency, then
the level of T4 remains lowered and the level of TSH remains elevated. Both these
measurements are used for the diagnosis of hypothyroidism at various stages in life, but
particularly in the neonate.

TSH secretion is also under the control of the brain through the thyrotropin releasing
hormone (TRH), which is released from the hypothalamus. TRH is released into the pituitary
portal system from where it goes direct to the pituitary. There, it influences the synthesis and
release of the pituitary (Figure 2). TRH is in turn under the influence of neurotransmitters.
Adrenaline, noradrenaline, and serotonin increase its output, and dopamine, which reduces it.

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 Figure 2: Thyroid Hormone Regulation

1.5) Physiologic Effects of Thyroid Hormones

The Physiologic Effects of Thyroid Hormones are summarized in Table 1.

Table 1: Physiologic effects of Thyroid Hormones1

Target tissue Effect Mechanism

Nervous system Developmental  Promote normal brain development
 Promote normal growth and skeletal
Bone Developmental
development
 Increase number and affinity of β-adrenergic
Heart Chronotropic
receptors
 Enhance responses to circulating
catecholamines
Ionotropic
 Increase proportion of α myosin heavy chain
(with ATPase activity)
Adipose tissue Catabolic  Stimulate lipolysis
Muscle Catabolic  Increase protein breakdown

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  Increase rate of carbohydrate absorption
Gut Metabolic

Lipoprotein Metabolic  Stimulate formation of LDL receptors

 Stimulate oxygen consumption by
metabolically active tissues (exceptions are
Other Calorigenic testes, uterus, lymph nodes, spleen, anterior
pituitary)
 Increase metabolic rate

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2. Iodine Deficiency – Mechanism

2.1) Iodine Deficiency: A Disease of the Soil

The causes for iodine deficiency in humans should be considered in light of the iodine
requirements in the human beings. The ideal iodine intake as recommended by
WHO/UNICEF/ICCIDD is shown in Table 2.

Table 2: Recommended daily Iodine intake in Human (WHO/UNICEF/ICCIDD)2

S.No. Age Group Iodine requirement (in mcg)

1 Infants (0-11 months) 50

2 Children () 90

3 12-59 months 120

4 School age children (6-12 years) 150

5 Pregnant and lactating women 200

Iodine deficiency occurs when iodine intake falls below recommended levels. Iodine
deficiency is an ecological phenomenon occurring naturally in many parts of the world.
Figure 4 depicts the effect of the environmental iodine deficiency on humans, who are
perched on top of the food chain.

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Figure 4: Iodine deficiency: A disease of soil

Effect on people HUMANS Health and Socioeconomic

Impact

Effect on Animals LIVESTOCK Clinical and reproductive

disorders
Decreased productivity

Low Availability of Iodine PLANTS Iodine Poor Feeds and Fodder,

Goitrogens

Soil Erosion WATER, SOIL Environmental Iodine Deficiency

Iodine deficiency is basically a disease of the soil. The atmosphere absorbs iodine from the
sea which then returns through the rain and snow to the mountainous regions. It is then
carried to the lowers hills and plains, eventually returning to the sea (the Iodine Cycle in
nature). The erosions of soils in reverie areas due to loss of vegetation from clearing for
agriculture production, overgrazing by livestock and tree-cutting for firewood, ensures a
continued and increasing loss of iodine from the soil. High rainfall and snow as well as flood
further increase the loss of iodine from the soil which has already been denuded.
The return of iodine is slow and small in amount compared to the original loss. Groundwater
and locally grown plants in these areas also lack iodine. Consequently, the animals feeding on
these plants also lack iodine. This results in iodine deficiency in human beings who are
dependent on these animals and plants for their dietary supply of iodine. In effect, iodine
deficiency is “a disease of the soil” where the environmental deficiency results in the
manifestation of the disease state in humans, perched on the top most level of the food chain.

11
3. Spectrum of IDD & its Consequences

3.1) Spectrum of Iodine deficiency disorders

When iodine intake falls below the recommended levels, the thyroid gland is no
longer able to synthesise sufficient amount of thyroid hormones. The resulting low
level of the thyroid hormones in the blood is the principal factor responsible for an
abnormal swelling in the neck called goitre, for damage done to developing brain and
for many other harmful effects, now described collectively as Iodine deficiency
disorders (IDD).
Iodine deficiency is the most common cause of preventable mental retardation. Lack
of iodine causes irreparable damage even before childbirth. Children of iodine
deficient mother suffer from brain damage while still in womb. Thyroxin plays a very
important role in the development of the foetal brain and body, in addition to its role
in the generation and utilization of body energy. The optimal development of human
brain requires thyroxin. The most critical period is from second trimester of
pregnancy to third year after birth as 90% of human brain growth occurs in this
period. Iodine and subsequent thyroxin deficiency during early stages of life leads to
permanent and irreversible damages. Many children may even be stillborn. Those
who survive can be permanently crippled with a spectrum of mental handicaps and
physical deformity – commonly referred to as endemic cretinism. It has been
estimated, on an average, school children living in iodine deficient areas have 13 IQ
points lower than those in iodine sufficient areas. Thus total loss accumulated to the
country is formidable.
The table 1 shows the spectrum of IDD at various stages of life. These can be divided
into two categories - reversible and irreversible. The irreversible manifestations
include still birth, abortion and endemic cretinism (a syndrome consisting of mental
retardation, defects in speech, hearing and stunted growth). The reversible
manifestation of iodine deficiency includes goitre and hypothyroidism.

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Table 3: The spectrum of iodine deficiency disorder
 Miscarriage
 Stillbirths
 Congenital anomalies
 Increased perinatal morbidity and mortality
 Endemic cretinism
 Neurological cretinism:
o Mental deficiency
Fetus o Deaf-mutism
o Spastic diplegia
o Squint
 Myxedematous cretinism
o Mental deficiency
o Dwarfism
 Psychomotor defects
 Hypothyroidism
 Neonatal goiter
 Neonatal hypothyroidism
Neonate  Endemic mental retardation
 Increased susceptibility of the thyroid gland to nuclear
radiation
 Goiter
 (Subclinical) hypothyroidism
Child and  Impaired mental function
adolescent  Retarded physical development
 Increased susceptibility of the thyroid gland to nuclear
radiation
 Goiter with its complications
 Hypothyroidism
 Impaired mental function
Adult  Spontaneous hyperthyroidism in the elderly
 Iodine-induced hyperthyroidism
 Increased susceptibility of the thyroid gland to nuclear
radiation

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4. IDD control: Why and how?
4.1) IDD Control: Why?

The effects of iodine supplementation are shown in Table 4 .The benefits of iodine
supplementation in human population are enormous. The consumption of iodised salt by the
life stalk will also add to economic productivity of the population. Thus the cost benefit ratio
of iodine supplementation programmes is significantly large making it a worthwhile
investment, not only for better human resource development but also to add to economic
contribution from livestock sector.

Table 4: Effects of iodine intervention and measurement of benefits

Human population
Effects Benefits
Reduction in 1. Value of higher work output in
1. Mental deficiency household and labour market.
2. Deaf mutism 2. Reduced cost of medical and
3. Spastic diplegia custodial care.
4. Squint 3. Reduced educational costs from
5. Dwarfism reduced absenteeism and grade
6. Motor deficiency repetition and higher academic
achievement by students.
Life stock population
Effects Benefits
Increase in :
1. Live births
2. Weight 1. Value of higher output of meat and
3. Strength other animal products
4. Health 2. Value of higher animal work output
5. Wool coats in sheep

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4.2) Iodine Deficiency Disorders (IDD) control: How?

4.2a) Survey methods

4.2a.i) Salt monitoring

An IDD control programme based on salt iodisation clearly cannot succeed unless all salt for
human consumption is being adequately iodised. Therefore the most important thing to
monitor is the salt itself, and the most important place to monitor it is at the site of
production. The various sites for monitoring of salt for iodine content are:

a) Site of production
b) Port of entry
c) Point of final packing
d) Wholesale and retail level
e) Community or household level

4.2a.ii) Iodine status assessment

Iodine status assessment requires carrying out a cross sectional survey of a representational
sample of the entire target population. The recommended survey method is multistage
“probability proportional to size” (PPS) cluster sampling. This method has been in use for
many years for evaluation of immunization (EPI) coverage, and can be applied to many other
health indicators. The target population for survey should be either school age children or
women of child bearing age. Survey should be either school-based or household based.

4.2a.iii) Sentinel surveillance

Large scale, representative cross sectional surveys are generally too costly to be used as a
regular monitoring of IDD control. To assess the change in iodine status of a defined
population over time, the method of monitoring which has proved most practical is the one
done through selection of sentinel districts. Such districts are chosen on the basis of they
being remote and being affected by moderate or severe IDD prior to implementation of the
IDD control programme. In each sentinel district, at least three rural schools should be chosen
at random for surveying. An urban area should also be included as control, and again at least
3 schools should be selected. Sentinel surveillance survey s should be performed at least
every two years in the early stages of an IDD control programme, and then reduced in
frequency to once every two or three years one every two or three years .once the situation

15
appears stable .It is important to be flexible when establishing a system for monitoring IDD
control.

4.2b) Survey indicators

4.2b.i) Thyroid size

Assessment of thyroid size by palpation is the time honoured method of assessing IDD
prevalence, but the long response time after the introduction of iodine supplementation means
that it is of limited use in assessing the impact of the programme. The term goitre refers to a
thyroid gland that is enlarged. The statement that “a thyroid gland each of whose lobes have a
volume greater than the terminal phalanges of thumb of the person will be considered
“goitrous” is empirical, but has been used in most epidemiological studies of endemic goitre.
Palpitation of thyroid is particularly useful in assessing the goitre prevalence.

Goitre is graded according to classification in Table 5 and this classification is followed in

the epidemiological surveys conducted under National Iodine Deficiency Disorders Control
Programme (NIDDCP) in India.

Table 5: Simplified classification of goitre by palpation

Grades Feature

Grade 0 No palpable or visible goitre

Grade 1 A goitre that is palpable but not visible when
neck is in normal position , even when the
thyroid is not visibly enlarged>Thyroid
nodule in a thyroid , which is otherwise not
enlarged fall in to this category.
Grade 2 A swelling in the neck that is clearly visible
when the neck is in normal position and is
consistent with an enlarged thyroid when the
neck is palpated.

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4.2b.ii) Urinary Iodine

Urinary iodine excretion is a good marker of very recent dietary intake as most iodine
absorbed in the body will eventually appears in urine. In individuals, urinary iodine excretion
can vary somewhat from day to day and even within a given day, but this variation tends to
dampen out in the populations. The cut off point proposed for classifying iodine nutrition into
different degrees of public health significance as shown in Table 6.

Frequency distribution curves are necessary for full interpretation. Urinary iodine values from
populations are usually not uniformly distributed, and therefore, the median should be used as
the measure of central tendency rather than mean. (Likewise percentiles should be used as
measures of spread rather than standard deviation).For a population to have no iodine
deficiency, at least 50% of sample collected should have median urinary iodine concentration
of 100microgram per litre and above. In addition no more than 20% of samples should be
below 50µg/L. Alternatively the first quintile (20th percentile) should be at least 50µg/L. In
adults, a urinary iodine concentration of 100µg/L corresponds to roughly to a daily iodine
intake of about 150µg/L under steady condition.

Urinary iodine concentration is currently the most practical biochemical marker for iodine
nutrition, when carried out with appropriate technology and sampling. It assesses iodine
nutrition at the time of measurement whereas thyroid size reflects iodine nutrition over
months or years. Therefore, populations may have attained iodine sufficiency by median
urinary iodine concentration, yet goitre may persist, even in children.

Table 6: Epidemiologic criteria for assessing iodine nutrition based on

median urinary iodine concentration in school aged children.
Median urinary Iodine intake Iodine nutrition
iodine(µg/L)

<20 Insufficient Severe iodine deficiency

20-49 Insufficient Moderate iodine deficiency

50-99 Insufficient susceptible group Mild iodine deficiency

100-199 Adequate Optimal

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200-299 More than adequate No documented adverse
health effect

>300 Surplus ? increased risk of iodine-

induced hyperthyroidism,
auto-immune thyroid
diseases)

4.2b.iii) Blood constituents

Two other indicators are included thyroid stimulating hormones (TSH) and
thyroglobulin(Tg). While TSH levels in neonates are particularly sensitive to iodine
deficiency, difficulties in interpretation remain and cost of implementing a screening
programme is too much for most developing countries to afford. The value of thyroglobulin
as an indicator of IDD status is yet to be fully explored and it is yet to gain wide acceptance.

4.2c) Indicators of Sustainable Elimination of IDD

Table 7 enlists the criteria for achieving sustainable elimination of iodine deficiency as public
health problem.

Table 7: Summary of criteria for monitoring progressing towards sustainable

elimination of IDD as a public health system
Indicators Goals
1.Thyroid size ( age group 6-12)
Proportion with enlarged thyroid <5%
2. Urinary iodine
Proportion below 100µg/L <50%
Proportion below 50 µg/L <20%
3.Salt iodisation
Proportion of households using adequately iodised salt >90%
4.Programmatic indicators
Attainment of indicators At least 8 out of 10

 With regards to populations iodine status :

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  The median urinary concentration should be at least 100 µg/l, with less than 20%
of values below 50 µg/l.
 The most recent monitoring data ( national or regional) should have been collected
in the last 2 years

 If iodised salt is the vehicle for elimination of iodine deficiency, as in almost all
countries, there must be guaranteed availability and consumption of adequately iodised
salt (>15ppm of iodine) in more than 90% of households. Precondition for this are:

 Local production and/or importation of iodised salt in a quantity that is sufficient

to satisfy human demand (about 4-5kg/person/year).
 95% of salt for human consumption must be iodised according to government
standards for iodine content, at production or imported level.
 The percent of food grade salt with iodine content of at least 15 ppm, in a
representative sample of households, must be equal to or greater than 90%.
 Iodine estimation at the point of production or importation and at whole sale an
detail levels must be made by titration, while at household level, it may be made
by either titration or certified kits.

 Programmatic indicators: At least 8 of the 10

1. National body responsible to the government for IDD elimination. It should
multidisciplinary, involving the relevant fields of nutrition, medicine, education,
the salt industry, the media, and consumers, with a chairman appointed by the
Minister of Health.
2. Evidence of political commitment to USI and elimination of IDD.
3. Appointment of a responsible executive officer for the IDD elimination program
4. Legislation or regulations on USI.
5. Commitment to regular of progress in IDD elimination, with access to
laboratories able to provide accurate data on salt and urinary iodine.
6. A program of public education and social mobilization on the importance of IDD
and the consumption of iodized salt.
7. Regular data on iodized salt at the factory, retail and household levels.
8. Regular laboratory data on urinary iodine in school-aged children, with
appropriate sampling for higher risk areas.

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9. Co-operation from the salt industry in maintenance of quality control.
10. A database for recording of results or regular monitoring procedures particularly
for salt iodine, urinary iodine and, if available, neonatal TSH, with mandatory
public reporting.

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5. Laboratory Procedure for Iodine Estimation of Salt

5.1) Background material:

Iodine is one of the first minerals recognized as essential for human health. Iodine
belongs to the family of halogens (Chlorine, bromine, iodine and fluorine) placed in the
seventh group of period table. Most of the iodine exists in the ocean. It was present during the
primordial development of the earth but large amounts were leached from the surface soil by
glaciations, snow, or rain and were carried by rivers, floods and winds into the sea.

Iodine is an essential micronutrient for humans and is required in a very small amount
i.e. 150 µg per day. The only role of iodine in the body, known at present is for the synthesis
of thyroid hormones. The human body contains 15 to 20 mg of iodine of which almost 80% is
in the thyroid gland. The iodine taken in the diet is absorbed throughout gastrointestinal tract
and circulates as plasma Inorganic Iodide (PII) in the body. The PII is mainly cleared by two
organs in the body - Thyroid and Kidney. The iodine taken up by the thyroid gland is used for
making thyroid hormones, thyroxine and triiodothyronine. Thyroxine is called T4 because it
has 4 iodine atoms in its structure while triiodothyronine is called T3 because it has 3 iodine
atoms in its structure. Iodine taken up by the kidney is excreted in the urine. The level of
excretion in urine correlates well with level of intake. Thus urinary iodine can be used to
assess the level of iodine intake.

Thyroxine has two major roles in the body one is for development of brain, central
nervous system and skeletal system during the early developmental stages and the second is
calorigenic effect i.e. increase in oxygen uptake by tissues thereby controlling all metabolic
processes in the body.

Iodine deficiency occurs mainly as an environmental deficiency. Iodine present in

upper crust of earth is washed/leached out by repeated flooding and glaciation. Thus making
the soil deficient in iodine. Crops grown on such soil will have less iodine and when
consumed by humans or animals lead to iodine deficiency. Thus it is the deficiency of soil
which is responsible for iodine deficiency in humans and animals.

21
 Iodine deficiency during pregnancy and first two to 3 years of life leads to
derangement in the development of brain and central nervous system & skeletal. These
changes are irreversible. No amount of iodine or thyroxine can revert back the damage done
during the developmental period leading to several disabilities like deaf mutism, squint, gait
defects etc. but the most important of these is the loss of learning ability with loss of 13 IQ
points. These can be easily prevented by supplying proper amount of iodine during this
period.

Since there is no natural food item which will help in supplying adequate amount of
iodine, it is essential to supplement or fortify the food item for meeting the iodine
requirements of the individual.

There are several modes of iodine supplementation used. These are: Iodised oil,
Iodised capsules, Iodised bread, Iodised water, Iodised salt

Out of these the most effective method is iodised salt. This is because salt is one food
item which is taken in a fixed amount everyday by everybody whether rich or poor, old or
young. Thus it is an ideal vehicle for supply of constant amount of iodine to everybody daily.
It is also most economical way of supplying iodine. Government of India had taken a
decision of supply of iodine via iodised salt from 1962 under the National Goitre Control
Programme.

The iodised salt may lose iodine due to moisture and heat during transportation. To
avoid this the PFA (Prevention of Food Adulteration) Act states that iodine content of salt
should not be less than 30 ppm at production and 15 ppm at consumer level. It is essential to
monitor the amount of iodine in salt by quantitative method (Iodometry). The details of
procedure are given.

22
5.2) Principle and Laboratory Procedures for Iodine Estimation in Salt

____________________________________________________________________

I) Principle

The iodine content in iodated salt is estimated by a process called iodometric titration.
Free iodine reacts with sodium thiosulphate solution as follows:

2Na2S2O3 + I2 = 2NaI + Na2S4O6

Sodium Iodine Sodium Sodium

thiosulphate iodide tetrathionate

II) Equipment and Chemicals

a) Equipment

1. Laboratory balance for preparing reagents

2. Beakers – 100, 200, 500 ml

3. Glass bottles with stoppers for reagents:

1,000 ml

250 ml

4. Open pan balance for weighing salt samples

23
5. Measuring cylinders with stopper 50 ml
6. Wash bottle 500 ml
7. Conical flasks with stopper 200 ml

8. Glass or plastic funnel

9. Auto dispensers

10. Burette 10 ml auto zeros

24
b) Chemicals

1. Sodium thiosulphate - Na2S2O3, Analytical Regent Grade (AR)

2. Concentrated sulphuric acid – H2SO4, (AR)

3. Potassium iodide – KI, (AR)

4. Soluble chemical starch

5. Boiled double-distilled water, pharmaceutical grade

The approximate cost of reagents would be Rs. 1200, which would analyze 100 salt
samples.

III) Preparation of Reagents

a) Sodium thiosulphate (Na2S2O3): Dissolve 1.24 grams in 1 litre double-distilled

water. Store the solution in a cool and dark place. Normality may change as
time progresses. It is advisable to prepare small quantity of 0.005 N Na2S2O3
each day as required to avoid change of normality and storage problems.

b) 2.N Sulphuric acid (2H2SO4): To 90 ml double-distilled water, add 5.56 ml

concentrated H2SO4 slowly. Add double-distilled water to make 100 ml. Store in
a cool dark place. The solution may be kept indefinitely.

Caution: To avoid violent and dangerous reaction always add acid to water,
never water to acid.

c) Potassium iodide (KI, AR): Dissolve 10 grams KI in 100 ml double-distilled

water. Store in a cool, dark place. Properly stored, the solution may be kept for 6
months.

d) Saturated Sodium Chloride (NaCl): To boiling double distilled water go on

adding sodium chloride while stirring until no more of it dissolves. Cool the
solution

25
 e) Soluble Chemical Starch: Weigh 1 gram of soluble starch and dissolve in 10 ml
double distilled water. Add 90 ml of saturated sodium chloride solution to make
it up to 100 ml. Add a pinch of sodium benzoate as a preservative.

IV) Procedure

Weigh 10 grams of salt and put it in a stoppered conical flask. Add 50 ml

of distilled water and dissolve the salt. Add 1 ml of 2N H2SO4 followed by 1 ml
of 10% KI solution with the help of an automatic dispenser and close the flask
with a stopper. If the iodine is present, the solution will turn yellow. Keep the
flask in the dark (for e.g. in a closed cupboard) for 10 minutes to avoid exposure
to light. Remove the flask and titrate against 0.005N Na2S203. During the
titration, when the yellow colour becomes pale, add 2 drops of the starch solution
as an external indicator. The solution will become purple. Continue titration till
the solution becomes colourless. Note the burette reading. To calculate the iodine
content in parts per million (PPM), refer to the Table – 8.

26
 Table 8: Iodine Content in Parts Per Million (PPM)
Burette reading Parts Per Million Burette reading Parts Per Million
(PPM) (PPM)

0.0 0.0 2.6 27.5

0.1 1.1 2.7 28.6

0.2 2.1 2.8 29.6

0.3 3.2 2.9 30.7

0.4 4.2 3.0 31.7

0.5 5.3 3.1 32.8

0.6 6.3 3.2 33.9

0.7 7.4 3.3 34.9

0.8 8.5 3.4 36.0

0.9 9.5 3.5 37.0

1.0 10.6 3.6 38.1

1.1 11.6 3.7 39.1

1.2 12.7 3.8 40.2

1.3 13.8 3.9 41.3

1.4 14.8 4.0 42.3

1.5 15.9 4.1 43.4

1.6 16.9 4.2 44.4

1.7 18.0 4.3 45.5

1.8 19.0 4.4 46.6

1.9 20.1 4.5 47.6

2.0 21.2 4.6 48.7

2.1 22.2 4.7 49.7

2.2 23.3 4.8 50.8

2.3 24.3 4.9 51.9

2.4 25.4 5.0 52.9

2.5 26.5 5.1 54.0

27
Burette reading Parts Per Million Burette reading Parts Per Million
(PPM) (PPM)

5.2 55.0 7.7 81.5

5.3 56.1 7.8 82.5

5.4 57.1 7.9 83.6

5.5 58.2 8.0 84.6

5.6 59.2 8.1 85.7

5.7 60.3 8.2 86.8

5.8 61.4 8.3 87.8

5.9 62.4 8.4 88.9

6.0 63.5 8.5 89.9

6.1 64.5 8.6 91.0

6.2 65.6 8.7 92.0

6.3 66.7 8.8 93.1

6.4 67.7 8.9 94.2

6.5 68.8 9.0 95.2

6.6 69.8 9.1 96.3

6.7 70.9 9.2 97.3

6.8 71.9 9.3 98.4

6.9 73.0 9.4 99.5

7.0 74.1 9.5 100.5

7.1 75.1 9.6 101.6

7.2 76.2 9.7 102.6

7.3 77.2 9.8 103.7

7.4 78.3 9.9 104.7

7.5 79.4 10.0 105.8

7.6 80.4

28
5.3) Calculations

The table of iodine content as determined by the burette reading has been prepared based
on the following:

1) 1 ml of 0.005N Na2S2O3 = 0.1058 mg of iodine

2) Thus, the burette reading X 0.1058 will give the amount of iodine in 10 gm of
salt

3) To get the iodine value in parts per million, one has to multiply by 1,00,000 to
either sides

Gms of salt I2 in mg

10 X 1,00,000 0.1058 X 1,00,000

4) To convert mg in to gm, divide by 1000

Equation becomes:

Gms of salt (I0,00,000) = Burette Reading X 0.1058X 100

= Burette Reading X 10.58

Thus Burette reading X 10.58 will give the iodine content in parts per million.

29
5.4) Precautions

Adding sulphuric acid to a solution of iodated salt liberates iodine, which is titrated with
sodium thiosulphate. Starch is used as an external indicator. Potassium iodide solution
is added to keep the iodine in the dissolved state.

1. The starch solution must be added near the end of the titration, when very
little iodine is left and the solution has a faint-yellow colour. If starch is
added earlier, the iodine starch complex becomes very strong and reacts too
slowly with sodium thiosulphate, resulting in false high readings.

2. The titration should be done in a comfortably cool room because iodine is

volatile and the sensitivity of the starch indicator diminishes as the
temperature rises.

3. Potassium iodide (KI) is used because of the low solubility of iodine. The
liberated iodine forms an unstable complex KI3 with KI:
KI + I2 = KI-3 and I- + I2 = I3

As free iodine is used up in the reaction with thiosulphate, the equilibrium

between I2 and I3 ions is disturbed and more iodine is dissolve in order to
maintain the equilibrium.

4. A few minutes should be allowed before titration, since the rate of reaction
between I- ions and the oxidant is slow.

5. The reaction mixture should be kept in the dark before titration because light
accelerates a side reaction in which iodide ions are oxidized to iodine by
atmospheric oxygen.

30
6) Laboratory Procedure for Urinary Iodine Estimation

6.1) Background

Iodine is ingested in food is in a variety of chemical forms. Most of it is broken in the gut and
absorbed into the blood stream where it circulates as Plasma Inorganic Iodide (PII). PII is
either taken up by thyroid gland or kidney. Only a fraction of PII goes into thyroid gland and
90-98% of PII comes out in the urine. Thus urinary iodide excretion reflects iodine intake of
an individual and is used an indicator of iodine nutrition of an individual. In epidemiological
surveys, urinary iodine excretion pattern indicates the iodine nutrition in the population and
has been effectively used to decide whether iodine deficiency exists in the population or not.

6.2) Urinary Iodine Estimation: Laboratory Procedure

Urinary iodine estimation include initial step in which urine is digested in strong acid, ashed
at high temperature or use of chemical agents such as ammonium per sulphate. Following
digestion step, iodine is measured by its catalytic action on the reduction of Cerric ion (Ce 4+)
to Cerrous ion (Ce +3) coupled to oxidation of arsenous (AS +3) to arsenic (AS +5). This
reaction is called Sandell –Kolthoff reaction:

2 Ce+4 + 2 I- → 2 Ce3+ + I2

I2 + AS+3 → AS +5 + 2I-

Cerric ion (Ce 4+) has yellow colour, while cerrous (Ce 3+) is colourless. The reaction can be
followed by the disappearance of yellow colour. The colour disappearance is directly
proportional to amount of iodide present in the system.

Excellent discussions of the Sandell-Kolthoff reaction have been published. The role of the
iodide ion is highly specific; iodate, chloride, and bromide have only slight catalytic activities.
Sulfuric acid and chloride are both important components of the reaction mixture; sulfuric
acid increases the speed of the reaction, and chloride stabilizes it by inhibiting oxidation of
iodide to iodate. Arsenite is present in large excess; some authors have recommended a ratio
of (Ce4+) to As3+ of 1:20.

31
Iodide is generally believed to be the chemical form of iodine in the urine and is also the form
that is catalytic in the Sandell-Kolthoff reaction. Therefore, it is reasonable to ask why a
digestion or ashing step is necessary to prepare urine samples for iodine determination by this
method. The answer is that digestion or ashing removes other substances, such as nitrite,
thiocyanate or ferrous iron that might interfere by reducing or oxidizing the ceric or arsenite
reactants. For example, thiocyanate in the urine can accelerate the Sandell-Kolthoff reaction,
thus arti-factually elevating the apparent iodide value. Thiocyanate is derived from the
ingestion of cassava which is consumed in many parts of the world, including those with
iodine deficiency, and its interference with the Sandell-Kolthoff reaction in undigested urine
can introduce serious error in iodine estimation. Cigarette smoking is another frequent source
of thiocyanate; heavy smokers (10 or more cigarettes per day) have urinary thiocyanate levels
as high as those of cassava eaters. Other interfering substances have not been as well
characterized, but most observers have noted that failure to incorporate a digestion or ashing
step can arti-factually raise or lower the apparent iodine concentration in the Sandell-Kolthoff
reaction, presumably by failing to remove such substances. On the other hand, Pineda
(Method F) routinely proceeds directly to the colorimetric assay without prior digestion or
ashing. On comparison, he found that the iodine concentration determined in the undigested
sample was about 72% the value found for the same sample when digested. He proposes a
correction factor to adjust for this difference.

Garrey et al. described a method using dialysis rather than digestion prior to colorimetric
determination for urinary iodine, and found that dialyzed samples gave essentially the same
values as digested ones. However, these samples came from children in iodine sufficient
areas of the United States, so virtually all samples were above 5 g/dl, and most were above
20 g/dl. May et al. reported that the dialysis technique gave arti-factually high iodine values
for urine samples from iodine – deficient areas of China, sometimes doubling the true values
obtained after ashing.

We conclude that at present the safest course is to include an ashing or digestion step prior to
colorimetric reaction for urinary iodine. However, if one can show that assays of digested
and undigested aliquots of the same urine samples consistently give the same iodine value
and that this result is homogeneous throughout a given study population, then omission of the
ashing or digestion step may be feasible.

32
 Other methods have occasionally been employed for urinary iodine determination, but they
are generally not practical for survey purposes. Ion specific electrodes can determine iodine
in urine, but currently are not sensitive enough to recognize the low levels encountered in
areas of iodine deficiency, and determination is not rapid. Neutron activation is sensitive and
specific, but cannot be done on large numbers of samples in a routine manner. Gas liquid
chromatography has also been used, but requires special equipment and is not as rapid as
some of the methods described here for epidemiologic purposes.

In summary, the Sandell-Kolthoff reaction, with some prior step involving ashing or
digestion, is usually the most practical approach for laboratory determination of urinary
iodine.

6.3) Overview of Available Urinary Iodine Estimation Methods

Table 9- General overview of the features of different methods for urinary iodine estimation.

Method
A B C D E F G
Sample preparation Mild Mild Vigorous Vigorous Ashing None Vigorous
Digest Digest Digest Digest Digest
Iodide analysis Colorimeter Stopwatch Auto- Colorimeter Colorimeter Colorimeter Auto-
analyzer analyzer
Removal of interfering Good Good Excellent Excellent Excellent None Excellent
substances

Samples/day/technician 150 150 100 50 25 150 50

Equipment cost Low Low High Low Low Low High
Operating cost Low Low Medium Medium High Low Medium
Equipment dependability Good Excellent Medium Good Good Good Medium

Ease of operation Excellent Excellent Medium Medium Medium Excellent Medium

Speed Excellent Excellent Good Fair Fair Excellent Fair
Precision Good Good Excellent Good Good Good Excellent
Safety Good Good Fair Fair Excellent Excellent Good

The two methods described in this chapter are (1) Ammonium Persulfate Digestion
Microplate (APDM) method, and (2) Conventional Ammonium Persulfate Digestion in Test
Tube and Spectrophotometer (NIN, Hyderabad) method. These two methods are commonly

33
being used in most of the iodine monitoring laboratories. These methods use ammonium
persulfate for digestion and avoid chloric acid which generates toxic fume.

6.4) Ammonium Persulfate Digestion Microplate (APDM) Method

Equipment, Apparatus and Glassware required:

1. Microtitre plates (Corning Costar Japan): Round and flat bottom.
2. Standard oven with stainless steel chamber which can maintain temperature of 95oC.
3. Sealing cassette specially designed by Hitachi Chemical Technoplant
4. Microplate reader (Elisa Reader)
5. Measuring devices (Micropipettes 0-100µl & 10-1000 µl, Multichannel 8 plate
micropipettes 0-100µl, Standard pipettes – 10ml, 5ml, 1ml)
6. Vortex Mixer
7. Digital Weighing machine one pan Balance (Accuracy up to .1mg)
8. UPS
9. Micro Tips (100 µl and 1000 µl) and Micro tip stand
10. Petri dish
11. Steel Spatula
12. Volumetric flask- 1000, 500, 250 and 100 ml
13. Measuring cylinder- 25, 50, 100, 250, 500 ml
14. Test tube – 1.5 * 15 cm
15. Amber colored Bottle- 1000, 500 ml
16. Funnel glass- 100mm
17. Para film roll
18. Filter paper- Quality circles 150 mm (Whatman)

Chemicals required: (All chemicals should be Analytical Grade)

1. Ammonium Per Sulphate
2. Arsenic trioxide
3. Tetra Ammonium Sulphate dehydrate
4. Concentrated Sulphuric Acid
5. Sodium Hydroxide
6. Potassium Iodate

34
Preparation of reagent:

1. Ammonium persulphate: Dissolve 5gm in 15 ml of double distilled water in a

measuring cylinder. Prepare fresh every day.
2. Arsenious acid solution: dissolve 5 gm of Arsenic trioxide in 100 ml of sodium
hydroxide (3.5 gm w/v) in a volumetric flask. Put in ice bath and add 16 ml of
concentrated Sulphuric Acid (add acid slowly in small amount). After cooling add
12.5 gm of sodium chloride to the volumetric flask and dilute up to 500 ml with cold
double distilled water. Filter and store in amber coloured reagent bottle. Store at 4oC.
3. Cerric Ammonium per sulphate: Prepare 3.5 N H2HSO4 solution by adding 24.25 ml
H2SO4 to 200 ml distilled water and dilute it to 250 ml. Dissolve 6 gm Cerric
Ammonium sulphate dehydrate in 3.5 N H2SO4 and dilute up to 500 ml with 3.5 N
H2SO4. Store in amber colored bottle at 4oC.
4. Standard iodide calibrators: dissolve168.6 mg of potassium iodate in 100 ml of double
distilled water in a volumetric flask. (100 mg/dL or 1mg/ml solution). Label this as
flask 1. Dilute flask 1 by 1000 times to get 1µg/ml solution. Label it as flask 2.
Prepare standard containing 10, 20, 100, 200, 300, 400 µg/l solution for standard
graph. The amount of flask 2 solution and water to dilute it to get respective strength
of standards can be calculated from following table (Table 10):

Standard Solution 10 20 50 100 150 200 300 400

Strength µg µg µg µg µg µg µg µg

Flask 2 solution 0.1 0.2 0.5 1.0 1.5 2.0 3.0 4.0

Water 9.9 9.9 9.5 9.0 8.5 8.0 7.0 6.0

Table 10: Composition of different standard iodide calibrators

35
Procedure:

1. In microplate pipette out 50 µl of standards known values samples and urine samples
in the wells as shown below.

1 2 3 4 5 6 7 8 9 10 11 12
A Blank 100 Urine Urine Urine Urine Urine Urine Urine Urine Urine Urine
Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample
B 0 200 Urine Urine Urine Urine Urine Urine Urine Urine Urine Urine
Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample
C 0 200 Urine Urine Urine Urine Urine Urine Urine Urine Urine Urine
Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample
D 10 300 Urine Urine Urine Urine Urine Urine Urine Urine Urine Urine
Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample
E 10 300 Urine Urine Urine Urine Urine Urine Urine Urine Urine Urine
Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample
F 20 400 Urine Urine Urine Urine Urine Urine Urine Urine Urine Urine
Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample
G 20 400 Urine Urine Urine Urine Urine Urine Urine Urine Urine Urine
Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample
H 100 Known Urine Urine Urine Urine Urine Urine Urine Urine Urine Urine
Value Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample
Sample

Blank A 1 – Nothing to be added.

In the first two columns – standard solutions of iodide as shown plus the known Value
Sample.

Column 3 to 12 urine sample so be analysed.

2. 50 µl of urine samples and standard are to be taken in respective microplate wells.

3. Add 100 µl of Ammonium Persulphate (freshly prepared) in each well using multi
channel micropipette.

4. Put the urine plate in heating cassette, seal the cassette and put it in oven at 95oC for
90 minutes.

5. Take out the cassette from oven and allow it to cool to room temperature.

36
 6. Take out the digested microplate and take another microplate (flat bottom) and
transfer 50 µl of digested standards and urine samples in corresponding wells using
multichannel pipette.

7. Add 100 µl of Arsenious Acid solution using multichannel micro pipette.

8. Wait for 15 minutes.

9. Add 50 µl of cerric Ammonium sulphate solution using multichannel micro pipette.

10. Measure Optical Density (O.D) using microplate reader at 405 nm.

11. Plot the graph using log of absorbance on Y-axis vs iodide concentration (µg/l) on X-
axis.

12. Extrapolate the values of urine sample from the graphs.

6.5) Conventional Ammonium Persulfate Digestion in Test Tube and

Spectrophotometer (NIN, Hyderabad) method

Equipment, Apparatus and Glassware required:

 Frigidaire with freezer compartment to store urine samples
 Reagent flasks and bottles
 Pipettes
 Weighing balance scale
 Oven / Heating block (vented fume hood not necessary)
 Colorimeter / Spectrophotometer

Chemicals required: (All chemicals should be Analytical Grade:

1. Potassium iodate (for calibrators)
2. Arsenic trioxide (analytical grade)
3. Potassium chlorate
4. Perchloric acid (700 g/L)

37
 5. Ammonium persulfate
6. Tetraammonium cerium (IV) sulfate dehydrate
7. Sodium chloride
8. Sulfuric acid
9. Glass distilled deionized water

Preparation of reagent:
a) Ammonium persulfate solution: Dissolve 14.1 g H2N2O8S2 in H2O, make up to
500 ml with H2O. Store away from light. Stable for at least one month.

b) 5 N H2SO4: Slowly add 139 ml concentrated (36 N) H2SO4 to about 700 ml

deionized water (careful – this generates heat!). When cool, adjust with
deionized water to a final volume of 1 litre.

c) Arsenious acid solution: In a 2000 ml Erlenmeyer flask, place 20 g As2O3 and

50 g NaCl, then slowly add 400 ml 5 N H2SO4. Add water to about 1 litre, heat
gently to dissolve, cool to room temperature, dilute with water to 2 litres, filter,
and store in a dark bottle away from light at room temperature. The solution is
stable for months.

d) Ceric ammonium sulfate: Dissolve 48 g ceric ammonium sulfate in 1 litre 3.5 N

H2SO4. (The 3.5 N H2SO4 is made by slowly adding 97 ml concentrated (36 N)
H2SO4 to about 800 ml deionized water (careful – this generates heat!), and when
cool, adjusting with deionized water to a final volume of 1 litre). Store in a dark
bottle away from light and keep at room temperature. The solution is stable for
months.

e) Iodine calibrators: In a 100-mL volumetric flask, 168.6 mg of potassium iodate

was dissolved in water to make a 7.88 mmol/L stock solution (1000 mg/L iodine).
The stock solution was diluted 100- and 10000-fold, and working solutions of
0.039-4.73 mol/L (5-600 g/L iodine) were prepared.

38
Procedure:
1. Mix urine to suspend sediment.

2. Pipette 250 l of each urine sample into a 13 x 100 mm test tube. Pipette
each iodine standard into a test tube, and then add H2O as needed to make a
final volume of 250 l. Duplicate iodine standards and a set of internal urine
standards should be included in each assay.

3. Add 1 ml 1.0 M ammonium persulfate to each tube.

4. Heat all tubes for 60 minutes at 100 C.

5. Cool tubes to room temperature.

6. Add 2.5 ml arsenious acid solution. Mix by inversion or vortex. Let stand
for 15 minutes.

7. Add 300 l of Ceric ammonium sulfate solution to each tube (quickly mixing)
at 15-30 second intervals between successive tubes. A stopwatch should be
used for this. With practice, a 15 second interval is convenient.

8. Allow to sit at room temperature. Exactly 30 minutes after addition of ceric

ammonium sulfate to the first tube, read its absorbance at 420 nm. Read
successive tubes at the same interval as when adding the ceric ammonium
sulfate.

9. Calculation of results: Construct a standard curve on graph paper by plotting

iodine concentration of each standard on the abscissa against its optical
density at 405 g/l (OD405) on the ordinate.

39
6.6) Precautions

An iodine-free work place: The methods described in this book detect minute
amounts of iodine. Any iodine in the environment or in reagents will grossly
distort analytic values for urinary iodine. Also, reducing or oxidizing substances
in the environment can interfere, for example, the thiocyanate in cigarette
smoke. It is difficult and unnecessary to identify all possible contaminants;
rather, the technician should exercise meticulous care and cleanliness in all
stages of the analysis, including preparation of standards and reagents.

Some important rules:

a. Weigh standard iodate samples well away from the area of subsequent
analysis, preferably in a different room. Carefully avoid carrying any
iodine back to the analytical area on clothing, spatulas, hands etc. Wash
hands carefully after preparing standards.

b. It is best to have a dedicated area or room for iodine analyses. Keep the
working area well separated from any other work with iodine, such as
making up iodine solutions for iodized water etc. Also, keep the area for
iodine analysis well protected from other laboratory procedures with
oxidizing or reducing agents, to avoid contamination.

c. The chemical reagents must be iodine-free. Use analytic grade only.

Reagents must also be free of moisture. For example, KI is hygroscopic,
and if used as a standard, care must be taken in weighing it to avoid
moisture accumulation, particularly in hot humid climates. In accordance
with general recommended laboratory procedure, do not pipette directly
from a reagent bottle, but instead pour into another vessel slightly more
than the approximate amount needed, and after pipetting the needed
amount, discard the excess rather than returning it to the reagent bottle.

d. The water used for reagents and dilution of samples must be iodine-free.
Most laboratories will have distilled and / or deionized water. This water

40
 should be routinely checked to make sure it is iodine free. Water can be
easily deionized by passing through an appropriate resin. Laboratory
supply houses list a variety of systems that can be connected to tap lines,
or through which water can be poured manually; typical costs are US
$100 for a cartridge; must be replaced when resin exhausts.

e. Much of the glassware in iodine analyses can be reused, but it must be

carefully washed, including a final wash with deionized iodine-free water.
Most disposable pipette tips and other plastic items as supplied by the
manufacturer are unlikely to have contaminating iodine.

f. Glassware and tips used for preparation for standard iodine solution
should be washed and kept separately from other glassware and tips
used for carrying out urinary iodine estimation.

g. The technician must scrupulously avoid any opportunity to introduce

contaminating iodine. Hands should be washed carefully and frequently.
Disposable gloves are useful. Eating, spitting, drinking, and smoking
should not take place in the iodine laboratory.

41
7) Laboratory Quality Assurance for Salt Iodine Content
Estimation

7.1) Introduction:

Monitoring of iodine content at production level forms one of the most critical components of
Universal Salt Iodization (USI) strategy to ensure elimination of IDD. The system to ensure
adequate levels of iodine content at production level requires a setup of laboratories. It is
imperative to standardize and establish a coordinated system of laboratories to contribute to
elimination of IDD in India. An appropriate quality assurance and quality control protocols is
essential to ensure this. Quality assurance is not a fault finding mission but a method to
ensure quality of results from each laboratory.

7.2) Difference between Quality Assurance (QA) and Quality control (QC)

The two terms quality assurance and quality control are used interchangeably though their
meanings differ albeit only marginally.

Quality assurance is a proactive and continuous process of monitoring a system for

reproducibility and reliability by:

a) Setting standards of performance & designating responsibility

b) Ensuring definitive corrective actions are taken when the criteria not met

c) Performing measurements within a stated level of confidence

A quality control system is an indicator system for documented performance and actions that:

a) Provides a record of consistency of performance

b) Records action taken when performance fails to meet standard

Thus quality control is tool by which the principle of quality assurance is achieved.

42
There are essentially two types of Quality Assurance protocols:

1) Internal Quality Assurance: steps taken by the implementing laboratory to

ensure that the process/system used by them to estimate salt iodine content
are consistent and operate with a specified level of confidence.

2) External Quality Assurance: steps taken by an external review agency to

ensure that the process/system used by the implementing laboratory to
estimate salt iodine content are consistent and operate with a specified
level of confidence.

7.3) Elements of QA system

a) An inventory agency responsible for coordinating QA/QC activities;

b) A QA/QC plan;
c) General QC procedures;
d) Functions of participants
e) Reporting, documentation, and archiving procedures.

a) An inventory agency responsible for coordinating QA/QC activities: an independent

agency to act as an inventory agency, which in collaboration with ICCIDD would
implement the QA system for all laboratories in country.

b) QA/QC plan:
1) Each Laboratory to have an internal quality assurance protocol.
2) For monitoring of internal quality assurance protocol and for external
quality assurance protocol all laboratories would be attached to main
reference laboratory at ICCIDD, New Delhi.

c) General QC procedures:

43
7.4) Steps in Internal Quality Assurance protocol:

Step1: Preparation of known value samples for Salt Iodine Estimation.

Step 2: Analysis of known value sample with every batch of salt to be tested.

Step 3: Plotting Levy Jenning plot.

Step 4: Monitoring and feedback based on results of Levy Jenning plot.

Step5: Six salt samples from participating laboratory to be collected randomly on six
different days and analyzed in duplicate.

Step6: Salt samples along with the results to be sent to the reference laboratory each month
(at least 50gms for each sample).

Step7: The reference laboratory will analyze these salt samples in duplicate and then compare
the results with the values obtained by laboratory which sent the samples.

Step 1: Preparation of known value samples for Salt Iodine Estimation

1. A sample of salt (1/2 – 1 kg) collected from the market

2. Analyze iodine in the salt samples for 10 times

3. Calculate mean  S.D and document them.

4. Store the samples in aliquot of 20 gms each in ziplock plastic bags

5. Preserve at room temperature away from heat and moisture

6. Take out one ziplock packet of salt each time for internal quality assessment, to be
run with the analysis of unknown samples

7. The ziplock bags are stable if stored properly, for 6-12 months

Step 2: Analysis of known value sample with every batch of salt to be tested.

1. By running minimum of one known value sample along with batch of test sample
at start of every analysis.

2. More than one known value sample can be tested in a day if different stock of
reagents is used during the day or a different batch of test samples is to be tested.

44
Step 3: Plotting Levy Jenning plot.

1. Quality control results can be reported by the system in several different ways.
2. Graphically, data can be presented in Levy-Jennings format.
3. Mean and standard deviations obtained from the initial 10 times analysis while
preparing the known value specimen are plotted on Y axis. (Fig 5)
4. The units and scale of y axis is the SD obtained from the initial 10 times
analysis while preparing the known value specimen.
5. Day of month is plotted on X axis and the value of known value salt sample
obtained on that particular day is plotted in the graph.

Fig. 5: Sample levy Jenning plot for Internal Quality assurance of salt Iodine
estimation.
Levy Jenning Plot

50
5 P
+ SD 40
3
30 P
Mean 1
20 M
- SD -1

-3
10 of I2
0
-5
0 1 2 3 4 5 6 7
Day of Month

Step 4: Monitoring and feedback based on results of Levy Jenning plot

1. If the value falls between  2 S.D. as shown in Fig. 5

 Indicates consistency of method
 Reagents quality
 Performers ability

45
 2. If the value falls outside  2 S.D. as in Fig.6
 Check aliquots for internal quality
 Check regents for contamination
 Pipetting error
 Any other

Fig. 6: Sample levy Jenning plot for Internal Quality assurance of salt Iodine
estimation with values falling outside  2 S.D.
Levy Jenning Plot

50
5 P
+ SD 40
3 30 P
Mean 1
20 M
- SD -1
-3
10 of I2
0
-5
0 1 2 3 4 5 6 7
Day of Month

Step5: Six salt samples from participating laboratory to be collected randomly on six
different days and analyzed in duplicate.

Step6: Salt samples along with the results to be sent to the reference laboratory each
month (at least 50gms for each sample).

Step7: The reference laboratory will analyze these salt samples in duplicate and then
compare the results with the values obtained by laboratory which sent the samples.

46
7.5) External Quality Assurance

External quality assurance is a process by which quality of results from each laboratory are
ensured by an external independent agency. The known value samples are exchanged
between the laboratories participating in the quality assurance program with a central
laboratories functioning as a reference laboratory. Samples to be analysed along with routine
test samples. No special treatment or precaution to be taken for external quality assurance
samples.

Steps in External Quality Assurance:

1. The reference laboratory will analyze six salt samples (different brands) purchased
from the market, six times each.
2. Find the Mean and the Standard Deviation for each salt sample.
3. Reference laboratory will send 50 gms of each salt sample in zip lock plastic bags to
the participating laboratory each month.
4. Participating laboratory will analyze these salt samples in duplicate and send the
results to reference laboratory.
5. Reference laboratory then will compile the results, compare the values, and advise
any remedial measures.

d) Functions of different laboratories involved:

Functions of Participating Laboratories

1) To follow IQA protocol and maintain Levy Jenning plot.

2) To send 6 salt samples analyzed in duplicate (collected on 6 different days of month)

to reference laboratory every month.

3) To analyze six salt samples in duplicate received from reference laboratory every
month.

4) Communicate the results IQA and EQA to reference laboratory once every month.

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Functions of Reference Laboratory (ICCIDD Laboratory, New Delhi)

1) To send six salt samples of 50 gm each to each participating Laboratory once a

month.

2) These salt samples will be of different brands obtained from local market and each
sample will be analyzed six times. The mean and standard deviation of each
sample will be calculated.

3) The results of the analysis will be sent to focal point in participating laboratories.

4) To analyse six salt samples received from each participating labs in duplicate and
communicate the results to focal point in participating labs.

5) To prepare consolidated chart of the results of IQA and EQA from all
participating laboratories.

6) After reviewing EQA and IQA results from each participating laboratory,
communicate to each laboratory lacunae if any with corrective measures.

7) To visit each laboratory initially to help them to establish IQA and EQA
programmes and subsequently once in six months to supervise the programme and
assist participating laboratories in solving day to day problems.

e) Reporting, documentation, and archiving procedures.

The monthly reporting to be filed by participating laboratory to be filled in

formats attached as follows:

Annexure 1: Monthly Reporting formats for participatory laboratory.

Annexure 2: Excel template file for generating Levy-Jenning plot.

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 Annexure 1: Monthly Reporting formats for participatory laboratory.

(To be dispatched on 15th of every month)

Quality assurance of salt production laboratories by ICCIDD

Month: Year

Salt Manufacturers name:

Internal Quality assurance

1: Enclose a copy of Levy Jenning plot

Table 1: Iodine content of 5 salt samples sent by production site laboratory to ICCIDD

(to be done in duplicate)

(to be collected randomly on different days during first 15 days of month)

Serial no. salt sample Iodine content 1 Iodine Content 2

S1
S2
S3
S4
S5

External quality assurance

Table 2: Iodine content of 5 salt samples sent by ICCIDD to production site laboratory

(to be done in duplicate)

Serial no. salt sample Iodine content 1 Iodine Content 2

I1
I2
I3
I4
I5

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 Annexure 2: Template for generating Levy-Jenning Plot
Name of Laboratory Reporting Month

Known value salt preparation

Day of
Test No. Value Value Mean-value/SD
Month
1 16 #DIV/0!
2 17 #DIV/0!
3 18 #DIV/0!
4 19 #DIV/0!
5 20 #DIV/0!
6 21 #DIV/0!
7 22 #DIV/0!
8 23 #DIV/0!
9 24 #DIV/0!
10 25 #DIV/0!
Known value Mean 0 26 #DIV/0!
Known value SD #DIV/0! 27 #DIV/0!
28 #DIV/0!
29 #DIV/0!

Mean-value/SD 30 #DIV/0!
31 #DIV/0!
4
1 #DIV/0!
3
2 #DIV/0!
2 3 #DIV/0!
4 #DIV/0!
1
5 #DIV/0!
0
6 #DIV/0!
-1 7 #DIV/0!

-2 8 #DIV/0!
9 #DIV/0!
-3
10 #DIV/0!
-4 11 #DIV/0!
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31
12 #DIV/0!
13 #DIV/0!
14 #DIV/0!
15 #DIV/0!

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8) Pictorial representation of salt iodine estimation

Step 1: Carefully weigh 10 grams of the salt sample to be tested using an electronic open
pan micro-balance

Step 2: Carefully pour the 10 grams of salt into a 250 ml conical flask, which has a
stopper

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 Step 3: Carefully measure 50 ml of double distilled water with a measuring cylinder

Step 4: Pour the 50 ml of double distilled water into the conical flask

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Step 5: Shake the flask till … Step 6: …all the salt dissolves

Step 7: Using the automatic dispensers*… Step 8: Add 1 ml of

(for 2N Sulphuric Acid on the left and 2N Sulphuric Acid
for 10% Potassium Iodide on the right)

* (One can also use a single dispenser if it is properly rinsed with double distilled water
between adding the two reagents)

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 Step 9: …and 5 ml of 10% Potassium Iodide & close mouth of the flask with a
stopper…

Step 10: …the solution will turn Step 11: Keep the flask in the dark (e.g

yellow (if iodine is present) in a closed cupboard) for 10 minutes to

avoid exposure to light

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 Step 12: Using the automatic Step 13: …fill the burette tube with
zero burette… 0.005N sodium thiosulphate,
with a pumping action…

Step14: …till the level of sodium thiosulphate Step 15: After 10 minutes, take

is at zero the flask out of the

dark…

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Step 16: Titrate the solution Step 17: … till the solution is pale (faint) yellow

in the flask with the in colour

sodium thiosulphate

Step 18: Add 2 drops of the Step 19: The solution will turn purple

1% starch solution… in colour.

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Step 19: Continue titration till the Step 20: …and eventually

solution colour starts fading… becomes colourless

Step 21: Note down the burette reading (in this case 3.9) and record the corresponding
iodine level as given in Table – 1 (in this case 41.3)

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“Fact finding is more effective than fault finding”

- Carl Lotus Becker (1873-1954),

Renowned American Historian and Philosopher.

58