|
|
Utility of Spot Testing Kit in the Assessment of
Iodine Content of Salt- A Multicentric
Study
Department
of Human Nutrition
All India
Institute of Medical Sciences
New
Delhi-110029
1999
Continued ..
POOLED ANALYSIS OF THE RESULTS OF THE EIGHT
CENTRES
Sensitivity and Specificity of STK against It
method
(n=6302)
Titration Method
|
|||
|
STK Method |
15 ppm & more |
<15 ppm |
Total |
|
15 ppm & more |
3505 |
824 |
4329 |
|
<15 ppm |
400 |
1573 |
1973 |
|
Total |
3905 |
2397 |
6302 |
Sensitivity = 3505/3905 X 100 = 89.8%
Specificity = 1573/2397 X 100 = 65.6%
Positive Predictive Value = 3505/4329 X
100 = 80.9%
Negative Predictive Value = 1573/1973 X 100 = 79.7%
Sensitivity, Specificity, PPV and NPV of STK
Against Iodometric Titration
Method at Different Centres
|
Centre |
Sensitivity (%) |
Specificity (%) |
PPV (%) |
NPV (%) |
|
(n=700) |
94.7 |
100.0 |
100.0 |
88.8 |
|
(n=700) |
81.4 |
85.5 |
84.8 |
82.2 |
|
Dehradun (n=797) |
95.3 |
71.8 |
84.7 |
90.4 |
|
(n-855) |
80.7 |
50.9 |
58.4 |
75.5 |
|
Kangra (n=746) |
90.2 |
51.0 |
96.3 |
26.8 |
|
(n=887) |
81.0 |
64.2 |
68.2 |
78.1 |
|
Udham Singh Nagar (n=736) |
95.5 |
54.7 |
87.7 |
78.6 |
|
|
92.6 |
50.4 |
69.0 |
85.3 |
|
Total (n=6302) |
89.8 |
65.6 |
80.9 |
79.7 |
7. References
1. Tyabji R, Karmarkar MG,
Pandav CS, Carriere CR, Acharya S. Estimation of iodine content in iodated
salt. In: A hand book of monitoring and quality control.
2. Gupta SD et al.
Evaluation of Universal Salt Iodisation in India - a mid term evaluation study,
Indian Institute of Health Management Research, Jaipur, India 1998, (personal
communication).
3. Kapil U, Bhanti T,
Saxena N, Nayar D, Dwivedi SN. Comparison of Spot Testing Kit in the estimation
of iodine content in salt. Indian Journal of Physiology and Pharmacology 1996,
40:276-280.
4. Hennekens CH, Burning
JE. Epidemiology in Medicine. Little Brown and Co. Publishers 1987, pp 331-332.
5. Bhasin SK and Dubey KK.
Validity of spot testing kit for estimation of iodine content of salt under
field conditions in national capital
Appendix
I
List of
different centres participating in the multicentric study for assessment of
utility of
Spot
Testing Kit
1. Dr.
Subhadra Seshadri,
Head,
Department
of Foods & Nutrition,
M S
University,
2. Dr.
S. S. Swami,
Head,
Department
of Preventive & Social Medicine,
3. Ms.
Beena
Mamta
Samajik Sanstha
Dehradun-248001
(UP)
4. Dr.
B.P. Mathur
Reader
Department
of Preventive and Social Medicine
5. Dr.
T. D. Sharma,
Principal,
Health
& Family Welfare Training Centre,
Kangra
(HP)
6. Dr.
Kumud Khanna
Director
New
Delhi-110059
7. Dr.
R.S. Raghuvanshi
Reader
Department
of Foods and Nutrition
GB
Pant
Pantnagar,
Distt.
Udham Singh Nagar (UP)
8. Dr.
Umesh Kapil
Additional
Professor
Department
of Human Nutrition
All
India Institute of Medical Sciences,
New
Delhi-110029
and
Dr.
S. N. Dwivedi
Associate
Professor,
Department
of Biostatistics,
All India
Institute of Medical Sciences,
New
Delhi-110029.
Appendix II
Research Methodology for Collection of Salt
Samples:
Each
centre selected one district keeping in view the operational feasibility. In
the district selected, one rural block
at least 30 km away from the district headquarters was selected. In the
block, one high school was further selected keeping in view the operational
feasibility.
In
each school, 750 children were included
for the study (It was expected that 50 children may not bring salt sample or
may bring less quantity of salt). These school children were briefed about the
study objectives and were given
auto-seal polythene pouches (airtight moisture proof) with an
identification slip. They were asked to bring about 20 g of salt from their
family kitchen. The iodine content of the salt samples collected were analysed
using the standard iodometric titration method.
Details for collection of salt samples:
I. Pre-requisites
i. Letter from
Department of Education was obtained to provide assistance to the research team members.
ii. Schools were
identified in block for inclusion in the survey.
iii. Letters were given to
the Principals of schools to be
surveyed.
II. Tools
for Data Collection
i. LDPE
carry bags
ii. Autoseal
polythene pouches
iii. Identification
slips
III. Methodology of data
collection/operationalisation of project
a) Day I:
i) The Principal/class teacher was contacted and
the objectives of survey, the importance of iodine in salt and it’s role in
prevention of ill effects of iodine deficiency, were explained to them.
ii) The methodology of collecting the salt
samples from school children was as follows:-
a) The students were explained the objectives of
the study, importance of iodine in salt and it’s role in the prevention of ill
effects of iodine deficiency.
b) The children were demonstrated how to open and
close the autoseal pouches.
c) Information to be written on the
identification slip was also
explained in detail. On each slip the student was asked to write his name,
class and section.
d) Each child was requested to bring four
spoonful of salt which was being consumed in the family from their kitchen the
next day.
e) One LDPE carry bag was given to the class
teacher so that the salt pouches could be collected from the students on the
next day.
b) Day II:
Research Team Members (RTM)
went to the schools and undertook the following activities
i) Collection of the salt samples from school
children/teachers and keeping them in the polythene carry bag.
ii) The results of iodine content of salt was
sent to school authorities after the
analysis.
Appendix
III
Estimation of Iodine Content in Salt by Iodometric Titration
1.1 Principle
The
iodine content in iodated salt is estimated by titrimetric procedure known as
iodometric titration. Free iodine reacts with sodium thiosulphate solution to
give a light yellow colour complex. This colour complex combines with soluble
chemical starch which indicates the presence of sodium iodide. The reaction
mechanism is as follows:
2Na2 S2O3 +
I2 ————> Na2S4O6. 2NaI
Sodium
thiosulphate + Iodine ———> unstable complex
Na2S2O6 2NaI Starch 2 NaI + Na2S4O6
Sodium
Iodide + Sodium tetrathionate
1.2 Equipment
and Chemicals
1.2.1 Equipment
i. Laboratory
balance
ii. Gas
burner or kerosene stove
iii. Reagent
bottles with stoppers—> 250 ml, 500 ml & 1000 ml
iv. Measuring
cylinder with stopper, 50 ml
v. Wash
bottle, 500 ml
vi. Glass
stirring rod
vii. Conical
flask with stopper, 100 ml
viii. Glass
or plastic funnel
ix. Burette,
10 ml
x. Burette,
stand
xi. Clock
or watch
xii. A
closed box, cupboard or drawer to keep the conical flask
1.2.2 Chemicals
i. Sodium
thiosulphate, (Na2S2O3 5H2O)
ii. Concentrated
sulfuric acid, (H2SO4)
iii. Potassium
iodide (KI)
iv. Soluble
chemical starch
All the
chemicals should be analytical grade and double distilled water to be used
should be free of iodine and other contaminants.
1.3 Preparation
of reagents
a) Sodium
thiosulphate (0.005 M)
Dissolve
1.24 g sodium thiosulphate in 1 litre hot double distilled water. This volume will be
sufficient for testing 200 salt samples. The solution should be stored in a
cool, dark place.
b) Sulfuric
acid (2N H2SO4)
Add
5.56 ml concentrated sulfuric acid drop wise into a 90 ml of chilled double
distilled water and make the final volume upto 100 ml with double distilled
water. This volume will be sufficient for testing 100 salt samples.
CAUTION: To avoid violent and
dangerous reaction, always add acid to water, never water to acid.
c) Potassium
iodide (KI)
Dissolve
100 g potassium iodide in 1000 ml double
distilled water. This volume will be sufficient for testing 200 salt
samples. This should be stored in a refrigerator.
d) Saturated
Salt Solution
Take
100 ml double distilled water in a conical flask and add sodium chloride until the
salt is insoluble. Heat the solution till the NaCl
crystals are formed on the sides of the vessels. After cooling down the
saturated salt solution at room temperature transfer the supernatant to a clean
bottle.
e) Soluble
Chemical Starch
Dissolve
10 g soluble chemical starch in 100 ml hot double distilled water. Adjust the
volume upto 1000 ml with the saturated sodium chloride solution. This will be
sufficient for testing 200 salt samples. The starch solution should be prepared
fresh every day.
1.4 Procedure
Dissolve
10 g of salt in 50 ml double distilled water. Then add 1.0 ml of 2 N sulfuric
acid and 5.0 ml of 10% potassium iodide to it. On shaking, the solution will
turn to a yellow colour. Incubate at room temperature in the dark for 10
minutes. During the incubation period pour the sodium thiosulphate solution
into the burette and the level of solution should be adjusted to zero. Remove
the samples from the dark and titrate against the sodium thiosulphate solution
until it turns into a very light yellow colour (pale yellow). Subsequently, add
a few drops (1-5 ml) of 1% starch solution. The solution will turn into a deep purple
colour. Finally, titrate until the solution becomes colourless and the final
reading is observed.
1.5 Result
From
table, the iodine content of the sample in parts per million was calculated.
The results were recorded in a register, under the following parameters:-
- Date of collection
- Date of testing
- Sample number
- Type of salt
- The level of iodine in the sample.
1.6 Precautions
i. The reaction mixture
should be kept in the dark for 10 minutes
before titration because light accelerates a side reaction in which
iodide ions are oxidized to iodine by atmospheric oxygen.
ii. Starch solution must
be prepared freshly.
iii. The starch solution
must be added near the end of the
titration, when very little amount of 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.
iv. The titration should
be done in a comfortable cool room
because iodine is volatile and the sensitivity of the starch indicator
diminishes as the temperature rises.
General Precautions Considered While
Undertaking Laboratory Procedures:
1. The laboratory
technicians were trained to estimate the iodine content of salt. The variations
in the iodine values for the same samples repeated for internal quality control
were within 5%.
2. The pH value of salt
determined the reaction between iodine in the salt and starch solution of STK.
Hence, pH of salt was determined for valid results.
Iodine Content in Parts Per Million
Burette
|
Parts per
million |
Burette
|
Parts per
million |
|
0.0 |
0.0 |
3.0 |
31.7 |
|
0.1 |
1.1 |
3.1 |
32.8 |
|
0.2 |
2.1 |
3.2 |
33.9 |
|
0.3 |
3.2 |
3.3 |
34.9 |
|
0.4 |
4.2 |
3.4 |
36.0 |
|
0.5 |
5.3 |
3.5 |
37.0 |
|
0.6 |
6.3 |
3.6 |
38.1 |
|
0.7 |
7.4 |
3.7 |
39.1 |
|
0.8 |
8.5 |
3.8 |
40.2 |
|
0.9 |
9.5 |
3.9 |
41.3 |
|
1.0 |
10.6 |
4.0 |
42.3 |
|
1.1 |
11.6 |
4.1 |
43.4 |
|
1.2 |
12.7 |
4.2 |
44.4 |
|
1.3 |
13.8 |
4.3 |
45.5 |
|
1.4 |
14.8 |
4.4 |
46.6 |
|
1.5 |
15.9 |
4.5 |
47.6 |
|
1.6 |
16.9 |
4.6 |
48.7 |
|
1.7 |
18.0 |
4.7 |
49.7 |
|
1.8 |
19.0 |
4.8 |
50.8 |
|
1.9 |
20.1 |
4.9 |
51.9 |
|
2.0 |
21.2 |
5.0 |
52.9 |
|
2.1 |
22.2 |
5.1 |
54.0 |
|
2.2 |
23.3 |
5.2 |
55.0 |
|
2.3 |
24.3 |
5.3 |
56.1 |
|
2.4 |
25.4 |
5.4 |
57.1 |
|
2.5 |
26.5 |
5.5 |
58.2 |
|
2.6 |
27.5 |
5.6 |
59.2 |
|
2.7 |
28.6 |
5.7 |
60.3 |
|
2.8 |
29.6 |
5.8 |
61.4 |
|
2.9 |
30.7 |
5.9 |
62.4 |
Burette
|
Parts
per
million
|
Burette
|
Parts per
million
|
|
6.0 |
63.5 |
8.0 |
84.6 |
|
6.1 |
64.5 |
8.1 |
85.7 |
|
6.2 |
65.6 |
8.2 |
86.8 |
|
6.3 |
66.7 |
8.3 |
87.8 |
|
6.4 |
67.7 |
8.4 |
88.9 |
|
6.5 |
68.8 |
8.5 |
89.9 |
|
6.6 |
69.8 |
8.6 |
91.0 |
|
6.7 |
70.9 |
8.7 |
92.0 |
|
6.8 |
71.9 |
8.8 |
93.1 |
|
6.9 |
73.0 |
8.9 |
94.2 |
|
7.0 |
74.1 |
9.0 |
95.2 |
|
7.1 |
75.1 |
9.1 |
96.3 |
|
7.2 |
76.2 |
9.2 |
97.3 |
|
7.3 |
77.2 |
9.3 |
98.4 |
|
7.4 |
78.3 |
9.4 |
99.5 |
|
7.5 |
79.4 |
9.5 |
100.5 |
|
7.6 |
80.4 |
9.6 |
101.6 |
|
7.7 |
81.5 |
9.7 |
102.6 |
|
7.8 |
82.5 |
9.8 |
103.7 |
|
7.9 |
83.6 |
9.9 |
104.7 |
Appendix
IV
Use of Spot Testing Kit
The use
of iodised salt has been evolved as a major strategy to combat Iodine
Deficiency Disorders (IDD). For the successful implementation of this strategy,
monitoring the quality of iodised salt is essential. The iodine content of
iodised salt is estimated routinely by
the standard “Iodometric Titration” (IT) method in the laboratory (1).
However, recently a simple kit has been developed for on the spot estimation of
iodine content. In the spot testing
method, estimation of iodine content in salt is done with the help of a
standard starch solution provided in the
Spot Testing Kit (STK). The STK has been advocated as a method for
semi-quantitative estimation of iodine in salt (2,3). The STK consists of a
test solution (A), and a recheck
solution (B). One drop of test solution (A) will be added to a pinch of salt
sample. The change in colour of salt sample will range from white to dark
violet depending on the iodine content of salt, i.e. Nil, less than
15ppm, and 15 ppm and above, which will be compared to the standard colour
chart provided with the kit. If on addition of test solution (A), no change in
colour of salt is observed, the recheck solution (B) will be added. This will
be done to make the salt medium acidic, in case the salt has alkaline constituents,
then the test solution (A) will be added again. The intensity of the blue
colour will be directly proportionate to the iodine content of salt.
Procedure for use of Spot Testing Kit
1. Take a spoonful of
iodated salt to be checked and spread it
flat.
2. Open the seal of the
ampule (white cap) by making a pin hole.
3. Discharge a drop of
the test solution on the surface of the
salt by gently pressing the ampule.
4. The salt will turn
light blue to dark violet depending on the iodine content of the salt.
5. Use the colour chart
given on plastic box to compare and
determine the iodine range in the iodated salt.
IF THE SALT IS ALKALINE OR MIXED WITH ALKALINE
FREE FLOW AGENTS, A DROP OF TEST SOLUTION WILL NOT DEVELOP ANY COLOUR EVEN IF
IODINE IS PRESENT, WHENEVER ONE GETS NO COLOUR INDICATION PLEASE ADOPT
PROCEDURE GIVEN BELOW:
1) Take a spoon of salt
on plate and level it
2) Add one drop of
Recheck solution (Red cap)
3) Over the same spot, put
one drop of test solution (white cap)
4) If iodine is present
colour will develop. If Iodine is not present no colour indication will be
there.
NOTE:
1. Shelf life of spot
testing kit is 18 months
2. Do not use an open
ampule of spot testing kit beyond
180 days (six months)
3. Shake well before use
4. Precise evaluation
analytical check (Iodometric Titration method) is recommended.
Appendix
V
Sensitivity is the proportion of trulu diseased persons in
the screened population who are identified as diseased by the screening test.
Sensitivity is a measure of the probability that any given case will be
identified by the test (Syn: true positive rate).
Specificity is the proportion of truly non-diseased persons who
are so identified by the screening test.
It is a measure of the probability of the correctly identifying a non=-diseased
person with a screening test (Syn : true negative rate).
The relationship are shown in the
following fourfold table, in which the letter a, b, c and d represent the
quantities specified below the table.
|
|
Results of
IT |
Total |
|
|
Results
of STK |
15 ppm & more |
<15 ppm |
|
|
15 ppm |
a |
b |
a+b |
|
<15
ppm |
c |
d |
c+d |
|
Total |
a+c |
b+d |
a+b+c+d |
a. True positives c. False Negatives
b. False positives d. True
Negatives
a d
Sensitivity =
Specificity =
a+c b+d
a
Predictive value (positive test result) =
a+b
d
Predictive value (negative test result) =
C+d
Appendix
VI
Internal Quality Control Procedure Adopted for IT Method During the
Multicentric Trial of each Centre
Once
the laboratory method for the estimations of iodine content of salt by IT
method has been standardised, it must establish and maintain ongoing quality
control (QC) data.
Known
positive iodized salt sample(s) should be obtained by the laboratory and stored
in sufficient quality for analysis every time salt titrations on unknown
samples are run e.g., daily or weekly. By performing multiple analyses
on these positive salt samples, a concentration range can be established and
used for operational quality control purposes. The following paragraphs
provides a description and worked example of how to calculate and establish a
quality control range and a quality control chart.
1. Establishing
and Interpreting a
Multiple
salt iodine analyses on a known positive salt sample should be performed until
approximately 15 to 20 titration results have been obtained. To establish the
control range, it is best if these results are obtained over a period of time (e.g.,
three to four tests per day), rather than all at once e.g., twenty tests
in one day), as this will give a more realistic estimate of true day-to-day and
analytic variability.
Once
a sufficient number of these tests results have been obtained, use standard
statistical formulae to calculate the sample mean concentration (X) in ppm, and
standard deviation (SD). The 95% confidence interval can then be calculated and used as the operating
control range, as follows :
Sample
Mean (X) ± 2 x SD
The
X-2(SD) = the lower confidence unit (L), and X+2(SD)= the upper confidence
limit (U). The operating control range is (L,U).
Unless
serious technical or performance errors occur during these initial analyses,
the above range should reasonably reflect the normal amount of variation
expected when using this method over time. Therefore, any future analysis of
the same sample should produce a result between the lower and upper limits (i.e.,
the L-U range), for 95% of test results. Values falling within this range are
considered to be “in control”. Results falling outside the established range
are considered potentially suspicious and therefore classed as
“out-of-control”.
2. Quality
Control Chart
Once
the above operating control range has been established, standard quality control
charts and rules should be used to interpret these control values, decide
acceptability of test results, and be kept as a permanent record to verify all
unknown sample results.
The
quality control chart should be prepared as follows:
· Use regular linear graph paper to prepare these plots.
· Enter
the salt iodine concentration (in ppm) scale for the control on the Y-axis extends from less
than the lower limit (i.e.,
<L), to above the upper limit (i.e., >U). For the example given above, which has a calculated
range of 27 to 37 ppm, the Y-axis could be scaled from 20 to 40 ppm.
· Find
the sample mean concentration value (i.e.,) on the Y-axis scale, and draw
a continuous horizontal line across the entire graph paper at this point. For
example this would be at 32 ppm.
· Find
the lower limit concentration value (i.e., L) on the Y-axis scale, and
draw a continuous horizontal line across the entire graph paper at this point.
For example this would be at 27 ppm.
· Find
the upper limit concentration value (i.e., U) on the Y- axis scale, and draw a continuous horizontal
line across the entire graph paper at this point. For the example this would be
37 ppm.
· The
X-axis is used to plot time, i.e., the date on which the control sample is analysed.
Once
prepared, this chart is used to plot the specific analysis date, and salt
iodine concentration obtained for the
control every time it is tested. If the control point obtained is between the
two limit lines, then the test is deemed
in control, and all results are accepted. Any control values that are plotted
outside the two limit lines should be considered as out-of-control, and the
results of any corresponding unknown salt samples tested at the same time
should also be rejected as unacceptable due to possible method error.
When
an out-of-control value is obtained, steps should be taken to identify the
possible reason for this event (e.g., use of old reagent, incorrect
procedure used or reagent mix-up), and correct the problem. Once resolved, and
control values have returned to normal, repeat the previously rejected unknown
salt samples to obtain acceptable results.
3. Other
Elements of Quality Assurance
Salt Sample Recording
Each
laboratory must maintain a logbook with sample details recorded in ink, such
as:
· Date/time collected
· Date/time received
· Same specific details (code #, brand, batch, expiry date)
· Date analysed
· Technician performing test
· Test result
· Supervisor’s signature
· Date result is reported
4. Reagent
Inventory Details
The
laboratory supervisor should ensure all relevant details regarding test
chemicals are recorded:
· Chemical
brand, quality, grade and batch/lot number
· Date
on which ordered and received
· Date
on which each “working” reagent is prepared, and by whom
· Give
each working reagent an “in-house” lot number.
Evaluation of Univesal Salt
Iodization in
1997
(Excerpts from the detailed report)
“Iodometric titration is the
standard method for the estimation if iodine content of iodised salt. Recently,
a simpler method, popularly known as STK, has been developed and widely
propagated foe determining the iodine content in edible salt. This method has
been recommended for the semi-quantitaive estimation of iodine. The present
study assessed the validity of the STK
using standard iodometric titration as gold standard. A total of 4317
samples were tested both by titration and STK. The results of the present study
are shown in the table. Sensitivity of the STK against the standard titration
method was 90.8 per cent and specificity 60.8 per cent, at all
|
State |
Sensitivity |
Specificity |
PPV |
|
|
95.4 |
66.1 |
64.0 |
|
M.P. |
94.9 |
42.2 |
82.6 |
|
|
76.9 |
54.3 |
54.9 |
|
Karnataka |
82.1 |
77.2 |
80.0 |
|
H.P. |
99.2 |
30.0 |
94.6 |
|
Manipur |
94.4 |
23.7 |
93.0 |
|
All |
90.8 |
60.8 |
77.0 |
* Source: Evaluation of Universal Salt
Iodisation in India (A Mid-Term Evaluation Study, Ministry of Industry,
Government of India, Indian Institute of Health and Management Research,
Jaipur, March, 1998.