|
|
Method for estimation of iodine in urine (Wet Digestion Method)
Principle
Urine is digested with
chloric acid under mild conditions and iodine is determined manually by its
catalytic role in the reduction of ceric ammonium sulfate in
the presence of arsenious acid. As the
reduction proceeds the intensity of colour decreases and this can be readily measured in a
spectrophotometer at 420 nm. The method is fast and inexpensive,
and the digestion is less harsh than
some other methods. This method can
measure urinary iodine concentrations in
the range of 0-150 mcg/liter but can be
extended further to cover a wider range of values.
Equipment and Chemicals
Equipment.
Oven with fan
exhaust, vented fume
hood on oven
for perchloric acid escape, UV spectrophotometer, thermometer, Timer (stop
watch reliable to 5 second, test tubes (15mm x
100mm), funnel (56x100 mm)
reagent flasks and bottles, pipettes
Whatman no 1 filter paper and a laboratory balance.
Chemicals
(analytical
grade AR /GR)
i) KCLO3 (potassium
chlorate),
ii) HCLO4 (perchloric
acid, 70%)
iii) As2O3 (arsenic
trioxide),
iv) NaOH (sodium
hydroxide),
v) H2SO4 (sulfuric acid)
vi) Ce(NH4)4 (SO4)4 2H2O
(ceric ammonium sulfate),
vii) KIO3 (potassium iodate),
viii) HCL (Hydrochloric
Acid)
ix) Double distilled
water (free of
iodine and other contaminants)
Preparation of reagents :
i)
Chloric acid solution:
In a 2000ml Erlenmeyer flask,
500g of potassium chlorate was dissolved
in 910ml hot double distilled water until the soluble state
(normally a little amount remains undissolved). 375 ml of 70% perchloric acid was added dropwise
(approx. 15 ml/min) while stirring
constantly. This preparation is carried out in a vented fume hood as it
produces toxic fumes. Subsequently, the
solution is kept in a freezer
of refrigerator overnight for
better separation. The next day
it is filtered through a filter paper,
(Whatman # 1) and stored in a refrigerator at 40C.
ii) Arsenious Acid Solution:
0.986 g arsenic trioxide
was taken in a 1000 ml volumetric
flask and dissolved in 10 ml of 0.5 N
hot sodium hydroxide.
This solution was
transferred into 750
ml chilled double distilled water. Then 20 ml concentrated HCL and
39.6 ml
conc. sulphuric acid (98%) was added dropwise with constant mixing. The
solution was stored in amber colour
bottle at room temperature.
(The solution
is stable for months).
Sulphuric Acid Solution (3.5N H2SO4) :
97 ml concentrated sulfuric
acid (98%) was added dropwise into
800 ml chilled double distilled
water (carefully as this generates heat) and
final volume was made up to 1
litre with double distilled water.
Ceric ammonium sulfate solution:
48g ceric ammonium sulfate was dissolved in 1 litre of 3.5N
H2SO4. This was
stored in a
amber colour bottle
at room temperature. (The
solution is stable for months).
Stock Iodine Standard (1mg/ml) :
168.5 mg KIO3 was dissolved
in double distilled water to make a final volume of 100 ml. This was stored in a amber colour bottled
(This solution is stable for months).
Dilute Iodine
Standard (1ug/ml) :
Take 100 ul of
Stock Iodine Standard and make a volume to 100 ml with double distilled
water.
Working Iodine
Standard :
Make the following
serial dilutions from diluted
Iodine Standard (1ug/ml) into volumetric
flasks (10 ml)
with double distilled water (diluent).
These dilutions are made freshly.
ug/dl Dilution
factors
5 ug :
0.5 ml of 1 ug/ml standard + 9.5 ml diluent
10 ug : 1.0 ml of 1 ug/ml standard + 9.0 ml diluent
15 ug : 1.5 ml of 1 ug/ml standard + 8.5 ml diluent
20 ug : 2.0 ml of 1 ug/ml standard + 8.0 ml diluent
PROCEDURE :
Step II : 750 micro liter of chloric acid solution was added to each
tube (samples, blank, internal quality
control sample, standards) and
mixed gently. All tubes were placed in the oven at 1100C-1200C for 75 minutes (with a
fume hood for the trapping of perchloric
acid). There will be very little
volume change during heating.
some samples may be faintly yellow. All the tubes were cooled
at room temperature for 15
minutes. Then, the decreased
volume was adjusted with double distilled
water to their original volume
(1.0 ml) and vortexed.
Step III: 3.5 ml of Arsenious
Acid was added to each test
tube and after mixing all test
tubes were kept for 15 minutes at room
temperature.
Step IV : 350 microliter of ceric
ammonium sulfate solution was added at a
fixed interval of time to each tube and quickly
mixed with help of a vortex. A stopwatch
was used to keep a constant interval between additions to
successive tubes, (30 seconds was a convenient interval). Exactly 20 minutes
after addition of ceric ammonium sulfate
to the first tube, the
reduction was read spectrophotometrically at 420 nm against
the reagent blank at the same interval. (Successive tubes were arranged in a
such a manner that the interval between the time of addition of ceric ammonium sulfate and the time of the reading was the
exactly 20 minutes for all samples, standards and
blanks).
Calculation of results: The exact value of
urine sample's iodine was calculated as
follows:
PRECAUTIONS:
Method for Internal Quality Control adopted:
A pooled
urine sample was prepared for internal quality control assessment. The internal
quality control sample was analysed
20-25 times with standards and blank in duplicate. The mean (X) and standard deviation (SD) of this internal
quality control sample was calculated and the sample was stored in refrigerator
and analysed with every batch of samples. The 95% confidence interval was then calculated and used as the
operating control range, as follows:
Sample Mean
(X) ± 2(SD)
The X - 2(SD) = the lower
confidence limit (L)
X + 2(SD) = the upper confidence
limit (U)
The operating control
range is (L,U).
A regular linear graph
paper was used to prepare these plots. The mean urinary iodine concentration
(in ppm) of the internal quality control sample was plotted as a continuous
horizontal line on the Y-axis. The lower
concentration value (L) was plotted below the mean line on the Y-axis scale and the upper concentration value (U)
was plotted above the mean line on the
Y-axis scale. The X-axis was used to
plot the date on which the internal quality control sample was analysed.
This chart was used to plot
the specific analysis date,
and urinary iodine concentration obtained for
the internal quality control sample every time it was tested. If the value of the internal quality control
sample was between the two limit lines, then the test was deemed
in control, and all results were
accepted. Any value of the internal
quality control sample that was plotted outside the two limit lines then, the
test was considered as out-of-control, and the entire batch was repeated.
+
2SD(U)
Mean
(X)
-
2SD(L)
Date 16 17 20 22