HYDROCHLOROTHIAZIDE SOP
1.0 OBJECTIVE:
To
lay down a procedure of analytical report for the active raw material of the Hydrochlorothiazide from the
Pharmacopoeial specifications.
2.0 SCOPE:
This
SOP shall be applicable in Q.C laboratory.
3.0 RESPONSIBILITY:
3.1
Q.C Pharmacist.
4.0 ACCOUNTABILITY:
4.1
Q.C Manager.
5.0 PROCEDURE:
5.1 Characters:
5.1.1
Appearance:
5.1.1.1
White or almost
white.
5.1.1.2
Crystalline
powder.
5.1.2
Solubility:
5.1.2.1
Material and equipment:
5.1.2.1.1
Glassware (test
tubes, spatula).
5.1.2.1.2
Ethanol (96%).
5.1.2.1.3
Acetone.
5.1.2.1.4
Purified water.
5.1.2.1.5
Dilute solution of
alkali hydroxide.
5.1.2.2
Sample:
5.1.2.2.1
Small quantity.
5.1.2.3
Method:
5.1.2.3.1
Take 4 test tubes
and add small quantity of sample for testing solubility according to B.P
specifications.
5.1.2.3.2
Add purified water
in test tube 1 and observe.
5.1.2.3.3
Add acetone in
test tube 2 and observe.
5.1.2.3.4
Add ethanol (96%)
in test tube 3 and observe.
5.1.2.3.5
Add dilute
solution of alkali hydroxide in test tube 4 and observe.
5.1.2.4
Observations:
5.1.2.4.1
The sample in test
tube 1 containing with water is very slightly soluble.
5.1.2.4.2
The sample in test
tube 2 containing with acetone is soluble.
5.1.2.4.3
The sample in test
tube 3 containing with ethanol (96%) is sparingly soluble.
5.1.2.4.4
The sample in test
tube 4 containing with dilute solution of alkali hydroxide is dissolved.
5.2 Identification
tests:
5.2.1
UV/VIS
absorption Spectrophotometry:
5.2.1.1
Material and equipment:
5.2.1.1.1
Glassware
(according to requirement).
5.2.1.1.2
UV/VIS
Spectrophotometer.
5.2.1.1.3
0.1M sodium
hydroxide.
5.2.1.1.4
0.01M sodium
hydroxide.
5.2.1.1.5
Purified water.
5.2.1.2
Sample:
5.2.1.2.1
50.0mg.
5.2.1.3
Spectral range:
5.2.1.3.1
250-350nm.
5.2.1.4
Absorption maxima:
5.2.1.4.1
At 273nm and
323nm.
5.2.1.5
Absorbance ratio A273/A323:
5.2.1.5.1
5.4 to 5.7.
5.2.1.6
Test solution:
5.2.1.6.1
Take a beaker of
100.0ml and add in it 50.0mg of the sample.
5.2.1.6.2
Dissolve it in
10.0ml of 0.1M sodium hydroxide.
5.2.1.6.3
And dilute it to
100.0ml with water.
5.2.1.6.4
Take 2.0ml of this
solution in another beaker.
5.2.1.6.5
And dilute it to
100.0ml with 0.01M sodium hydroxide.
5.2.1.7
Method:
5.2.1.7.1
Switch ON the
UV/VIS spectrophotometer and operate it according to SOP
5.2.1.7.2
Observe the test
solution on above given spectral range.
5.2.1.7.3
Note down the
absorbance at maxima λ i.e. 273nm & 323nm.
5.2.1.7.4
Calculate the
absorbance ratio by using formula:
Absorbance ratio= A273/A323
5.2.1.8
Observations:
5.2.1.8.1
Absorbance ratio
must lie in 5.4 to 5.7.
5.2.2
5.2.2.1
Material and equipment:
5.2.2.1.1
Glassware
(according to requirement).
5.2.2.1.2
Water bath.
5.2.2.1.3
0.5g/L solution of
chromotropic acid.
5.2.2.1.4
Sodium salt.
5.2.2.1.5
Sulfuric acid.
5.2.2.1.6
Purified water.
5.2.2.2
Sample:
5.2.2.2.1
1.0mg.
5.2.2.3
Method:
5.2.2.3.1
Take a test tube
and add 1.0mg of sample in it.
5.2.2.3.2
Gently heat it
with 2.0ml of freshly prepared 0.5g/L solution of chromotropic acid, sodium
salt in a cooled mixture of 35 volumes of water and 65 volumes of sulfuric
acid.
5.2.2.3.3
Observe the change
in colour.
5.2.2.4
Observations:
5.2.2.4.1
A violet colour
develops.
5.3 Assay:
5.3.1
Apparatus:
5.3.1.1
HPLC apparatus.
5.3.1.2
Glassware
(according to the requirement).
5.3.1.3
Sonicator.
5.3.2
Material
and reagents:
5.3.2.1
Octadecylsilyl silica
gel for chromatography (3μm).
5.3.2.2
Acetonitrile.
5.3.2.3
Methanol.
5.3.2.4
Phosphate buffer
solution pH 3.2.
5.3.2.5
Chlorothiazide
CRS.
5.3.2.6
Hydrochlorothiazide
CRS.
5.3.2.7
Tetrahydrofuran.
5.3.3
Requirements:
5.3.3.1
Solvent
mixture:
5.3.3.1.1
Take a beaker of
1000.0ml and add in it 50.0ml of a mixture of equal volumes of acetonitrile and
methanol.
5.3.3.1.2
Dilute it to
200.0ml with phosphate buffer solution pH 3.2.
5.3.3.2
Sample:
5.3.3.2.1
30.0mg
5.3.3.3
Test
solution:
5.3.3.3.1
Test solution (a):
5.3.3.3.1.1 Take
100ml of beaker and dissolve 30.0mg of the substance to be examined in 5.0ml of
a mixture of equal volumes of acetonitrile and methanol, using sonication if
necessary.
5.3.3.3.1.2 And
dilute to 20.0ml with phosphate buffer solution pH 3.2.
5.3.3.3.2
Test solution (b):
5.3.3.3.2.1 Take
a beaker of 100ml and dilute 1.0ml of test solution (a) to 20.0ml with phosphate
buffer solution pH 3.2.
5.3.3.4
Reference
solutions:
5.3.3.4.1
Reference solution
(a):
5.3.3.4.1.1 Take
100ml beaker and dissolve 3.0mg of Chlorothiazide CRS and 3.0mg of
hydrochlorothiazide CRS in 5.0ml of a mixture of equal volumes of acetonitrile
and methanol, using sonication if necessary.
5.3.3.4.1.2 Dilute
to 20.0ml with phosphate buffer solution pH 3.2.
5.3.3.4.1.3 Take
5.0ml of this solution in another beaker dilute to 100.0ml with the solvent
mixture.
5.3.3.4.2
Reference solution
(c):
5.3.3.4.2.1 Take
100ml beaker and dissolve 30.0mg of hydrochlorothiazide CRS in 5.0ml of a
mixture of equal volumes of acetonitrile and methanol, using sonication if
necessary.
5.3.3.4.2.2 Dilute
to 20.0ml with phosphate buffer solution pH 3.2.
5.3.3.4.2.3 Take
1.0ml of this solution in another beaker dilute to 20.0ml with phosphate buffer
solution pH 3.2.
5.3.3.5
Column:
5.3.3.5.1
Size:
5.3.3.5.1.1 Length=0.1m,
5.3.3.5.1.2 θ=4.6mm.
5.3.3.5.2
Stationary
phase:
5.3.3.5.2.1 Octadecylsilyl
silica gel for chromatography (3μm).
5.3.3.6
Mobile
phase:
5.3.3.6.1
Mobile
phase A:
5.3.3.6.1.1 Take
1000.0ml of beaker and add 940.0ml of phosphate buffer solution pH 3.2 and
60.0ml of methanol and 10.0ml of tetrahydrofuran.
5.3.3.6.1.2 Mix
it properly.
5.3.3.6.2
Mobile
phase B:
5.3.3.6.2.1 Take
1000.0ml of beaker and add in it a mixture of 500.0ml of methanol and 500.0ml
of phosphate buffer solution pH 3.2 and 50.0ml of tetrahydrofuran.
5.3.3.6.2.2 Mix
it properly.
|
Time
(min)
|
Mobile phase A
(percent v/v)
|
Mobile phase B
(percent v/v)
|
|
0-4
|
80
|
20
|
|
4-10
|
80®20
|
20®80
|
5.3.3.7
Flow
rate:
5.3.3.7.1
1.6ml/min.
5.3.3.8
Detection:
5.3.3.8.1
Spectrophotometer
at 224nm.
5.3.3.9
Injection:
5.3.3.9.1
20μL of the test
solution (b) and reference solution (a) and (c).
5.3.3.10 Relative retention:
5.3.3.10.1 With
reference to hydrochlorothiazide (retention time=about 2.2min): impurity A=
about 0.9.
5.3.3.11 System suitability
5.3.3.11.1 Reference
solution (a):
5.3.3.11.1.1 Resolution:
minimum 2.0 between the peaks due to impurity A and hydrochlorothiazide.
5.3.4
Method
of analysis:
5.3.4.1 Firstly
prepare the test solution, reference solution and mobile phase according to the
requirements.
5.3.4.2 The
solutions must be free from solid particles.
5.3.4.3 Prepare
the apparatus.
5.3.4.4 The
mobile phase solvent mixtures must be deaerated prior to use either by boiling
or by applying a partial vacuum to the solvent reservoir.
5.3.4.5 Equilibrate
the column with the prescribed mobile phase, flow rate and at temperature
specified until a suitable baseline is achieved.
5.3.4.6 Test
solution of the mixture to be separated is now introduced into the mobile phase
with the help of an injector just before entering the separating column.
5.3.4.7 As
the eluate leaves the column it enters a detector, where it is continuously
monitored at the specified λ.
5.3.4.8 The
electrical signal obtained from detector is amplified and routes to recorder
which record the developed chromatogram.
5.3.4.9 Calculate
the percentage content of Hydrochlorothiazide (C7H8ClN3O4S2)
taking into account the assigned content of hydrochlorothiazide CRS.
5.3.5
Limit:
5.3.5.1 97.5%
to 102.0% (dried substance).
6.0 REVISION LOG:
|
Revision No.
|
Effective Date
|
Reason
|
|
00
|
|
New SOP
|
7.0 REFERENCES:
7.1
The British
Pharmacopoeia. Vol I.,
Official Monograph /Hydrochlorothiazide: 2015, pp. 1151-1153.
8.0 ANNEXURES:
Annexure 1: Calculations
of UV/VIS spectrophotometer.
Annexure 2: Observations
and calculations of HPLC method.
Annexure: 1
Calculations
of UV/VIS spectrophotometer
|
UV/VIS spectrophotometer
Model:
_____________________________ Date:
_________________
OBSERVATIONS:
CALCULATIONS:
Absorbance
ratio A273/A323 =
Results:
_______________
Remarks:
______________________________________________________________
|
Annexure:
2
Observations
and calculations of HPLC method
|
Analysis
on HPLC
Instrument:
___________________
Date: _________________
Model:
___________________
Sample
solution: _______________________
Reference
standard solution: ______________
Impurities:
____________________________
(calculate
each component calculation separately)
OBSERVATIONS:
Attach
chromatogram.
CALCULATIONS:
1.
Retention time:
n= no. of peak
Retention time of unretained peak (tm)=
_____________
2.
Retention volume:
Flow rate= _______________ml/min.
3.
Retention factor:
Retention time of unretained peak (tm)=
_____________
4.
Separation factor (α):
5.
Resolution:
Retention time of unretained peak (tm)=
_____________
6.
Efficiency:
7.
Height equivalent to a theoretical plate (HETP):
Length of column = ________________________
8.
Symmetry factor (tailing factor):
9.
Response factor & Relative response factor:
Conc. (mg/ml)= ___________________
10. Relative standard deviation (%RSD):
Use formula of relative standard deviation where it is
required i.e.,
pic
11. Percentage of content:
Percentage content = (rU/rS) x (CS/CU)
x 100.
rU= peak response of substance from the sample
solution.
rS= peak response of substance from the standard
solution.
CS= concentration of substance in the standard
solution (mg/mL).
CU= concentration of substance in the sample
solution (mg/mL).
RESULTS:
________________________________________________________________________________________________________________________________________________
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9.0 ABBREVIATIONS:
|
Abbreviation
|
Expanded Form
|
|
SOP
|
Standard
operating procedure
|
|
&
|
And
|
|
No.
|
Number
|
|
Ltd.
|
Limited
|
|
Sr.#
|
Serial
number
|
|
%
|
Percentage
|
|
B.P
|
British
pharmacopoeia
|
|
UV/VIS
|
Ultraviolet/
visible
|
|
M
|
Molar
|
|
mg
|
Milligram
|
|
nm
|
Nanometer
|
|
ml
|
Milliliter
|
|
λ
|
Lambda
|
|
g/L
|
Grams
per liter
|
|
μm
|
Micron
|
|
CRS
|
Chemical
reference standard
|
|
θ
|
Theta
|
|
min
|
Minute
|
|
ml/min
|
Milliliter
per minute
|
|
v/v
|
Volume
by volume
|
|
Vol
|
Volume
|
|
QCA
|
Quality
control active ingredient
|
|
F
|
Format
|