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:
Thickness of cell:

Spectral range:
250 t0 350nm.
Maxima absorption wavelength:
273nm and 323nm.
Sample:

Other reagent used:


No. of obs.
Concentration
Wavelength
Absorbance


273nm



323nm


CALCULATIONS:
Absorbance ratio A273/A323 =





Results: _______________
Remarks: ______________________________________________________________



Annexure: 2
Observations and calculations of HPLC method
Analysis on HPLC
Instrument: ___________________                                           Date: _________________
Model: ___________________
Column size:
Length=
θ=
Stationary phase:

Temperature:

Mobile phase:

Flow rate:

Injection size:

Detector:

Wavelength:
λ=

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)= _____________
No. of peaks
Retention time of peak of interest
(tr)n
Height of peak of interest
(h)n
Width of peak of interest
(w)n
Area of peak of interest
A=1/2(h x w)




















2.      Retention volume:
Flow rate= _______________ml/min.
No. of peaks
Retention time of peak of interest
(tr)n
Retention volume = retention time x flow rate












3.      Retention factor:
Retention time of unretained peak (tm)= _____________
No. of peaks
Retention time of peak of interest
(tr)n
Retention factor of a component
k= (tr-tm)/tm














4.      Separation factor (α):
No. of peaks
Retention factor of a component
(kn)
Relative retention of two adjacent peaks
α = k2/k1












5.      Resolution:
Retention time of unretained peak (tm)= _____________
No. of peaks
Retention time of peak of interest
(tr)n
Width of peak of interest
(w)n
Resolution
Rs = 2 (tr2-tr1)
        (w1-w2)
















6.      Efficiency:
No. of peaks or components
Retention time of peak of interest
(tr)n
Width of peak of interest
(w)n
Efficiency
(No. of theoretical plates)
N= 16 (tr/w)2


















7.      Height equivalent to a theoretical plate (HETP):
Length of column = ________________________
No. of peaks or components
No. of theoretical plates
(N)
Height equivalent to a theoretical plate HETP = L/N












8.      Symmetry factor (tailing factor):
No. of peaks or components
Distance from the peak max. to leading edge of the peak
(f)
Width w
Symmetry factor
At 5%
At 10%
As = w5%
       2f
As = w10%
       2f
























9.      Response factor & Relative response factor:
Conc. (mg/ml)= ___________________
No. of peak
Peak area
Response factor = (peak area/conc.)
Relative response factor = (response factor of impurity/response factor of API)


















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:
________________________________________________________________________________________________________________________________________________


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


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