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PYRAZINAMIDE METHOD OF ANALYSIS SOP



1.0  OBJECTIVE:
To lay down a procedure of analytical report for the active raw material of the Pyrazinamide from the Pharmacopoeial specifications.
2.0  SCOPE:
This SOP shall be applicable in Q.C laboratory.
3.0  RESPONSIBILITY:
3.1  Q.C Analysts.
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 (3 test tubes, 1 spatula, 1 pipette).
5.1.2.1.2        Ethanol (96%).
5.1.2.1.3        Purified water.
5.1.2.1.4        Methylene chloride.
5.1.2.2  Sample:
5.1.2.2.1        Small quantity.
5.1.2.3  Method:
5.1.2.3.1        Take 3 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 ethanol (96%) in test tube 2 and observe.
5.1.2.3.4        Add Methylene chloride in test tube 3 and observe.
5.1.2.4  Observations:
5.1.2.4.1        The sample in test tube 1 containing with water is sparingly soluble.
5.1.2.4.2        The sample in test tube 2 & 3 containing with ethanol (96%) and Methylene chloride are slightly soluble.
5.2  Identification tests:
5.2.1        Melting point determination:
5.2.1.1  Material and equipment:
5.2.1.1.1        Glassware (according to requirement).
5.2.1.1.2        Melting point apparatus.
5.2.1.1.3        Capillary tubes.
5.2.1.1.4        Purified water.
5.2.1.2  Sample:
5.2.1.2.1        Sufficient quantity of sample.
5.2.1.3  Method:
5.2.1.3.1        Introduce the sufficient quantity of sample into a capillary tube.
5.2.1.3.2        Set the apparatus and immerse the capillary tube into the apparatus such that the closed end is near the centre of the bulb of thermometer.
5.2.1.3.3        Switch on the melting point apparatus.
5.2.1.3.4        Operate the melting point apparatus according to the SOP
5.2.1.3.5        Raise the temperature of the apparatus.
5.2.1.3.6        Record the temperature at which the last particle passes into the liquid phase.
5.2.1.3.7        Record measurements in annexure-1.
5.2.1.4  Observations:
5.2.1.4.1        The melting point is 188oC-191oC.
5.2.2        UV/VIS absorption Spectrophotometry:
5.2.2.1  Material and equipm4ent:
5.2.2.1.1        UV/VIS Spectrophotometer.
5.2.2.1.2        Glassware (according to requirement).
5.2.2.1.3        Purified water.
5.2.2.2  Sample:
5.2.2.2.1        50.0mg.
5.2.2.3  Method:
5.2.2.3.1        Test solution (a):
5.2.2.3.1.1  Take a beaker of 100.0ml and add 50.0mg of sample in it.
5.2.2.3.1.2  Dissolve it in sufficient quantity of purified water.
5.2.2.3.1.3  And dilute it to 100.0ml with the same solvent.
5.2.2.3.2        Test solution (b):
5.2.2.3.2.1  Take a beaker and add 1.0ml of test solution (a) in it.
5.2.2.3.2.2  Dilute it to 10.0ml with purified water.
5.2.2.3.3        Test solution (c):
5.2.2.3.3.1  Take a beaker and add 2.0ml of test solution (a) in it.
5.2.2.3.3.2  Dilute it to 100.0ml with purified water.
5.2.2.3.4        Spectral range:
5.2.2.3.4.1  290-350nm for the test solution (b).
5.2.2.3.4.2  230-290nm for the test solution (c).
5.2.2.3.5        Absorption maximum:
5.2.2.3.5.1  At 310nm for test solution (b).
5.2.2.3.5.2  At 268nm for test solution (c).
5.2.2.3.6        Operate the UV/VIS spectrophotometer according to the SOP
5.2.2.3.7        Measure the absorption maximum of the resulting solution (b) & (c) at the given spectral range.
5.2.2.3.8        Note down values of absorption maximum for the test solution (b) & (c) in Annexure-2.
5.2.2.3.9        Measure the absorbance of the resulting solution at the absorption maximum at 268nm
5.2.2.3.10    Note down values of absorbance in annexure-2.
5.2.2.3.11    Calculate the specific absorbances at the absorption maximum by using formula:
1cmA1% = __a__  
                       b.c
5.2.2.4  Observations:
5.2.2.4.1        Specific absorbance at absorption maximum at 268nm:
5.2.2.4.1.1  640 to 680nm for the test solution (c).
5.2.3         
5.2.3.1  Material and equipment:
5.2.3.1.1        Glassware (according to requirement).
5.2.3.1.2        5.0ml of Purified water.
5.2.3.1.3        1.0ml of ferrous sulphate solution R2.
5.2.3.1.4        1.0ml of dilute sodium hydroxide solution R.
5.2.3.2  Sample:
5.2.3.2.1        0.1g.
5.2.3.3  Method:
5.2.3.3.1        Take a test tube add in it 0.1g of the sample in it.
5.2.3.3.2        Add 5.0ml of Purified water R. dissolve it thoroughly.
5.2.3.3.3        Add 1.0ml of ferrous sulphate solution R2
5.2.3.3.4        Observe the changes.
5.2.3.3.5        The solution becomes orange.
5.2.3.3.6        Add 1.0ml of dilute sodium hydroxide solution R.
5.2.3.3.7        Again observe the changes.
5.2.3.4  Observations:
5.2.3.4.1        The solution becomes dark blue.
5.3  Assay:
5.3.1        Apparatus:
5.3.1.1  Glassware (according to requirement).
5.3.1.2  Potentiometer.
5.3.1.3  Magnetic stirrer.
5.3.2        Material and reagents:
5.3.2.1  50.0ml of acetic anhydride R.
5.3.2.2  0.1M Perchloric acid.
5.3.2.3  Crystal violet solution.
5.3.3        Sample:
5.3.3.1  0.100g.
5.3.4        Method of analysis:
5.3.4.1  Take a 50.0ml of beaker and add 0.100g of sample in it.
5.3.4.2  Add 50.0ml of acetic anhydride R in it and dissolve by using magnetic stirrer i.e.
5.3.4.3  Fill the right hand side burette with titrant 0.1M Perchloric acid.
5.3.4.4  Carry out a Potentiometric titration using crystal violet solution as an indicator.
5.3.4.5  Operate potentiometer according to SOP
5.3.4.6  To neutralize analyte add titrant fixed volume (1ml, 0.5ml or 0.1ml) from burette every time note the reading of change in potential difference (millivolts) for each addition in given annexure-3.
5.3.4.7  Plot a graph, volume used v/s millivolts.
5.3.4.8  Find out the END POINT.
5.3.4.9  Peak of graph indicates END POINT i.e. the point at which maximum millivolts. Note down volume used at that point.
5.3.4.10    Perform blank titration without using sample. Similarly, as sample titration performed. Record observations in annexure-3.
5.3.4.11    Calculate volume used by substance by using formula:
Volume used by substance = Blank titration - Sample titration.
5.3.4.12    Calculate percentage purity of the sample by using formula:
%age purity = volume used by substance x factor x 100
                         Weight of sample
5.3.5        Factor:
5.3.5.1  1ml of 0.1M Perchloric acid is equivalent to 12.31mg of Pyrazinamide C5H5N3O.
5.3.6        Limit:
5.3.6.1  99.0% to 101.0% (anhydrous substance).
6.0  REVISION LOG:
Revision No.
Effective Date
Reason
00

New SOP

7.0  REFERENCES:
7.1  The British Pharmacopoeia. Vol II., Official Monograph / Pyrazinamide: 2015, pp. 702-703.
8.0  ANNEXURES:
Annexure 1: Observations of Melting point apparatus.
Annexure 2: Observations of UV/VIS spectrophotometer.
Annexure 3: Assay observations and calculations (Potentiometric titration).











Annexure: 1
Observations of Melting point apparatus
Sample = _____________
Time period = _____________
Sr.#
Initial (Ti)
(oC)
Final (Tf)
(oC)
Tf - Ti
(oC)












Average: _____________

Result: _________________

Remarks: _______________________________________________________________









                                                                                                                                   




Annexure: 2
Observations and Calculations of UV/VIS spectrophotometer
UV/VIS spectrophotometer
Model: _____________________________                             Date: _________________
OBSERVATIONS:
Thickness of cell:

Spectral range:
220-320nm.
Maxima absorption wavelength:
265nm.
Sample:

Other reagent used:


No. of obs.
Concentration
(c)
Wavelength
(λ)
Absorbance
(a)
Specific absorbance
A (1%,1cm)











CALCULATIONS:
1cmA1% =  __a__
                   b.c





Results: _______________
Remarks: ______________________________________________________________


Annexure: 3
Assay observations and calculations (Potentiometric titration)
Potentiometric titration
Reference electrode: ___________________
Indicator electrode: ____________________
Speed of magnetic stirrer: _______________
Titrant used: __________________________
Indicator: ____________________________
Blank titration:
Sr.#
Volume used
(ml)
Voltmeter
(mV)












Plot a graph, volume used v/s millivolts and find out peak of graph i.e. END POINT of blank titration.
Sample titration:
Sr.#
Volume used
(ml)
Voltmeter
(mV)












Plot a graph, volume used v/s millivolts and find out peak of graph i.e. END POINT of sample titration:
Volume used by Blank titration: __________________
Volume used by Sample titration: _________________
Volume used by substance = Blank titration - Sample titration.


mV used by Blank titration: __________________
mV used by Sample titration: _________________
mV used by substance = Blank titration - Sample titration.

Volume used by substance: _______________________
Voltmeter (mV) used by substance: _________________


RESULT: ____________________________________________________________
















9.0  ABBREVIATIONS:
Abbreviation
Expanded Form
SOP
Standard operating procedure
&
And
No.
Number  
Ltd.
Limited
Q.C
Quality control
%
Percentage
B.P
British pharmacopoeia
mg
Milligram
ml
Milliliter
g
Grams
M
Molar
Vol
Volume
QCA
Quality control active ingredient
F
Format
v/s
Verses
mV
Millivolts
oC
Degree centigrade
R
Reagent
Ti
Initial temperature
Tf
Final temperature
Temp.
Temperature
λ
Lambda
UV/VIS
Ultraviolet/ visible
nm
Nanometer
h
Hours
A
Absorbance


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