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:
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:
Plot a graph, volume used v/s millivolts and find out peak
of graph i.e. END POINT of blank titration.
Sample
titration:
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
|