METRONIDAZOLE BENZOATE SOP


1.0  OBJECTIVE:
To lay down a procedure of analytical report for the active raw material of the Metronidazole Benzoate 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 slightly yellowish.
5.1.1.2  Crystalline powder or flakes.
5.1.2        Solubility:
5.1.2.1  Material and equipment:
5.1.2.1.1        Glassware (4 test tubes, 1 spatula, 1 pipette).
5.1.2.1.2        Acetone.
5.1.2.1.3        Alcohol.
5.1.2.1.4        Purified water.
5.1.2.1.5        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 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 Methylene chloride in test tube 2 and observe.
5.1.2.3.4        Add acetone in test tube 3 and observe.
5.1.2.3.5        Add alcohol in test tube 4 and observe.
5.1.2.4  Observations:
5.1.2.4.1        The sample in test tube 1 containing with purified water is practically insoluble.
5.1.2.4.2        The sample in test tube 2 containing with methylene chloride is freely soluble.
5.1.2.4.3        The sample in test tube 3 containing with acetone is soluble.
5.1.2.4.4        The sample in test tube 4 containing with alcohol is 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 99oC-102oC.
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        103g/L solution of hydrochloric acid R.
5.2.2.2  Sample:
5.2.2.2.1        0.100g.
5.2.2.3  Method:
5.2.2.3.1        Test solution:
5.2.2.3.1.1  Take a beaker of 100.0ml and add 0.100g of sample in it.
5.2.2.3.1.2  Add in it 103g/L solution of hydrochloric acid R.
5.2.2.3.1.3  Dissolve it by using magnetic stirrer i.e. SOP
5.2.2.3.1.4  And dilute it to 100.0ml with the same acid.
5.2.2.3.1.5  Take 1.0ml of this solution in 100.0ml of beaker.
5.2.2.3.1.6  And dilute it to 100.0ml with 103g/L solution of hydrochloric acid R.
5.2.2.3.2        Spectral range:
5.2.2.3.2.1  220-350nm.
5.2.2.3.3        Absorption maximum:
5.2.2.3.3.1  At 232nm and 275nm.
5.2.2.3.4        Operate the UV/VIS spectrophotometer according to the SOP.
5.2.2.3.5        Measure the absorption maximum at given spectral range.
5.2.2.3.6        Measure the absorbance of the resulting solution at the maximum wavelength 232nm and 275nm.
5.2.2.3.7        Note down values of absorbance in annexure-2.
5.2.2.3.8        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 232nm:
5.2.2.4.1.1  525 to 575nm.
5.2.3        Primary aromatic amines test:
5.2.3.1  Material and equipment:
5.2.3.1.1        Glassware (1 test tube, 1 pipette, 1 spatula).
5.2.3.1.2        Water bath.
5.2.3.1.3        10.0mg of zinc chloride.
5.2.3.1.4        1.0ml of purified water.
5.2.3.1.5        0.3ml of hydrochloric acid R.
5.2.3.1.6        0.2ml of sodium nitrite solution R.
5.2.3.1.7        1.0ml of β-naphthol solution R.
5.2.3.2  Sample:
5.2.3.2.1        10.0mg.
5.2.3.3  Method:
5.2.3.3.1        Take a test tube add in it 10.0mg of sample, 10.0mg of zinc chloride R, 1.0ml of purified water R and 0.3ml of hydrochloric acid R. Dissolve it.
5.2.3.3.2        Heat it on water bath for 5 minutes. And after that cool it.
5.2.3.3.3        Acidify the solution with dilute hydrochloric acid R and add 0.2ml of sodium nitrite solution R.
5.2.3.3.4        After 1 minute to 2 minute, add 1.0ml of β-naphthol solution R.
5.2.3.3.5        Observe the changes.
5.2.3.4  Observations:
5.2.3.4.1        An intense orange or red colour and usually a ppt of the same colour are produced.
5.3  Loss on drying:
5.3.1        Material and equipment:
5.3.1.1  Glassware (according to requirement).
5.3.1.2  Analytical weighing balance.
5.3.1.3  Oven.
5.3.2        Sample:
5.3.2.1  1.0g.
5.3.3        Method:
5.3.3.1  Weigh 1.0g of the test sample.
5.3.3.2  Set the oven apparatus. Operate it according to the SOP.
5.3.3.3  Place the sample into the tray and dry it.
5.3.3.4  Set the temperature of oven at 80oC for at least 3hours.
5.3.3.5  And wait till the sample loses its moisture.
5.3.3.6  After 3hours weigh the sample again by using analytical weighing balance i.e. the final weight.
5.3.3.7  Note down readings on given Annexure-3.
5.3.4        Observation:
5.3.4.1  Maximum 0.5%.
5.4  Assay:
5.4.1        Apparatus:
5.4.1.1  Glassware (according to requirement).
5.4.1.2  Potentiometer.
5.4.1.3  Magnetic stirrer.
5.4.2        Material and reagents:
5.4.2.1  50.0ml of anhydrous acetic acid R.
5.4.2.2  0.1M Perchloric acid.
5.4.2.3  Crystal violet solution (as an indicator).
5.4.3        Sample:
5.4.3.1  0.250g.
5.4.4        Method of analysis:
5.4.4.1  Take a 50.0ml of beaker and add 0.250g of sample in it.
5.4.4.2  Add 50.0ml of anhydrous acetic acid R in it and dissolve by using magnetic stirrer i.e. SOP.
5.4.4.3  Fill the right hand side burette with titrant 0.1M Perchloric acid.
5.4.4.4  Carry out a Potentiometric titration using crystal violet solution as an indicator.
5.4.4.5  Operate potentiometer according to SOP.
5.4.4.6  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-4.
5.4.4.7  Plot a graph, volume used v/s millivolts.
5.4.4.8  Find out the END POINT.
5.4.4.9  Peak of graph indicates END POINT i.e. the point at which maximum millivolts. Note down volume used at that point.
5.4.4.10 Perform blank titration without using sample. Similarly, as sample titration   performed. Record observations in annexure-4.
5.4.4.11 Calculate volume used by substance by using formula:
Volume used by substance = Blank titration - Sample titration.
5.4.4.12 Calculate percentage purity of the sample by using formula:
%age purity = volume used by substance x factor x 100
                         Weight of sample
5.4.5        Factor:
5.4.5.1  1ml of 0.1M Perchloric acid is equivalent to 27.53mg of Metronidazole benzoate C13H13N3O4.
5.4.6        Limit:
5.4.6.1  98.5% to 101.0% (dried 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 / Metronidazole: 2015, pp. 281-282.
8.0  ANNEXURES:
Annexure 1: Observations of Melting point apparatus.
Annexure 2: Observations of UV/VIS spectrophotometer.
Annexure 3: Observations of Percentage Loss of drying by using oven.
Annexure 4: 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:
220-350nm.
Maxima absorption wavelength:
232nm and 275nm.
Sample:
0.100g.
Other reagent used:


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


232nm




275nm



CALCULATIONS:
1cmA1% =  __a__
                   b.c



Results: _______________
Remarks: ______________________________________________________________



Annexure: 3
Observations of percentage loss of drying by using Oven
Percentage loss of drying by using Oven
Apparatus: ____________________
Temperature: __________________
Weight of Sample = _____________
Time period = _____________
Pressure= _________________
Sr.#
Time (min)
Weight of sample (g)
% Loss of Moisture
Initial weight
Final weight















Average % Loss of Moisture: _____________

% Loss of Moisture:







Remarks: _______________________________________________________________



Annexure: 4
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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