RIFAMPICIN METHOD OF ANALYSIS SOP

1.0  OBJECTIVE:
To lay down a procedure of analytical report for the active raw material of the Rifampicin 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  Reddish brown or brownish-red.
5.1.1.2  Crystalline powder.
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        Methanol.
5.1.2.1.3        Purified water.
5.1.2.1.4        Acetone.
5.1.2.1.5        Ethanol (96%).
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 methanol 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 Ethanol (96%) 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 slightly soluble.
5.1.2.4.2        The sample in test tube 2 containing with methanol is soluble.
5.1.2.4.3        The sample in test tube 3 and 4 containing with acetone and Ethanol (96%) are slightly soluble respectively.
5.2  Identification tests:
5.2.1        UV/VIS absorption Spectrophotometry:
5.2.1.1  Material and equipm4ent:
5.2.1.1.1        UV/VIS Spectrophotometer.
5.2.1.1.2        Glassware (according to requirement).
5.2.1.1.3        50.0ml of methanol.
5.2.1.1.4        Phosphate buffer solution pH at 7.4 R.
5.2.1.2  Sample:
5.2.1.2.1        50.0mg.
5.2.1.3  Method:
5.2.1.3.1        Test solution:
5.2.1.3.1.1  Take a beaker of 50.0ml and add 50.0mg of sample in it.
5.2.1.3.1.2  Dissolve it in 50.0ml of methanol.
5.2.1.3.1.3  Take another beaker and add 1.0ml of this solution in it.
5.2.1.3.1.4  Dilute it to 50.0ml with phosphate buffer solution pH 7.4 R.
5.2.1.3.2        Spectral range:
5.2.1.3.2.1  220-500nm.
5.2.1.3.3        Absorption maxima:
5.2.1.3.3.1  At 237nm, 254nm, 334nm and 475nm.
5.2.1.3.4        Operate the UV/VIS spectrophotometer according to the SOP .
5.2.1.3.5        Measure the absorption maximum at given spectral range.
5.2.1.3.6        Measure the absorbance of the resulting solution at the maximum wavelength 334nm and 475nm.
5.2.1.3.7        Note down values of absorbance in annexure-1.
5.2.1.3.8        Calculate the absorbance ratio by using formula:
A334/ A475
5.2.1.4  Observations:
5.2.1.4.1        Absorbance ratio A334/ A475:
5.2.1.4.1.1  Absorbance ratio A334/ A475 = is about 1.75.
5.2.2         
5.2.2.1  Material and equipment:
5.2.2.1.1        Glassware (1 test tube, 1 pipette, 1 spatula).
5.2.2.1.2        Vortex mixer.
5.2.2.1.3        25.0ml of purified water.
5.2.2.1.4        1.0ml of 100g/L solution of ammonium persulfate R.
5.2.2.1.5        Phosphate buffer pH at 7.4 R
5.2.2.2  Sample:
5.2.2.2.1        25.0mg.
5.2.2.3  Method:
5.2.2.3.1        Take a test tube and suspend in it 25.0mg of the sample with 25.0ml of purified water.
5.2.2.3.2        Shake it for 5 minutes and filter it by using filtration apparatus.
5.2.2.3.3        Take 5.0ml of the filtrate in another test tube and add 1.0ml of a 100g/L solution of ammonium persulfate R in phosphate buffer solution pH 7.4 R.
5.2.2.3.4        Shake it for a few minutes.
5.2.2.3.5        Observe the changes.
5.2.2.4  Observations:
5.2.2.4.1        The colour changes from orange-yellow to violet-red and no ppt is formed.
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 at a pressure not exceeding 0.67kPa for at least 4 hours.
5.3.3.5  And wait till the sample loses its moisture.
5.3.3.6  After 4 hours weigh the sample again by using analytical weighing balance i.e. the final weight.
5.3.3.7  Note down readings on given Annexure-2.
5.3.4        Observation:
5.3.4.1  Maximum 1.0%.
5.4  Assay:
5.4.1        Apparatus:
5.4.1.1  Glassware (according to requirement).
5.4.1.2  UV/VIS Spectrophotometer.
5.4.1.3  Magnetic stirrer.
5.4.2        Material and reagents:
5.4.2.1  Methanol.
5.4.2.2  Phosphate buffer solution pH 7.4.
5.4.3        Sample:
5.4.3.1  0.100g.
5.4.4        Method of analysis:
5.4.4.1  Take a beaker of 100.0ml and add 0.100g of sample in it.
5.4.4.2  Add sufficient quantity of methanol and dissolve it by using magnetic stirrer i.e. SOP.
5.4.4.3  Dilute it to 100.0ml with the same solvent.
5.4.4.4  Take another beaker and add in it 2.0ml of this solution and dilute it to 100.0ml with phosphate buffer solution pH 7.4.
5.4.4.5  Operate the UV/VIS spectrophotometer according to the SOP.
5.4.4.6  Measure the absorbance at the absorption maximum at 475nm using Phosphate buffer solution pH 7.4 R as the compensation liquid.
5.4.5        Determination of content:
5.4.5.1  Calculate the content of C43H58N4O12, taking the specific absorbance to be 187.
5.4.6        Limit:
5.4.6.1  97.0% 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 II., Official Monograph / Rifampicin: 2015, pp. 748-749.
8.0  ANNEXURES:
Annexure 1: Observations of UV/VIS spectrophotometer.
Annexure 2: Observations of Percentage Loss of drying by using oven.
Annexure 3: Assay observations and calculations of UV/VIS spectrophotometer.


Annexure: 1
Observations and Calculations of UV/VIS spectrophotometer
UV/VIS spectrophotometer
Model: _____________________________                             Date: _________________
OBSERVATIONS:
Thickness of cell:

Spectral range:
220nm to 500nm.
Maxima absorption wavelength:
237nm, 254nm, 334nm and 475nm
Sample:

Other reagent used:


No. of obs.
Concentration
(c)
Wavelength
(λ)
Absorbance
(a)

















CALCULATIONS:
A334/ A475 ratio =



Results: _______________
Remarks: ______________________________________________________________



Annexure: 2
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: 3
Assay observations and calculations of UV/VIS spectrophotometer
UV/VIS spectrophotometer
Model: _____________________________                             Date: _________________
OBSERVATIONS:
No. of tablets:

Sample:

Thickness of cell (b):

Concentration solution (c):

At A(1%, 1cm):

Wavelength (λ):


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










CALCULATIONS:
By using formulas:
1.      1cmA1% =  __a__
                             b.c

2.      %age purity=     Absorbance of sample     x 100
                                      Absorbance of standard

Results: _______________
Remarks: ______________________________________________________________



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
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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