CLARITHROMYCIN SOP


CLARITHROMYCIN SOP


1.0  OBJECTIVE:
To lay down a procedure of analytical report for the active raw material of the Clarithromycin 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 (test tubes, spatula).
5.1.2.1.2        Purified water.
5.1.2.1.3        Methanol.
5.1.2.1.4        Acetone.
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, acetone, methylene chloride and methanol in each test tube separately in a small volume and observe the solubility of the sample.
5.1.2.4  Observations:
5.1.2.4.1        The sample in test tube 1 containing with water is practically insoluble.
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 methylene chloride is soluble.
5.1.2.4.4        The sample in test tube 4 containing with methanol is slightly soluble.
5.2  Solution S:
5.2.1        Material and equipment:
5.2.1.1  Analytical weighing balance.
5.2.1.2  Glassware (1 beaker of 50.0ml, 1 stirrer, 1 spatula).
5.2.2        Sample:
5.2.2.1  0.500g.
5.2.3        Preparation of solution S:
5.2.3.1  Take a beaker of 50.0ml and add 0.5g of the sample in it.
5.2.3.2  Dissolve the sample in sufficient quantity of methylene chloride.
5.2.3.3  Dilute it to 50.0ml with the same solvent.
5.3  Specific optical rotation:
5.3.1        Material and equipment:
5.3.1.1  Polarimeter.
5.3.1.2  Analytical weighing balance.
5.3.1.3  Glassware (1 beaker of 50.0ml, 1 stirrer, 1 spatula).
5.3.1.4  Methanol.
5.3.2        Sample:
5.3.2.1  Solution S.
5.3.3        Method:
5.3.3.1  Firstly clean the Polarimeter with clean dry cloth, according to
5.3.3.2  Operate the Polarimeter according to
5.3.3.3  Fill the Polarimeter tube with blank solution and determine the observed optical rotation.
5.3.3.4  Similarly, fill the Polarimeter tube with sample solution (i.e. solution S) and determine the observed optical rotation.
5.3.3.5  Note down the values in annexure-1.
5.3.3.6  Calculate the specific optical rotation by using formula:
[α]λ T = α/lc
5.3.4        Observations:
5.3.4.1  -94 to -102 (anhydrous substance).
5.4  Assay:
5.4.1        Apparatus:
5.4.1.1  HPLC apparatus.
5.4.1.2  Glassware (according to the requirement).
5.4.2        Material and reagents:
5.4.2.1  Octadecylsilyl silica gel for chromatography (3.5μm).
5.4.2.2  C18 filtration kit.
5.4.2.3  Acetonitrile.
5.4.2.4  4.76g/L solution of potassium dihydrogen phosphate.
5.4.2.5  Phosphoric acid.
5.4.2.6  45g/L solution of potassium hydroxide.
5.4.2.7  Purified water.
5.4.3        Requirements:
5.4.3.1  Sample:
5.4.3.1.1        75.0mg (sample to be examined).
5.4.3.1.2        75.0mg (clarithromycin CRS) for reference solution.
5.4.3.2  Test solution:
5.4.3.2.1        Test solution:
5.4.3.2.1.1  Take 100ml of beaker and dissolve 75.0mg of the substance to be examined in 25ml of acetonitrile.
5.4.3.2.1.2  And dilute to 50.0ml with water.
5.4.3.3  Reference solutions:
5.4.3.3.1        Reference solution (a):
5.4.3.3.1.1  Take 100ml beaker and dissolve 75.0mg of clarithromycin CRS in 25ml of acetonitrile.
5.4.3.3.1.2  And dilute to 50.0 ml with water.
5.4.3.4  Blank solution:
5.4.3.4.1        Take a 50ml beaker and dilute 25ml of acetonitrile to 50.0ml with water and mix.
5.4.3.5  Column:
5.4.3.5.1        Size:
5.4.3.5.1.1  Length=0.10m,
5.4.3.5.1.2  θ=4.6mm.
5.4.3.5.2        Stationary phase:
5.4.3.5.2.1  Octadecylsilyl silica gel for chromatography (3.5μm).
5.4.3.5.3        Temperature:
5.4.3.5.3.1  40oC.
5.4.3.6  Mobile phase:
5.4.3.6.1        Mobile phase A:
5.4.3.6.1.1  A 4.76g/L solution of potassium dihydrogen phosphate adjusted to pH 4.4 with dilute phosphoric acid.
5.4.3.6.1.2  Or a 45g/L solution of potassium hydroxide filtered through a C18 filtration kit.
5.4.3.6.2        Mobile phase B:
5.4.3.6.2.1  Acetonitrile.



Time
(min)
Mobile phase A
(Percent V/V)
Mobile phase B
(Percent V/V)
0-32
75®40
25®60
32-34
40
60

5.4.3.7  Flow rate:
5.4.3.7.1        1.1ml/min.
5.4.3.8  Detection:
5.4.3.8.1        Spectrophotometer at 205nm.
5.4.3.9  Injection:
5.4.3.9.1        10μL of blank solution, test solution and reference solution.
5.4.3.10    Relative retention:
5.4.3.10.1    With reference to clarithromycin about 11min.
5.4.4        Method of analysis:
5.4.4.1  Firstly prepare the test solution, reference solution and mobile phase according to the requirements.
5.4.4.2  The solutions must be free from solid particles.
5.4.4.3  Prepare the apparatus.
5.4.4.4  The mobile phase solvent mixtures must be de-aerated prior to use either by boiling or by applying a partial vacuum to the solvent reservoir.
5.4.4.5  Equilibrate the column with the prescribed mobile phase, flow rate and at temperature specified until a suitable baseline is achieved.
5.4.4.6  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.
5.4.4.7  As the eluate leaves the column it enters a detector, where it is continuously monitored at the specified λ.
5.4.4.8  The electrical signal obtained from detector is amplified and routes to recorder which record the developed chromatogram.
5.4.4.9  Calculate the percentage content of Clarithromycin (C38H69NO13).
5.4.5        Observations:
5.4.5.1  96% to 102% (anhydrous 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 /Clarithromycin: 2015, pp. 573-575.
8.0  ANNEXURES:
Annexure 1: Observations and calculations of specific optical rotation.
Annexure 2: Observations and calculations of HPLC method.















Annexure: 1
Observations and calculations of specific optical rotation
Instrument: ___________________                                              Date: _______________
Model: _______________________        Length of Polarimeter tube: ________________
Sample: ________________________________g.
Solvent: ________________________________ml.
Concentration of sample solution: ____________g/ml.
Blank solution:
Sr.#
Blank solution
Temperature
Optical rotation
(α)












                                                                                                 Average: _______________
Optical rotation of blank solution: _______________
Sample solution:
Sr.#
Sample solution
Temperature
Optical rotation
(α)












                                                                                                 Average: _______________
Optical rotation of sample solution: ______________
Optical rotation of substance = Blank solution - Sample solution.




Specific optical rotation of sample solution by using formula:
[α]λ T = α/lc





                                                                      Result: ________________
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 spectrum.







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.,

 

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
Q.C
Quality control
B.P
British pharmacopoeia
C18
Carbon-18
μm
Micron/ micrometer
g/L
Gram per liter
ml
Milliliter
mg
Milligram
CRS
Chemical reference solution
m
Meter
θ
Theta
mm
Millimeter
oC
Degree Celsius
ml/min
Milliliter per minute
nm
Nanometer
μL
Microliter
Min
Minute
λ
Lamda
%
Percentage
Vol
Volume
QCA
Quality control active ingredient
F
Format
o
Degree (angle)
l
Length
g
Grams
c
Concentration (g/ml)
g/ml
Gram per milliliter
α
Alpha
λ
Lambda
T
Temperature



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