AZITHROMYCIN SOP



AZITHROMYCIN SOP

1.0  OBJECTIVE:
To lay down a procedure of analytical report for the active raw material of the Azithromycin dihydrate 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 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        Anhydrous ethanol.
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, anhydrous ethanol and methylene chloride 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 anhydrous ethanol is freely soluble.
5.1.2.4.3        The sample in test tube 3 containing with methylene chloride is freely soluble.
5.2  Assay:
5.2.1        Apparatus:
5.2.1.1  HPLC apparatus.
5.2.1.2  Glassware (according to the requirement).
5.2.1.3  Spectrophotometer (detector).
5.2.2        Material and reagents:
5.2.2.1  Azithromycin dihydrate sample.
5.2.2.2  Octadecylsilyl vinyl polymer for chromatography (5μm).
5.2.2.3  Acetonitrile.
5.2.2.4  6.7g/L solution of dipotassium hydrogen phosphate.
5.2.2.5  Phosphoric acid.
5.2.2.6  560g/L solution of potassium hydroxide.
5.2.3        Requirements:
5.2.3.1  Solution A:
5.2.3.1.1        Take 1000ml of beaker and mix 60 volumes of acetonitrile and 40 volumes of a 6.7g/L solution of dipotassium hydrogen phosphate adjusted to pH 8.0 with phosphoric acid.
5.2.3.2  Sample:
5.2.3.2.1        53.0mg (sample to be examined).
5.2.3.2.2        53.0mg (Azithromycin CRS) for reference solution.
5.2.3.3  Test solution:
5.2.3.3.1        Test solution:
5.2.3.3.1.1  Take 100ml of beaker and dissolve 53.0mg of the substance to be examined in 2ml of acetonitrile.
5.2.3.3.1.2  And dilute to 100.0ml with the solution A.
5.2.3.4  Reference solutions:
5.2.3.4.1        Reference solution:
5.2.3.4.1.1  Take 100ml beaker and dissolve 53.0mg of Azithromycin CRS in 2ml of acetonitrile.
5.2.3.4.1.2  And dilute to 100.0 ml with solution A.
5.2.3.5  Column:
5.2.3.5.1        Size:
5.2.3.5.1.1  Length=0.25m,
5.2.3.5.1.2  θ=4.6mm.
5.2.3.5.2        Stationary phase:
5.2.3.5.2.1  Octadecylsilyl vinyl polymer for chromatography (5μm).
5.2.3.5.3        Temperature:
5.2.3.5.3.1  40oC.
5.2.3.6  Mobile phase:
5.2.3.6.1        Mix 60 volumes of acetonitrile and 40 volumes of a 6.7g/L solution of dipotassium hydrogen phosphate adjusted to pH 11.0 with 560g/L solution of potassium hydroxide.
5.2.3.7  Flow rate:
5.2.3.7.1        1.0ml/min.
5.2.3.8  Detection:
5.2.3.8.1        Spectrophotometer at 210nm.
5.2.3.9  Injection:
5.2.3.9.1        10μL.
5.2.3.10 Run time:
5.2.3.10.1    1.5 times the retention time of Azithromycin.
5.2.3.11 Retention time Azithromycin:
5.2.3.11.1    About 10min.
5.2.4        Method of analysis:
5.2.4.1  Firstly prepare the test solution, reference solution and mobile phase according to the requirements.
5.2.4.2  The solutions must be free from solid particles.
5.2.4.3  Prepare the apparatus.
5.2.4.4  The mobile phase solvent mixtures must be deaerated prior to use either by boiling or by applying a partial vacuum to the solvent reservoir.
5.2.4.5  Equilibrate the column with the prescribed mobile phase, flow rate and at temperature specified until a suitable baseline is achieved.
5.2.4.6  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.
5.2.4.7  As the eluate leaves the column it enters a detector, where it is continuously monitored at the specified λ.
5.2.4.8  The electrical signal obtained from detector is amplified and routes to recorder which record the developed chromatogram.
5.2.4.9  Calculate the percentage content of Azithromycin (C38H72N2O12) from the declared content of Azithromycin CRS.
5.2.5        Observations:
5.2.5.1  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 /Azithromycin: 2015, pp. 225-227.
8.0  ANNEXURES:
Annexure 1: Observations and calculations of HPLC method.

Annexure: 1
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 chromatogram.

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
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


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