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:
___________________
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)= _____________
2.
Retention volume:
Flow
rate= _______________ml/min.
3.
Retention factor:
Retention
time of unretained peak (tm)= _____________
4.
Separation factor (α):
5.
Resolution:
Retention
time of unretained peak (tm)= _____________
6.
Efficiency:
7.
Height equivalent to a theoretical plate
(HETP):
Length
of column = ________________________
8.
Symmetry factor (tailing factor):
9.
Response factor & Relative response
factor:
Conc.
(mg/ml)= ___________________
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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