MONTELUKAST SODIUM SOP

1.0  OBJECTIVE:
To lay down a procedure of analytical report for the active raw material of the Montelukast sodium from the Pharmacopoeial specifications.
2.0  SCOPE:
This SOP shall be applicable in Q.C laboratory.
3.0  RESPONSIBILITY:
3.1  Q.C Analyst.
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  Hygroscopic powder.
5.1.2        Solubility:
5.1.2.1  Material and equipment:
5.1.2.1.1        Glassware (3 test tubes, spatula).
5.1.2.1.2        Ethanol (96.0%).
5.1.2.1.3        Methylene chloride.
5.1.2.1.4        Purified water.
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 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 ethanol (96.0%) in test tube 3 and observe.
5.1.2.4  Observations:
5.1.2.4.1        The sample in test tubes 1 & 2 containing with water and methylene chloride is freely soluble.
5.1.2.4.2        The sample in test tube 3 containing with ethanol (96.0%) is freely soluble to very soluble.
5.2  Identification tests:
5.2.1        Sodium test:
5.2.1.1  Material and equipment:
5.2.1.1.1        Glassware (1 crucible, 1 beaker, 2 test tubes, 1 glass rod, 1 funnel).
5.2.1.1.2        Bunsen burner.
5.2.1.1.3        Filter paper.
5.2.1.1.4        2.0ml of 150g/L solution of potassium carbonate.
5.2.1.1.5        4.0ml of potassium pyroantimonate solution.
5.2.1.1.6        Ice water.
5.2.1.1.7        Purified water (q.s)
5.2.1.2  Sample:
5.2.1.2.1        0.1g.
5.2.1.3  Method:
5.2.1.3.1        Take a crucible and add 0.1g of sample in it and ignite on Bunsen burner, until an almost white residue is obtained.
5.2.1.3.2        Take up the residue in test tube and add 2.0ml of purified water in it.
5.2.1.3.3        Filter it by using filter paper, funnel and another beaker for collection of filtrate carefully.
5.2.1.3.4        Take 2.0ml of the above filtrate in test tube.
5.2.1.3.5        2.0ml of the 150g/L solution of potassium carbonate
5.2.1.3.6        Heat it on Bunsen burner till boiling.
5.2.1.3.7        No ppt is formed.
5.2.1.3.8        Add 4.0ml of potassium pyroantimonate solution heat on burner till boiling.
5.2.1.3.9        Take a beaker of ice water. And cool the test tube into it, if necessary rub inside of the test tube with a glass rod.
5.2.1.3.10    Observe the changes.
5.2.1.4  Observations:
5.2.1.4.1        A dense white precipitate is formed.
5.3  Assay:
5.3.1        Apparatus:
5.3.1.1  HPLC apparatus.
5.3.1.2  Glassware (according to the requirement).
5.3.1.3  Magnetic stirrer.
5.3.2        Material and reagents:
5.3.2.1  Phenylsilyl silica gel for chromatography (1.8μm).
5.3.2.2  65.0mg of Montelukast dicyclohexylamine CRS.
5.3.2.3  Methanol.
5.3.2.4  Trifluroacetic acid.
5.3.2.5  1000.0ml of acetonitrile.
5.3.2.6  Purified water (q.s).
5.3.3        Requirements:
5.3.3.1  Sample:
5.3.3.1.1        50.0mg
5.3.3.2  Solvent mixture: Water and methanol (10:90 v/v).
5.3.3.3  Test solution:
5.3.3.3.1        Test solution (a):
5.3.3.3.1.1  Take 50.0ml of beaker and dissolve 50.0mg of the substance to be examined in the solvent mixture.
5.3.3.3.1.2  Dilute it to 50.0ml with the solvent mixture.
5.3.3.3.2        Test solution (b):
5.3.3.3.2.1  Take 10.0ml of test solution (a) in 100.0ml of beaker.
5.3.3.3.2.2  Dilute it to 100.0ml with the solvent mixture.
5.3.3.4  Reference solutions:
5.3.3.4.1        Reference solution (d):
5.3.3.4.1.1  Take 50.0ml beaker and dissolve 65.0mg of Montelukast dicyclohexylamine CRS in sufficient solvent mixture. And make up the volume up to 50.0ml with the solvent mixture.
5.3.3.4.1.2  Take 10.0ml of this solution in another 100.0ml beaker.
5.3.3.4.1.3  Dilute it to 100.0ml with the solvent mixture.
5.3.3.5  Column:
5.3.3.5.1        Size:
5.3.3.5.1.1  Length=0.05m,
5.3.3.5.1.2  θ=4.6mm.
5.3.3.5.2        Stationary phase:
5.3.3.5.2.1  Phenylsilyl silica gel for chromatography (1.8μm).
5.3.3.5.3        Temperature:
5.3.3.5.3.1  30oC.
5.3.3.6  Mobile phase:
5.3.3.6.1        Mobile phase A:
5.3.3.6.1.1  Take 1000.0ml of purified water in a beaker of 1000.0ml.
5.3.3.6.1.2  Add 1.5ml of trifluroacetic acid with the help of pipette in beaker.
5.3.3.6.1.3  Mix it by using magnetic stirrer i.e SOP.
5.3.3.6.2        Mobile phase B:
5.3.3.6.2.1  Take 1000.0ml of acetonitrile in a beaker of 1000.0ml.
5.3.3.6.2.2  Add 1.5ml of trifluroacetic acid with the help of pipette in beaker.
5.3.3.6.2.3  Mix it by using magnetic stirrer.
Time
(min)
Mobile phase A
(percent v/v)
Mobile phase B
(percent v/v)
0-3
60
40
3-16
60®49
40®51

5.3.3.7  Flow rate:
5.3.3.7.1        1.2ml/min.
5.3.3.8  Detection:
5.3.3.8.1        Spectrophotometer at 238nm.
5.3.3.9  Injection:
5.3.3.9.1        20μL of the test solution (b) and reference solution (d).
5.3.4        Method of analysis:
5.3.4.1  Carry out the test protected from light. Prepare the solutions in amber flasks.
5.3.4.2  Firstly prepare the test solution, reference solution and mobile phase according to the requirements.
5.3.4.3  The solutions must be free from solid particles.
5.3.4.4  Prepare the apparatus, use normal procedure of liquid chromatography.
5.3.4.5  The mobile phase solvent mixtures must be deaerated prior to use either by boiling or by applying a partial vacuum to the solvent reservoir.
5.3.4.6  Equilibrate the column with the prescribed mobile phase, flow rate and at temperature specified until a suitable baseline is achieved.
5.3.4.7  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.
5.3.4.8  As the eluate leaves the column it enters a detector, where it is continuously monitored at the specified λ.
5.3.4.9  The electrical signal obtained from detector is amplified and routes to recorder which record the developed chromatogram.
5.3.4.10    Calculate the percentage content of Montelukast sodium (C35H3 ClNNaO3S) using the following expression:
A1 x m2 x 79.24 x p
A2 x m1 x (100-a)
A1 = area of the principal peak in the chromatogram obtained with test solution (b);
A2 = area of the principal peak in the chromatogram obtained with reference solution (d);
m1 =mass of the substance to be examined used to prepare test solution (a), in mg.
m2 =mass of Montelukast dicyclohexylamine CRS used to prepare reference solution (d), in mg.
p=declared percentage content of Montelukast dicyclohexylamine CRS.
a=percentage content of water in the substance to be examined.
5.3.5        Limit:
5.3.5.1  98.0% to 102.0% (anhydrous 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 /Montelukast sodium: 2015, pp. 311-314.
7.2  The British Pharmacopoeia. Vol V., Official Monograph / Liquid Chromatography: 2015, Appendix: IIID pp. 202-204.
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
%
Percentage
g/L
Gram per liter
ml
Milliliter
q.s
Quantity sufficient
g
Grams
UV/VIS
Ultraviolet/ visible
ppt
Precipitate
μm
Micron
mg
Milligram
CRS
Chemical reference substance
v/v
Volume by volume
m
Meters
θ
Theta
λ
Lambda
oC
Degree centigrade
Min
Minutes
ml/min
Milliliter per minute
nm
Nanometer
μL
Microliter
B.P
British pharmacopoeia
Vol
Volume
QCA
Quality control active ingredient
F
Format


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