FLUOXETINE HYDROCHLORIDE SOP

1.0  OBJECTIVE:
To lay down a procedure for the active raw material of the Fluoxetine hydrochloride 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  Crystalline powder.
5.1.2        Solubility:
5.1.2.1  Material and equipment:
5.1.2.1.1        Glassware (3 test tubes, 1 spatula).
5.1.2.1.2        Methanol.
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 methanol in test tube 2 and observe.
5.1.2.3.4        Add methylene chloride in test tube 3 and observe.
5.1.2.4  Observations:
5.1.2.4.1        The sample in test tube 1and 3 containing with purified water and methylene chloride are sparingly soluble, respectively.
5.1.2.4.2        The sample in test tube 2 containing with methanol is freely soluble.
5.2  Identification tests:
5.2.1        Chlorides test:
5.2.1.1  Material and equipment:
5.2.1.1.1        Glassware (according to requirement).
5.2.1.1.2        Centrifuge machine.
5.2.1.1.3        Dilute nitric acid.
5.2.1.1.4        Purified water (q.s).
5.2.1.1.5        0.4ml of Silver nitrate R1.
5.2.1.1.6        1.5ml of ammonia.
5.2.1.2  Sample:
5.2.1.2.1        Quantity of substance to be examined equivalent to about 2.0mg of chloride.
5.2.1.3  Method:
5.2.1.3.1        Take a test tube add in it 2.0ml of water with the help of pipette.
5.2.1.3.2        Dissolve in 2.0ml of water a quantity of the substance to be equivalent to about 2.0mg of chloride.
5.2.1.3.3        Acidify with dilute nitric acid.
5.2.1.3.4        And add 0.4ml of silver nitrate R1.
5.2.1.3.5        Shake and allow it to stand.
5.2.1.3.6        A curdled, white ppt is formed.
5.2.1.3.7        Centrifuge it in centrifugation machine, according to SOP.
5.2.1.3.8        The obtained ppt is wash with 3 quantities, each of 1ml, of water.
5.2.1.3.9        Carry out this operation rapidly is subdued light, degrading the fact that the supernatant solution may not become perfectly clear.
5.2.1.3.10    Suspend the precipitate in 2.0ml of water and add 1.5ml of ammonia.
5.2.1.4  Observations:
5.2.1.4.1        The precipitate dissolves easily with the possible exception of a few large particles which dissolves slowly.
5.3  Solution S:
5.3.1        Material and equipment:
5.3.1.1  Glassware (1 100.0ml of beaker, 1 spatula, 1 glass rod, 1 pipette).
5.3.1.2  Analytical weighing balance.
5.3.1.3  Magnetic stirrer.
5.3.1.4  85 volumes of methanol.
5.3.1.5  Purified water.
5.3.2        Sample:
5.3.2.1  2.0g.
5.3.3        Preparation of solution S:
5.3.3.1  Take a beaker of 100.0ml and add sample 2.0g in it.
5.3.3.2  Dissolve it in a mixture of 15 volumes of purified water and 85 volumes of methanol.
5.3.3.3  Dilute it to 100.0ml with the same mixture of solvent.
5.4  Optical rotation:
5.4.1        Material and equipment:
5.4.1.1  Polarimeter.
5.4.1.2  Magnetic stirrer.
5.4.1.3  Glassware (1 beaker of 50.0ml, 1 stirrer, 1 spatula).
5.4.2        Sample:
5.4.2.1  Solution S.
5.4.3        Method:
5.4.3.1  Firstly clean the Polarimeter with clean dry cloth, according to SOP No. BM/QCEC/SOP031-00.
5.4.3.2  Operate the Polarimeter according to SOP.
5.4.3.3  Fill the Polarimeter tube with blank solution and determine the observed optical rotation.
5.4.3.4  Similarly, fill the Polarimeter tube with sample solution and determine the observed optical rotation.
5.4.3.5  Note down the values in annexure-1.
5.4.4        Observations:
5.4.4.1  The optical rotation is -0.05o to +0.05o.
5.5  Assay:
5.5.1        Apparatus:
5.5.1.1  HPLC apparatus.
5.5.1.2  Glassware (according to the requirement).
5.5.2        Material and reagents:
5.5.2.1  55.0mg of Fluoxetine hydrochloride CRS.
5.5.2.2  Methanol.
5.5.2.3  Tetrahydrofuran.
5.5.2.4  Triethylamine.
5.5.2.5  Purified water.
5.5.2.6  Phosphoric acid.
5.5.3        Sample:
5.5.3.1  55.0mg.
5.5.4        Test solution:
5.5.4.1  Take a beaker of 50.0ml and add 55.0mg of the substance to be examined in it.
5.5.4.2  Add in sufficient quantity of mobile phase and dissolved it by using magnetic stirrer operate according to SOP.
5.5.4.3  And dilute it to 50.0ml with the mobile phase.
5.5.4.4  Take 10.0ml of this solution in another beaker.
5.5.4.5  And dilute it to 100.0ml with the mobile phase.
5.5.5        Reference solutions:
5.5.5.1  Take a beaker of 50.0ml and add 55.0mg of Fluoxetine hydrochloride CRS in it.
5.5.5.2  Add in sufficient quantity of mobile phase and dissolved it by using magnetic stirrer operate according to SOP.
5.5.5.3  And dilute it to 50.0ml with the mobile phase.
5.5.5.4  Take 10.0ml of this solution in another beaker.
5.5.5.5  And dilute it to 100.0ml with the mobile phase.
5.5.6        Column:
5.5.6.1  Size:
5.5.6.1.1        Length=0.25m,
5.5.6.1.2        θ=4.6mm.
5.5.6.2  Stationary phase:
5.5.6.2.1        Octylsilyl silica gel for chromatography R (5μm).
5.5.7        Mobile phase:
5.5.7.1  Mix 8 volumes of methanol R, 30 volumes of tetrahydrofuran R and 62 volumes of a solution of triethylamine R prepared as follows: to 10.0ml of triethylamine R, and add 980.0ml of purified water R, mix and adjust to pH 6.0 with phosphoric acid R (about 4.5ml) and dilute to 1000.0ml with purified water R.
5.5.8        Flow rate:
5.5.8.1  1.0ml/min.
5.5.9        Detection:
5.5.9.1  Spectrophotometer at 227nm.
5.5.10    Injection:
5.5.10.1    10μL.
5.5.11    Run time:
5.5.11.1    3 times the retention time of fluoxetine.
5.5.12    Retention time:
5.5.12.1    Fluoxetine=10min to 18min; if necessary, adjust the volumes of methanol and of the solution of triethylamine in the mobile phase.
5.5.13    System suitability: reference solution:
5.5.13.1    Symmetry factor: Maximum 2.0 calculated at 10% of the height of the peak due to fluoxetine.
5.5.14    Method of analysis:
5.5.14.1    Firstly prepare the test solution, reference solution and mobile phase according to the requirements.
5.5.14.2    The solutions must be free from solid particles.
5.5.14.3    Prepare the apparatus.
5.5.14.4    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.
5.5.14.5    Equilibrate the column with the prescribed mobile phase, flow rate and at temperature specified until a suitable baseline is achieved.
5.5.14.6    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.
5.5.14.7    As the eluate leaves the column it enters a detector, where it is continuously monitored at the specified λ.
5.5.14.8    The electrical signal obtained from detector is amplified and routes to recorder which record the developed chromatogram.
5.5.14.9    Calculate the percentage content of Fluoxetine hydrochloride (C17H19ClF3NO) from the declared content of Fluoxetine hydrochloride CRS.
5.5.15    Observations:
5.5.15.1    98.0% to 102.0% (anhydrous substance).
6.0  REFERENCES:
6.1  The British Pharmacopoeia. Vol I., Official Monograph / Fluoxetine hydrochloride: 2015, pp. 995-996.
6.2  The British Pharmacopoeia. Vol V., Official Monograph /Qualitative Reactions and Tests: 2015, pp. 266-270.
7.0  ANNEXURES:
Annexure 1: Optical rotation observations and calculations.
Annexure 2: Observations and calculations of HPLC method.

Annexure: 1
Specific optical rotation observations and calculations
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 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:
________________________________________________________________________________________________________________________________________________


8.0  ABBREVIATIONS:
Abbreviation
Expanded Form
SOP
Standard operating procedure
&
And
No.
Number  
Ltd.
Limited
QCA
Quality control active ingredient
F
Format
Q.C
Quality control
Vol
Volume
g
Grams
ml
Milliliter
oC
Degree centigrade
mg
Milligram
M
Molar
%
Percentage
R
Reagent
μm
Micron/ micrometer
g/L
Gram per liter
CRS
Chemical reference solution
m
Meter
θ
Theta
mm
Millimeter
ml/min
Milliliter per minute
nm
Nanometer
μL
Microliter
Min
Minute
λ
Lamda


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