ALENDRONATE SODIUM SOP



ALENDRONATE SODIUM SOP
1.0  OBJECTIVE:
To lay down a procedure of analytical report for the active raw material of Alendronate 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  Identification test:
5.1.1        Sodium test:
5.1.1.1  Material and equipment:
5.1.1.1.1        Glassware (spatula, glass rod).
5.1.1.1.2        Bunsen burner.
5.1.1.2  Sample:
5.1.1.2.1        Small amount of sample.
5.1.1.3  Method:
5.1.1.3.1        Take a small quantity of sample over the flame of the Bunsen burner.
5.1.1.3.2        Observe the changes.
5.1.1.4  Observations:
5.1.1.4.1        Sodium compounds impart an intense yellow color to a non-luminous flame.
5.2  Assay:
5.2.1        Apparatus:
5.2.1.1  HPLC apparatus.
5.2.1.2  Analytical weighing balance.
5.2.1.3  Glassware (according to the requirement).
5.2.1.4  Centrifugation machine.
5.2.1.5  Magnetic stirrer.
5.2.1.6  UV/VIS Spectrophotometer.
5.2.2        Material and reagents:
5.2.2.1  14.7g of sodium citrate dihydrate.
5.2.2.2  7.05g of anhydrous dibasic sodium phosphate.
5.2.2.3  Phosphoric acid.
5.2.2.4  29.4g of sodium citrate dihydrate.
5.2.2.5  19.1g of sodium borate.
5.2.2.6  0.5mg of 9-Fluorenylmethyl chloroformate.
5.2.2.7  Acetonitrile.
5.2.2.8  Methanol.
5.2.2.9  USP Alendronate Sodium RS.
5.2.2.10    Methylene chloride.
5.2.2.11    25.0mg of the alendronate sodium.
5.2.2.12    Purified water.
5.2.3        Requirements:
5.2.3.1  Buffer solution:
5.2.3.1.1        Take a volumetric flask of 1000.0ml with sufficient quantity of purified water and add 14.7g of sodium citrate dihydrate and 7.05g of anhydrous dibasic sodium phosphate in it, stir by using magnetic stirrer.
5.2.3.1.2        Dilute it with purified water to the volume.
5.2.3.1.3        Adjust with phosphoric acid to a pH of 8.0.
5.2.3.2  Diluent:
5.2.3.2.1        Take a volumetric flask of 1000.0ml with sufficient quantity of purified water.
5.2.3.2.2        Add 29.4g of sodium citrate dihydrate, mix it by using magnetic stirrer.
5.2.3.2.3        And finally dilute it with purified water to volume.
5.2.3.3  Borate solution:
5.2.3.3.1        Take a volumetric flask of 1000.0ml with sufficient quantity of purified water.
5.2.3.3.2        Add 19.1g of sodium borate, mix it by using magnetic stirrer.
5.2.3.3.3        And finally dilute it with purified water to volume.
5.2.3.4  9-Fluorenylmethyl chloroformate solution:
5.2.3.4.1        Prepare a solution in acetonitrile containing about 0.5mg of 9-Fluorenylmethyl chloroformate per ml.
5.2.3.4.2        Prepare this solution fresh just prior to use.
5.2.3.5  Mobile phase:
5.2.3.5.1        Prepare a filtered and degassed mixture of buffer solution, acetonitrile and methanol (70:25:5).
5.2.3.5.2        Make adjustments if necessary.
5.2.3.6  Standard stock solution:
5.2.3.6.1        Prepare a solution of USP Alendronate Sodium RS in Diluent having a known concentration of about 0.1mg per ml.
5.2.3.6.2        Calculate the concentration, Cs, of anhydrous alendronate sodium in this solution.
5.2.3.7  Standard preparation:
5.2.3.7.1        Take a 50-ml polypropylene screw-cap centrifuge tube and add 5.0ml of Borate solution and 5.0 ml of the standard stock solution.
5.2.3.7.2        Add 5.0ml of 9-Fluorenylmethyl chloroformate solution, shake it for 30 seconds.
5.2.3.7.3        Allow it to stand at room temperature for 25minutes.
5.2.3.7.4        Add 25.0ml of methylene chloride and shake vigorously for 1 minute.
5.2.3.7.5        Centrifuge for 5 to 10 minutes by using centrifugation machine.
5.2.3.7.6        Use a portion of the clear upper aqueous layer.
5.2.3.8  Reagent blank:
5.2.3.8.1        Take a 50-ml polypropylene screw-cap centrifuge tube and add 5.0ml of Borate solution and 5.0 ml of diluent.
5.2.3.8.2        Add 5.0ml of 9-Fluorenylmethyl chloroformate solution, shake it for 30 seconds.
5.2.3.8.3        Allow it to stand at room temperature for 25minutes.
5.2.3.8.4        Add 25.0ml of methylene chloride and shake vigorously for 1 minute.
5.2.3.8.5        Centrifuge for 5 to 10 minutes by using centrifugation machine.
5.2.3.8.6        Use a portion of the clear upper aqueous layer.
5.2.3.9  Assay stock preparation:
5.2.3.9.1        Accurately weigh 25.0mg of the alendronate sodium by using analytical weighing balance.
5.2.3.9.2        Take a 250.0ml of volumetric flask and transfer about 25.0mg of the alendronate sodium in sufficient quantity of diluent, mix it by using magnetic stirrer.
5.2.3.9.3        Dilute it with diluent up to the volume.
5.2.3.10    Assay preparation:
5.2.3.10.1    Take a 50-ml polypropylene screw-cap centrifuge tube and add 5.0ml of Borate solution and 5.0 ml of assay stock preparation.
5.2.3.10.2    Add 5.0ml of 9-Fluorenylmethyl chloroformate solution, shake it for 30 seconds.
5.2.3.10.3    Allow it to stand at room temperature for 25minutes.
5.2.3.10.4    Add 25.0ml of methylene chloride and shake vigorously for 1 minute.
5.2.3.10.5    Centrifuge for 5 to 10 minutes by using centrifugation machine.
5.2.3.10.6    Use a portion of the clear upper aqueous layer.
5.2.3.11    Chromatographic system:
5.2.3.11.1    Mode: Liquid chromatography.
5.2.3.11.2    Detector: 266nm.
5.2.3.11.3    Column: 4.1mm x 25cm; column that containing packing L21.
5.2.3.11.4    Column temperature: Maintain at about 35o.
5.2.3.11.5    Flow rate: 1.2ml/min.
5.2.3.11.6    Injection size: 10.0μL.
5.2.3.11.7    Chromatograph the standard preparation, and record the peak responses as directed for procedure:
5.2.3.11.7.1 Column efficiency: Not less than 1500 theoretical plates.
5.2.3.11.7.2    Tailing factor: Not more than 1.5.
5.2.3.11.7.3    Relative standard deviation for replicate injections: Not more than 2.0%.
5.2.4        Procedure:
5.2.4.1  Equilibrate the column and detector with mobile phase at specified flow rate until a constant signal is received.
5.2.4.2  Separately inject equal volumes (about 10.0μL) of the standard preparation, the assay preparation, and the reagent blank, or use an auto-sampler.
5.2.4.3  Begin the gradient program.
5.2.4.4  Record the chromatogram and measure the responses for the major peaks.
5.2.4.5  Analyze as directed in the monograph.
5.2.4.6  Calculate the quantity, in mg, of C4H14NNaO7P2 in the portion of alendronate sodium taken by formula:
DCs (rU/rS)
In which,
D= is the dilution factor for the assay stock preparation;
Cs= is as defined under the standard stock solution preparation;
rU and rS= are the peak area responses for alendronic acid obtained from the assay preparation and the standard preparation, respectively.
5.2.5        Limit:
5.2.5.1  90.0%-110.0%.
6.0  REVISION LOG:
Revision No.
Effective Date
Reason
00

New SOP

7.0  REFERENCES:
7.1  USP38NF33 Volume-3 Official Monograph/ Alendronate sodium: 2015, pp.: 2082-2083.
7.2  USP38NF33 Volume-1 Official Monograph/ Identification tests-General (Sodium test): 2015, pp.: 216-219.
7.3  USP38NF33 Volume-1 Official Monograph/ Chromatography: 2015, pp.: 424-434.
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 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
QCA
Quality control active ingredient
F
Format
Q.C
Quality control
ml
Milliliter
ppt
Precipitate
L
Length
g/L
Gram per liter
mg/ml
Milligram per milliliter
μg/ml
Microgram per milliliter
UV/VIS
Ultra violet/ Visible
μg
Microgram
mg
Milligram
RS
Reference standard
UV
Ultra violet
USP
United states pharmacopoeia
nm
Nanometer
mm
Millimeter
cm
Centimeter
μm
Micron
oC
Degree centigrade
ml/min.
Milliliter per minute
μL
Microliter
%
Percentage
NF
National formulary


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