ALFACALCIDOL SOP



ALFACALCIDOL SOP

1.0  OBJECTIVE:
To lay down a procedure of analytical report for the active raw material of Alfacalcidol 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 crystals.
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%).
5.1.2.1.3        Fatty oil.
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, ethanol (96%) in test tube 2 and fatty oil in test tube 3 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 purified water is practically insoluble.
5.1.2.4.2        The sample in test tube 2 containing with ethanol (96%) is freely soluble.
5.1.2.4.3        The sample in test tube 3 containing with fatty oil is soluble.
5.2  Assay:
5.2.1        Apparatus:
5.2.1.1  Liquid chromatography 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  1.0mg of Alfacalcidol CRS.
5.2.2.2  Vial of alfacalcidol.
5.2.2.3  Ammonia R.
5.2.2.4  Acetonitrile.
5.2.2.5   Purified water.
5.2.3        Sample:
5.2.3.1  1.0mg.
5.2.4        Test solution:
5.2.4.1  Test solution:
5.2.4.1.1        Take 50.0ml of beaker and add 1.0mg of the substance to be examined in it.
5.2.4.1.2        Add 10.0ml of mobile phase and dissolve it by using magnetic stirrer without heating.
5.2.5        Reference solutions:
5.2.5.1  Reference solution (a):
5.2.5.1.1        Take 50.0ml of beaker and add 1.0mg of Alfacalcidol CRS in it.
5.2.5.1.2        Add 10.0ml of mobile phase and dissolve it by using magnetic stirrer without heating.
5.2.5.2  Reference solution (c):
5.2.5.2.1        In order to prepare pre-alfacalcidol in situ, take a beaker and add the contents of a vial of alfacalcidol for system suitability CRS (containing impurities A and B).
5.2.5.2.2        Add 25.0ml of the mobile phase and dissolve it by using magnetic stirrer.
5.2.5.2.3        Heat it in a water-bath at 80oC under a reflux condenser for 2 hours.
5.2.5.2.4        Cool it.
5.2.6        Column:
5.2.6.1  Size:
5.2.6.1.1        Length=0.25m,
5.2.6.1.2        θ=4.6mm.
5.2.6.2  Stationary phase:
5.2.6.2.1        End-capped octadecylsilyl silica gel for chromatography R (5μm).
5.2.7        Mobile phase:
5.2.7.1  Ammonia R, purified water R, acetonitrile R (1:200:800 V/V/V).
5.2.8        Flow rate:
5.2.8.1  2.6ml/min.
5.2.9        Detection:
5.2.9.1  Spectrophotometer at 265nm.
5.2.10    Injection:
5.2.10.1    100μL of the test solution and reference solution (a) & (c).
5.2.11    Run time:
5.2.11.1    Twice the retention time of alfacalcidol.
5.2.12    Identification of impurities:
5.2.12.1    Use the chromatogram supplied with alfacalcidol for system suitability CRS and the chromatogram obtained with reference solution (c) to identify the peaks due to impurities A and B.
5.2.13    Relative retention:
5.2.13.1    With reference to alfacalcidol (retention time=about 21 min): pre-alfacalcidol= about 0.88; impurity A= about 0.93; impurity B= about 1.1.
5.2.14    System suitability: Reference solution (c).
5.2.14.1    Repeatability: Maximum relative standard deviation of 1 per cent for the peak due to alfacalcidol after 6 injections.
5.2.15    Method of analysis:
5.2.15.1    Firstly prepare the test solution, reference solution and mobile phase according to the requirements.
5.2.15.2    The solutions must be free from solid particles.
5.2.15.3    Prepare the apparatus.
5.2.15.4    The mobile phase solvent mixtures must be deaerated prior to use either by boiling or by applying a partial vacuum to the solvent reservoir.
5.2.15.5    Equilibrate the column with the prescribed mobile phase, flow rate and at temperature specified until a suitable baseline is achieved.
5.2.15.6    Test solution and reference 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.
5.2.15.7    As the eluate leaves the column it enters a detector, where it is continuously monitored at the specified λ.
5.2.15.8    The electrical signal obtained from detector is amplified and routes to recorder which record the developed chromatogram.
5.2.15.9    Calculate the percentage content of Alfacalcidol (C27H44O2) taking into account the assigned content of alfacalcidol CRS and, if necessary, the peak due to pre-alfacalcidol.
5.2.16    Limit:
5.2.16.1    97.0% 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 I., Official Monograph /Alfacalcidol: 2015, pp. 87-88.
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 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
Q.C
Quality control
B.P
British pharmacopoeia
Vol
Volume
QCA
Quality control active ingredient
F
Format
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
Lambda
%
Percentage


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