FOLIC ACID U.S.P. SOP

1.0  OBJECTIVE:
To lay down a procedure for the active raw material of the Folic acid 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 tests:
5.1.1        UV/VIS Spectrophotometer analysis:
5.1.1.1  Sample:
5.1.1.1.1        10μg/ml in 0.1N sodium hydroxide solution.
5.1.1.2  Method:
5.1.1.2.1        Take a beaker of 100.0ml and prepare 10μg/ml in 0.1N sodium hydroxide solution.
5.1.1.3  Spectral range:
5.1.1.3.1        256-365nm.
5.1.1.4  Operate the UV/VIS spectrophotometer according to the SOP No. BM/QCEO/SOP027-00.
5.1.1.5  Measure the absorbance of the resulting solution at the maxima and minima wavelength.
5.1.1.6  Note down values of absorbance in annexure-1.
5.1.1.7  Calculate the absorbances at the absorption maximum and minimum by using formula:
A256/A365
5.1.1.8  Observations:
5.1.1.8.1        Absorbance ratio at A256/A365: The absorbance ratio is A256/A365 = 2.80-3.00.
5.2  Assay:
5.2.1        Material:
5.2.1.1  98g of phosphoric acid.
5.2.1.2  40.0ml of ammonium hydroxide.
5.2.1.3  2.0g of monobasic potassium phosphate.
5.2.1.4  0.5M tetrabutylammonium hydroxide.
5.2.1.5  Methanol.
5.2.1.6  Methylparaben.
5.2.1.7  USP folic acid RS.
5.2.1.8  10% ammonium hydroxide.
5.2.1.9  Purified water.
5.2.2        3N Phosphoric acid:
5.2.2.1  Take 1000.0ml of beaker and add 98g of phosphoric acid in sufficient quantity of purified water.
5.2.2.2   Dissolve it by using magnetic stirrer operate according to SOP No. And finally make the volume up to 1000.0ml.
5.2.3        6N ammonium hydroxide:
5.2.3.1  Take 100.0ml of beaker and add 40.0ml of ammonium hydroxide in sufficient quantity of purified water.
5.2.3.2   Dissolve it by using magnetic stirrer operate according to SOP No.. And finally make the volume up to 100.0ml.
5.2.4        Mobile phase:
5.2.4.1  Take 1000.0ml of volumetric flask and transfer 2.0g of monobasic potassium phosphate.
5.2.4.2  Dissolve it in 650.0ml of purified water and mix it by using magnetic stirrer operate according to SOP.
5.2.4.3  Add 15.0ml of a solution of 0.5M tetrabutylammonium hydroxide in methanol, 7.0ml of 3N phosphoric acid, and 270.0m of methanol.
5.2.4.4  Cool it room temperature.
5.2.4.5  Adjust with 3N phosphoric acid or 6N ammonium hydroxide to a pH of 5.0.
5.2.4.6  Dilute it with purified water to volume.
5.2.4.7  Recheck the pH before use.
5.2.5        Internal standard solution:
5.2.5.1  Take a beaker of 100.0ml and prepare 2.0mg/ml of Methylparaben in mobile phase.
5.2.5.2  Dissolve the Methylparaben first with methanol (about 4% of the final volume), and dilute with mobile phase to the volume.
5.2.6        Standard stock solution:
5.2.6.1  Take a beaker of 100.0ml and prepare 1.0mg/ml of USP folic acid RS in mobile phase.
5.2.6.2  Dissolve the folic acid with the aid of 10% ammonium hydroxide (about 1% of the final volume), and dilute with mobile phase to the volume.
5.2.7        Standard solution:
5.2.7.1  Take a 50.0ml of volumetric flask and add 4.0ml of standard stock solution and 4.0ml of internal standard solution in sufficient quantity of mobile phase.
5.2.7.2  Dissolve it by using magnetic stirrer operate according to SOP
5.2.7.3  Dilute it with mobile phase up to the volume.
5.2.8        Sample stock solution:
5.2.8.1  Take a 100.0ml of volumetric flask and transfer 100.0mg of folic acid in it.
5.2.8.2  Dissolve it in 40.0ml of mobile phase and 1.0ml of 10% ammonium hydroxide, by using magnetic stirrer operate according to SOP.
5.2.8.3  Dilute it with mobile phase to the volume.
5.2.9        Sample solution:
5.2.9.1  Take a 50.0ml of volumetric flask and add 4.0ml of sample stock solution and 4.0ml of internal standard solution in sufficient quantity of mobile phase.
5.2.9.2  Dissolve it by using magnetic stirrer operate according to SOP.
5.2.9.3  Dilute it with mobile phase up to the volume.
5.2.10    Chromatographic system:
5.2.10.1    Mode: Liquid chromatography.
5.2.10.2    Detector: UV 280nm.
5.2.10.3    Column: 4.0mm x 25cm; packing L1.
5.2.10.4    Flow rate: 1.2ml/min.
5.2.10.5    Injection size: 10µL.
5.2.11    System suitability:
5.2.11.1    Samples: Standard solution.
5.2.12    Suitability requirements:
5.2.12.1    Resolution: NLT 3.6 between Methylparaben and folic acid.
5.2.12.2    Relative standard deviation: NMT 2.0% for the ratios of the folic acid peak area to the internal standard peak area.
5.2.13    Procedure:
5.2.13.1    Equilibrate the column and detector with mobile phase at specified flow rate until a constant signal is received.
5.2.13.2    Separately inject equal volumes (about 10μL) of the standard solution and the sample solution into the chromatograph.
5.2.13.3    Record the chromatograms, and measure the responses for the peaks. Note down in annexure-2.
5.2.13.4    Calculate the percentage of folic acid (C19H19N7O6) in the sample taken:
Result = (RU/RS) × (CS/CU) × 100
RU= internal standard ratio (peak response of folic acid/peak response of the internal standard) from the sample solution.
RS= internal standard ratio (peak response of folic acid/peak response of the internal standard) from the standard solution.
CS= concentration of USP Folic acid RS in the standard stock solution (mg/ml).
CU= concentration of folic acid in the sample stock solution (mg/ml).
5.2.14    Acceptance criteria:
5.2.14.1    97.0%-102.0% on the anhydrous basis.
6.0  REVISION LOG:
Revision No.
Effective Date
Reason
00

New SOP

7.0  REFERENCES:
7.1  USP38NF33 Volume-4 Official Monograph/ Folic acid: 2015, pp.: 3601-3602.
7.2  The British Pharmacopoeia. Vol V., Official Monograph / Ultraviolet and Visible Absorption Spectrophotometry: 2015, Appendix: IIB pp. 169-170.
8.0  ANNEXURES:
Annexure 1: Observations and calculations of UV/VIS spectrophotometer analysis.
Annexure 2: Observations and calculations of HPLC method.


Annexure: 1
Observations and calculations of UV/VIS spectrophotometer analysis.
UV/VIS spectrophotometer
Model: _____________________________                             Date: _________________
OBSERVATIONS:
Thickness of cell:

Spectral range:

Sample:

Other reagent used:


No. of obs.
Concentration
(c)
Wavelength
(λ)
Absorbance
(a)


256



365

CALCULATIONS:
The absorbance ratio is A256/A365 =








Results: _______________
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 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
RM
Raw-Material 
%
Percentage
B.P
British pharmacopoeia
ml
Milliliter
mg
Milligram
g
Grams
M
Molar
N
Normal
Vol
Volume
QCA
Quality control active ingredient
F
Format
RS
Reference standard
μm
Micron/ micrometer
g/L
Gram per liter
m
Meter
θ
Theta
mm
Millimeter
ml/min
Milliliter per minute
μg/ml
Microgram per milliliter
nm
Nanometer
μL
Microliter
Min
Minute
λ
Lambda
UV/VIS Spectrophotometer
Ultraviolet/Visible spectrophotometer


No comments:

Post a Comment