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:
CALCULATIONS:
The
absorbance ratio is A256/A365 =
Results:
_______________
Remarks:
______________________________________________________________
|
Annexure: 2
Observations
and calculations of HPLC method
Analysis
on HPLC
Instrument:
___________________
Date: _________________
Model: ___________________
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)=
_____________
2.
Retention volume:
Flow rate= _______________ml/min.
3.
Retention factor:
Retention time of unretained peak (tm)=
_____________
4.
Separation factor (α):
5.
Resolution:
Retention time of unretained peak (tm)=
_____________
6.
Efficiency:
7.
Height equivalent to a theoretical plate (HETP):
Length of column = ________________________
8.
Symmetry factor (tailing factor):
9.
Response factor & Relative response factor:
Conc. (mg/ml)= ___________________
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
|