FOLIC ACID U.S.P. SOP

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/QCE_O/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 = (R_U/R_S) × (C_S/C_U) × 100

R_U= internal standard ratio (peak response of folic acid/peak response of the internal standard) from the sample solution.

R_S= internal standard ratio (peak response of folic acid/peak response of the internal standard) from the standard solution.

C_S= concentration of USP Folic acid RS in the standard stock solution (mg/ml).

C_U= 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