Folic Acid

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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C₁₉H₁₉N₇O₆ 441.40

L-Glutamic acid, N-[4-[[(2-amino-1,4-dihydro-4-oxo-6-pteridinyl)methyl]amino]benzoyl]-;

N-[p-[[(2-Amino-4-hydroxy-6-pteridinyl)methyl]amino]benzoyl]-L-glutamic acid CAS RN^®: 59-30-3; UNII: 935E97BOY8.

1 DEFINITION

Folic Acid contains NLT 97.0% and NMT 102.0% of folic acid (C₁₉H₁₉N₇O₆), calculated on the anhydrous basis.

2 IDENTIFICATION

Change to read:

A. SPECTROSCOPIC IDENTIFICATION TESTS (197), Ultraviolet-Visible Spectroscopy: 197U (CN 1-MAY-2020)

Sample solution: 10 µg/mL in 0.1 N sodium hydroxide solution

Acceptance criteria: Meets the requirements. The ratio A₂₅₆/A₃₆₅ is 2.80-3.00.

3 ASSAY

3.1 PROCEDURE

[NOTE-Use low-actinic glassware throughout the following procedure.]

3 N phosphoric acid: 98 g/L of phosphoric acid in water

6 N ammonium hydroxide: Dilute 40 mL of ammonium hydroxide with water to 100 mL.

Mobile phase: Transfer 2.0 g of monobasic potassium phosphate into a 1000-mL volumetric flask, and dissolve in 650 mL of water. Add 15.0 mL of a solution of 0.5 M tetrabutylammonium hydroxide in methanol, 7.0 mL of 3 N phosphoric acid, and 270 mL of methanol. Cool to room temperature, adjust with 3 N phosphoric acid or 6 N ammonium hydroxide to a pH of 5.0, and dilute with water to volume. Recheck the pH before use.

Internal standard solution: 2 mg/mL of methylparaben in Mobile phase. Dissolve the methylparaben first with methanol (about 4% of the final volume), and dilute with Mobile phase to volume.

Standard stock solution: 1 mg/mL of USP Folic Acid RS in Mobile phase. Dissolve the folic acid with the aid of 10% ammonium hydroxide (about 1% of the final volume), and dilute with Mobile phase to volume.

Standard solution: Transfer 4.0 mL of Standard stock solution and 4.0 mL of Internal standard solution to a 50-mL volumetric flask, and dilute with Mobile phase to volume.

Sample stock solution: Transfer 100 mg of Folic Acid to a 100-mL volumetric flask, and dissolve in 40 mL of Mobile phase and 1 mL of 10% ammonium hydroxide. Dilute with Mobile phase to volume.

Sample solution: Transfer 4.0 mL of Sample stock solution and 4.0 mL of Internal standard solution to a 50-mL volumetric flask, and dilute with Mobile phase to volume.

3.2 Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 280 nm

Column: 4.0-mm x 25-cm; packing L1

Flow rate: 1.2 mL/min

Injection size: 10 µL

System suitability

Sample: Standard solution

Suitability requirements

Resolution: NLT 3.6 between methylparaben and folic acid

Relative standard deviation: NMT 2.0% for the ratios of the folic acid peak area to the internal standard peak area

3.3 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of folic acid (C₁₉H₁₉N₇O₆) in the sample taken:

Result = (R_U/R_S) x (C_S/C_U) x 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)

Acceptance criteria: 97.0%-102.0% on the anhydrous basis

4 IMPURITIES

4.1 RESIDUE ON IGNITION (281)

NMT 0.3%

4.2 RELATED COMPOUNDS

3 N phosphoric acid, 6 N ammonium hydroxide, Internal standard solution, Standard stock solution, Standard solution, and

Chromatographic system: Proceed as directed in the Assay.

Sample solution: Use the Sample stock solution, prepared as directed in the Assay.

Analysis

Sample: Sample solution

Allow the Sample solution to elute for NLT 2 times the retention time of folic acid. Record the chromatogram, and measure the areas of all the peaks.

Calculate the percentage of total secondary peaks in the portion of Folic Acid taken:

Result = (r_U/r_T) x 100

r_U = sum of the areas of all the peaks except that of the folic acid peak

r_T = sum of the areas of all the peaks

Acceptance criteria: NMT 2.0%

5 SPECIFIC TESTS

WATER DETERMINATION, Method (921)

Acceptance criteria: NMT 8.5%

Analysis : Proceed as directed in the chapter, except stir the methanol solvent before and during the addition of the test specimen and during the titration.

6 ADDITIONAL REQUIREMENTS

6.1 PACKAGING AND STORAGE

Preserve in well-closed, light-resistant containers.

6.2 USP REFERENCE STANDARDS (11)

USP Folic Acid RS