L-Glutathione Reduced CAS 70-18-8 Assay 98.0~101.0% (Titration)

Glutathione
C10H17N3O6S 307.32
Pentanoic acid, 2-amino-5-[(R)-1-(carboxymethylamino)-3-mercapto-1-oxopropan-2-ylamino]-5-oxo, (S);
N-(N-l--Glutamyl-l-cysteinyl)glycine [70-18-8].
DEFINITION
Glutathione contains NLT 98.0% and NMT 101.0% of C10H17N3O6S, as glutathione, calculated on the dried basis.
IDENTIFICATION
• A. Infrared Absorption <197K>
• B. Optical Rotation, Specific Rotation <781S>
Sample solution: 40 mg/mL in water
Acceptance criteria: 15.5° to 17.5°, at 20℃
ASSAY
• Procedure
Sample: 500 mg of glutathione previously dried
Titrimetric system
(See Titrimetry <541>.)
Mode: Direct titration
Titrant: 0.1 N iodine VS
Endpoint detection: Visual
Blank: 50 mL of metaphosphoric acid (1 in 50)
Analysis: Dissolve the Sample in 50 mL of metaphosphoric acid (1 in 50) and titrate with the Titrant.
Calculate the percentage of glutathione (C10H17N3O6S) in the portion of Glutathione taken:
Result = [(V B) × N × F × 100]/W
V = titrant volume of the Sample (mL)
B = titrant volume of the Blank (mL)
N = titrant normality (mEq/mL)
F = equivalency factor, 307.32 mg/mEq
W = weight of the Sample (mg)
Acceptance criteria: 98.0%~101.0% on the dried basis
IMPURITIES
Inorganic Impurities
• Ammonium
Standard solution: 10 µg of ammonium from a diluted ammonium chloride solution
Sample solution: 50 mg of Glutathione
Analysis: Transfer the Sample solution and the Standard solution to separate 25-mL jars fitted with caps, and dissolve in 1 mL of water. Add 0.30 g of magnesium oxide. Close immediately after placing a piece of silver manganese paper 5-mm square, wetted with a few drops of water, under the caps. Swirl, avoiding projections of liquid, and allow to stand at 40 for 30 min.
Acceptance criteria: If the silver manganese paper shows a gray color, it is not more intense than the standard (NMT 200 ppm).
• Arsenic <211>: NMT 2 ppm
• Chloride and Sulfate, Chloride <221>: Dissolve 0.7 g with water to make 15 mL. The solution shows no more chloride than corresponds to 0.20 mL of 0.020 N hydrochloric acid (NMT 200 ppm).
• Chloride and Sulfate, Sulfate 221: Dissolve 0.8 g in water to make 15 mL. The solution shows no more sulfate than corresponds to 0.25 mL of 0.020 N sulfuric acid (NMT 300 ppm).
• Heavy Metals, Method I <231>: NMT 10 ppm
• Iron <241>: NMT 10 ppm
• Residue on Ignition <281>: NMT 0.1%
Organic Impurities
• Procedure
Mobile phase: 6.8 g/L of potassium dihydrogen phosphate with 2.02 g/L of sodium 1-heptane sulfonate. Adjust with phosphoric acid to a pH of 3.0. Mix 970 mL of this solution with 30 mL of methanol.
System suitability solution: 0.1 mg/mL of USP l-Phenylalanine RS, 0.5 mg/mL of USP Glutathione RS, and 0.5 mg/mL of USP Ascorbic acid RS in Mobile phase
Standard solution: 0.01 mg/mL of USP Glutathione RS in Mobile phase. [Note—This solution has a concentration equivalent to 2.0% of that of the Sample solution. ]
Sample solution: 50 mg of glutathione in 100 mL of Mobile phase. [Note—Allow the solution to stand for 5 min before use. ]
Chromatographic system
(See Chromatography <621>, System Suitability.)
Mode: LC
Detector: UV 210 nm
Column: 4.6-mm × 15-cm; 5-µm packing L1
Column temperature: 30
Flow rate: Adjust so that the retention time of glutathione is about 5 min.
Injection size: 10 µL
System suitability
Sample: System suitability solution
Suitability requirements
Resolution: NLT 5.0 between the ascorbic acid and glutathione peaks; and NLT 5.0 between the glutathione and l-phenylalanine peaks
Relative standard deviation: NMT 1.5% for replicate injections
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of any impurity in the portion of Glutathione taken:
Result = (rU/rS) × (CS/CU) × 100
rU = peak response of any peak from the Sample solution other than glutathione
rS = peak response of the glutathione peak from the Standard solution
CS = concentration of USP Glutathione RS in the Standard solution (mg/mL)
CU = concentration of Glutathione in the Sample solution (mg/mL)
Acceptance criteria
Individual impurity: NMT 1.5% for the impurity with the relative retention time of about 4
Total impurities: NMT 2.0%
SPECIFIC TESTS
• Clarity and Color of Solution
Sample solution: 0.1 g/mL in water
Analysis: Using identical tubes of colorless, transparent, neutral glass with a flat base and an internal diameter of 15-25 mm, compare the liquid to be examined with water, the depth of the layer being 40 mm. Compare the colors in diffused daylight, viewing vertically against a white background.
Acceptance criteria: The solution is clear and colorless.
• Loss on Drying <731>: NMT 0.5%, 105℃ for 3 h
ADDITIONAL REQUIREMENTS
• Packaging and Storage: Preserve in tight containers.
• USP Reference Standards <11>
USP Ascorbic Acid RS
USP Glutathione RS
USP l-Phenylalanine RS