Amifostine for Injection

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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1 DEFINITION

Amifostine for Injection is a sterile, crystalline substance suitable for parenteral use. It contains NLT 90.0% and NMT 110.0% of the labeled amount of amifostine (C₅H₁₅N₂O₃PS).

2 IDENTIFICATION

A. Spectroscopic Identification Tests 〈197〉, Infrared Spectroscopy: 197K

B. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.

3 ASSAY

3.1 Procedure

Buffer: 0.94 g/L of sodium 1-hexanesulfonate. Adjust with phosphoric acid to a pH of 3.0.

Mobile phase: Methanol and Buffer (7:18)

Standard solution: 3 mg/mL of USP Amifostine RS in water. [Note—Inject immediately after preparation, or refrigerate until use.]

Sample solution: 3 mg/mL of amifostine from Amifostine for Injection, in water. [Note—Inject immediately after preparation, or refrigerate until use.]

3.2 Chromatographic system

Mode: LC

Detector: UV 220 nm

Column: 4.6-mm × 25-cm; 5-μm packing L7

Autosampler temperature: 4°

Flow rate: 1.0 mL/min

Injection size: 10 μL

3.3 System suitability

Sample: Standard solution

3.4 Suitability requirements

Column efficiency: NLT 1000 theoretical plates

Tailing factor: NMT 2.0

Relative standard deviation: NMT 2.0%

3.5 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of C₅H₁₅N₂O₃PS in the portion of Amifostine for Injection taken:

Result = (r_u/r_s) × (C_s/C_u) × 100

r_u = peak responses from the Sample solution

r_s = peak responses from the Standard solution

C_s = concentration of USP Amifostine RS in the Standard solution (mg/mL)

C_u = nominal concentration of amifostine in the Sample solution (mg/mL)

Acceptance criteria: 90.0%–110.0%

4 PERFORMANCE TESTS

Uniformity of Dosage Units 〈905〉: Meets the requirements

5 IMPURITIES

Organic Impurities

5.1 Procedure 1

Mobile phase and Chromatographic system: Proceed as directed in the Assay.

Standard solution 1: 70 μg/mL of USP Amifostine Thiol RS in water

Standard solution 2: 15 μg/mL of sodium thiophosphate and 13 μg/mL of N,N-dimethylformamide in water. [Note-The retention times of sodium thiophosphate and N,N-dimethylformamide are about 2 min and about 3.6 min, respectively.]

Sample solution: 2.4 mg/mL of amifostine from Amifostine for Injection in water. [Note—Inject immediately after preparation.]

5.1.1 System suitability

Samples: Standard solution 1 and Standard solution 2

5.1.2 Suitability requirements

Relative standard deviation: NMT 10.0%, Standard solution 1; NMT 4.0%, Standard solution 2

5.1.3 Analysis

Samples: Standard solution 1, Standard solution 2, and Sample solution

Calculate the percentage of amifostine thiol in the portion of sample taken:

Result = (r_u/r_s) × (C_s/C_u) × (M_r1/M_r2) × 100

r_u = peak response of amifostine thiol from the Sample solution

r_s = peak response of amifostine thiol from Standard solution 1

C_s = concentration of USP Amifostine Thiol RS in Standard solution 1 (mg/mL)

C_u = concentration of amifostine in the Sample solution (mg/mL)

M_r1 = molecular weight of amifostine thiol, 134.24

M_r2 = molecular weight of amifostine thiol dihydrochloride, 207.17

Calculate the percentage of sodium thiophosphate or N,N-dimethylformamide in the portion of sample taken, if present:

Result = (r_u/r_s) × (C_s/C_u)× 100

r_u = peak response of sodium thiophosphate or N,N-dimethylformamide from the Sample solution

r_s = peak response of sodium thiophosphate or N,N-dimethylformamide from Standard solution 2

C_s = concentration of sodium thiophosphate or N,N-dimethylformamide in Standard solution 2 (mg/mL)

C_u = concentration of amifostine in the Sample solution (mg/mL)

Calculate the percentage of any other individual, unspecified impurity in the portion of sample taken:

Result = (r_U/r_T) × 100

r_U = peak response of each individual impurity in the Sample solution

r_T = total of all peak responses in the Sample solution

Acceptance criteria: NMT 0.1% of sodium thiophosphate; NMT 0.088% of N,N-dimethylformamide; NMT 0.1% of any other individual unspecified impurity

5.2 Procedure 2

Buffer: 0.4 g/L of sodium 1-octanesulfonate. Adjust with trifluoroacetic acid to a pH of 2.5 ± 0.1.

Mobile phase: Acetonitrile and Buffer (1:3)

Standard solution: 46 μg/mL of USP Amifostine Disulfide RS in water

Sample solution: Dilute a quantity of Amifostine for Injection in water to prepare a solution equivalent to 10 mg/mL. [Note-Inject immediately after preparation.]

5.2.1 Chromatographic system

Mode: LC

Detector: UV 247 nm

Column: 4.6-mm × 25-cm; 5-μm packing L1

Autosampler temperature: 4°

Flow rate: 1.0 mL/min

Injection size: 10 μL

5.2.2 System suitability

Sample: Standard solution

5.2.3 Suitability requirements

Tailing factor: NMT 2.5

Relative standard deviation: NMT 4.0%

5.2.4 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of amifostine disulfide in the portion of sample taken:

Result = (r_u/r_s) × (C_s/C_u) × (M_r1/M_r2) × 100

r_u = peak response of amifostine disulfide from the Sample solution

r_s = peak response of amifostine disulfide from the Standard solution

C_s = concentration of USP Amifostine Disulfide RS in the Standard solution (mg/mL)

C_u = concentration of amifostine in the Sample solution (mg/mL)

M_r1 = molecular weight of amifostine disulfide, 266.47

M_r2 = molecular weight of amifostine disulfide tetrahydrochloride, 412.31

Acceptance criteria: NMT 2.0% of total impurities, including amifostine thiol and amifostine disulfide

6 SPECIFIC TESTS

Constituted Solution: At the time of use, it meets the requirements for Injections and Implanted Drug Products 〈1〉, Specific Tests, Completeness and clarity of solutions. When constituted with 0.9% Sodium Chloride Injection, the solution must completely dissolve in 45 s.

Change to read:

X-Ray Powder Diffraction 〈941〉 (CN 1-May-2022) : Its X-ray diffraction pattern conforms to that of USP Amifostine RS, similarly determined.

Sterility Tests 〈71〉: It meets the requirements when tested as directed for Test for Sterility of the Product to be Examined, Membrane Filtration.

pH 〈791〉: 6.5–7.5, in a solution constituted as directed in the labeling

Water Determination, Method Ic〈921〉

Sample solution: To 100.0 mg of Amifostine for Injection, contained in a stoppered centrifuge tube, add 10.0 mL of a solution of N-ethylmaleimide in methanol (4 in 100), and sonicate for 15 min. Shake to disperse, and sonicate for an additional 15 min. Use 1.0 mL of the supernatant.

Acceptance criteria: 18.0%-22.0%

Particulate Matter in Injections 〈788〉: Meets the requirements for small-volume injections

Bacterial Endotoxins Test 〈85〉: Contains NMT 0.2 USP Endotoxin Unit/mg of amifostine

Other Requirements: Meets the requirements for Labeling 〈7〉, Labels and Labeling for Injectable Products.

7 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve as described in Packaging and Storage Requirements 〈659〉, Injection Packaging, and store at controlled room temperature.

USP Reference Standards 〈11〉

USP Amifostine RS

USP Amifostine Disulfide RS

1,3-Propanediamine, N,N-(dithiodi-2,1-ethanediyl)bis, tetrahydrochloride.

C₁₀H₃₀N₄S₂Cl₄ 412.32

USP Amifostine Thiol RS

Ethanethiol, 2-[(3-aminopropyl)amino]-, dihydrochloride.

C₅H₁₆N₂SCl₂ 207.17