Amifostine

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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C₅H₁₅N₂O₃PS.3H₂O 268.27

Ethanethiol, 2-[(3-aminopropyl)amino]-, dihydrogen phosphate (ester), trihydrate;

S-[2-(3-Aminopropyl)amino]ethyl]dihydrogen phosphorothioate, trihydrate CAS RN®: 112901-68-5; UNII: M487QF2F4V.

1 DEFINITION

Amifostine contains NLT 78.0% and NMT 82.0% of C₅H₁₅N₂O₃PS, calculated on the as-is basis.

2 IDENTIFICATION

Change to read:

A. Spectroscopic Identification Tests 〈197〉, Infrared Spectroscopy: 197K (CN 1-May-2020)

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.]

Sample solution: 3 mg/mL of Amifostine in water.

[Note-Inject immediately after preparation.]

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

Tailing factor: NMT 2.0

Column efficiency: NLT 1000 theoretical plates

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 taken:

Result = (r_u/r_s) × (C_s/C_u) × 100

r_u = peak response from the Sample solution

r_s = peak response from the Standard solution

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

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

Acceptance criteria: 78.0% - 82.0% on the as-is basis

4 IMPURITIES

Organic Impurities

4.1 Procedure

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

Standard solution: 70 μg/mL of USP Amifostine Thiol RS and 16 μg/mL of USP Amifostine RS in water. [Note-Inject immediately after preparation.]

System suitability solution: Use the Standard solution as described in the Assay. [Note-Inject immediately after preparation.]

Sample solution: 15 mg/mL of Amifostine in water.

[Note-Inject immediately after preparation.]

4.2 System suitability

Samples: Standard solution and System suitability solution

4.3 Suitability requirements

Column efficiency: NLT 1000 theoretical plates, System suitability solution

Tailing factor: NMT 2.0, System suitability solution

Relative standard deviation: NMT 15.0%, Standard solution

4.4 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of amifostine thiol in the portion of Amifostine 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 the Standard solution

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

C_u = nominal 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 any other individual impurity in the portion of Amifostine taken:

Result = (r_u/r_s) × (C_s/C_u)× 100

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

r_s = peak response of amifostine in the Standard solution

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

C_u = concentration of the Sample solution (μg/mL)

4.5 Acceptance criteria

Amifostine thiol: NMT 0.3%

Any individual impurity, excluding amifostine thiol: NMT 0.1%

Total impurities including amifostine thiol: NMT 0.3%

5 SPECIFIC TESTS

pH 〈791〉: 6.5–7.5, in a solution (5 in 100)

Water Determination, Method Ic〈921〉

Sample solution: To 100.0 mg of Amifostine, contained in a stoppered centrifuge tube, add 10.0 mL of the 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: 19.2%–21.2%

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in tight, light-resistant containers, and store in a refrigerator.

USP Reference Standards 〈11〉

USP Amifostine RS

USP Amifostine Thiol RS

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

C₅H₁₆N₂SCl₂ 207.17