Amifostine for Injection
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).
IDENTIFICATION
• B.
The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.
ASSAY
• 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. ]
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
System suitability
Sample:
Standard solution
Suitability requirements
Column efficiency:
NLT 1000 theoretical plates
Tailing factor:
NMT 2.0
Relative standard deviation:
NMT 2.0%
Analysis
Samples:
Standard solution and Sample solution
Calculate the percentage of C5H15N2O3PS in the portion of Amifostine for Injection taken:
Result = (rU/rS) × (CS/CU) × 100
| rU | = | = peak responses from the Sample solution |
| rS | = | = peak responses from the Standard solution |
| CS | = | = concentration of USP Amifostine RS in the Standard solution (mg/mL) |
| CU | = | = nominal concentration of amifostine in the Sample solution (mg/mL) |
Acceptance criteria:
90.0%–110.0%
PERFORMANCE TESTS
• Uniformity of Dosage Units 
905
:
Meets the requirements
905:
Meets the requirements
IMPURITIES
Organic Impurities
• 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. ]
System suitability
Samples:
Standard solution 1 and Standard solution 2
Suitability requirements
Relative standard deviation:
NMT 10.0%, Standard solution 1; NMT 4.0%, Standard solution 2
Analysis
Samples:
Standard solution 1, Standard solution 2, and Sample solution
Calculate the percentage of amifostine thiol in the portion of sample taken:
Result = (rU/rS) × (CS/CU) × (Mr1/Mr2) × 100
| rU | = | = peak response of amifostine thiol from the Sample solution |
| rS | = | = peak response of amifostine thiol from Standard solution 1 |
| CS | = | = concentration of USP Amifostine Thiol RS in Standard solution 1 (mg/mL) |
| CU | = | = concentration of amifostine in the Sample solution (mg/mL) |
| Mr1 | = | = molecular weight of amifostine thiol, 134.24 |
| Mr2 | = | = 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 = (rU/rS) × (CS/CU) × 100
| rU | = | = peak response of sodium thiophosphate or N,N-dimethylformamide from the Sample solution |
| rS | = | = peak response of sodium thiophosphate or N,N-dimethylformamide from Standard solution 2 |
| CS | = | = concentration of sodium thiophosphate or N,N-dimethylformamide in Standard solution 2 (mg/mL) |
| CU | = | = 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 = (rU/rT) × 100
| rU | = | = peak response of each individual impurity in the Sample solution |
| rT | = | = 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
• 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. ]
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
System suitability
Sample:
Standard solution
Suitability requirements
Tailing factor:
NMT 2.5
Relative standard deviation:
NMT 4.0%
Analysis
Samples:
Standard solution and Sample solution
Calculate the percentage of amifostine disulfide in the portion of sample taken:
Result = (rU/rS) × (CS/CU) × (Mr1/Mr2) × 100
| rU | = | = peak response of amifostine disulfide from the Sample solution |
| rS | = | = peak response of amifostine disulfide from the Standard solution |
| CS | = | = concentration of USP Amifostine Disulfide RS in the Standard solution (mg/mL) |
| CU | = | = concentration of amifostine in the Sample solution (mg/mL) |
| Mr1 | = | = molecular weight of amifostine disulfide, 266.47 |
| Mr2 | = | = molecular weight of amifostine disulfide tetrahydrochloride, 412.31 |
Acceptance criteria:
NMT 2.0% of total impurities, including amifostine thiol and amifostine disulfide
SPECIFIC TESTS
• Constituted Solution:
At the time of use, it meets the requirements for Injections 1, Constituted Solutions. When constituted with 0.9% Sodium Chloride Injection, the solution must completely dissolve in 45 s.
1, Constituted Solutions. When constituted with 0.9% Sodium Chloride Injection, the solution must completely dissolve in 45 s.
• X-Ray Diffraction 941:
Its X-ray diffraction pattern conforms to that of USP Amifostine RS, similarly determined.
941:
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.
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
791:
6.5–7.5, in a solution constituted as directed in the labeling
• Water Determination, Method Ic 921
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
788:
Meets the requirements for small-volume injections
• Bacterial Endotoxins Test 85:
Contains NMT 0.2 USP Endotoxin Unit/mg of amifostine
85:
Contains NMT 0.2 USP Endotoxin Unit/mg of amifostine
• Other Requirements:
Meets the requirements for Injections 1, Labeling.
1, Labeling.
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Preserve in tight Containers for Sterile Solids as described under Injections 1, and store at controlled room temperature.
1, and store at controlled room temperature.
• USP Reference Standards 11
11
USP Amifostine RS 
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USP Amifostine Disulfide RS
1,3-Propanediamine, N,N-(dithiodi-2,1-ethanediyl)bis, tetrahydrochloride.
C10H30N4S2Cl4 412.32
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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
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Ethanethiol, 2-[(3-aminopropyl)amino]-, dihydrochloride.
C5H16N2SCl2 207.17
USP Endotoxin RS
Auxiliary Information—
Please check for your question in the FAQs before contacting USP.
| Topic/Question | Contact | Expert Committee |
|---|---|---|
| Monograph | Sujatha Ramakrishna, Ph.D.
Senior Scientific Liaison 1-301-816-8349 |
(SM22010) Monographs - Small Molecules 2 |
| 71 |
Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison 1-301-816-8339 |
(GCM2010) General Chapters - Microbiology |
| 85 |
Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison 1-301-816-8339 |
(GCM2010) General Chapters - Microbiology |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
USP35–NF30 Page 2157
Pharmacopeial Forum: Volume No. 36(1) Page 63