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
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.
•  X-Ray Diffraction 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.
•  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 Injections 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.
•  USP Reference Standards 11
USP Amifostine RS Click to View Structure
USP Amifostine Disulfide RS Click to View Structure
1,3-Propanediamine, N,N-(dithiodi-2,1-ethanediyl)bis, tetrahydrochloride.
    C10H30N4S2Cl4         412.32
USP Amifostine Thiol RS Click to View Structure
Ethanethiol, 2-[(3-aminopropyl)amino]-, dihydrochloride.
    C5H16N2SCl2        207.17
USP Endotoxin RS
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