Related compounds—
Mobile phase, Blank solution, and Test solution—
Prepare as directed in the test for Related compounds under Amifostine.
Standard thiol solution—
Transfer about 40.1 mg of
USP Amifostine Thiol RS, accurately weighed, to a 50-mL volumetric flask. Dissolve in and dilute with water to volume, and mix.
[NOTE—Inject immediately after preparation, or refrigerate until use. The solution is stable for 48 hours if maintained at about 5
.
]
Standard disulfide solution—
Transfer about 18.6 mg of
USP Amifostine Disulfide RS, accurately weighed, to a 100-mL volumetric flask. Dissolve in and dilute with water to volume, and mix.
[NOTE—Inject immediately after preparation, or refrigerate until use. The solution is stable for 48 hours if maintained at about 5
.
]
System suitability solution—
Dissolve about 5.0 mg of
USP Amifostine RS, accurately weighed, in 1 mL of
Standard thiol solution, and mix.
[NOTE—Inject immediately after preparation, or refrigerate until use. The solution is stable for 12 hours if maintained at about 5
.
]
Chromatographic system (see Chromatography 
621
)—
The liquid chromatograph is equipped with a detector capable of recording at both 220 nm and 247 nm, and a 4.6-mm × 25-cm column that contains packing L1. The column temperature is maintained at 30
, and the temperature of solutions to be injected is maintained at between 2
and 8
. The flow rate is about 1.0 mL per minute. Chromatograph the
System suitability solution, the
Standard disulfide solution, and the
Standard thiol solution, and record the peak responses as directed for
Procedure: the column efficiency calculated for the amifostine thiol peak is not less than 2300 theoretical plates; the tailing factor is not more than 4.0; the capacity factor,
k¢, is more than 0.5; and the relative standard deviation for replicate injections is not more than 4.0%. The column efficiency calculated for the amifostine disulfide peak is not less than 2000 theoretical plates; the tailing factor is not more than 4.5; the capacity factor,
k¢, is more than 2.2; and the relative standard deviation for replicate injections is not more than 4.0%.
Procedure—
Separately inject equal volumes (about 10 µL) of the
Standard thiol solution, the
Standard disulfide solution, the
Test solution, and the
Blank solution into the chromatograph, record the chromatograms, and measure the responses of all the peaks except the peaks corresponding to those obtained from the
Blank solution. Calculate the percentage of amifostine thiol in the portion of Amifostine for Injection taken by the formula:
(134.24/207.17)1000(C/W)(rU / rS),
in which 134.24 and 207.17 are the molecular weights of amifostine thiol and amifostine thiol dihydrochloride, respectively;
C is the concentration, in mg per mL, of amifostine thiol dihydrochloride in the
Standard thiol solution; W is the weight, in mg, of amifostine taken to prepare the
Test solution; and
rU and
rS are the amifostine thiol peak responses recorded at 220 nm, obtained from the
Test solution and the
Standard thiol solution, respectively. Calculate the percentage of amifostine disulfide in the portion of Amifostine for Injection taken by the formula:
(266.47/412.31)(10C)(rU / rS),
in which 266.47 and 412.31 are the molecular weights of amifostine disulfide and amifostine disulfide tetrahydrochloride, respectively;
C is the concentration, in mg per mL, of
USP Amifostine Disulfide RS in the
Standard disulfide solution; and
rU and
rS are the peak responses recorded at 247 nm, obtained from the
Test solution and the
Standard disulfide solution, respectively: not more than 2.0% of total impurities, including amifostine thiol and amifostine disulfide, is found. Calculate the percentage of each of the other impurities in the portion of Amifostine for Injection taken by the formula:
100(ri / rA),
in which
ri and
rA are the peak responses for each impurity and amifostine, respectively, obtained from the
Test solution: not more than 0.1% of any individual impurity except amifostine thiol is found.
Assay—
Mobile phase, Standard preparation, and Chromatographic system—
Prepare as directed in the
Assay under
Amifostine.
Assay preparation—
Transfer an accurately weighed quantity of Amifostine for Injection, equivalent to about 500 mg of amifostine, to a 50-mL volumetric flask, dissolve in 10 mL of water, dilute with water to volume, and mix. Transfer 6.0 mL of this solution to a 25-mL volumetric flask, add 6.5 mL of water, dilute with methanol to volume, and mix.
Procedure—
Proceed as directed in the
Assay under
Amifostine. Calculate the quantity, in mg, of amifostine (C
5H
15N
2O
3PS) in the portion of Amifostine for Injection taken by the formula:
208.33C(rU / rS),
in which
C is the concentration, in mg per mL, of
USP Amifostine RS in the
Standard preparation; and
rU and
rS are the peak responses obtained from the
Assay preparation and the
Standard preparation, respectively.
