Mobile phase, Blank solution, and System suitability 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.]

Test solution— Transfer about 50 mg of Amifostine for Injection, accurately weighed, to a 1-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.]

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: 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: 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: 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.

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 (C5H15N2O3PS) in the portion of Amifostine for Injection taken by the formula: 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.