A: Infrared Absorption 197K.

B: The retention time of the major peak in the chromatogram of the Assay preparation corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay.

Mobile phase— Dissolve 1.0 mL of nonafluorobutane sulfonic acid in 1200 mL of HPLC grade water, add 400 µL of trifluoroacetic acid, and adjust with triethylamine to a pH of 2.5. Prepare a degassed mixture of this solution and acetonitrile (68:32).

Blank solution— Use water.

Standard thiol solution— Transfer about 12.4 mg of USP Amifostine Thiol 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.]

Test solution— Transfer about 50 mg of Amifostine, 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 220-nm detector and a 4.6-mm × 25-cm column that contains packing L1. The column temperature is maintained at 30, and the temperature of the solutions to be injected is maintained at 2 to 8. The flow rate is about 1.0 mL per minute. Chromatograph the System suitability solution and the Standard thiol solution, and record the peak responses as directed for Procedure: the resolution, R, between amifostine and amifostine thiol is not less than 2.0; 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%.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard thiol solution, the Test solution, and the Blank solution into the chromatograph, record the chromatograms, and measure the responses of all the peaks, excluding the peaks corresponding to those obtained from the Blank solution. Calculate the percentage of amifostine thiol in the portion of Amifostine 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 obtained from the Test solution and the Standard thiol solution, respectively. Calculate the percentage of each of the other impurities in the portion of Amifostine 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, excluding amifostine thiol, is found; and not more than 0.3% of total impurities, including amifostine thiol, is found.

Mobile phase— Dissolve 1.0 mL of nonafluorobutane sulfonic acid in 1200 mL of HPLC grade water. Prepare a degassed mixture of this solution and acetonitrile (90:10).

Standard preparation— Transfer about 30 mg of USP Amifostine RS, accurately weighed, to a 10-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.]

Assay preparation— Transfer about 30 mg of Amifostine, accurately weighed, to a 10-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 220-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L1. The column temperature is maintained at 30, and the temperature of the solutions to be injected is maintained at 2 – 8. The flow rate is about 1.0 mL per minute. Chromatograph the Standard preparation and the Assay preparation, and record the peak responses as directed for Procedure: the column efficiency is not less than 7500 theoretical plates; the tailing factor is not more than 2; and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of C5H15N2O3PS in the portion of Amifostine 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.