Assay—
Buffer solution—
Dissolve 790 mg of ammonium carbonate in 1000 mL of water. Adjust, if necessary, with a solution of formic acid (1 in 10) or ammonium hydroxide (1 in 10) to a pH of 8.0 ± 0.05.
Diluent—
Prepare a mixture of methanol and water (65:35).
Mobile phase—
Prepare a filtered and degassed mixture of methanol and
Buffer solution (65:35). Make adjustments if necessary (see
System Suitability under
Chromatography 
621
).
Standard preparation—
Transfer about 25 mg of
USP Altretamine RS, accurately weighed, to a 50-mL volumetric flask. Dissolve in 35 mL of methanol, dilute with water to volume, and mix. Transfer 5.0 mL of this solution to a 50-mL volumetric flask, dilute with
Diluent to volume, and mix. This solution contains about 0.05 mg per mL.
Assay preparation—
Transfer about 25 mg of Altretamine, accurately weighed, to a 50-mL volumetric flask. Dissolve in 35 mL of methanol, dilute with water to volume, and mix. Transfer 5.0 mL of this solution to a 50-mL volumetric flask, dilute with Diluent to volume, and mix.
Chromatographic system—
The liquid chromatograph is equipped with a 227-nm detector and a 4.6-mm × 30-cm column that contains packing L1. The flow rate is about 2 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the tailing factor is not greater than 1.5, 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 C
9H
18N
6 in the portion of Altretamine taken by the formula:
500C(rU / rS),
in which
C is the concentration, in mg per mL, of
USP Altretamine RS in the
Standard preparation; and
rU and
rS are the peak responses obtained from the
Assay preparation and the
Standard preparation, respectively.
;)
