Assay—
Mobile phase—
Prepare a suitable filtered and degassed mixture of water and acetonitrile (9:1). Make adjustments if necessary (see
System Suitability under
Chromatography 
621
).
Standard preparation—
Dissolve an accurately weighed quantity of
USP Thiotepa RS in
Mobile phase to obtain a solution having a known concentration of about 1.5 mg per mL.
Assay preparation—
Transfer about 75 mg of Thiotepa, accurately weighed, to a 50-mL volumetric flask, dissolve in
Mobile phase, dilute with
Mobile phase to volume, and mix.
Resolution solution—
Transfer about 10 mg of
USP Thiotepa RS to a 4-mL vial, add 2 mL of methanol, and mix. Add 50 µL of 0.1% phosphoric acid solution. Place a cap on the vial, and heat at 65
for 50 seconds. Cool the solution, add 1 mL of methanol, and mix.
Chromatographic system
(see
Chromatography 621)—The liquid chromatograph is equipped with a 215-nm detector and a 4-mm × 15-cm column that contains packing L1. The flow rate is about 0.8 mL per minute. Chromatograph the
Resolution solution, and record the peak responses as directed for
Procedure: the relative retention times are about 1.25 for methoxythiotepa and 1.0 for thiotepa, and the resolution,
R, between the methoxythiotepa peak and the thiotepa peak is not less than 3.0. Chromatograph the
Standard preparation, and record the responses as directed for
Procedure: the tailing factor for the thiotepa peak is not more than 1.8, the column efficiency is not less than 2600 theoretical plates, 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
6H
12N
3PS in the portion of Thiotepa taken by the formula:
50C(rU / rS),
in which
C is the concentration, in mg per mL, of
USP Thiotepa RS in the
Standard preparation, and
rU and
rS are the thiotepa peak responses obtained from the
Assay preparation and the
Standard preparation, respectively.
;)
