Assay—
Diluent—
Prepare a mixture of 500 mL of water, 450 mL of methanol, and 50 mL of acetonitrile.
Mobile phase—
Dissolve 6 g of sodium 1-heptanesulfonate in 500 mL of water, add 12 mL of glacial acetic acid, 2.0 mL of triethylamine, 450 mL of methanol, and 50 mL of acetonitrile, and mix. Pass through a suitable filter of 0.5 µm or finer porosity. Make adjustments if necessary (see
System Suitability under
Chromatography 
621
).
Standard preparation—
Dissolve an accurately weighed quantity of
USP Amprolium RS in
Diluent to obtain a solution having a known concentration of about 0.5 mg per mL.
Assay preparation—
Transfer about 50 mg of Amprolium, accurately weighed, to a 100-mL volumetric flask, add Diluent to volume, and mix.
Resolution solution—
Prepare a solution in
Diluent containing about 0.5 mg of
USP Amprolium RS and 0.2 mg of 2-picoline per mL.
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 25-cm column containing packing L13. The flow rate is about 0.6 mL per minute. Chromatograph the
Resolution solution, and record the responses as directed for
Procedure: the resolution,
R, between amprolium and 2-picoline is not less than 7; the column efficiency determined from the amprolium peak is not less than 6500 theoretical plates; the tailing factor for the analyte peak is not more than 2.3; and the relative standard deviation of the amprolium responses for replicate injections is not more than 1.0%.
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 areas for the major peaks. Calculate the quantity, in mg, of amprolium (C
14H
19ClN
4·HCl) in the portion of Amprolium taken by the formula:
100C(rU / rS),
in which
C is the concentration, in mg per mL, of
USP Amprolium RS in the
Standard preparation; and
rU and
rS are the amprolium peak responses obtained from the
Assay preparation and the
Standard preparation, respectively.
;)
for 3 hours: it loses not more than 1.0% of its weight.