Chloride 
221
—
Boil 2.0 g with 60 mL of water for 5 minutes, cool, and filter. To a 30-mL portion of the filtrate add 1 mL of 2 N nitric acid and 1 mL of
silver nitrate TS: the filtrate shows no more chloride than corresponds to 0.10 mL of 0.020 N hydrochloric acid (0.007%).
Assay—
Acetate buffer—
Transfer 2.72 g of sodium acetate to a 1000-mL beaker, and dissolve in about 700 mL of water. Adjust with glacial acetic acid to a pH of 4.1. Filter through a 0.5-µm filter, dilute with filtered water to 1000 mL, and mix.
Mobile phase—
Prepare a filtered and degassed mixture of acetonitrile with 560 mL of
Acetate buffer (440:560). Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Internal standard solution—
Dissolve 300 mg of desoxycorticosterone acetate in 200 mL of acetonitrile, and mix.
Standard preparation—
Dissolve an accurately weighed quantity of
USP Phenylbutazone RS in acetonitrile, with the aid of sonication, and dilute quantitatively with acetonitrile to obtain a solution having a concentration of about 1.4 mg per mL. Pipet 10 mL of this solution into a 50-mL volumetric flask, add 10.0 mL of
Internal standard solution, dilute with acetonitrile to volume, and mix.
[N
OTE—Use this solution within 8 hours of its preparation.
]
Assay preparation—
Transfer about 140 mg of Phenylbutazone, accurately weighed, to a 100-mL volumetric flask, add 75 mL of acetonitrile, and sonicate to dissolve. Dilute with acetonitrile to volume, and mix. Pipet 10 mL of this solution into a 50-mL volumetric flask, add 10.0 mL of Internal standard solution, dilute with acetonitrile to volume, and mix. [NOTE—Use this solution within 8 hours of its preparation.]
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 25-cm column that contains packing L7, preceded by a pre-column that contains packing L2. The flow rate is about 2.4 mL per minute. Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the resolution,
R, of phenylbutazone and the internal standard is not less than 3.5, and the relative standard deviation of the ratio of their peak responses in replicate injections is not more than 2.0%.
Procedure—
Separately inject equal volumes (about 25 µL) of the
Standard preparation and the
Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. The relative retention times are about 1.0 for the internal standard and 0.7 for phenylbutazone. Calculate the quantity, in mg, of C
19H
20N
2O
2 in the portion of Phenylbutazone taken by the formula:
500C(RU / RS),
in which
C is the concentration, in mg per mL, of
USP Phenylbutazone RS in the
Standard preparation; and
RU and
RS are the ratios of the peak response of the phenylbutazone to that of the internal standard for the
Assay preparation and the
Standard preparation, respectively.
;)
for 4 hours: it loses not more than 0.5% of its weight.