Identification—
To a 60-mL separator transfer 10 mL of pH 9.0 buffer (prepared by mixing equal volumes of 0.1
M monobasic potassium phosphate and 0.1 N sodium hydroxide and, using a pH meter, adjusting to a pH of 9.0 by adding, as necessary, more of either solution) add 1 mL of Injection, and mix. Add 2 mL of chloroform, shake vigorously for 1 minute, filter the chloroform extract through a pledget of cotton, and mix the filtrate with 500 mg of potassium bromide. Evaporate the chloroform, carefully removing the last trace of solvent in a small vacuum flask: the IR absorption spectrum of a potassium bromide dispersion of the isoxsuprine so obtained exhibits maxima only at the same wavelengths as that of a similar preparation of
USP Isoxsuprine Hydrochloride RS that has been treated in the same manner.
Assay—
pH 4.0 Citrate buffer—
Mix equal volumes of 0.5 M citric acid and 0.5 M sodium citrate, and adjust, by the addition of either solution as necessary, the pH of the solution to 4.0 ± 0.2.
Mixed solvent—
Shake 40 mL of ether, 160 mL of isooctane, and 10 mL of water in a separator, remove and discard the water phase, and pass the solvent phase through a large pledget of cotton to remove excess water.
Standard preparation—
Transfer about 40 mg of
USP Isoxsuprine Hydrochloride RS, accurately weighed, to a 50-mL volumetric flask, add 2 N sulfuric acid to volume, and mix. Transfer 10.0 mL of this solution to a 100-mL volumetric flask, dilute with 2 N sulfuric acid to volume, and mix. The concentration of
USP Isoxsuprine Hydrochloride RS in the
Standard preparation is about 80 µg per mL.
Chromatographic column—
Proceed as directed under
Column Partition Chromatography (see
Chromatography 
621
), packing a chromatographic tube with two segments of packing material. The lower segment is a mixture of 2 g of
Solid Support and 1 mL of
pH 4.0 Citrate buffer, and the upper segment is a mixture prepared as directed under
Assay preparation.
Assay preparation—
Transfer an accurately measured volume of Injection, equivalent to about 4 mg of isoxsuprine hydrochloride, to a 100-mL beaker, add 1 mL of dimethyl sulfoxide, and allow to stand for about 10 minutes, with occasional swirling. Add 1 mL of
pH 4.0 Citrate buffer and 3 g of
Solid Support, mix as directed under
Chromatographic column, and transfer to the column. Pass 75 mL of
Mixed solvent through the column, and discard the eluate. Elute the column with a solution prepared by mixing 0.2 mL of bis(2-ethylhexyl)phosphoric acid with 75 mL of
Mixed solvent, and collect the eluate in a 125-mL separator. Extract the eluate with two 20-mL portions of 2 N sulfuric acid. Transfer the extracts to a 50-mL volumetric flask, dilute with 2 N sulfuric acid to volume, and mix.
Procedure—
Concomitantly determine the absorbances of the
Assay preparation and the
Standard preparation in 1-cm cells at the wavelength of maximum absorbance at about 275 nm, with a suitable spectrophotometer, using a column blank, prepared with
Mixed solvent, to set the instrument. Calculate the quantity, in mg, of C
18H
23NO
3·HCl in the portion of Injection taken by the formula:
0.05C(AU / AS),
in which
C is the concentration, in µg per mL, of
USP Isoxsuprine Hydrochloride RS in the
Standard preparation; and
AU and
AS are the absorbances of the
Assay preparation and the
Standard preparation, respectively.
