Assay—
Ion-pair solution—
Dissolve 3.38 g of sodium 1-octanesulfonate in 1000 mL of water, pipet 3 mL of triethylamine into the solution, and mix. Adjust the solution with phosphoric acid to a pH of 2.5.
Mobile phase—
Prepare a filtered and degassed mixture of the Ion-pair solution, acetonitrile, and methanol (58:28:14). Make adjustments if necessary (see
System Suitability under
Chromatography 
621
).
[NOTE—The ratio of acetonitrile to methanol is critical to the elution order of the
System suitability solution components.
]
System suitability solution—
Dissolve suitable quantities of 4-(4-hydroxyphenyl)-2-butanone and
USP Dobutamine Hydrochloride RS in
Mobile phase to obtain a solution containing about 0.3 and 0.56 mg per mL, respectively.
Standard preparation—
Dissolve an accurately weighed quantity of
USP Dobutamine Hydrochloride RS in
Mobile phase and dilute quantitatively, and stepwise if necessary, with
Mobile phase to obtain a solution having a known concentration of about 0.56 mg per mL (equivalent to about 0.5 mg of dobutamine per mL).
Assay preparation—
Transfer an accurately measured volume of Injection, equivalent to about 25 mg of dobutamine, to a 50-mL volumetric flask. Dilute with Mobile phase to volume, and mix.
Chromatographic system
(see
Chromatography 621)—The liquid chromatograph is equipped with a 280-nm detector and a 4.6-mm × 25-cm column that contains 5-µm, base-deactivated packing L1. The flow rate is about 1 mL per minute. Chromatograph the
System suitability solution, and record the peak responses as directed under
Procedure: the relative retention times are about 0.9 for 4-(4-hydroxyphenyl)-2-butanone and 1.0 for dobutamine, the resolution,
R, between 4-(4-hydroxyphenyl)-2-butanone and dobutamine is not less than 1.5; the tailing factor for dobutamine is not more than 1.5; and the relative standard deviation for replicate injections is not more than 2.0%.
Procedure—
Separately inject equal volumes (about 20 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
18H
23NO
3 in the portion of Injection taken by the formula:
(301.39 / 337.84)(50C)(rU / rS),
in which 301.39 is the molecular weight of dobutamine; 337.84 is the molecular weight of dobutamine hydrochloride;
C is the concentration, in mg per mL, of
USP Dobutamine Hydrochloride RS in the
Standard preparation; and
rU and
rS are the peak responses obtained from the
Assay preparation and the
Standard preparation, respectively.
