A: Infrared Absorption 197K.

B: It responds to the test for dry chlorides in Chloride 191.

Solution A— Dissolve 2.60 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. Filter and degas before use.

Solution B— Prepare a filtered and degassed mixture of methanol and acetonitrile (82:18). Make adjustments if necessary (see System Suitability under Chromatography 621).

Diluting solution— Prepare a mixture of Solution A and Solution B (1:1).

Mobile phase— Use variable mixtures of Solution A and Solution B as directed for Chromatographic system. Make adjustments to either Solution if necessary (see System Suitability under Chromatography 621).

Standard solution— Dissolve an accurately weighed quantity of USP Dobutamine Hydrochloride RS in Diluting solution and dilute quantitatively, and stepwise if necessary, with Diluting solution to obtain a solution having a known concentration of about 0.05 mg per mL.

Test solution— Transfer about 50.0 mg of Dobutamine Hydrochloride, accurately weighed, to a 10-mL volumetric flask, dissolve in and dilute with Diluting solution to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 280-nm detector and a 4.6-mm × 15-cm column that contains packing L1. The flow rate is about 1 mL per minute. The chromatograph is programmed as follows. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the tailing factor is not more than 2.0; 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 solution and the Test solution into the chromatograph, record the chromatograms, and measure all of the peak responses. Calculate the percentage of each impurity in the portion of Dobutamine Hydrochloride taken by the formula: in which C is the concentration, in mg per mL, of USP Dobutamine Hydrochloride RS in the Standard solution; D is the concentration, in mg per mL, of Dobutamine Hydrochloride in the Test solution; ri is the peak response for each impurity found in the Test solution; and rS is the dobutamine response obtained from the Standard solution: not more than 0.5% of any individual impurity is found, and not more than 1.0% of total impurities is found.

Phosphate buffer— Transfer about 23 g of monobasic ammonium phosphate to a 2-liter volumetric flask, add 1900 mL of water, and mix. Adjust with phosphoric acid to a pH of 2.2, dilute with water to volume, and mix.

Mobile phase— Prepare a filtered and degassed mixture of Phosphate buffer and acetonitrile (4:1). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve an accurately weighed quantity of USP Dobutamine Hydrochloride RS in water, and dilute quantitatively, and stepwise if necessary, with water to obtain a solution having a known concentration of about 0.5 mg per mL. [note—Prepare fresh daily, and refrigerate until injected.]

System suitability solution— Dissolve suitable quantities of 5-(hydroxymethyl)furfural and USP Dobutamine Hydrochloride RS in water to obtain a solution containing about 0.01 and 0.5 mg per mL, respectively.

Assay preparation— Transfer about 50 mg of Dobutamine Hydrochloride, accurately weighed, to a 100-mL volumetric flask, dissolve in and dilute with water to volume, and mix. [note—Refrigerate until injected, and use within 8 hours.]

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 280-nm detector and a 3.9-mm × 30-cm column that contains packing L1. The flow rate is about 1.5 mL per minute. Chromatograph the System suitability solution, and record the peak responses as directed under Procedure: the relative retention times are 1.0 for dobutamine and not more than 0.62 for 5-(hydroxymethyl)furfural, and the retention time of dobutamine is not more than 5.3 minutes. Chromatograph the Standard preparation, and record the peak responses as directed under Procedure: the tailing factor is not more than 2.0, 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 C18H23NO3·HCl in the portion of Dobutamine Hydrochloride taken by the formula: in which 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.