Ion-pair solution— Dissolve 3.38g of sodium 1-octanesulfonate in 1000mLof water,pipet 3mLof triethylamine into the solution,and mix.Adjust the solution with phosphoric acid to a pHof 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 Suitabilityunder Chromatography á621ñ).[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 RSin Mobile phaseto obtain a solution containing about 0.3and 0.56mg per mL,respectively.

Standard preparation— Dissolve an accurately weighed quantity of USP Dobutamine Hydrochloride RSin Mobile phaseand dilute quantitatively,and stepwise if necessary,with Mobile phaseto obtain a solution having a known concentration of about 0.56mg per mL(equivalent to about 0.5mg of dobutamine per mL).

Assay preparation— Transfer an accurately measured volume of Injection,equivalent to about 25mg of dobutamine,to a 50-mLvolumetric flask.Dilute with Mobile phaseto volume,and mix.

Chromatographic system (see Chromatography á621ñ)—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 1mLper minute.Chromatograph the System suitability solution,and record the peak responses as directed under Procedure:the relative retention times are about 0.9for 4-(4-hydroxyphenyl)-2-butanone and 1.0for 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 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of C18H23NO3in the portion of Injection taken by the formula: in which 301.39is the molecular weight of dobutamine;337.84is the molecular weight of dobutamine hydrochloride;Cis the concentration,in mg per mL,of USP Dobutamine Hydrochloride RSin the Standard preparation;and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.