A: The chromatogram obtained from the Assay preparationin the Assayexhibits a major peak for doxapram,the retention time of which corresponds to that of the doxapram peak in the chromatogram of the Standard preparation.

B: Transfer a volume of Injection,equivalent to about 50mg of doxapram hydrochloride hydrate,to a separator containing 5mLof water.Add 1mLof a saturated solution of sodium chloride to the separator,insert the stopper,and mix.Add 5mLof 2.5Nsodium hydroxide,and extract with three 15-mLportions of chloroform.Pass each extract through a pledget of glass wool,combine the filtrates in a 50-mLvolumetric flask,dilute with chloroform to volume,and mix.Evaporate to dryness about 5mLof this solution.Dissolve the residue in 0.01Nsulfuric acid,dilute with the same solvent to 100mL,and mix:the UVabsorption spectrum of the solution so obtained exhibits maxima and minima at the same wavelengths as a solution similarly prepared,about 50mg of USP Doxapram Hydrochloride RS,instead of Doxapram Hydrochloride Injection,being used.

Mobile phase— Dissolve 2.8g of monobasic potassium phosphate in 1liter of water,adjust with 50%phosphoric acid or 1Npotassium hydroxide to a pHof 3.0±0.1,and filter through a 0.5-µm or finer porosity filter.Prepare a suitable mixture of this solution and acetonitrile (65:35).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Internal standard solution— Prepare a solution of diphenhydramine hydrochloride in water containing about 1.5mg per mL.

Standard preparation— Dissolve an accurately weighed quantity of USP Doxapram Hydrochloride RSin water to obtain a solution having a known concentration of about 2mg per mL.Transfer 5.0mLof this solution and 5.0mLof Internal standard solutionto a 50-mLvolumetric flask,dilute with water to volume,and mix.

Assay preparation— Transfer an accurately measured volume of Injection,equivalent to about 100mg of doxapram hydrochloride monohydrate,to a 50-mLvolumetric flask,dilute with water to volume,and mix.Transfer 5.0mLof this solution and 5.0mLof Internal standard solutionto a 50-mLvolumetric flask,dilute with water to volume,and mix.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 225-nm detector and a 4.6-mm ×15-cm column containing 5-µm packing L10,and is maintained at 40.The flow rate is about 1.5mLper minute.Chromatograph the Standard preparation,and record the responses as directed under Procedure:the relative retention times are about 1.0for doxapram and 1.2for diphenhydramine,the resolution,R,between the doxapram and diphenhydramine peaks is not less than 3.0;the tailing factor for the peaks 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 5µL)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 doxapram hydrochloride hydrate (C24H30N2O2·HCl·H2O)in each mLof the Injection taken by the formula: in which 432.98and 414.98are the molecular weights of doxapram hydrochloride monohydrate and anhydrous doxapram hydrochloride,respectively,Cis the concentration,in mg per mL,of USP Doxapram Hydrochloride RSin the Standard preparation,Vis the volume,in mL,of Injection taken to prepare the Assay preparation,and RUand RSare the ratios of the peak responses of doxapram and diphenhydramine obtained from the Assay preparationand the Standard preparation,respectively.