A:Infrared Absorption á197Mñ.

B:Ultraviolet Absorption á197Uñ—

C: Asolution (1in 4000)responds to the tests for Chloride á191ñ.

Benzaldehyde solution— Transfer 1.0mLof benzaldehyde to a 100-mLvolumetric flask,dilute with a mixture of methanol and water (9:1)to volume,and mix.

Acetonitrile solution— Transfer 300mLof water to a 1000-mLvolumetric flask,dilute with acetonitrile to volume,and mix.

Phosphate buffer— Dissolve 5.82g of dibasic sodium phosphate and 3.81g of monobasic potassium phosphate in 1000mLof water,and adjust with either 1Nsodium hydroxide or 1Nphosphoric acid to a pHof 7.0±0.1.

Mobile phase— Dissolve 300mg of edetate disodium in 300mLof water in a 1000-mLvolumetric flask.Dilute with acetonitrile to volume,mix,filter,and degas.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard solution— Transfer about 65mg of hydrazine dihydrochloride,accurately weighed,to a 100-mLvolumetric flask,dissolve in and dilute with water to volume,and mix to obtain a solution having a known concentration of about 0.65mg per mL.Dilute this solution quantitatively,and stepwise if necessary,with water to obtain a Standard solutionhaving a known concentration of about 0.325µg of hydrazine dihydrochloride per mL.Transfer 1.0mLof the Standard solutionto a 10-mLreaction vessel.Add 4.0mLof Benzaldehyde solution,and shake by mechanical means for 20minutes.Transfer 2.0mLof this solution to a 5-mLvolumetric flask,dilute with Acetonitrile solutionto volume,and mix.

Test solution— [NOTE—Condition the extraction column specified in this procedure in the following manner.Wash the column with two 2.0-mLportions of hexanes,and dry with the aid of vacuum for two minutes.Wash the column with two 2.0-mLportions of methanol,two 2.0-mLportions of water,and two 2.0-mLportions of pH7.0phosphate buffer.At no time after the hexanes wash should the column be allowed to dry out.]Transfer about 20mg of Hydralazine Hydrochloride,accurately weighed,to a 10-mLreaction vessel,and dissolve in 1.0mLof water.Add 4.0mLof Benzaldehyde solution,and shake by mechanical means for 20minutes.Pipet 2.0mLof this solution into a freshly conditioned solid phase extraction column containing benzenesulfonic acid strong cation-exchange packing with a sorbent-mass to column volume ratio of 500mg per 3mL,or equivalent,and elute into a 5-mLvolumetric flask.Wash the column with two 1.5-mLportions of Acetonitrile solution,collecting the washings with the eluate,dilute with Acetonitrile solutionto volume,and mix.

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a 310-nm detector and a 4.0-mm ×25-cm column that contains 10-µm packing L1.The flow rate is about 1mLper minute.Chromatograph the Standard solution,and record the peak responses as directed for Procedure:the relative retention times are about 1.0for hydralazine derivative and 1.5for hydrazine derivative;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 20µL)of the solution from the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the percentage of hydrazine in the portion of Hydralazine Hydrochloride taken by the formula: in which 32.05and 104.97are the molecular weights of hydrazine and hydrazine dihydrochloride,respectively;Cis the concentration,in µg per mL,of hydrazine dihydrochloride in the Standard solution;Wis the weight,in mg,of Hydralazine Hydrochloride taken for the Test solution;and rUand rSare the hydrazine peak responses obtained from the Test solutionand from the solution from the Standard solution,respectively:not more than 0.001%of hydrazine is found.

Mobile phaseand Resolution solution— Prepare as directed in the Assay.

Test solution— Transfer about 25mg of Hydralazine Hydrochloride,accurately weighed,to a 50-mLvolumetric flask.Add about 30mLof 0.1Nacetic acid,and sonicate to dissolve.Cool,dilute with 0.1Nacetic acid to volume,and mix.

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a 230-nm detector and a 4.0-mm ×25-cm column that contains 10-µm packing L10.The flow rate is about 1mLper minute.Chromatograph the Resolution solution,and record the peak responses as directed for Procedure:the relative retention times are about 0.65for phthalazine and 1.0for hydralazine hydrochloride;and the resolution,R,between the phthalazine peak and the hydralazine peak is not less than 4.0.

Procedure— Inject about 20µLof the Test solutioninto the chromatograph,record the chromatogram,and measure the peak responses.Calculate the percentage of each peak,other than the solvent peak and the hydralazine peak,in the portion of Hydralazine Hydrochloride taken by the same formula: in which riis the response of each peak;and rtis the sum of the responses of all the peaks,excluding that of the solvent peak:not more than 1.0%total impurities is found.

Mobile phase— Dissolve 1.44g of sodium dodecyl sulfate and 0.75g of tetrabutylammonium bromide in 770mLof water,and add 230mLof acetonitrile.Adjust with 0.1Nsulfuric acid to a pHof 3.0,mix,filter,and degas.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Dissolve an accurately weighed quantity of USP Hydralazine Hydrochloride RSin 0.1Nacetic acid to obtain a solution having a known concentration of about 0.4mg per mL.Transfer 10.0mLof this solution to a 100-mLvolumetric flask,dilute with 0.1Nacetic acid to volume,and mix to obtain a solution having a known concentration of about 40µg per mL.

Resolution solution— Prepare a solution in 0.1Nacetic acid containing about 0.25mg of USP Hydralazine Hydrochloride RSand 0.05mg of phthalazine per mL.Transfer 5.0mLof this solution to a 50-mLvolumetric flask,dilute with 0.1Nacetic acid to volume,and mix to obtain a solution containing about 25µg of USP Hydralazine Hydrochloride RSand 5µg of phthalazine per mL.

Assay preparation— Transfer about 100mg of Hydralazine Hydrochloride,accurately weighed,to a 250-mLvolumetric flask.Dissolve in and dilute with 0.1Nacetic acid to volume,and mix.Transfer 10.0mLof this solution to a 100-mLvolumetric flask,dilute with 0.1Nacetic acid to volume,mix,and filter,discarding the first 10mLof the filtrate.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 230-nm detector and a 4.0-mm ×25-cm column that contains 10-µm packing L10.The flow rate is about 1mLper minute.Chromatograph the Resolution solution,and record the peak responses as directed for Procedure:the relative retention times are about 0.65for phthalazine and 1.0for hydralazine hydrochloride;and the resolution,R,between the phthalazine and hydralazine peaks is not less than 4.0.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 25µL)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 C8H8N4·HCl in the portion of Hydralazine Hydrochloride taken by the formula: in which Cis the concentration,in µg per mL,of USP Hydralazine Hydrochloride RSin the Standard preparation;and rUand rSare the hydralazine hydrochloride peak responses obtained from the Assay preparationand the Standard preparation,respectively.