A: Evaporate a volume of Injection,equivalent to about 2mg of histamine phosphate,on a steam bath to dryness,dissolve the residue in 0.5mLof water,and add 0.5mLof 1Nsodium hydroxide.Add 2drops of sodium nitrite solution (1in 10),and add 1mLof a solution prepared by mixing 50mg of sulfanilic acid with 10mLof water containing 2drops of hydrochloric acid:an orange-red color is produced.

B: To 1mLof Injection,equivalent to not less than 1mg of histamine phosphate (concentrate a larger volume by evaporation,if necessary),add ammonium molybdate TSdropwise:a yellow precipitate,which is soluble in ammonia TS,is formed.

Standard preparation— Dissolve an accurately weighed quantity of USP Histamine Dihydrochloride RSin water,and quantitatively dilute with water to obtain a solution having a known concentration of 20µg per mL,equivalent to 33.4µg of histamine phosphate.

Assay preparation— Dilute an accurately measured volume of Injection,equivalent to about 1.65mg of histamine phosphate,with water in a 50-mLvolumetric flask to volume,and mix.

If phenol is present ,prepare the Assay preparationas follows.Dilute an accurately measured volume of Injection,equivalent to about 1.65mg of histamine phosphate,with water to about 25mL.Heat the solution on a steam bath until the odor of phenol is no longer perceptible,adding water as required to maintain a volume of about 15mL.Transfer to a 50-mLvolumetric flask,cool,dilute with water to volume,and mix.

Procedure— Pipet 5mLeach of the Standard preparationand the Assay preparationinto separate,10-mLvolumetric flasks,to each add 1mLof sodium borate solution (1in 100),followed by 1mLof a freshly prepared solution of 50mg of b-naphthoquinone-4-sodium sulfonate in 10mLof water.Place the flasks in boiling water for 10minutes,then immerse them for 5minutes in water maintained between 5and 10.To each flask,add 1mLof acid-formaldehyde (made by adding 0.5mLof formaldehyde TSto a mixture of 45mLof 1Nhydrochloric acid and 10mLof glacial acetic acid and diluting with water to 80mL),mix,add 1mLof 0.1Nsodium thiosulfate,then dilute with water to volume,and mix.Concomitantly and immediately determine the absorbances of both solutions at the wavelength of maximum absorbance at about 460nm,with a suitable spectrophotometer,against a reagent blank.Calculate the quantity,in mg,of C5H9N3·2H3PO4in each mLof the Injection taken by the formula: in which Cis the concentration,in µg per mL,of USP Histamine Dihydrochloride RSin the Standard preparation;Vis the volume,in mL,of Injection taken;and AUand ASare the absorbances of the solutions from the Assay preparationand the Standard preparation,respectively.