A: Place 10mLof water in a small beaker,and deliver 20sprays from the Aerosol under the surface of the water,actuating the valve by pressing the tip against the bottom of the beaker.To 5mLof the solution add 1drop of dilute sulfuric acid (1in 200),add 0.5mLof 0.1Niodine,allow to stand for 5minutes,and add 1mLof 0.1Nsodium thiosulfate:a red-brown color is produced.

B: To the balance of the solution obtained in Identificationtest Aadd 3mLof Mercuric sulfate solution,prepared as directed in the test for Dose uniformity over the entire contents.Heat on a steam bath for 5minutes,cool,and add 3mLof sodium nitrite solution (1in 80):a deep red color is produced.

Ferro-citrate solution and Buffer solution—Prepare as directed under Epinephrine Assay á391ñ.

Mercuric sulfate solution— While stirring a mixture of 15g of yellow mercuric oxide and 80mLof water,slowly add 20mLof sulfuric acid,and stir until completely dissolved.

Isoproterenol hydrochloride standard preparation— Dissolve an accurately weighed quantity of USP Isoproterenol Hydrochloride RSin dilute sulfuric acid (1in 1000),and dilute quantitatively and stepwise with the same dilute sulfuric acid as necessary to obtain a solution having a known concentration of about 8µg per mL.

Phenylephrine hydrochloride standard preparation— Dissolve an accurately weighed quantity of USP Phenylephrine Hydrochloride RSin dilute sulfuric acid (1in 1000),and dilute quantitatively and stepwise with the same dilute sulfuric acid as necessary to obtain a solution having a known concentration of about 12µg per mL.

Test preparation for isoproterenol hydrochloride— Discharge the minimum recommended dose into the sampling apparatus,and detach the inhaler as directed.Rinse the apparatus (filter and interior)with four 5.0-mLportions of dilute sulfuric acid (1in 1000),and transfer the resulting solutions quantitatively to a 50-mLcentrifuge tube.Add 10mLof chloroform,insert the stopper,shake vigorously for 1minute,and centrifuge for 20minutes.Use the clear supernatant as directed in the Procedure for isoproterenol hydrochloride.

Test preparation for phenylephrine bitartrate— Proceed as directed for Test preparation for isoproterenol hydrochlorideand use the clear supernatant as directed in the Procedure for phenylephrine bitartrate.

Procedure for isoproterenol hydrochloride— Into three separate flasks transfer the Test preparation for isoproterenol hydrochloride,20.0mLof the Isoproterenol hydrochloride standard preparation,and 20.0mLof dilute sulfuric acid (1in 1000)to provide the blank.To each flask add 100µLof Ferro-citrate solutionand 1.0mLof Buffer solution,and mix.Concomitantly determine the absorbances of the Test preparationand the Standard preparationagainst the blank at the wavelength of maximum absorbance at about 530nm,in 5-cm cells,with a suitable spectrophotometer.Calculate the quantity,in µg,of C11H17NO3·HCl contained in the minimum dose taken by the formula: in which Cis the concentration,in µg per mL,of USP Isoproterenol Hydrochloride RSin the Isoproterenol hydrochloride standard preparation;Nis the number of sprays discharged to obtain the minimum dose;and AUand ASare the absorbances of the solutions from the Test preparationand the Standard preparation,respectively.

Procedure for phenylephrine bitartrate— Into three separate flasks transfer the Test preparation for phenylephrine bitartrate,20.0mLof the Phenylephrine hydrochloride standard preparation,and 20.0mLof dilute sulfuric acid (1in 1000)to provide the blank.To each flask add 3.0mLof Mercuric sulfate solution,and mix.Immerse in a water bath maintained between 35and 40for 10minutes,remove,and allow to stand at room temperature for 30minutes.Add 0.25mLof a sodium nitrite solution (1in 80),mix,and allow to stand,with occasional swirling,for 30minutes.Concomitantly determine the absorbances of the Test preparationand the Standard preparationagainst the blank at the wavelength of maximum absorbance at about 495nm,in 5-cm cells,with a suitable spectrophotometer.Calculate the quantity,in µg,of C9H13NO2·C4H6O6in the minimum dose taken by the formula: in which 317.30and 203.67are the molecular weights of phenylephrine bitartrate and phenylephrine hydrochloride,respectively;Cis the concentration,in µg per mL,of USP Phenylephrine Hydrochloride RSin the Phenylephrine hydrochloride standard preparation;Nis the number of sprays discharged to obtain the minimum dose;and AUand ASare the absorbances of the solutions from the Test preparationand the Standard preparation,respectively.

Ferro-citrate solution ,Buffer solution,and Mercuric sulfate solution—Prepare as directed in the test for Dose uniformity over the entire contents.

Isoproterenol hydrochloride standard preparation— Transfer about 50mg of USP Isoproterenol Hydrochloride RS,accurately weighed,to a 100-mLvolumetric flask,add dilute sulfuric acid (1in 1000)to volume,and mix.Transfer 10.0mLof this solution to a 50-mLvolumetric flask,add dilute sulfuric acid (1in 1000)to volume,and mix.The concentration of USP Isoproterenol Hydrochloride RSin the Isoproterenol hydrochloride standard preparationis about 100µg per mL.

Phenylephrine hydrochloride standard preparation— Transfer about 50mg of USP Phenylephrine Hydrochloride RS,accurately weighed,to a 100-mLvolumetric flask,add dilute sulfuric acid (1in 1000)to volume,and mix.Transfer 10.0mLof this solution to a 50-mLvolumetric flask,add dilute sulfuric acid (1in 1000)to volume,and mix.The concentration of USP Phenylephrine Hydrochloride RSin the Phenylephrine hydrochloride standard preparationis about 100µg per mL.

Assay preparation— [NOTE—Valve actuation during sampling is to be performed in a manner that will deliver freely the spray into the solvent in the bottom of the sampling beaker,but with minimal lateral pressure on the valve stem to avoid possible side-of-stem leakage.Any devices and techniques designed to accomplish these objectives may be used (e.g.,actuating the valve by pressing the tip into an indentation in the bottom of the beaker or using a special adapter to spray at right angles to the valve held at the bottom of the beaker).]Place 20mLof chloroform in a suitable 100-mLbeaker.Prime the valve of the Aerosol by alternately shaking and firing it 10times through its oral inhalation actuator.Accurately weigh the Aerosol,shake it,and immediately deliver a single spray under the surface of the chloroform.Raise the Aerosol above the surface of the chloroform,and shake it gently preparatory to delivering another spray similarly under the surface of the chloroform.Deliver a total of 6sprays in this manner.Rinse the valve stem and ferrule with about 2mLof chloroform,collecting the rinsing with the sample in the beaker.Allow the Aerosol to dry,weigh it,and determine the total weight of the 6sprays.Transfer the solution to a centrifuge tube with the aid of two 3-mLportions of chloroform,and add 10.0mLof dilute sulfuric acid (1in 1000).Insert the stopper,shake vigorously for 1minute,centrifuge for 20minutes,and use the clear supernatant.

Procedure for isoproterenol hydrochloride— Transfer 3.0mLeach of the Isoproterenol hydrochloride standard preparationand the Assay preparationto separate test tubes.To each tube add 0.10mLof Ferro-citrate solutionand 1.0mLof Buffer solution,and mix.Concomitantly determine the absorbances of the solutions in 1-cm cells at the wavelength of maximum absorbance at about 530nm,with a suitable spectrophotometer,using water as the blank.Calculate the quantity,in mg,of isoproterenol hydrochloride (C11H17NO3·HCl)in each mLof the Aerosol taken by the formula: in which Cis the concentration,in µg per mL,of USP Isoproterenol Hydrochloride RSin the Isoproterenol hydrochloride standard preparation;dis the density,in g per mL,of Aerosol taken;Wis the weight,in g,of the sample taken;and AUand ASare the absorbances of the solutions from the Assay preparationand the Isoproterenol hydrochloride standard preparation,respectively.[The density,d,is determined as follows.Weigh a known volume (v)of the Aerosol in a suitable 5-mLgas-tight syringe equipped with a linear valve.Calibrate the volume of the syringe by filling to the 5-mLmark with dichlorotetrafluoroethane withdrawn from a plastic-coated glass vial sealed with a neoprene multiple-dose rubber stopper and an aluminum seal,using 1.456g per mLas the density of the calibrating liquid.Maintain the dichlorotetrafluoroethane,the Aerosol sample,and the syringe (protected from becoming wet)at 25in a water bath.Obtain the sample,equivalent to the same volume as that obtained during the sampling procedure,from the Aerosol by means of a sampling device consisting of a replaceable rubber septum engaged in the plate threads at one end of a threaded fitting,the opposite end of which contains a sharpened tube capable of puncturing the aerosol container,and a rubber gasket around the tube to prevent leakage of the container contents after puncture.*Calculate the density taken by the formula: in which wis the weight of the volume,v,of Aerosol taken.]

Procedure for phenylephrine bitartrate— Transfer 5.0mLeach of the Phenylephrine hydrochloride standard preparation,the Assay preparation,and a blank consisting of dilute sulfuric acid (1in 1000),to separate test tubes.To each tube add 3.0mLof Mercuric sulfate solution,and mix.Immerse in a water bath maintained between 35and 40for 10minutes,remove,and allow to stand at room temperature for 30minutes.Add 0.25mLof sodium nitrite solution (1in 80),mix,and allow to stand,with occasional swirling,for 30minutes.Concomitantly determine the absorbances of the solutions against the blank in 1-cm cells at the wavelength of maximum absorbance at about 495nm,with a suitable spectrophotometer.Calculate the quantity,in mg,of phenylephrine bitartrate (C9H13NO2·C4H6O6)in each mLof the Aerosol taken by the formula: in which 317.30and 203.67are the molecular weights of phenylephrine bitartrate and phenylephrine hydrochloride,respectively;Cis the concentration,in µg per mL,of USP Phenylephrine Hydrochloride RSin the Phenylephrine hydrochloride standard preparation;dis the density,in g per mL,of Aerosol taken;Wis the weight,in g,of the sample taken;and AUand ASare the absorbances of the solutions from the Assay preparationand the Phenylephrine hydrochloride standard preparation,respectively.