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

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

Ferro-citrate solution and Buffer solution—Prepare as directed under Epinephrine Assay 391.

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

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

Phenylephrine hydrochloride standard preparation— Dissolve an accurately weighed quantity of USP Phenylephrine Hydrochloride RS in dilute sulfuric acid (1 in 1000), and dilute quantitatively and stepwise with the same dilute sulfuric acid as necessary to obtain a solution having a known concentration of about 12 µg 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-mL portions of dilute sulfuric acid (1 in 1000), and transfer the resulting solutions quantitatively to a 50-mL centrifuge tube. Add 10 mL of chloroform, insert the stopper, shake vigorously for 1 minute, and centrifuge for 20 minutes. 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 hydrochloride and 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, 20.0 mL of the Isoproterenol hydrochloride standard preparation, and 20.0 mL of dilute sulfuric acid (1 in 1000) to provide the blank. To each flask add 100 µL of Ferro-citrate solution and 1.0 mL of Buffer solution, and mix. Concomitantly determine the absorbances of the Test preparation and the Standard preparation against the blank at the wavelength of maximum absorbance at about 530 nm, 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 C is the concentration, in µg per mL, of USP Isoproterenol Hydrochloride RS in the Isoproterenol hydrochloride standard preparation; N is the number of sprays discharged to obtain the minimum dose; and AU and AS are the absorbances of the solutions from the Test preparation and the Standard preparation, respectively.

Procedure for phenylephrine bitartrate— Into three separate flasks transfer the Test preparation for phenylephrine bitartrate, 20.0 mL of the Phenylephrine hydrochloride standard preparation, and 20.0 mL of dilute sulfuric acid (1 in 1000) to provide the blank. To each flask add 3.0 mL of Mercuric sulfate solution, and mix. Immerse in a water bath maintained between 35 and 40 for 10 minutes, remove, and allow to stand at room temperature for 30 minutes. Add 0.25 mL of a sodium nitrite solution (1 in 80), mix, and allow to stand, with occasional swirling, for 30 minutes. Concomitantly determine the absorbances of the Test preparation and the Standard preparation against the blank at the wavelength of maximum absorbance at about 495 nm, in 5-cm cells, with a suitable spectrophotometer. Calculate the quantity, in µg, of C9H13NO2·C4H6O6 in the minimum dose taken by the formula: in which 317.30 and 203.67 are the molecular weights of phenylephrine bitartrate and phenylephrine hydrochloride, respectively; C is the concentration, in µg per mL, of USP Phenylephrine Hydrochloride RS in the Phenylephrine hydrochloride standard preparation; N is the number of sprays discharged to obtain the minimum dose; and AU and AS are the absorbances of the solutions from the Test preparation and the Standard preparation, respectively.

(Official January 1, 2007)

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

Phenylephrine hydrochloride standard preparation— Transfer about 50 mg of USP Phenylephrine Hydrochloride RS, accurately weighed, to a 100-mL volumetric flask, add dilute sulfuric acid (1 in 1000) to volume, and mix. Transfer 10.0 mL of this solution to a 50-mL volumetric flask, add dilute sulfuric acid (1 in 1000) to volume, and mix. The concentration of USP Phenylephrine Hydrochloride RS in the Phenylephrine hydrochloride standard preparation is about 100 µg 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 20 mL of chloroform in a suitable 100-mL beaker. Prime the valve of the Aerosol by alternately shaking and firing it 10 times 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 6 sprays in this manner. Rinse the valve stem and ferrule with about 2 mL of chloroform, collecting the rinsing with the sample in the beaker. Allow the Aerosol to dry, weigh it, and determine the total weight of the 6 sprays. Transfer the solution to a centrifuge tube with the aid of two 3-mL portions of chloroform, and add 10.0 mL of dilute sulfuric acid (1 in 1000). Insert the stopper, shake vigorously for 1 minute, centrifuge for 20 minutes, and use the clear supernatant.

Procedure for isoproterenol hydrochloride— Transfer 3.0 mL each of the Isoproterenol hydrochloride standard preparation and the Assay preparation to separate test tubes. To each tube add 0.10 mL of Ferro-citrate solution and 1.0 mL of Buffer solution, and mix. Concomitantly determine the absorbances of the solutions in 1-cm cells at the wavelength of maximum absorbance at about 530 nm, with a suitable spectrophotometer, using water as the blank. Calculate the quantity, in mg, of isoproterenol hydrochloride (C11H17NO3·HCl) in each mL of the Aerosol taken by the formula: in which C is the concentration, in µg per mL, of USP Isoproterenol Hydrochloride RS in the Isoproterenol hydrochloride standard preparation; d is the density, in g per mL, of Aerosol taken; W is the weight, in g, of the sample taken; and AU and AS are the absorbances of the solutions from the Assay preparation and 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-mL gas-tight syringe equipped with a linear valve. Calibrate the volume of the syringe by filling to the 5-mL mark with dichlorotetrafluoroethane withdrawn from a plastic-coated glass vial sealed with a neoprene multiple-dose rubber stopper and an aluminum seal, using 1.456 g per mL as the density of the calibrating liquid. Maintain the dichlorotetrafluoroethane, the Aerosol sample, and the syringe (protected from becoming wet) at 25 in 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 w is the weight of the volume, v, of Aerosol taken.]

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