A: The retention time of the major peak in the chromatogram of the Assay preparation corresponds to that observed in the chromatogram of the Standard preparation, as obtained in the Assay.

Mobile phase, Sensitivity solution, and Standard solution— Proceed as directed for Enantiomeric purity and chiral identity under Levalbuterol Hydrochloride.

Test solution— All sample concentrations are injected neat.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 225-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L63. The flow rate is about 1.0 mL per minute. The minimum run time is 30 minutes. Inject 10 µL of the Sensitivity solution. The resolution, R, between levalbuterol and (S)-albuterol is not less than 3.0; and the tailing factor for levalbuterol and (S)-albuterol are not more than 1.6 and 2.0, respectively. Inject the Sensitivity solution three times, and relative standard deviation calculated from the peak area of (S)-albuterol is not greater than 20%.

Procedure— Inject equal volumes of the Standard solution and the Test solution such that approximately 4.2 µg of levalbuterol are injected. The percentage of (S)-albuterol is calculated using the following equation: in which ri is the peak response for (S)-albuterol, and rs is the sum of the responses of both levalbuterol and (S)-albuterol peaks. The Test solution contains not more than 2.50% (S)-albuterol.

Solution A, Solution B, Mobile phase, and Chromatographic system— Proceed as directed in the Assay under Levalbuterol Hydrochloride.

Diluent— Dissolve about 9.0 ± 0.05 g of sodium chloride in 950 mL of water. Adjust with dilute sulfuric acid to a pH of 4.0, and dilute with water to 1000 mL. Mix, and pass through 0.45-µm filter.

Standard preparation— Dissolve an accurately weighed portion of USP Levalbuterol Hydrochloride RS in Diluent to obtain a solution having a known concentration of 100 µg per mL.

Assay preparation— Transfer an accurately measured volume of Inhalation Solution to a suitable volumetric flask, and quantitatively dilute with Diluent to obtain a solution having a concentration of about 100 µg of levalbuterol hydrochloride per mL.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the concentration, in µg per mL, of C13H21NO3·HCl per ampul by the formula: in which D is the dilution factor; C is the concentration of USP Levalbuterol Hydrochloride RS in the Standard preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.1S (USP31)