Add the following:

Solution A, Solution B, Mobile phase, and Diluent —Proceed as directed in the Assay.

Test solution— Use the Assay preparation.

Standard solution— Dissolve accurately weighed quantities of USP Levalbuterol Hydrochloride RS, USP Levalbuterol Related Compound A RS, USP Levalbuterol Related Compound B RS, USP Levalbuterol Related Compound C RS, USP Levalbuterol Related Compound D RS, USP Levalbuterol Related Compound E RS, and USP Levalbuterol Related Compound F RS in Diluent to obtain a solution having known concentrations of about 0.05 µg per mL of each related compound and 100 µg per mL of USP Levalbuterol Hydrochloride RS.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 220-nm detector and a 4.6-mm × 15-cm column that contains 5-µm packing L1. The column temperature is maintained at 45. The flow rate is about 1 mL per minute. The chromatograph is programmed as follows. Chromatograph the Standard solution, and record the peak areas as directed for Procedure: the resolution, R, between levalbuterol and levalbuterol related compound A is not less than 4.9, and between levalbuterol related compound B and levalbuterol related compound C is not less than 1.5; the column efficiency determined from the levalbuterol peak is not less than 4000; the tailing factor for the levalbuterol peak is not greater than 4.0; and the relative standard deviation for replicate injection is less than 20%, determined from any of the six related compound peaks.

Procedure— Separately inject equal volumes (about 50 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the peak responses. Determine the area of the levalbuterol peak, and integrate all peaks with an area greater than 0.05% of the area corresponding to the levalbuterol peak. Calculate the percentage of each impurity in the portion of Levalbuterol Hydrochloride taken by the formula: in which ri is the peak response for each impurity obtained from the Test solution; rs is the sum of the responses of all the peaks (see Table 1 for limits of individual impurities); and F is the relative response factor for each impurity. Not more than 0.5% of total impurities is found. USP32

Mobile phase— Prepare a degassed mixture of acetonitrile, methanol, acetic acid, and triethylamine (500: 500: 3: 1).

Diluent— Use the Mobile phase.

Sensitivity solution— Dissolve about 10 mg of USP Levalbuterol Hydrochloride RS and 40 µg of USP Albuterol RS in 100 mL of Diluent, and mix.

Standard solution— Dissolve an accurately weighed quantity of USP Albuterol RS in Diluent to obtain a solution having a known concentration of about 1.5 mg per mL.

Test solution— Transfer an accurately weighed quantity of Levalbuterol Hydrochloride to a suitable volumetric flask, and quantitatively dilute with Diluent to obtain a solution having a concentration of about 0.8 mg per mL of levalbuterol hydrochloride.

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 mL per minute. Chromatograph the Sensitivity solution, and record the peak responses as directed for Procedure: the resolution, R, between levalbuterol and (S)-albuterol is not less than 2.0; the column efficiency calculated from either peak is not less than 4000; the tailing factor for levalbuterol and (S)-albuterol is not more than 2.2. The Sensitivity solution is injected three times, and the relative standard deviation for replicate injections is not more than 20% for (S)-albuterol.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard solution and the Test solution. 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: not more than 0.2% of (S)-albuterol is found.

Solution A— Prepare a 1 in 1000 solution of phosphoric acid in water, and degas.

Solution B— Prepare a degassed mixture of acetonitrile, methanol, water, and phosphoric acid (700: 700: 600: 2).

Mobile phase— Use variable mixtures of Solution A and Solution B as directed for Chromatographic system. Make adjustments if necessary (see System Suitability under Chromatography 621).

Diluent— Use Solution A.

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

Assay preparation— Transfer about 10 mg of Levalbuterol Hydrochloride, accurately weighed, to a 100-mL volumetric flask. Dissolve in and dilute with Diluent to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 220-nm detector and a 4.6-mm × 15-cm column that contains 5-µm packing L1. The column temperature is maintained at 35. The flow rate is about 1 mL per minute. The chromatograph is programmed as follows: Chromatograph the Standard preparation, and record the peak areas as directed for Procedure: the column efficiency is greater than 5500 theoretical plates; the tailing factor is less than 2.3; and the relative standard deviation for replicate injections is not more than 2.0%.

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 quantity in mg of C13H21NO3·HCl in the portion of Levalbuterol Hydrochloride taken by the formula: in which CS is the concentration, in µg per mL, of USP Levalbuterol Hydrochloride RS in the Standard preparation; and rU and rS are the peak responses of levalbuterol hydrochloride obtained from the Assay preparation and the Standard preparation, respectively.