A: Infrared Absorption 197M.

B: It responds to the tests for Chloride 191.

Detection reagent— Transfer 2.5 g of cadmium acetate to a 500-mL volumetric flask, add 10 mL of glacial acetic acid, dilute with alcohol to volume, and mix. Just prior to use, prepare a 0.2 in 100 solution of ninhydrin in the cadmium acetate solution for use as the Detection reagent.

Solvent mixture— Prepare a solution of methanol and water (4:1), and mix.

Ammonium chloride reference solution— Dissolve 60 mg of ammonium chloride in 10.0 mL of water, and mix.

Standard stock solution— Dissolve USP Labetalol Hydrochloride RS in Solvent mixture, and mix to obtain a solution having a known concentration of 40 mg per mL.

Standard solution 1— Quantitatively dilute a portion of the Standard stock solution with Solvent mixture to obtain a solution having a known concentration of 0.2 mg per mL.

Standard solution 2— Quantitatively dilute a portion of the Standard solution 1 with Solvent mixture to obtain a solution having a known concentration of 0.1 mg per mL.

Test solution— Dissolve 200 mg of Labetalol Hydrochloride in 5.0 mL of Solvent mixture, and mix.

Total impurities— The sum of the intensities of all secondary spots (other than those due to ammonium chloride) observed in the chromatograms of the Test solution from both Procedure I and Procedure II does not exceed 1.0%.

1-Butaneboronic acid solution— Dissolve 1-butaneboronic acid in pyridine, previously dried over a suitable molecular sieve, and mix to obtain a solution having a known concentration of 20 mg per mL.

System suitability solution— Dissolve an accurately weighed quantity of USP Labetalol Hydrochloride RS in 1-Butaneboronic acid solution, and dilute quantitatively and stepwise with 1-Butaneboronic acid solution to obtain a solution having a known concentration of about 1.4 mg of USP Labetalol Hydrochloride RS per mL. Allow the solution to stand at room temperature for 20 minutes before using.

Test solution— Transfer about 1 mg of Labetalol Hydrochloride to a 1-mL reaction vial, add 0.7 mL of 1-Butaneboronic acid solution, and mix until the labetalol hydrochloride is completely dissolved. Allow the solution to stand at room temperature for 20 minutes before using.

Chromatographic system (see Chromatography 621)— The gas chromatograph is equipped with a flame-ionization detector and a 2-mm × 1.8-m glass column packed with 10% phase G3 on 100- to 120-mesh support S1AB. The column temperature is maintained at about 320, and the injection port and the detector block temperatures are maintained at about 340. Nitrogen is used as the carrier gas at the flow rate of about 30 mL per minute. Chromatograph the System suitability solution, and record the peak responses as directed for Procedure: the relative retention times are about 0.8 for the diastereoisomer B 1-butaneboronate derivative and 1.0 for the diastereoisomer A 1-butaneboronate derivative; the resolution, R, between the diastereoisomer A 1-butaneboronate derivative and diastereoisomer B 1-butaneboronate derivative peaks is not less than 1.5; and the relative standard deviation of the ratios of the peak areas of the diastereoisomers for replicate injections is not more than 2.0%.

Procedure— Inject about 2 µL of the Test solution into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the diastereoisomer A content, in percentage, taken by the formula: in which rA is the peak area of the diastereoisomer A 1-butaneboronate derivative peak; and rB is the peak area of the diastereoisomer B 1-butaneboronate derivative peak. The diastereoisomer A content is not less than 45.0% and not more than 55.0%.

Mobile phase— Prepare a suitable filtered and degassed mixture of 0.1 M monobasic sodium phosphate and methanol (65:35). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve an accurately weighed quantity of USP Labetalol Hydrochloride RS in Mobile phase to obtain a solution having a known concentration of about 0.4 mg per mL.

Assay preparation— Transfer about 40 mg of Labetalol Hydrochloride, accurately weighed, to a 100-mL volumetric flask, dilute with Mobile phase to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 230-nm detector and a 4.6-mm × 20-cm column that contains packing L1 and is maintained at 60 ± 1. The flow rate is about 1.5 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the column efficiency determined from the analyte peak is not less than 700 theoretical plates; the tailing factor for the analyte peak is not more than 2.0; and the relative standard deviation for replicate injections is not more than 1.5%.

Procedure— Separately inject equal volumes (about 5 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the area responses for the major peaks. Calculate the quantity, in mg, of labetalol hydrochloride (C19H24N2O3·HCl) in the portion of Labetalol Hydrochloride taken by the formula: in which C is the concentration, in mg per mL, of USP Labetalol Hydrochloride RS in the Standard preparation; and rU and rS are the peak area responses obtained from the Assay preparation and the Standard preparation, respectively.