A: Infrared Absorption 197K—

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

Mobile phase and Diluent— Proceed as directed in the Assay.

Standard solution— Transfer 10 mg of USP Dantrolene Related Compound B RS, accurately weighed, into a 50-mL volumetric flask, and dissolve in 2.5 mL of dimethylformamide. Add 2.5 mL of glacial acetic acid, and dilute with acetone to volume to obtain a solution having a known concentration of about 0.2 mg per mL. Dilute with Diluent to obtain a solution having a known concentration of about 0.002 mg per mL of dantrolene related compound B.

Test solution— Use the Assay preparation.

Chromatographic system— Proceed as directed in the Assay. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the relative standard deviation for replicate injections for dantrolene related compound B is not more than 5.0%.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the peak responses. Calculate the percentage of dantrolene related compound B in the portion of Dantrolene Sodium for Injection taken by the formula: in which rU is the peak response for dantrolene related compound B obtained from the Test solution; rS is the corresponding peak response in the Standard solution; CS is the concentration, in mg per mL, of dantrolene related compound B in the Standard solution; and CT is the concentration, in mg per mL, of dantrolene sodium hydrate in the Test solution. Not more than 8% of dantrolene related compound B is found.

Buffer— Dissolve 3.3 g of ammonium acetate in 1 L of water, and adjust with acetic acid to a pH of 4.5 ± 0.1.

Solution A— Prepare a filtered and degassed mixture of Buffer, acetonitrile, and glacial acetic acid (120:80:7).

Solution B— Prepare a filtered and degassed mixture of acetonitrile and water (70:30).

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— Prepare a mixture of acetonitrile and water (60:40).

Standard preparation— Transfer 40 mg of USP Dantrolene RS, accurately weighed, into a 50-mL volumetric flask, and dissolve in 2.5 mL of dimethylformamide. Add 2.5 mL of glacial acetic acid, and dilute with acetone to volume to obtain a solution having a known concentration of about 0.8 mg per mL. Dilute this solution with Diluent to obtain a solution having a known concentration of about 0.08 mg per mL of dantrolene.

Assay preparation— Using 70 mL of water for each vial, transfer the entire contents of the required number of vials to a suitable flask necessary to obtain a solution having a known concentration of about 0.1 mg of dantrolene sodium hydrate per mL. Sonicate for 2 to 5 minutes to dissolve the sample. Dilute with Diluent to volume.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 365-nm detector and a 4.6-mm × 15-cm column that contains 5-µm packing L1. The flow rate is about 1.5 mL per minute. The chromatograph is programmed as follows. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the tailing factor is not more than 1.5; and the relative standard deviation for replicate injections for dantrolene is not more than 1.5%.

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 peak responses for dantrolene. Calculate the percentage of C14H9N4NaO5·3½H2O in the portion of Dantrolene Sodium for Injection taken by the formula: in which 399.29 and 314.25 are the molecular weights of dantrolene sodium hydrate and dantrolene, respectively; rU and rS are the peak responses for dantrolene obtained from the Assay preparation and the Standard preparation, respectively; CS is the concentration, in mg per mL, of dantrolene in the Standard preparation; and CU is the concentration, in mg per mL, of dantrolene sodium hydrate in the Assay preparation.