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.

C: Ignite about 200 mg: the residue meets the requirements of the flame test for Sodium 191.

Test stock solution— Prepare as directed for the Assay stock preparation as directed in the Assay.

Mobile phase— Prepare a filtered and degassed mixture of acetonitrile and water (80:20). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard solution— Dissolve an accurately weighed quantity of USP Dantrolene Related Compound A RS and USP Dantrolene Sodium RS in dimethylformamide to obtain a solution having known concentrations of 17.5 µg per mL of dantrolene related compound A and 50 µg per mL of dantrolene sodium. Dilute with acetonitrile to obtain a solution having concentrations of about 0.35 µg per mL of dantrolene related compound A and 1 µg per mL of dantrolene sodium.

Test solution— Dilute the Test stock solution with acetonitrile to obtain a solution having a concentration of about 0.175 mg per mL of dantrolene sodium.

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 1.0 mL per minute. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the tailing factor for dantrolene related compound A is not more than 1.5; and the relative standard deviation for replicate injections is not more than 5% for dantrolene related compound A. [note—The dantrolene peak elutes at void volume at approximately 1.5 minutes.]

Procedure— Inject equal volumes (about 20 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the response for the dantrolene related compound A peak. Calculate the percentage of dantrolene related compound A in the portion of Dantrolene Sodium taken by the formula: in which rU and rS are the peak responses of dantrolene related compound A obtained from the Test solution and the Standard solution, respectively; CS is the concentration, in mg per mL, of dantrolene related compound A in the Standard solution; and CT is the concentration, in mg per mL, of Dantrolene Sodium in the Test solution: not more than 0.15% of dantrolene related compound A is found.

Mobile phase, System suitability stock solution B, Diluent, and Chromatographic system— Prepare as directed in the Assay.

Standard solution— Dilute quantitatively the System suitability stock solution B with Diluent to obtain a solution having a known concentration of 0.25 µg per mL each of dantrolene related compound B and dantrolene related compound C.

Test solution— Use the Assay preparation.

Procedure— Inject equal volumes (about 20 µL) of the Standard solution and Test solution into the chromatograph, record the chromatograms, and measure the peak responses. Calculate the percentage of each of the relevant dantrolene related compounds in the portion of Dantrolene Sodium taken by the formula: in which CS is the concentration, in mg per mL, of dantrolene related compound B or dantrolene related compound C in the Standard solution; CT is the concentration, in mg per mL, of Dantrolene Sodium in the Test solution; rU is the individual peak response for dantrolene related compound B or dantrolene related compound C obtained from the Test solution; and rS is the response of the corresponding peak obtained from the Standard solution. Not more than 0.50% of dantrolene related compound B is found; and not more than 0.30% of dantrolene related compound C is found.

Buffer— Dissolve 3.85 g of ammonium acetate in 1.0 L of water, and adjust with glacial acetic acid to a pH of 4.5 ± 0.1.

Diluent— Prepare a mixture of water and acetonitrile (50:50).

Solution A— Prepare a filtered and degassed mixture of water, Buffer, and acetonitrile (70:20:10).

Solution B— Prepare a filtered and degassed mixture of acetonitrile and Buffer (80:20).

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).

System suitability stock solution A— Transfer 62.5 mg of USP Dantrolene Sodium 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 1.25 mg per mL of dantrolene sodium.

System suitability stock solution B— Transfer 6.3 mg each of USP Dantrolene Related Compound B RS and USP Dantrolene Related Compound C 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 known concentrations of about 0.125 mg per mL each of dantrolene related compound B and dantrolene related compound C.

System suitability solution— Quantitatively dilute suitable volumes of System suitability stock preparation A and System suitability stock preparation B with Diluent to obtain a solution having concentrations of about 0.125 mg per mL of dantrolene sodium and 2.5 µg per mL each of dantrolene related compound B and dantrolene related compound C.

Standard stock preparation— Transfer 50 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 1.0 mg per mL of dantrolene.

Standard preparation— Dilute the Standard stock preparation with Diluent to obtain a solution having a known concentration of about 0.1 mg per mL of dantrolene.

Assay stock preparation— Transfer an accurately weighed quantity of dantrolene sodium into a 100-mL volumetric flask, and dissolve in 5 mL of dimethylformamide. Add 5 mL of glacial acetic acid, and dilute with acetone to volume to obtain a solution having a concentration of about 1.25 mg per mL of dantrolene sodium.

Assay preparation— Dilute the Assay stock preparation with Diluent to obtain a solution having a concentration of about 0.125 mg per mL of dantrolene sodium.

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 L1 packing. The flow rate is about 2.0 mL per minute. The chromatograph is programmed as follows. Chromatograph the System suitability solution, and record the peak responses as directed for Procedure: the resolution, R, between dantrolene and dantrolene related compound C is not less than 8. 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 is not more than 1.0%. [note—For the purpose of identification, the approximate relative retention times are 0.68 for dantrolene related compound B, 1.24 for dantrolene related compound C, and 1.0 for dantrolene.]

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the dantrolene peaks. Calculate the percentage of dantrolene (C14H10N4O5) in the portion of Dantrolene Sodium taken by the formula: in which CS is the concentration, in mg per mL, of dantrolene in the Standard preparation; CU is the concentration, in mg per mL, of Dantrolene Sodium in the Assay preparation; and rU and rS are the peak responses for dantrolene obtained from the Assay preparation and the Standard preparation, respectively.