A: Infrared Absorption 197K.

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

C: Examine in visible light the thin-layer chromatograph prepared as directed in the test for Related glycosides: the RF value of the principal blue spot obtained from the Test solution corresponds to that obtained from the Standard solution.

Chloramine T–trichloroacetic acid reagent— Mix 10 mL of a freshly prepared solution of chloramine T (3 in 100) and 40 mL of a 1 in 4 solution of trichloroacetic acid in dehydrated alcohol.

Spotting solvent— Prepare a mixture of chloroform and methanol (2:1).

Standard solution— Dissolve an accurately weighed quantity of USP Digoxin RS in Spotting solvent to obtain a solution containing 10 mg per mL.

Gitoxin standard solution— Dissolve an accurately weighed quantity of USP Gitoxin RS in Spotting solvent to obtain a solution containing 0.30 mg per mL.

Test solution— Transfer 250.0 mg of Digoxin to a 25-mL volumetric flask, dissolve in and dilute with Spotting solvent to volume, and mix.

Procedure— Apply 10 µL of the Test solution, 10 µL of the Standard solution, and 10 µL of the Gitoxin standard solution on a line parallel to and about 2.5 cm from the bottom edge of a reversed-phase thin-layer chromatographic plate coated with a 0.25-mm layer of chromatographic silica gel mixture to which is permanently bonded octadecylsilane (C18). Allow the spots to dry, and place the plates in a developing chamber containing a mixture of methanol and water (7:3). Develop the chromatogram until the solvent front has moved about 15 cm above the line of application. Remove the plate, and allow the solvent to evaporate. Spray the plate with Chloramine T–trichloroacetic acid reagent, freshly mixed, and heat in an oven at 110 for 10 minutes. Examine the plate under long-wavelength UV light: no spot from the Test solution except that due to digoxin is more intense than the spot from the Gitoxin standard solution (not more than 3% of any related glycoside as gitoxin).

Solvent— Use dimethyl sulfoxide.

(Official January 1, 2007)

Mobile phase— Prepare a suitable degassed and filtered mixture of water and acetonitrile (37:13), making adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve an accurately weighed quantity of USP Digoxin RS in diluted alcohol, and dilute quantitatively and stepwise with diluted alcohol to obtain a solution having a known concentration of about 250 µg per mL. Use a sonic bath to aid dissolution.

Assay preparation— Transfer about 50 mg of Digoxin, accurately weighed, to a 200-mL volumetric flask. Dissolve in about 150 mL of diluted alcohol by sonication, dilute with diluted alcohol to volume, and mix.

System suitability preparation— Prepare a solution in diluted alcohol of USP Digoxin RS and digoxigenin having concentrations of about 40 µg of each per mL.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 218-nm detector and a 4.2-mm × 25-cm column that contains packing L1 and a 3.2-mm × 15-mm guard column that contains packing L1. The flow rate is about 3.0 mL per minute. Chromatograph the System suitability preparation, and record the peak responses as directed for Procedure: the resolution, R, between digoxin and digoxigenin is not less than 4.0; the column efficiency determined from the digoxin peak is not less than 1200 theoretical plates; the tailing factor for the digoxin peak is not more than 2.0; and the relative standard deviation for replicate injection 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 C41H64O14 in the portion of Digoxin taken by the formula: in which C is the concentration, in µg per mL, of USP Digoxin RS in the Standard preparation; and rU and rS are the responses for the digoxin peaks obtained from the Assay preparation and the Standard preparation, respectively.