Identification—
A:
Chloramine T–trichloroacetic acid reagent—Prepare as directed for
Identification test
B under
Digoxin Oral Solution.
Spotting solvent—
Use dehydrated alcohol.
Test solution—
Transfer an accurately weighed portion of finely powdered Tablets, equivalent to 0.5 mg of digoxin, to a 10-mL centrifuge tube. Add 2 mL of Spotting solvent, sonicate for 10 to 15 minutes, and centrifuge. Decant and use the supernatant.
Standard solution—
Dissolve an accurately weighed quantity of
USP Digoxin RS in
Spotting solvent to obtain a solution having a known concentration of about 0.25 mg per mL.
Procedure—
Proceed as directed for
Procedure in the test for
Related glycosides under
Digoxin, except to omit the use of the
Gitoxin standard solution. Examine the plate under long-wavelength UV light: the
RF value of the principal spot obtained from the
Test solution corresponds to that obtained from the
Standard solution.
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.
Dissolution 
711
—
[NOTE—Throughout this procedure, use scrupulously clean glassware, which previously has been rinsed successively with hydrochloric acid, water, and alcohol, and carefully dried. Take precautions to prevent contamination from fluorescent particles and from metal and rubber surfaces.
]
Medium:
0.1 N hydrochloric acid; 500 mL. [NOTE—Use the same batch of Dissolution Medium throughout the test.]
Apparatus 1:
120 rpm.
Time:
60 minutes.
Ascorbic acid–methanol solution—
Prepare a solution containing 2 mg of ascorbic acid per mL of methanol.
Hydrogen peroxide–methanol solution—
On the day of use, dilute 2.0 mL of recently assayed 30 percent hydrogen peroxide with methanol to 100 mL. Store in a refrigerator. Just prior to use, dilute 2.0 mL of this solution with methanol to 100 mL.
Standard solutions—
Transfer about 25 mg of
USP Digoxin RS, accurately weighed, to a 500-mL volumetric flask. Dissolve in a minimum amount of alcohol, add dilute alcohol (4 in 5) to volume, and mix. Dilute 10.0 mL of this solution with dilute alcohol (4 in 5) to 100.0 mL, and mix. Just prior to use, dilute suitable aliquots of the resulting solution with
Dissolution Medium to 50.0 mL to prepare
Standard solutions equivalent to 20%, 40%, 60%, 80%, and 100%, respectively, of the labeled amount of digoxin in 500 mL.
Test solution—
Promptly after withdrawal, pass a portion of the solution under test through a filter having a 0.8-µm or finer porosity, discarding the first 10 mL of the filtrate. This is the Test solution.
Procedure—
Transfer to individual glass-stoppered flasks duplicate 1.0-mL portions of the Test solution, 1.0-mL portions of each of the Standard solutions, and 1.0 mL of the Medium to provide a blank. Begin with the Standard solutions, and keep all flasks in the same sequence throughout, so that the elapsed time from addition of reagents to reading of fluorescence is the same for each flask in the set. Treating one flask at a time, add the following three reagents, in the order named, in as rapid a sequence as possible, swirling after each addition: 1.0 mL of Ascorbic acid–methanol solution, 5.0 mL of hydrochloric acid, and 1.0 mL of Hydrogen peroxide–methanol solution. Insert the stoppers in the flasks, and after 2 hours, measure the fluorescence at about 485 nm, the excitation wavelength being about 372 nm. To check the stability of the fluorometer, repeat the measurement of fluorescence on one or more treated Standard solutions. Correct each reading for the blank, and plot a standard curve of fluorescence versus percentage dissolution. Determine the percentage dissolution of digoxin in the Test solution by reading from the standard graph.
Tolerances—
Not less than 80%
(Q) of the labeled amount of C
41H
64O
14 is dissolved in 60 minutes. The requirement is met if the quantities dissolved from the Tablets tested conform to the accompanying acceptance table instead of the table shown under
Dissolution 711.
Acceptance Table
| Stage |
Number
Tested |
Acceptance
Criteria |
| S1 |
6 |
Each unit is not less than Q + 5%. |
| S2 |
6 |
Average of 12 units (S1 + S2) is equal to or greater than Q, and no unit is less than Q  5%. |
Assay—
Mobile phase, Chromatographic system, and System suitability preparation—
Proceed as directed in the
Assay under
Digoxin.
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 40 µg per mL. Use a sonic bath to aid dissolution.
Assay preparation—
Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 1 mg of digoxin, to a glass-stoppered, 50-mL conical flask. Add 25.0 mL of diluted alcohol with swirling, sonicate for about 30 minutes, and cool. Filter a portion of this solution through a 0.8-µm porosity membrane filter, discarding the first 10 mL of the filtrate.
Procedure—
Separately inject equal volumes (about 50 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 C
41H
64O
14 in the portion of Tablets taken by the formula:
25C(rU / rS),
in which
C is the concentration, in mg 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.
