Test solution— Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 120 mg of isosorbide mononitrate, to a suitable container, add 50.0 mL of absolute alcohol, sonicate for 10 minutes, and then centrifuge. Quantitatively dilute the supernatant (10 in 50) with absolute alcohol.

Standard solution: a solution of USP Diluted Isosorbide Mononitrate RS in absolute alcohol containing 0.5 mg of isosorbide mononitrate per mL.

Application volume: 20 µL.

Developing solvent system: a mixture of chloroform and methanol (95:5).

Spray reagent— Dissolve 1 g of soluble starch in 100 mL of boiling water. Cool, add 0.5 g of potassium iodide, and mix to dissolve.

Procedure— Examine the plate under short-wavelength UV light, marking any observed spots. Visualize nitrates on the plate by spraying with Spray reagent and illuminating with short-wavelength UV light for about 10 minutes. Isosorbide mononitrate and other nitrates appear as a violet spot on a white to light violet background.

Determine the amount of C6H9NO6 dissolved by employing the following method.

Adsorbent, Standard solution 1, Standard solution 2, Standard solution 3, Application volume, and Developing solvent system— Prepare as directed in Related compounds, Test 1 under Diluted Isosorbide Mononitrate.

Test solution— Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 100 mg of isosorbide mononitrate, to a suitable container, add 20.0 mL of absolute alcohol, sonicate for 10 minutes, and then centrifuge. Use the supernatant.

Procedure— Proceed as directed for Thin-Layer Chromatography under Chromatography 621. After developing, dry the plate with warm air for about 10 minutes, dip the plate in a solution prepared by dissolving 1.25 g of potassium permanganate and 10.0 g of sodium hydroxide in 500 mL of water (prepared fresh for each plate), and heat at 105 for 5 minutes. Any spot in the chromatogram obtained from the Test solution and corresponding to the RF value of the spots obtained from the Standard solutions is not more intense than the spot in the chromatogram obtained from Standard solution 3: not more than 1.0% of any individual impurity is found. If the spot in the chromatogram obtained from the Test solution is nearly as intense as the spot obtained from Standard solution 3, further dilute the Test solution (1:1) with absolute alcohol, repeat the test, and compare the intensity of the isosorbide spot in the diluted Test solution with the intensity of the spots obtained from the Standard solutions, correcting the percentage level for the additional dilution of the Test solution.

Mobile phase and Resolution solution— Proceed as directed in the Assay.

Isosorbide mononitrate related compound A standard stock solution— Dissolve an accurately weighed quantity of USP Diluted Isosorbide Mononitrate Related Compound A RS in methanol, and dilute quantitatively, and stepwise if necessary, with methanol to obtain a solution having a known concentration of about 0.1 mg of isosorbide mononitrate related compound A per mL.

Isosorbide dinitrate standard stock solution— Dissolve an accurately weighed quantity of USP Diluted Isosorbide Dinitrate RS in methanol, and dilute quantitatively, and stepwise if necessary, with methanol to obtain a solution having a known concentration of about 0.1 mg of isosorbide dinitrate per mL.

Standard solution— Transfer a quantity of USP Diluted Isosorbide Mononitrate RS, accurately weighed, to a suitable volumetric flask. Dissolve in water, quantitatively add a volume of Isosorbide mononitrate related compound A standard stock solution and a volume of Isosorbide dinitrate standard stock solution, and dilute with water to volume to obtain a solution having a known concentration of about 0.1 mg of isosorbide mononitrate per mL, 0.0005 mg of isosorbide mononitrate related compound A per mL, and 0.0005 mg of isosorbide dinitrate per mL. Filter a portion of the solution, discarding the first few mL of the filtrate.

Test solution— Use the Assay preparation, prepared as directed in the Assay.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 220-nm detector and a 4.6-mm × 25-cm column that contains packing L1. The flow rate is about 1.0 mL per minute. Chromatograph the Resolution solution, and record the peak responses as directed for Procedure: the resolution, R, between isosorbide mononitrate related compound A and isosorbide mononitrate is not less than 1.5. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the relative standard deviation for replicate injections is not more than 10% for the isosorbide mononitrate related compound A and isosorbide dinitrate peaks.

Procedure— Separately inject equal volumes (about 50 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the areas for the major peaks. Compare the peak areas of the corresponding impurity obtained from the Test solution and the Standard solution, respectively. The average peak area of the impurity in the Test solution is less than or equal to the average peak area of the corresponding peak in the Standard solution: not more than 0.5% of isosorbide mononitrate related compound A is found; and not more than 0.5% of isosorbide dinitrate is found.

200C(rU / rS)