A: The RF value of the principal spot in the chromatogram of the Identification test preparation corresponds to that in the chromatogram of the Standard preparation, as obtained in the test for Chromatographic purity.

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, obtained as directed in the Assay.

Standard solution— Dissolve an accurately weighed quantity of USP Diluted Nitroglycerin RS in methanol, and dilute quantitatively with methanol to obtain a solution having a concentration of 400 µg of nitroglycerin per mL.

Identification test solution— Prepare a clear solution in methanol containing an amount of Diluted Nitroglycerin equivalent to about 400 µg of nitroglycerin per mL.

Test solution— Transfer an accurately weighed portion, equivalent to 100 mg of nitroglycerin, to a 5-mL volumetric flask. Dissolve (or suspend) in methanol, dilute with methanol to volume, and mix. Centrifuge a portion, if necessary, to obtain a clear liquid phase.

Procedure— Apply separately 20 µL of the Test solution, 5, 10, 15, and 20 µL of the Standard solution, and 20 µL of the Identification test solution to a suitable thin-layer chromatographic plate (see Chromatography 621) coated with a 0.25-mm layer of chromatographic silica gel mixture. Allow the spots to dry, position the plate in a chromatographic chamber, and develop the chromatograms in a solvent system consisting of a mixture of toluene and ethyl acetate (4:1) until the solvent front has moved about three-fourths of the length of the plate. Remove the plate from the developing chamber, mark the solvent front, and allow the solvent to evaporate. Spray the plate with a 1 in 100 solution of diphenylamine in methanol, and irradiate the plate with short- and long-wavelength UV light for about 15 minutes, and examine the chromatogram: any spot obtained from the Test solution, other than the principal spot, is not more intense than the spot in the chromatogram from the 20-µL application of the Standard solution. Compare the intensities of any secondary spots observed in the chromatogram of the Test solution with those of the principal spots in the chromatograms of the Standard solution (corresponding to 0.5%, 1.0%, 1.5%, and 2.0%, respectively): the sum of the intensities of the secondary spots obtained in the Test solution is not more than 3%. [note—Nitrates of glycerin typically have RF values of about 0.21, 0.37, and 0.61 for mono-, di-, and tri-substituted glycerins, respectively.]

Mobile phase— Prepare a degassed solution containing equal volumes of methanol and water, making adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve an accurately weighed quantity of USP Diluted Nitroglycerin RS in Mobile phase to obtain a solution having a known concentration of about 0.075 mg of nitroglycerin per mL.

Assay preparation— Transfer an accurately weighed portion of Diluted Nitroglycerin, equivalent to about 7.5 mg of nitroglycerin, to a 100-mL volumetric flask, and dissolve in about 75 mL of Mobile phase. If necessary, sonicate for 2 minutes or until the solid is totally dispersed, then shake by mechanical means for 30 minutes. Dilute with Mobile phase to volume, mix, and filter.

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, and if necessary a short precolumn that contains packing L1. The flow rate is about 1 mL per minute. Chromatograph replicate injections of the Standard preparation, and record the peak responses as directed for Procedure: the column efficiency is not less than 3000 theoretical plates, the tailing factor for the analyte peak is not more than 2.5, and the relative standard deviation is not more than 3.0%.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph by means of a microsyringe or sampling valve, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of C3H5N3O9 in the portion of Diluted Nitroglycerin taken by the formula: in which C is the concentration, in mg per mL, of nitroglycerin in the Standard preparation, and rU and rS are the peak responses of nitroglycerin obtained from the Assay preparation and the Standard preparation, respectively.