A: The RFvalue of the principal spot in the chromatogram of the Identification test preparationcorresponds 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 preparationcorresponds 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 RSin methanol,and dilute quantitatively with methanol to obtain a solution having a concentration of 400µg of nitroglycerin per mL.

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

Test solution— Transfer an accurately weighed portion,equivalent to 100mg of nitroglycerin,to a 5-mLvolumetric 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 20µLof the Test solution,5,10,15,and 20µLof the Standard solution,and 20µLof the Identification test solutionto a suitable thin-layer chromatographic plate (see Chromatography á621ñ)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 1in 100solution of diphenylamine in methanol,and irradiate the plate with short-and long-wavelength UVlight for about 15minutes,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-µLapplication of the Standard solution.Compare the intensities of any secondary spots observed in the chromatogram of the Test solutionwith 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 solutionis not more than 3%.[NOTE—Nitrates of glycerin typically have RFvalues of about 0.21,0.37,and 0.61for 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 Suitabilityunder Chromatography á621ñ).

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

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

Chromatographic system(see Chromatography á621ñ)— 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 1mLper minute.Chromatograph replicate injections of the Standard preparation,and record the peak responses as directed for Procedure:the column efficiency is not less than 3000theoretical 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 20µL)of the Standard preparationand the Assay preparationinto 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 C3H5N3O9in the portion of Diluted Nitroglycerin taken by the formula: in which Cis the concentration,in mg per mL,of nitroglycerin in the Standard preparation,and rUand rSare the peak responses of nitroglycerin obtained from the Assay preparationand the Standard preparation,respectively.