The following chromatographic procedures are provided to confirm the identity of Pharmacopeial drug substances that are of the tetracycline type,such as doxycycline,oxytetracycline,and tetracycline,and to confirm the identity of such compounds in their respective Pharmacopeial dosage forms.Two procedures are provided,one based on paper chromatography (Method I)and the other on thin-layer chromatography (Method II).Method Iis to be used unless otherwise directed in the individual monograph.

METHOD I

pH3.5Buffer— Dissolve 13.4g of anhydrous citric acid and 16.3g of dibasic sodium phosphate in 1000mLof water,and mix.

Developing Solvent— On the day of use,mix 10volumes of chloroform,20volumes of nitromethane,and 3volumes of pyridine.

Mixed Test Solution— Mix equal volumes of the Standard Solutionand the Test Solution.

Chromatographic Sheet— Draw a spotting line 2.5cm from one edge of a 20-cm ×20-cm sheet of filter paper (Whatman No.1,or equivalent).Impregnate the sheet with pH3.5Bufferby passing it through a trough filled with pH3.5Buffer,and remove the excess solvent by firmly pressing the sheet between nonfluorescent blotting papers.

Procedure— To a suitable chromatographic chamber,prepared for ascending chromatography (see Chromatography á621ñ)add Developing Solventto a depth of 0.6cm.Apply at 1.5-cm intervals 2µLeach of the Standard Solution,the Test Solution,and the Mixed Test Solutionto the spotting line of the Chromatographic Sheet.Allow the sheet to dry partially,and while still damp place it in the chromatographic chamber with the bottom edge touching the Developing Solvent.When the solvent front has risen about 10cm,remove the sheet from the chamber,and expose the sheet to ammonia vapor.Examine the chromatogram under long-wavelength UVlight.Record the positions of the major yellow fluorescent spots:the RFvalue of the principal spot obtained from the Test Solutionand from the Mixed Test Solutioncorresponds to that obtained from the Standard Solution.

METHOD II

Resolution Solution— Unless otherwise directed in the individual monograph,prepare a solution in methanol containing 0.5mg each of USP Chlortetracycline Hydrochloride RS,USP Doxycycline Hyclate RS,USP Oxytetracycline RS,and USP Tetracycline Hydrochloride RSper mL.

Developing Solvent— Prepare a mixture of 0.5Moxalic acid,previously adjusted with ammonium hydroxide to a pHof 2.0,acetonitrile,and methanol (80:20:20).

Chromatographic Plate— Use a suitable thin-layer chromatographic plate (see Thin-layer Chromatographyunder Chromatography á621ñ)coated with a 0.25-mm layer of octylsilanized chromatographic silica gel mixture.Activate the plate by heating it at 130for 20minutes,allow to cool,and use while still warm.

Procedure— Separately apply 1µLeach of the Standard Solution,the Test Solution,and the Resolution Solutionto the Chromatographic Plate.Allow the spots to dry,and develop the chromatogram in the Developing Solventuntil 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 to air-dry.Expose the plate to ammonia vapors for 5minutes,and promptly locate the spots on the plate by viewing under long-wavelength UVlight:the chromatogram of the Resolution Solutionshows clearly separated spots,and the principal spot obtained from the Test Solutioncorresponds in RFvalue,intensity,and appearance to that obtained from the Standard Solution.