á201ñTHIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST

GENERAL PROCEDURE
The following procedure is applicable as an aid in verifying the identities of many compendial drug substances as such and in their respective dosage forms.
Prepare a test solution as directed in the individual monograph.On a line parallel to and about 2cm from the edge of a suitable thin-layer chromatographic plate,coated with a 0.25-mm layer of chromatographic silica gel mixture (see Chromatography á621ñ)apply 10µLof this solution and 10µLof a Standard solution prepared from the USP Reference Standard for the drug substance being identified,in the same solvent and at the same concentration as the test solution,unless otherwise directed in the individual monograph.Allow the spots to dry,and develop the chromatogram in a solvent system consisting of a mixture of chloroform,methanol,and water (180:15:1),unless otherwise directed in the individual monograph,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.Unless otherwise directed in the individual monograph,locate the spots on the plate by examination under short-wavelength UVlight.The RFvalue of the principal spot obtained from the test solution corresponds to that obtained from the Standard solution.

PROCEDURE FOR BACITRACIN,NEOMYCIN,AND POLYMYXIN B
The following thin-layer chromatographic procedure is applicable as an aid in verifying the identities of bacitracin,neomycin,and polymyxin Bactive ingredients and in dosage forms when present singly and in two-and three-component mixtures.The reference á201BNPñin a monograph signifies that this procedure is intended.
Prepare aTest Solution as follows,unless otherwise directed in the individual monograph.
Test Solution
FOR DRUG SUBSTANCES— Dissolve a portion of Bacitracin,Bacitracin Zinc,Neomycin Sulfate,or Polymyxin B Sulfate in 0.1Nhydrochloric acid to obtain a solution containing about 500USP Bacitracin Units per mL,3.5mg of neomycin (base)per mL,or 10,000USP Polymyxin B Units per mL.
FOR SOLUTIONS— Where the Solution contains neomycin and polymyxin B,dilute a portion of it with 0.1Nhydrochloric acid to obtain a solution containing the equivalent of about 3.5mg of neomycin (base)per mL.Where the Solution contains polymyxin Bbut not neomycin,dilute a portion of it with 0.1Nhydrochloric acid to obtain a solution containing about 10,000USP Polymyxin B Units per mL.
FOR CREAMS,LOTIONS,AND OINTMENTS— Where the Cream,Lotion,or Ointment contains Bacitracin or Bacitracin Zinc,transfer a portion of it equivalent to about 500USP Bacitracin Units,to a 15-mLcentrifuge tube.Where the Cream,Lotion,or Ointment contains neomycin,but not Bacitracin or Bacitracin Zinc,transfer a portion of it equivalent to about 3.5mg of neomycin (base)per mLto a 15-mLcentrifuge tube.Add 4mLof chloroform to the centrifuge tube,and shake well to disperse the Cream,Lotion,or Ointment.Add 1mLof 0.1Nhydrochloric acid,vortex for 4minutes,centrifuge,and use the clear supernatant.
NOTE—The Modified Test Solutionas described below in the Modified Procedure may be used in lieu of the Test Solution.
Standard Bacitracin Solution— Dissolve a portion of USP Bacitracin Zinc RSin 0.1Nhydrochloric acid to obtain a solution containing 500USP Bacitracin Units per mL.
Standard Neomycin Solution— Dissolve a portion of USP Neomycin Sulfate RSin 0.1Nhydrochloric acid to obtain a solution containing the equivalent of 3.5mg of neomycin (base)per mL.
Standard Polymyxin B Solution— Dissolve a portion of USP Polymyxin B Sulfate RSin 0.1Nhydrochloric acid to obtain a solution containing 10,000USP Polymyxin B Units per mL.Where the article under test also contains Bacitracin or Bacitracin Zinc,dissolve a portion of USP Polymyxin B Sulfate RSin 0.1Nhydrochloric acid to obtain a solution containing 500JUSP Polymyxin B Units per mL,Jbeing the ratio of the labeled amount of USP Polymyxin B Units to the labeled amount of USP Bacitracin Units in each g of Cream,Lotion,or Ointment.
Developing Solvent Solution— Prepare a mixture of methanol,isopropyl alcohol,methylene chloride,ammonium hydroxide,and water (4:2:2:2:1.5).
Procedure— Apply 10µLof the Test Solutionand each of the relevant Standard Solutionsto a suitable thin-layer chromatographic plate (see Chromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel.Place the plate in a presaturated chromatographic chamber,and develop the chromatogram with the Developing Solvent Systemuntil the solvent front has moved about three-fourths of the length of the plate.Remove the plate from the chamber,and dry at 105for 10minutes.Spray the plate with a 0.2%solution of ninhydrin in butyl alcohol,and heat at 105for 5minutes.The RFvalue of each principal spot in the chromatogram of the Test Solutioncorresponds to that of the principal spot in the chromatogram obtained from each relevant Standard Solutionas appropriate for the labeled active ingredient or ingredients specified on the label.If the chromatogram of the Test Solution yields excessive streaking,proceed as directed for Modified Procedure.
Modified Procedure— Transfer the Test Solutionto a 15-mLcentrifuge tube,add 10mLof saturated aqueous picric acid solution (1.2%,w/v),vortex for 1minute,centrifuge for 10minutes,and discard the supernatant.Wash the residue with 1-mLportions of water until no yellow color is observed in the washing.Discard the washings,and dry the residue under a stream of nitrogen at 50.Dissolve the residue in 1mLof acetone,add 1mLof a freshly prepared solution of sulfuric acid in acetone (1in 100),shake,centrifuge for 5minutes,and discard the supernatant.Rinse the residue with 1mLof acetone,centrifuge briefly,and discard the washing.Repeat the washing until no yellow color is observed.Dry the residue under a stream of nitrogen at 50.Dissolve the residue in 0.5mLof 0.1Nhydrochloric acid (Modified Test Solution).Repeat the Procedureusing this Modified Test Solution instead of the Test Solution.The RFvalue of each principal spot in the chromatogram of the Modified Test Solutioncorresponds to that of the principal spot in the chromatogram obtained from each relevant Standard Solutionas appropriate for the active ingredient or ingredients specified on the label.