This test,where called for in the individual monograph,is provided to evaluate the impurity profile of an article.See Chromatography á621ñfor a general discussion of the thin-layer chromatographic technique.Unless otherwise specified in the individual monograph,use the following method.
Test Solution Prepare,in the solvent specified in the monograph,a solution of the substance under test having an accurately known final concentration of about 10mg per mL.[NOTEHeat or sonication may be used to dissolve the drug substance where use of such does not adversely affect the compound.]
Standard Solutions Prepare,in the solvent specified in the monograph,solutions of the USP Reference Standard or designated substance having accurately known concentrations of 0.01mg per mL,0.05mg per mL,0.1mg per mL,and 0.2mg per mL.[NOTEHeat or sonication may be used to dissolve the drug substance where use of such does not adversely affect the compound.]
Procedure Use a thin-layer chromatographic plate coated with a 0.25-mm layer of chromatographic silica gel mixture,and the Eluantspecified in the monograph.Apply equal volumes (20µL)of the Test Solutionand Standard Solutionsto the plate,using a stream of nitrogen to dry the spots.
Allow the chromatogram to develop in a pre-equilibrated chamber until the solvent front has moved about three-fourths of the length of the plate.Remove the plate from the chamber,and air-dry.View the plate using the visualization technique(s)specified.Locate any spots other than the principal spot,in the chromatogram of the Test Solution,and determine their relative intensities by comparison with the chromatograms of the appropriate Standard Solutions.The total of any ordinary impurities observed does not exceed 2.0%,unless otherwise specified in the individual monograph.
(1)Use UVlight at 254nm and at about 366nm.
(2)Use Iodoplatinate TS.
(3)Solution AMix 850mg of bismuth subnitrate with 40mLof water and 10mLof glacial acetic acid.
Solution BDissolve 8g of potassium iodide in 20mLof water.Mix Aand Btogether to obtain a Stock Solution which can be stored for several months in a dark bottle.Mix 10mLof the Stock Solution with 20mLof glacial acetic acid,and dilute with water to make 100mL,to prepare the spray reagent.
(4)Ninhydrin SprayDissolve 200mg of ninhydrin in 100mLof alcohol.Heat the plate after spraying.
(5)Acid SprayIn an ice bath,add slowly and cautiously,with stirring,10mLof sulfuric acid to 90mLof alcohol.Spray the plate,and heat until charred.
(6)Acid-Dichromate SprayAdd sufficient potassium dichromate to 100mLof sulfuric acid to make a saturated solution.Spray the plate,and heat until charred.
(7)VanillinDissolve 1g of vanillin in 100mLof sulfuric acid.
(8)Chloramine T-Trichloroacetic AcidMix 10mLof a 3%aqueous solution of chloramine Twith 40mLof a 25%alcoholic solution of trichloroacetic acid.Prepare immediately before use.
(9)Folin-CAdd 10g of sodium tungstate and 2.5g of sodium molybdate to 70mLof water,add 5mLof 85%phosphoric acid and 10mLof 36%hydrochloric acid,and reflux this solution for 10hours.
(10)KMnO4Dissolve 100mg of Potassium Permanganate in 100mLof water.
(11)DABMix 1g of p-dimethylaminobenzaldehyde in 100mLof 0.6Nhydrochloric acid.
(12)DACMix 100mg of p-dimethylaminocinnamaldehyde in 100mLof 1Nhydrochloric acid.
(13)FerricyanideMix equal volumes of a 1%ferric chloride solution and a 1%potassium ferricyanide solution.Use immediately.
(14)Fast Blue BReagent ADissolve 500mg of Fast Blue B Salt in 100mLof water.
Reagent B0.1Nsodium hydroxide.
Spray first with A,then with B.
(15)Alkaline Ferric CyanideDilute 1.5mLof a 1%potassium ferricyanide solution with water to 20mL,and add 10mLof 15%sodium hydroxide solution.
(16)Iodine SprayPrepare a 0.5%solution of iodine in chloroform.
(17)Expose the plate for 10minutes to iodine vapors in a pre-equilibrated closed chamber,on the bottom of which there are iodine crystals.
(18)Solution ADissolve 0.5g of potassium iodide in 50mLof water.
Solution BPrepare a solution of 0.5g of soluble starch in 50mLof hot water.
Just prior to use,mix equal volumes of Solution Aand Solution B.
(19)PTSSDissolve 20g of p-toluenesulfonic acid in 100mLof alcohol,spray the plate,dry for 15minutes at 110,and view under UVlight at 366nm.
(20)o-Tolidine SprayDissolve 160mg of o-tolidine in 30mLof glacial acetic acid,dilute with water to make 500mL,add 1g of potassium iodide,and mix until the potassium iodide has dissolved.
(21)Mix 3mLof chloroplatinic acid solution (1in 10)with 97mLof water,followed by the addition of 100mLof potassium iodide solution (6in 100)to prepare the spray reagent.
(22)IodineMethanol SprayPrepare a mixture of iodine TSand methanol (1:1).