á521ñSULFONAMIDES

Identification of Individual Sulfonamides in Mixed Sulfonamides
NOTE—The following instructions for preparations and procedure are applicable to all sulfonamides except sulfadiazine.When testing for sulfadiazine proceed in the same manner,except to use sulfadiazine preparations having one-half the designated concentration,and apply twice the designated volumes of sulfadiazine preparations to the chromatographic plates.
Standard Preparation— Transfer a quantity of the pertinent USP Reference Standard to a suitable glass-stoppered,conical flask,dissolve in methanol to obtain a solution having a concentration of about 2mg per mL,and mix.Aseparate Standard Preparationis required for each sulfonamide present in mixed sulfonamides.
Test Preparation— Transfer a portion of the thoroughly mixed suspension or finely powdered tablets,equivalent to about 100mg of each sulfonamide,to a 50-mLvolumetric flask containing 10mLof ammonia TS,and swirl.Add methanol to volume,mix,filter,and use the filtrate in the Procedure.
Preparation of Chromatographic Plates— Prepare three identical chromatographic plates according to the following directions.Apply separately,and 2cm apart along a spotting line 1.5cm from the bottom of the plate and parallel to it,2µLof each Standard Preparationand 2µLof the Test Preparationto a suitable thin-layer chromatographic plate (see Chromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel mixture.On another spot,2cm along the spotting line from the application of the Test Preparation,apply,successively,2µLof each Standard Preparationto obtain a mixed standard.Dry the spots immediately with the aid of a stream of nitrogen.
Procedure— Prepare a chromatographic chamber lined with filter paper and containing a solvent system consisting of ethyl acetate,methanol,and a 1in 4aqueous solution of ammonium hydroxide (17:6:5),and allow to equilibrate for 1hour.Similarly prepare a second chamber to contain a solvent system consisting of solvent hexane,chloroform,and butyl alcohol (1:1:1),and a third chamber to contain a solvent system consisting of chloroform and methanol (95:5).Place one prepared chromatographic plate in each equilibrated chamber,and develop the chromatograms until the solvent front has moved about three-fourths of the length of each plate.Remove each plate from its developing chamber,mark the solvent front,and allow the solvent to evaporate.Locate the spots on the plates by viewing under short-wavelength UVlight.Spray the plates with a 1in 100solution of p-dimethylaminobenzaldehyde in dilute hydrochloric acid (1in 20),and heat at 110for 5minutes or until bright yellow spots become visible.The RFvalues of the yellow spots obtained from each Test Preparationcorrespond to those obtained from the mixed Standard Preparationson the respective plates.The individual sulfonamides may be identified by comparison of the RFvalues of the yellow spots obtained from the Test Preparationsand individual Standard Preparationson the respective plates.

Determination of Individual Sulfonamides in Mixed Sulfonamides
Standard Preparation— Aseparate Standard Preparationis required for each sulfonamide being determined.Transfer about 50mg,accurately weighed,of the pertinent USP Reference Standard to a 50-mLvolumetric flask containing 1.5mLof ammonium hydroxide,add methanol,dissolve in methanol,dilute with methanol to volume,and mix.Transfer 1.0mLof this solution to a 100-mLvolumetric flask,add dilute hydrochloric acid (1in 100)to volume,and mix.[NOTE—Retain the methanol solutions for the Mixed Standard Preparation.The methanol solutions are stable for at least 1week,and the acid solutions for at least 1month.]
Mixed Standard Preparation— Transfer 1.0mLof each methanol solution,prepared as required for each Standard Preparation,to a small glass-stoppered flask,and mix.[NOTE—This Standard is used to identify the components of the Assay Preparationon the chromatogram.]
Assay Preparation— Prepare as directed in the individual monograph.
Procedure— Prepare the necessary number of chromatographic sheets (Whatman No.1filter paper,or equivalent),about 20×20cm in size,by drawing a pencil line parallel to and 2.5cm from one edge of the paper.Mark the line at points 2.5and 5cm from each edge of the paper.Impregnate the paper by dipping it in the immobile solvent (prepared fresh by dissolving 30mLof redistilled formamide in 70mLof acetone)for 30seconds.Remove the paper,drain for 10seconds,and blot between filter paper.Place the impregnated paper on dry filter paper,and air-dry for 3to 5minutes.With a micropipet,and with repeated applications,streak 100µLof the Assay Preparationalong the starting line,applying the volume in five streaks of about 20µLeach and evaporating the solvent with a gentle stream of nitrogen between applications.[NOTE—Make the streak as narrow as possible along the starting line,and keep within the 5-cm border.]Rinse the tip of the pipet with a drop of methanol-ammonia TSmixture (9:1),and then streak the rinse along the starting line between the 5-and 2.5-cm points at the right edge.Repeat the rinsing with two additional drops,and then blow out the pipet.
Apply 10µLof the Mixed Standard Preparationat the mark 2.5cm from the left edge.
Place 50mLof methylene chloride (mobile solvent)in a tray in a 23-×23-×7.5-cm chromatographic chamber arranged for ascending chromatography (see Chromatography á621ñ),and allow the chamber to equilibrate for about 15minutes.Remove the cover,place from 7to 10mLof water in a second tray,and without delay,suspend the prepared chromatographic paper sheet so that it dips into the mobile solvent.Cover and seal the chamber,and allow the chromatogram to develop for 1hour.Remove the paper from the chamber,and allow to air-dry for 5minutes.Place the chromatogram on a dry sheet of filter paper,and view it under short-wavelength UVlight.[NOTE—Conduct the following identification and marking without delay to avoid excessive exposure of the sulfonamide spots to UVirradiation.]Identify and mark the respective spots by matching RFvalues with those of the spots produced by the Mixed Standard Preparation.[NOTE—Sulfadiazine and sulfamerazine are chromatographed with increasing RF,respectively.]
Cut the marked zones from the paper,cut each zone into five or six pieces,and place the pieces from each spot in separate,glass-stoppered,50-mLflasks.Add 20.0mLof dilute hydrochloric acid (1in 100)to each flask,and allow to stand for about 30minutes,swirling each flask at least five times during this period.Filter the solutions through dry glass wool into separate test tubes,discarding the first 5mLof the filtrate.Transfer 5.0mLof the subsequent filtrate from each solution into separate 10-mLvolumetric flasks.Transfer 3.0mLof each required Standard Preparationinto separate,10-mLvolumetric flasks.To each flask,and to a blank flask containing 5mLof dilute hydrochloric acid (1in 100),add 1.0mLof sodium nitrite solution (1in 1000)and 0.10mLof hydrochloric acid,and allow to stand for 5minutes with frequent swirling.To each flask add 1.0mLof ammonium sulfamate solution (1in 200),and allow to stand for 5minutes,swirling frequently.Finally,to each flask add 1.0mLof freshly prepared N-(1-naphthyl)ethylenediamine dihydrochloride solution (1in 1000),mix,dilute with water to volume,and mix.Allow each solution to stand between 15and 60minutes,and then concomitantly determine the absorbances of the solutions,in 1-cm cells,recording the spectra from 440to 700nm,with a suitable spectrophotometer,using the blank to set the instrument.Draw a baseline,and determine the corrected absorbance for each solution at the wavelength of maximum absorbance at about 545nm.
Calculate the concentration,in mg per mL,of each sulfonamide in the Assay Preparationby the formula:
0.12C(AU/AS),
in which Cis the concentration,in µg per mL,of the pertinent USP Reference Standard in the Standard Preparation;AUis the corrected absorbance of the Assay Preparation;and ASis the corrected absorbance of the pertinent Standard Preparation.From the concentration of the Assay Preparationthus determined,and applying appropriate dilution factors,calculate the percentage of sulfonamide in the specimen taken.