Identification of Individual Sulfonamides in Mixed Sulfonamides
noteThe 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 2 mg per mL, and mix. A separate Standard Preparation is 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 100 mg of each sulfonamide, to a 50-mL volumetric flask containing 10 mL of 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 2 cm apart along a spotting line 1.5 cm from the bottom of the plate and parallel to it, 2 µL of each Standard Preparation and 2 µL of the Test Preparation to a suitable thin-layer chromatographic plate (see Chromatography 621) coated with a 0.25-mm layer of chromatographic silica gel mixture. On another spot, 2 cm along the spotting line from the application of the Test Preparation, apply, successively, 2 µL of each Standard Preparation to 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 1 in 4 aqueous solution of ammonium hydroxide (17:6:5), and allow to equilibrate for 1 hour. 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 UV light. Spray the plates with a 1 in 100 solution of p-dimethylaminobenzaldehyde in dilute hydrochloric acid (1 in 20), and heat at 110 for 5 minutes or until bright yellow spots become visible. The RF values of the yellow spots obtained from each Test Preparation correspond to those obtained from the mixed Standard Preparations on the respective plates. The individual sulfonamides may be identified by comparison of the RF values of the yellow spots obtained from the Test Preparations and individual Standard Preparations on the respective plates.
Determination of Individual Sulfonamides in Mixed Sulfonamides
Standard Preparation A separate Standard Preparation is required for each sulfonamide being determined. Transfer about 50 mg, accurately weighed, of the pertinent USP Reference Standard to a 50-mL volumetric flask containing 1.5 mL of ammonium hydroxide, add methanol, dissolve in methanol, dilute with methanol to volume, and mix. Transfer 1.0 mL of this solution to a 100-mL volumetric flask, add dilute hydrochloric acid (1 in 100) to volume, and mix. [noteRetain the methanol solutions for the Mixed Standard Preparation. The methanol solutions are stable for at least 1 week, and the acid solutions for at least 1 month.]
Mixed Standard Preparation Transfer 1.0 mL of each methanol solution, prepared as required for each Standard Preparation, to a small glass-stoppered flask, and mix. [noteThis Standard is used to identify the components of the Assay Preparation on the chromatogram.]
Assay Preparation Prepare as directed in the individual monograph.
Procedure Prepare the necessary number of chromatographic sheets (Whatman No. 1 filter paper, or equivalent), about 20 × 20 cm in size, by drawing a pencil line parallel to and 2.5 cm from one edge of the paper. Mark the line at points 2.5 and 5 cm from each edge of the paper. Impregnate the paper by dipping it in the immobile solvent (prepared fresh by dissolving 30 mL of redistilled formamide in 70 mL of acetone) for 30 seconds. Remove the paper, drain for 10 seconds, and blot between filter paper. Place the impregnated paper on dry filter paper, and air-dry for 3 to 5 minutes. With a micropipet, and with repeated applications, streak 100 µL of the Assay Preparation along the starting line, applying the volume in five streaks of about 20 µL each and evaporating the solvent with a gentle stream of nitrogen between applications. [noteMake 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 TS mixture (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 10 µL of the Mixed Standard Preparation at the mark 2.5 cm from the left edge.
Place 50 mL of methylene chloride (mobile solvent) in a tray in a 23- × 23- × 7.5-cm chromatographic chamber arranged for ascending chromatography (see Chromatography 621), and allow the chamber to equilibrate for about 15 minutes. Remove the cover, place from 7 to 10 mL of 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 1 hour. Remove the paper from the chamber, and allow to air-dry for 5 minutes. Place the chromatogram on a dry sheet of filter paper, and view it under short-wavelength UV light. [noteConduct the following identification and marking without delay to avoid excessive exposure of the sulfonamide spots to UV irradiation.] Identify and mark the respective spots by matching RF values with those of the spots produced by the Mixed Standard Preparation. [noteSulfadiazine 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-mL flasks. Add 20.0 mL of dilute hydrochloric acid (1 in 100) to each flask, and allow to stand for about 30 minutes, swirling each flask at least five times during this period. Filter the solutions through dry glass wool into separate test tubes, discarding the first 5 mL of the filtrate. Transfer 5.0 mL of the subsequent filtrate from each solution into separate 10-mL volumetric flasks. Transfer 3.0 mL of each required Standard Preparation into separate, 10-mL volumetric flasks. To each flask, and to a blank flask containing 5 mL of dilute hydrochloric acid (1 in 100), add 1.0 mL of sodium nitrite solution (1 in 1000) and 0.10 mL of hydrochloric acid, and allow to stand for 5 minutes with frequent swirling. To each flask add 1.0 mL of ammonium sulfamate solution (1 in 200), and allow to stand for 5 minutes, swirling frequently. Finally, to each flask add 1.0 mL of freshly prepared N-(1-naphthyl)ethylenediamine dihydrochloride solution (1 in 1000), mix, dilute with water to volume, and mix. Allow each solution to stand between 15 and 60 minutes, and then concomitantly determine the absorbances of the solutions, in 1-cm cells, recording the spectra from 440 to 700 nm, 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 545 nm.
Calculate the concentration, in mg per mL, of each sulfonamide in the Assay Preparation by the formula:
0.12C(AU / AS)
in which C is the concentration, in µg per mL, of the pertinent USP Reference Standard in the Standard Preparation; AU is the corrected absorbance of the Assay Preparation; and AS is the corrected absorbance of the pertinent Standard Preparation. From the concentration of the Assay Preparation thus determined, and applying appropriate dilution factors, calculate the percentage of sulfonamide in the specimen taken.