A: Dissolve about 100 mg in 5 mL of sodium hydroxide solution (1 in 20), evaporate the solution to dryness, and gently fuse the residue over a small flame until it no longer evolves ammonia. Allow the residue to cool, dissolve it in 20 mL of water, neutralize the solution with 3 N hydrochloric acid, and filter: the addition of a drop of ferric chloride TS to the filtrate produces a violet color.

B: Mix 20 mg with 40 mg of resorcinol, add 10 drops of sulfuric acid, and heat the mixture in a suitable liquid bath at 200 for 3 minutes. Allow it to cool, and add 10 mL of water and an excess of 1 N sodium hydroxide: a fluorescent green liquid results.

Internal standard solution— Place 10 mg of n-tricosane in a 10-mL volumetric flask, dissolve in n-heptane, dilute with n-heptane to volume, and mix.

Standard stock solution— Transfer 20 mg each, accurately weighed, of USP o-Toluenesulfonamide RS and of USP p-Toluenesulfonamide RS to a 10-mL volumetric flask, dissolve in methylene chloride, dilute with methylene chloride to volume, and mix.

Standard preparations— Transfer 100, 150, 200, and 250 µL, respectively, of Standard stock solution to each of four 10-mL volumetric flasks. Add 250 µL, accurately measured, of Internal standard solution to each flask, dilute each with methylene chloride to volume, and mix. These preparations contain, in each mL, 25 µg of n-tricosane and, respectively, 20, 30, 40, and 50 µg of each toluenesulfonamide isomer.

Test preparation— Prepare as directed under Column Partition Chromatography (see Chromatography 621), employing a chromatographic tube fitted with a porous glass disk in its base, a plastic stopcock on the delivery tube, and a reservoir at the top. Add a mixture consisting of 12 g of Solid Support and a solution of 2.0 g, accurately weighed, of Saccharin with 12 mL of filtered sodium bicarbonate solution (1 in 11). Add about 200 mg of sodium bicarbonate to effect complete solution of the saccharin. Pack the contents of the tube by tapping the column on a padded surface, and then by tamping firmly from the top. Place 100 mL of methylene chloride in the reservoir, and adjust the stopcock so that 50 mL of eluate is collected in 20 to 30 minutes. To the eluate add 25 µL of Internal standard solution, mix, and concentrate the solution, by suitable means, to a volume of 1.0 mL.

Chromatographic system (see Chromatography 621)— Under typical conditions, the instrument is equipped with a flame-ionization detector, and contains a 3.2-mm. × 1.8-m glass column packed with 10% liquid phase G3 on 100- to 120-mesh support S1AB, utilizing a glass-lined sample introduction system or on-column injection. The injector port, column, and detector block are maintained at temperatures of about 225, 210, and 250, respectively, and dry helium is used as the carrier gas at a flow rate of about 30 mL per minute.

Procedure— Inject portions (about 2.5 µL) of the Standard preparations, successively, into a gas chromatograph, and record each chromatogram so as to obtain at least 50% of maximum recorder response. Measure the areas under the first (o-toluenesulfonamide), second (p-toluenesulfonamide), and third (n-tricosane) peaks, and for each chromatogram record the values as Ao, Ap, and AN, respectively. Calculate the ratios Ro and Rp taken by the equations: and prepare standard curves by plotting the concentrations, in µg per mL, of USP o-Toluenesulfonamide RS and of USP p-Toluenesulfonamide RS in the Standard preparations versus Ro and Rp, respectively. [NOTE—Relative retention times are, approximately, 0.39 for o-toluenesulfonamide, 0.46 for p-toluenesulfonamide, and 1.0 for n-tricosane.] Similarly inject a portion (about 2.5 µL) of the Test preparation, and record the chromatogram. Measure the areas under the first (o-toluenesulfonamide), second (p-toluenesulfonamide), and third (n-tricosane) peaks, and record the values as ao, ap, and aN, respectively. Calculate the ratios ro and rp taken by the equations: and, from the standard curve, determine the concentration, in µg per mL, of each toluenesulfonamide isomer in the Test preparation: the total amount of toluenesulfonamides in the specimen taken is not more than 0.0025%.

Solvent— Use dimethyl sulfoxide.

(Official January 1, 2007)