Hydrazine solution— Transfer 1.0 g of hydrazine sulfate to a 100-mL volumetric flask, dissolve in and dilute with water to volume, and mix. Allow to stand for 4 to 6 hours.

Methenamine solution— Transfer 2.5 g of methenamine to a 100-mL glass-stoppered flask, add 25.0 mL of water, insert the glass stopper, and mix to dissolve.

Primary opalescent suspension— [note—This suspension is stable for 2 months, provided it is stored in a glass container free from surface defects. The suspension must not adhere to the glass and must be well mixed before use.] Transfer 25.0 mL of Hydrazine solution to the Methenamine solution in the 100-mL glass-stoppered flask. Mix, and allow to stand for 24 hours.

Opalescence standard— [note—This suspension should not be used beyond 24 hours after preparation.] Transfer 15.0 mL of the Primary opalescent suspension to a 1000-mL volumetric flask, dilute with water to volume, and mix.

Reference suspensions— Transfer 5.0 mL of the Opalescence standard to a 100-mL volumetric flask, dilute with water to volume, and mix to obtain Reference suspension A. Transfer 10.0 mL of the Opalescence standard to a second 100-mL volumetric flask, dilute with water to volume, and mix to obtain Reference suspension B.

Test solution— Dissolve 5.0 g of test material in about 20 mL of a 200 g per L solution of sodium acetate, dilute with the same solution to 25 mL, and mix.

Procedure— Transfer a sufficient portion of the Test solution to a test tube of colorless, transparent, neutral glass with a flat base and an internal diameter of 15 mm to 25 mm to obtain a depth of 40 mm. Similarly transfer portions of Reference suspension A, Reference suspension B, water, and a 200 g per L solution of sodium acetate to separate matching test tubes. Compare the Test solution, Reference suspension A, Reference suspension B, water, and a 200 g per L solution of sodium acetate in diffused daylight, viewing vertically against a black background (see Visual Comparison under Spectrophotometry and Light-Scattering 851). [note—The diffusion of light must be such that Reference suspension A can readily be distinguished from water and that Reference suspension B can readily be distinguished from Reference suspension A.] The Test solution shows the same clarity as that of water, or the 200 g per L solution of sodium acetate, or its opalescence is not more pronounced than that of Reference suspension A.

Standard stock solution— Combine 3.0 mL of ferric chloride CS, 3.0 mL of cobaltous chloride CS, 2.4 mL of cupric sulfate CS, and 1.6 mL of dilute hydrochloric acid (10 g per L).

Standard solution— [note—Prepare the Standard solution immediately before use.] Transfer 1.0 mL of Standard stock solution to a 100-mL volumetric flask, dilute with dilute hydrochloric acid (10 g per L) to volume, and mix.

Test solution— Use the Test solution from Clarity of solution.

Procedure— Transfer a sufficient portion of the Test solution to a test tube of colorless, transparent, neutral glass with a flat base and an internal diameter of 15 mm to 25 mm to obtain a depth of 40 mm. Similarly transfer portions of the Standard solution, a 200 g per L solution of sodium acetate, and water to separate matching test tubes. Compare the Test solution, the Standard solution, a 200 g per L solution of sodium acetate, and water in diffused daylight, viewing vertically against a white background (see Visual Comparison under Spectrophotometry and Light-Scattering 851). The Test solution has the appearance of water or the 200 g per L solution of sodium acetate, or is not more intensely colored than the Standard solution.

Internal standard solution— Dissolve 25 mg of caffeine in methylene chloride, and dilute with the same solvent to 100 mL.

Reference solution— Dissolve 20.0 mg of USP o-Toluenesulfonamide RS and 20.0 mg of USP p-Toluenesulfonamide RS in methylene chloride, and dilute with the same solvent to 100.0 mL. Dilute 5.0 mL of the solution with methylene chloride to 50.0 mL. Evaporate 5.0 mL of the final solution to dryness in a stream of nitrogen. Dissolve the residue in 1.0 mL of the Internal standard solution.

Test solution— Suspend 10.0 g of the substance to be examined in 20 mL of water, and dissolve using 5 mL to 6 mL of 10 N sodium hydroxide. If necessary, adjust the solution with 1 N sodium hydroxide or 1 N hydrochloric acid to a pH of 7 to 8, and dilute with water to 50 mL. Shake the solution with four quantities each of 50 mL of methylene chloride. Combine the lower layers, dry over anhydrous sodium sulfate, and filter. Wash the filter and the sodium sulfate with 10 mL of methylene chloride. Combine the solution and the washings, and evaporate almost to dryness in a water bath at a temperature not exceeding 40. Using a small quantity of methylene chloride, quantitatively transfer the residue into a suitable 10-mL tube, evaporate to dryness in a stream of nitrogen, and dissolve the residue in 1.0 mL of the Internal standard solution.

Blank solution— Evaporate 200 mL of methylene chloride to dryness in a water bath at a temperature not exceeding 40. Dissolve the residue in 1 mL of methylene chloride.

Chromatographic system (see Chromatography 621)— The gas chromatograph is equipped with a flame-ionization detector and contains a 0.53-mm × 10-m fused silica column, coated with G3 phase (film thickness 2 µm). The injection port, column, and detector temperatures are maintained at about 250, 180, and 250, respectively; and nitrogen is used as the carrier gas at a flow rate of about 10 mL per minute. The injector employs a split ratio of 1:2.

Procedure— Inject about 1 µL of the Reference solution. Adjust the sensitivity of the detector so that the height of the peak due to caffeine is not less than 50% of the full scale of the recorder. The substances are eluted in the following order: o-toluenesulfonamide, p-toluenesulfonamide, and caffeine. The test is not valid unless the resolution between the peaks due to o-toluenesulfonamide and p-toluenesulfonamide is at least 1.5. Inject about 1 µL of the Blank solution. In the chromatogram obtained, verify that there are no peaks with the same retention times as the internal standard, o-toluenesulfonamide, and p-toluenesulfonamide. Inject about 1 µL of the Test solution and 1 µL of the Reference solution. If any peaks due to o-toluenesulfonamide, and p-toluenesulfonamide appear in the chromatogram obtained with the Test solution, the ratio of their areas to that of the internal standard is not greater than the corresponding ratio in the chromatogram obtained with the Reference solution (10 ppm of o-toluenesulfonamide and 10 ppm of p-toluenesulfonamide).