Test solution— In a 25-mL volumetric flask mix 1.25 g with 1 mL of edetate disodium solution (1 in 100), add 7.5 mL of sodium hydroxide solution (1 in 25), and mix to dissolve. Dilute to volume with pH 7.0 buffer (prepared by mixing 29.5 mL of 1 N sodium hydroxide, 50 mL of 1 M monobasic potassium phosphate, and sufficient water to make 100 mL and, using a pH meter, adjusting to a pH of 7.0 ± 0.1 by adding, as necessary, more of either solution).

(Official January 1, 2007)

Mobile phase— Dissolve 6.8 g of monobasic potassium phosphate in 1000 mL of water, pass through a membrane filter having a 0.45-µm porosity, and degas. Adjust with phosphoric acid to a pH of 3.0.

Internal standard solution— Dissolve about 1 g of DL-phenylalanine in 200 mL of freshly prepared sodium metabisulfite solution (1 in 2000).

Standard preparation— Dissolve an accurately weighed quantity of USP Acetylcysteine RS in sodium metabisulfite solution (1 in 2000) to obtain a solution having a known concentration of about 10 mg per mL. Pipet 10.0 mL of this solution and 10.0 mL of Internal standard solution into a 200-mL volumetric flask, dilute with sodium metabisulfite solution (1 in 2000) to volume, and mix to obtain a Standard preparation having a known concentration of about 0.5 mg per mL.

Assay preparation— Transfer about 1000 mg of Acetylcysteine, accurately weighed, to a 100-mL volumetric flask. Dissolve in sodium metabisulfite solution (1 in 2000), dilute with the same solvent to volume, and mix. Pipet 10.0 mL of this solution and 10.0 mL of Internal standard solution into a 200-mL volumetric flask, dilute with sodium metabisulfite solution (1 in 2000) to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 214-nm detector and a 3.9-mm × 30-cm column that contains packing L1. The flow rate is about 1.5 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative standard deviation for replicate injections is not more than 2.0%; and the resolution, R, between acetylcysteine and DL-phenylalanine is not less than 6.

Procedure— Separately inject equal volumes (about 5 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. The relative retention times are about 0.5 for acetylcysteine and 1.0 for DL-phenylalanine. Calculate the quantity, in mg, of C5H9NO3S in the Acetylcysteine taken by the formula: in which C is the concentration, in mg per mL, of USP Acetylcysteine RS in the Standard preparation; and RU and RS are the ratios of the peak response of acetylcysteine to that of DL-phenylalanine obtained from the Assay preparation and the Standard preparation, respectively.