A: The retention time of the major peak in the chromatogram of the Assay preparation corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay.

B: It meets the requirements of the test for Specific rotation 781S.

C: Mix 0.2 g with 2 mL of iodine TS, warm in a water bath to dissolve the test specimen, and allow to stand at room temperature: a yellow-brown precipitate is formed.

Test solution: 10 mg per mL, in water.

Potassium chloride solution— Transfer 22.4 g of potassium chloride into a 100-mL volumetric flask, and dilute with water to volume.

Cupric solution— Dissolve 15 g of cupric sulfate in water to make 100 mL.

Tartrate solution— Dissolve 2.5 g of anhydrous sodium carbonate, 2.5 g of potassium sodium tartrate, 2.0 g of sodium bicarbonate, and 20 g of anhydrous sodium sulfate in water to make 100 mL.

Cupric–tartaric solution— Immediately before use, mix 1 part of Cupric solution with 25 parts of Tartrate solution.

Ammonium molybdate reagent— Mix 10 mL of a solution of disodium arsenate (6 in 100), 50 mL of a solution of ammonium molybdate (1 in 10), and 90 mL of diluted sulfuric acid, and dilute with water to 200 mL.

Test solution— Transfer about 1.0 g of Alfadex, accurately weighed, to a 100-mL volumetric flask, dissolve in and dilute with water that has been previously boiled and cooled to room temperature, to volume, and mix. To 1 mL of this solution add 1 mL of Cupric–tartaric solution. Heat on a water bath for 10 minutes, then cool to room temperature. Add 10 mL of Ammonium molybdate reagent, and allow to stand for 15 minutes.

Standard solution— Prepare as directed for the Test Solution, at the same time, except to use 1 mL of a solution containing 20 mg of glucose per L.

Procedure— Concomitantly measure the absorbance of the Test solution and the Standard solution at the wavelength of maximum absorbance at 740 nm relative to that of water, with a suitable spectrophotometer. The absorbance of the Test solution is not greater than that of the Standard solution (0.2%).

System suitability solution— Prepare as directed for System suitability preparation in the Assay.

Standard solution— Transfer 5.0 mL of the System suitability solution into a 50-mL volumetric flask, and dilute with water to volume.

Test solution— Use the Assay stock preparation prepared as directed in the Assay.

Chromatographic system (see Chromatography 621)— Proceed as directed in the Assay.

Procedure— Separately inject equal volumes (about 50 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the responses for the major peaks. For the Test solution, the areas of any peaks corresponding to beta cyclodextrin or to gamma cyclodextrin are not greater than half of the area of the corresponding peaks in the chromatogram of the Standard solution (0.25%), and the sum of the areas of all the peaks, excluding the principal peak and the peaks corresponding to beta cyclodextrin or to gamma cyclodextrin, is not greater than half of the area of the peak corresponding to alpha cyclodextrin in the chromatogram of the Standard solution (0.5%).

Test solution— Transfer about 1.0 g of Alfadex, accurately weighed, to a 100-mL volumetric flask, dissolve in and dilute with water, which has been previously boiled and cooled to room temperature, to volume, and mix.

Procedure— Determine the absorbance of the Test solution in a 1-cm cell with a suitable spectrophotometer, after correcting for the blank: between 230 nm and 350 nm, the absorbance is not greater than 0.10; and between 350 nm and 750 nm, the absorbance is not greater than 0.05.

(Official January 1, 2007)

Mobile phase— Prepare a filtered and degassed mixture of water and methanol (90:10). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Transfer 25 mg of USP Alpha Cyclodextrin RS, accurately weighed, to a 25-mL volumetric flask, and dissolve in and dilute with water to volume.

System suitability preparation— Transfer 25 mg of USP Beta Cyclodextrin RS, 25 mg of USP Gamma Cyclodextrin RS, and 50 mg of USP Alpha Cyclodextrin RS, accurately weighed, to a 50-mL volumetric flask, dissolve in and dilute with water to volume, and mix.

Assay stock preparation— Transfer 250 mg of Alfadex, accurately weighed, to a 25-mL volumetric flask, and dissolve in water with the aid of heat. Cool, and dilute with water to volume.

Assay preparation— Transfer 5.0 mL of the Assay stock preparation to a 50-mL volumetric flask, and dilute with water to volume.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a refractive index detector and a 4.6-mm × 25-cm column that contains 10-µm packing L1. The flow rate is about 1.5 mL per minute. Chromatograph the System suitability preparation, and record the chromatograms for about 3.5 times the retention time of alpha cyclodextrin. Record the peak responses as directed for Procedure: the retention time of alpha cyclodextrin is about 4.5 minutes; the relative retention times are about 1.0 for alpha cyclodextrin, about 2.2 for beta cyclodextrin, and about 0.7 for gamma cyclodextrin; the resolution, R, between the gamma cyclodextrin and alpha cyclodextrin peaks is not less than 1.5; and for the alpha cyclodextrin peak, the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 50 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the percentage of (C6H10O5)6 in the portion of Alfadex taken by the formula: in which C is the concentration, in mg per mL, of alpha cyclodextrin in the Standard preparation, calculated on the dried basis, as determined from the concentration of USP Alpha Cyclodextrin RS corrected for the declared moisture content; W is the weight, in mg, of alpha cyclodextrin taken to prepare the Assay stock preparation; and RU and RS are the peak responses of the alpha cyclodextrin peak obtained from the Assay preparation and the Standard preparation, respectively.