Test solution— Transfer a quantity of Gamma Cyclodextrin, equivalent to 2.5 g on the dried basis, into a 25-mL volumetric flask, dissolve in and dilute with water that 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: at 420 nm, the absorbance is not greater than 0.20; and the solution is clear.

A: Infrared Absorption 197K.

B: 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.

C: It meets the requirements of the test for Specific rotation.

Test solution: 10 mg per mL, in water.

Mobile phase and Chromatographic system— Prepare as directed in the Assay.

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.

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 alfadex (alpha cyclodextrin) or to betadex (beta cyclodextrin) are not greater than the area of the corresponding peaks in the chromatogram of the Standard solution (0.5%); and the sum of the areas of all the peaks, excluding the principal peak, the peaks corresponding to alfadex or to betadex, and artifact peaks, is not greater than the area of the peak corresponding to gamma cyclodextrin in the chromatogram of the Standard solution (0.5%).

Dextrose standard solution— Dissolve an accurately weighed quantity of USP Dextrose RS in water to obtain a solution having a known concentration of about 10.0 mg per mL for USP Dextrose RS, calculated on the anhydrous basis.

Procedure— Transfer a quantity of Gamma Cyclodextrin, equivalent to 1.0 g on the dried basis, to a 500-mL conical flask, dissolve in 10 mL of water, and add 25 mL of alkaline cupric citrate TS2. Cover the flask with aluminum foil, and boil the solution for 5 minutes. Cool in an ice bath to room temperature. Add 25 mL of 0.6 N acetic acid, 10 mL of 3 N hydrochloric acid, and 10 mL of 0.1 N iodine solution. [note—The addition of these solutions must be in the order given.] Titrate the solution with 0.1 N sodium thiosulfate VS, and determine the endpoint potentiometrically. Perform a blank determination (see Residual Titrations under Titrimetry 541). Calculate the difference in volumes required. Create a calibration curve by similarly titrating 0.25, 0.5, 0.75, and 1.0 mL of Dextrose standard solution. Plot the amount, in mg, of dextrose in each titrated Dextrose standard solution versus the volume consumed, in mL, of 0.1 N sodium thiosulfate VS in the titration, and draw a straight line through the four points. From the line so obtained and the volume of 0.1 N sodium thiosulfate VS required in the titration of Gamma Cyclodextrin, determine the amount, W, in mg, of dextrose in the portion of Gamma Cyclodextrin taken. Calculate the percentage of the reducing substances by the formula: in which WG is the weight, in g, of Gamma Cyclodextrin taken: not more than 0.5% is found.

Mobile phase— Prepare a filtered and degassed mixture of water and methanol (93:7). Make adjustments if necessary (see System Suitability under Chromatography 621).

System suitability preparation— Dissolve accurately weighed quantities of USP Alpha Cyclodextrin RS, USP Beta Cyclodextrin RS, and USP Gamma Cyclodextrin RS in water, and dilute if necessary, to obtain a solution having known concentrations of 0.5 mg of each per mL for USP Alpha Cyclodextrin RS and USP Beta Cyclodextrin RS, each calculated on the anhydrous basis, and 0.5 mg per mL for USP Gamma Cyclodextrin RS, calculated on the dried basis.

Standard preparation— Dissolve an accurately weighed quantity of USP Gamma Cyclodextrin RS in water to obtain a solution having a known concentration of about 1.0 mg per mL, calculated on the dried basis.

Assay stock preparation— Transfer 250 mg of Gamma Cyclodextrin, accurately weighed, to a 25-mL volumetric flask, and dissolve in water, with the aid of heat if necessary. 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 × 15-cm column that contains 5-µm packing L1. The temperature for the refractive index detector is maintained at 40, and the temperature for the analytical column is maintained at 30. The flow rate is about 1.5 mL per minute. Chromatograph the System suitability preparation, and identify the components based on their relative retention times, which are 0.8, 1.0, and 1.9 for gamma cyclodextrin, alfadex, and betadex, respectively. Record the peak responses as directed for Procedure: the resolution, R, between the gamma cyclodextrin and alfadex peaks is not less than 1.5; the tailing factors for the three cyclodextrins are between 0.8 and 2.0; and the relative standard derivation 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)8 in the portion of Gamma Cyclodextrin taken by the formula: in which 10 is the dilution factor for preparing the Assay preparation from the Assay stock preparation; 25 is the volume, in mL, of the Assay stock preparation; C is the concentration, in mg per mL, of USP Gamma Cyclodextrin RS in the Standard preparation; W is the weight, in mg, of Gamma Cyclodextrin taken to prepare the Assay stock preparation; A is the percentage Loss on drying of the Gamma Cyclodextrin taken; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.