Identification—
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:
Prepare a test solution in water containing 15 mg per mL. Separately apply 2 µL each of the test solution and a Standard solution of
USP Beta Cyclodextrin RS in water containing 5 mg per mL to a suitable thin-layer chromatographic plate (see
Chromatography 
621
) coated with a 0.25-mm layer of chromatographic silica gel. Allow the applications to dry, and develop the chromatogram in a solvent system consisting of a mixture of
n-propyl alcohol, water, ethyl acetate, and ammonium hydroxide (6:3:1:1) until the solvent front has moved about three-fourths of the length of the plate. Remove the plate from the developing chamber, mark the solvent front, and allow the solvent to evaporate. Locate the spots on the plate by lightly spraying with iodine and potassium iodide TS 1: the
RF value of the principal spot obtained from the test solution corresponds to that obtained from the Standard solution.
D:
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.
Reducing substances—
Transfer 1.0 g to a 250-mL conical flask, dissolve in 10 mL of water, and add 25 mL of
alkaline cupric citrate TS. Cover the flask, boil gently for 5 minutes, accurately timed, and cool rapidly to room temperature. Add 25 mL of 0.6 N acetic acid, 10.0 mL of 0.1 N iodine VS, and 10 mL of 3 N hydrochloric acid, and titrate with 0.1 N sodium thiosulfate VS, adding 3 mL of
starch TS as the endpoint is approached (see
Titrimetry 541 ). Perform a blank determination, omitting the specimen, and note the difference in volumes required. Each mL of the difference in volume of 0.1 N sodium thiosulfate consumed is equivalent to 2.7 mg of reducing substances (as dextrose): the limit is 1.0%.
Assay—
Mobile phase—
Prepare a filtered and degassed mixture of acetonitrile and water (65:35). Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Internal standard solution—
Dissolve 2.0 g of glycerol in water contained in a 100-mL volumetric flask, dilute with water to volume, and mix. Pass through a 0.45-µm membrane filter. Use fresh, or store in a freezer, thaw in hot water, and mix.
Standard preparation—
Dissolve an accurately weighed quantity of
USP Beta Cyclodextrin RS in water, and dilute quantitatively with water to obtain a solution having a known concentration of about 10 mg per mL. Use fresh, or store in a freezer, thaw in hot water, and mix. Mix 1.0 mL of this solution with 1.0 mL of
Internal standard solution.
Assay preparation—
Transfer about 1 g of Betadex, accurately weighed, to a 100-mL volumetric flask, dissolve in and dilute with water to volume, and mix. Pass this solution through a 0.45-µm membrane filter. Mix 1.0 mL with 1.0 mL of Internal standard solution.
Chromatographic system (see Chromatography 621)—
The chromatograph is equipped with a refractive index detector, a 4.6-mm × 25-cm column that contains 10-µm packing L8, and a guard column that contains packing L8. The columns and, if necessary, the detector are maintained at a constant temperature of about 25 ± 2
, and the flow rate is about 2.0 mL per minute. If the detector or the columns are operated at a temperature other than 25 ± 2
, the system also must be shown to meet all system suitability requirements.Chromatograph the
System suitability preparation, and record the peak responses as directed for
Procedure: the alpha cyclodextrin and beta cyclodextrin peaks exhibit baseline separation, the relative retention times being about 0.8 and 1.0, respectively; and the relative standard deviation for replicate injections is not more than 2.0%.
Procedure—
Separately inject equal volumes (about 20 µL) of the
Assay preparation and the
Standard preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of (C
6H
10O
5)
7 in the portion of Betadex taken by the formula:
100C(RU / RS),
in which
C is the concentration, in mg per mL, of anhydrous beta cyclodextrin in the
Standard preparation, as determined from the concentration of
USP Beta Cyclodextrin RS, corrected for moisture content by a titrimetric water determination; and
RU and
RS are the peak response ratios of the beta cyclodextrin peak to the internal standard peak obtained from the
Assay preparation and the
Standard preparation, respectively.
