ANNEXSupplementary provisions
ANNEX SPECIFICATIONS OF ALPHA-CYCLODEXTRIN Synonyms α-cyclodextrin, α-dextrin, cyclohexaamylose, cyclomaltohexaose, α-cycloamylase Definition A non-reducing cyclic saccharide consisting of six α-1,4-linked D-glucopyranosyl units produced by the action of cyclodextrin glucosyltransferase (CGTase, EC 2.4.1.19) on hydrolyzed starch. Recovery and purification of α-cyclodextrin may be carried out using one of the following procedures: precipitation of a complex of α-cyclodextrin with 1-decanol, dissolution in water at elevated temperature and re-precipitation, steam-stripping of the complexant, and crystallisation of α-cyclodextrin from the solution; or chromatography with ion-exchange or gel filtration followed by crystallisation of α-cyclodextrin from the purified mother liquor; or membrane separation methods such as ultra-filtration and reverse osmosis. Chemical name Cyclohexaamylose CAS. number 10016-20-3 Chemical formula (C 6 H 10 O 5 ) 6 Structural formula Formula weight 972,85 Assay Not less than 98 % (dry basis) Description Virtually odorless, white or almost white crystalline solid. Characteristics Identification Melting range Decomposes above 278 °C Solubility Freely soluble in water; very slightly soluble in ethanol Specific rotation [α] D 25 : Between +145° and +151° (1 % solution) Chromatography The retention time for the major peak in a liquid chromatogram of the sample corresponds to that for α-cyclodextrin in a chromatogram of reference α-cyclodextrin (available from Consortium für Elektrochemische Industrie GmbH, München, Germany or Wacker Biochem Group, Adrian, MI, USA) using the conditions described in the METHOD OF ASSAY Purity Water Not more than 11 % (Karl Fischer Method) Residual complexant Not more than 20 mg/kg (1-decanol) Reducing substances Not more than 0,5 % (as glucose) Sulfated ash Not more than 0,1 % Lead Not more than 0,5 mg/kg Method of assay Determine by liquid chromatography using the following conditions: Sample solution: Weigh accurately about 100 mg of test sample into a 10-ml volumetric flask and add 8 ml of deionised water. Dissolve the sample completely using an ultra-sonification bath (10-15 min) and dilute to the mark with purified deionised water. Filter through a 0,45-micrometer filter. Reference solution: Weigh accurately about 100 mg of α-cyclodextrin into a 10-ml volumetric flask and add 8 ml of deionised water. Dissolve the sample completely using an ultra-sonification bath and dilute to the mark with purified deionised water. Chromatography: Liquid chromatograph equipped with a refractive index detector and an integrating recorder. Column and packing: Nucleosil-100-NH2 (10 μm) (Macherey & Nagel Co. Düren, Germany) or similar. Length: 250 mm Diameter: 4 mm Temperature: 40 °C Mobile phase: acetonitrile/water (67/33, v/v) Flow rate: 2,0 ml/min Injection volume: 10 μl Procedure: Inject the sample solution into the chromatograph, record the chromatogram, and measure the area of the α-CD peak. Calculate the percentage of α-cyclodextrin in the test sample as follows: % α-cyclodextrin (dry basis) = 100 × (A S /A R ) (W R /W S ) where A s and A R are the areas of the peaks due to α-cyclodextrin for the sample solution and reference solution, respectively. W s and W R are the weights (mg) of the test sample and reference α-cyclodextrin, respectively, after correcting for water content.