| Compound toad skin capsule is a preparation of traditional Chinese medicine made fromthe skin of Angelica and toad. Toad skin tastes spicy, cool and slightly toxic, can detoxify, bulkaccumulation, eliminate swelling. Angelica tastes sweet, acrid, warm, can enrich people’sblood, relive women’s menstruation pain and moisten intestines. Compound toad skin capsulemainly used for the adjuvant treatment of esophageal cancer, advanced primary liver cancer andadvanced gastric cancer, which can improve clinical symptoms and the quality of life.The content of toad skin in Compound toad skin capsule plays a key role to pharmacology.Low content of toad skin will weaken the auxiliary treatment effect of compound toad skincapsule, otherwise, excessive content of toad skin will bring greater side effects to cancerpatients. Methods for extraction of active ingredients in Angelica and the content determinationhas matured both at home and abroad, but research of separation and content determination ofactive ingredients for Angelica and toad skin preparations is rare. In order to control the qualityof compound toad skin capsules better, the paper studied the quality control of toad skin incompound toad skin capsules. Cinobufagin and Resibufogenin were extracted and separatedfrom compound toad skin capsule.The method of content determination of Cinobufagin andResibufogenin was studied.In terms of extraction and separation, the extraction method of Active ingredient inCompound Toad skin capsule was methanol reflux. In order to ensure the purity of the samplesanalyzed by liquid chromatography, the reflux extraction was further purified. The refluxextraction was mixed well with1g silica gel, and dried in105℃.[Method of colum: The size ofsilica gel was (200-300mesh). The size of colum was1.7*30cm.] The eluent was Cyclohexane-chloroform-acetone (4:3:3). The impact of the separation of silica was analyzed. Content ofCinobufagin and Resibufogenin in the collected liquid1-50ml and51-100ml were determined.In the aspect of determination of sample analysis, the condition of reference sample waspreliminary explored, which laid the foundation for the content analysis of toad skin incompound toad skin capsule. In this paper, Agilent ZORBAX SB-C18(4.6×250mm,5μm)column was used to analyse the content of Cinobufagin and Resibufogenin.0.5%potassiumdihydrogen phosphate solution–acetonitrile that adjusted the pH of the mobile phase with acarboxylic acid was chosen as the mobile phase. The optimal column temperature and flow rateto the detected peak were also determined. Method validation(negative control, detection limit,quantification limit, the standard curve, recovery, precision, reproducibility) was made and12months of stability of three batches of test samples were also investigated.The method of extraction and determination of compound toad skin capsule wasdetermined. The test product was extracted by methanol reflux for1hour, filtered, then theresidue washed with a little methanol, and the filtrate was evaporated to dryness. The residuedissolved in5ml of methanol, and stirred with silica gel1g sample homogeneity which dried in105℃. The dried sample was prepared for the silica gel colum[Method of colum: The size ofsilica gel was (200-300mesh). The size of colum was1.7*30cm.].The eluent was Cyclohexane-chloroform-acetone (4:3:3). The former eluent50ml was collected and evaporated to dryness sample that was dissolved by methanol to10ml volumetric flask. Agilent ZORBAX SB-C18(4.6×250mm,5μm) column was used in HPLC to analyse the content of test product. Themobile phase was0.5%potassium dihydrogen phosphate solution-acetonitrile (64:34)(adjustedto pH3.2with formic acid). Flow rate was1ml·min-1.Detection wavelength was296nm.Column temperature was40℃. Theoretical plates number of Cinobufagin and Resibufogeninshould not be less than4000, respectively. In the aspect of method validation, Cinobufagin andResibufogenin showed a good linear relationship within the concentration range5μg/ml-25μg/ml（.Cinobufagin: R=0.9995， n=5, Resibufogenin: R=0.9995, n=5）, The averagerecovery was99.12%, n=6, RSD=0.2%, Content limit of Compound toad skin capsule was5-15μg/ml.12months for the three batches of constant stability was investigated, the resultsobtained by the method established stable and suitable, good durability.The method is suitable for the separation and determination of Cinobufagin andResibufogenin content in compound toad skin capsule, which is simple and feasible, showedgood durability and reproducibility. Limited toxicity ingredients of products can be wellcontrolled, which provides a theoretical basis for the standard upgrade of compound toad skincapsule.This research provide enterprises a quality control standards of product. Modernization ofindustrial enterprises was promoted which can make contribution to upgrade and enhance thecore competitiveness of enterprises. It is necessary to improve the security of the national drugby using this study, which provide technical support for the international development ofChinese medicine. |
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