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       【关键词】  Zhenxian Tablets Bilirubin TLC RP-HPLC
       Tablets［1］ is a pure formulated preparation of traditional Chinese medicine(略)
        
        According to the literatures, the quality evaluation standards of Zhenxian Tablets are not very scientific and reasonable. The major identical method is micro-identification, which is not all-round, so we also set up the TLC method for the identification of Rhizoma Acori Tatarinowii, Radix Curcumae and Radix Polygalae. And in the study of quality control of ZhenXian Tablets, we choose the Bilirubin as quality control criteria and establish an RP-HPLC method for determining the content of bilirubin in Zhenxian Tablets. Bilirubin is the major active constituent of Calculus Bovis and also one of the main effective contents in Zhenxian Tablets. So we choose Bilirubin as the major marker substance. The analytical results are satisfying and will provide a qualitative and quantitative evaluation in the production of Zhenxian Tablets.
       1  Apparutus and chemicals
        Chromatographic analysis was performed on a Model LC-20AT high performance liquid chromatograph (Shimadzu, Japan) equipped with a Model SPD-20A wavelength UV detector and an LC Solution data processing system.
        Rhizoma Acori Tatarinowii, Radix Curcumae Radix Polygalae and Calculus Bovis were all supplied from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, P.R.China). Zhenxian Tablets was researched and developed in our research center. All reagents and chemicals used for the preparation and for the RP-HPLC analysis were of analytical grade. The water used for the preparation and for the RP-HPLC analysis was freshly deionized and redistilled.
       2  Experimental methods and results
       2.1  TLC methods
       2.1.1  TLC method for the identification of Rhizoma Acori TatarinowiiAbout 3 g of the sample was weighed and dispersed in 50 ml of diethyl ether, extracted ultrasonically for 30min. After cooling, the layer of diethyl ether was obtained and evaporated to dryness, the residue was dissolved in 20 ml water, then extracted twice by 15ml ethyl acetate separately, complicated the ethyl acetate solution and evaporated to dryness, the residue was dissolved in 1ml of methanol, a test solution was obtained. In addition, Rhizoma Acori Tatarinowii was weighed accurately 2 g, and dispersed in 50 ml of diethyl ether, extracted ultrasonically for 30 min, and filtered at room temperature, the filtrate was evaporated to dryness, the residue was dissolved in 1ml methanol to produce a reference solution. The thin layer chromatography was adopted for the quality identification of Rhizoma Acori Tatarinowii. 10 μl of the test solution and 5 μl of the reference solution were applied separately to the silica gel G plate, using petroleum ether(60～90℃)- ethyl acetate(8∶2) as the mobile phases. After developing in this mobile phases, the plate was removed, dried in air and examined under ultra-violet light (365 nm). The TLC chromatograms showed that the fluorescent spots in the chromatograms obtained with the test solutions corresponded in color and position to the spots in the chromatograms obtained with the reference solution. In addition, there was no interference in blank solution for the quality identification of Rhizoma Acori Tatarinowii.
       2.1.2   TLC method for the identification of Radix Curcumae Ten Tablets were obtained and dispersed in 10 ml of alcohol, extracted ultrasonically for 30 min, and filtered at room temperature. The filtrate was evaporated to dryness, the residue was dissolved in 2 ml of ethyl alcohol, a test solution was obtained; In addition, Radix Curcumae was weighed accurately 1g and it was treated by the same method to prepare the reference solution. The thin layer chromatography was adopted for the quality identification of Radix Curcumae. 8 μl of the test solution and 4  μl of the reference solution were applied separately to the silica gel G plate, using cyclohexane- ethyl acetate (9∶3) as the mobile phases. After developing in this mobile phases, the plate was removed, dried in air and examined under ultra-violet light  (365 nm). The TLC chromatograms showed that the hepatic spots in the chromatograms obtained with the test solutions corresponded in color and position to the spots in the chromatograms obtained with the reference solution. In addition, there was no interference in blank solution for the quality identification of Radix Curcumae.
       2.1.3  TLC method for the identification of Radix PolygalaeTen tablets were obtained and dispersed in 10 ml of a solution which was composed of 5%of hydrochloric acid of 70% of ethyl alcohol, extracted under refluxing on a water bath for 1h, and filtered at room temperature. The filtrate was put in the separating funnel, added 40 ml of water, mixed, and extracted by 10 ml of chloroform, then the chloroform solution was obtained and evaporated to dryness, the residue was dissolved in 1 ml of methanol, a test solution was obtained. In addition, Radix Polygalae was weighed accurately 1 g and it was treated by the same method to prepare the reference solution. The thin layer chromatography was adopted for the quality identification of Radix Polygalae. 8 μl of the test solution and 4 μl of the reference solution were applied separately to the silica gel G plate, using chloroform-acetone-N-hexane-glacial acetic acid (8:4:0.5:0.5) as the mobile phases. After developing in this mobile phases, the plate was removed, dried in air and consperged the vanillin ethyl alcohol solution to coloration. The TLC chromatograms showed that the fluorescent spots in the chromatograms obtained with the test solutions corresponded in colour and position to the spots in the chromatograms obtained with the reference solution. In addition, there was no interference in blank solution for the quality identification of Radix Polygalae.
       2.1.4  TLC chromatograms of Rhizoma Acori Tatarinowii(A), Radix Curcumae(B) and Radix Polygalae(C) Three TLC chromatograms were shown in Figure 1.
       2.2  HPLC methods
       2.2.1  Chromatographic conditions and systematic compatibility test  Separation were obtained on a C18 analytical column(4.6 mm×150 mm,5 μm) with methanol-1% of phosphoric acid solution-tetrahydrofuran (80∶8∶12) as mobile phase. The UV detection wavelength was 450 nm and the flow rate was 1 ml·min-1 .The column temperature was 28℃ and injection volume was 10 μl. Under the condition used, the number of theoretical plates calculated on the peak of Bilirubin was no less than 3000.
       2.2.2  The preparation of standard solution   2 mg of Bilirubin was accurately weighed and dispersed in methanol in a brown constant-volumn of 25 ml, mixed( cold-storage in dark place)as the stock solution. Then diluted by the mixed solution which was composed of chloroform-methanol-water (90∶10∶0.3), a 10 μg per milliliter standard solution was obtained. 
       2.2.3  The preparation of sample solution   About 2 g of the sample ( comminuted powder) was accurately weighed and put in a brown constant-volumn of 100 ml, dispersed to the scale in the mixed solution which was composed of chloroform-methanol-water-hydrochloric acid(90∶10∶0.3∶0.015), extracted ultrasonically for 15min. After cooling, filtered by the micropore film(0.45 μm), the   filtrate was obtained as a sample solution.
       2.2.4  The preparation of blank solution  According to the proportion of prescription, other crude herbal materials in the prescription were weighed in the suitable amounts except Calculus Bovis. Using preparation technology of Zhenxian Tablets and preparation method of sample solution, a blank solution was obtained.
       2.2.5  Interference test of blank solution   10 μl of standard solution, sample solution and blank solution were injected respectively into the RP-HPLC column under the chromatographic conditions described above. The results(Figure 2)showed that there was a same peak of Bilirubin appeared in the same retention time between standard solution and sample solution, whereas there was no peak of Bilirubin appeared in this retention time in blank solution, thus there was no interference for the determination of the content of Bilirubin in sample solution.
       2.2.6  Calibration curve and linear range  A suitable amount of Bilirubin was accurately weighed and it was treated by the same method to prepare the standard stock solution, then 4.39 μg·ml-1, 8.78 μg·ml-1, 13.17 μg·ml-1, 17.56 μg·ml-1, 21.95 μg·ml-1, 26.34 μg·ml-1and 30.73 μg·ml-1of standard solutions were obtained .Under the chromatographic conditions described above, 10ul of each standard solution was taken for measuring and the chromatograms were recorded. After regression analysis of linearity, the calibration curve was described by regression equation A=4 647 159.12C+798.87, r=0.999 8, where A was the peak area of Bilirubin(as longitudinal coordinate), C was the concentration of Bilirubin (as horizontal coordinate), and r was the correlation coefficient. The results showed that the peak area of Bilirubin and the concentration of Bilirubin had fine linear response in the concentration range of 0.043 9～0.307 3 μg.
       2.2.8  Chromatographic test of precision  10 μl of a same concentration of Bilirubin standard solution was taken to continuously analyze for six times, the RSD of peak area of Bilirubin was 1.07%. The results showed that the chromatographic method was quite precise.
       2.2.9  Chromatographic test of stability  According to the method for the determination of the content of  Bilirubin , a sample of the same batch number was accurately weighed and the sample solution was prepared, and 10 μl of this solution was measured in 0, 1, 2, 5, 8h within a day, the RSD of peak area of Bilirubin was 1.17%. The results showed that the sample solution was stable within 8h.
       2.2.10  Chromatographic test of reproducibilitySix of a suitable amount of samples(comminuted powder) were accurately weighed, and the content of Bilirubin was determined after treated like the method of determination of the content of sample, the average content of Bilirubin was 0.072 0%, and the RSD was 1.67%. The results showed that the chromatographic method had a good reproducibility.
       2.2.11  Recovery of the chromatographic method   Six of a suitable amount of Bilirubin were accurately weighed and added to the sample (comminuted powder) in which the content of Bilirubin had been determined (the content of Bilirubin was 0.072 0%), and the content of Bilirubin were determined after treated like the method of determination of the content of sample, the recovery was calculated, the average recovery of Bilirubin was 98.62%, and the RSD was 1.09%(n=6). The results were shown in Table 1. Tab 1  Recovery test of Bilirubin（略）
       2.2.12  Determination of the content of sample Ten of a suitable amount of samples (comminuted powder) were accurately weighed, and treated like the method of determination of the content of sample, ten of sample solutions were obtained. 10ul of each sample solution was taken for measuring and the content of Bilirubin was calculated based on calibration curve. The results were shown in Table 2.Tab 2   Results of sample analysis（略）
       3  Discussion
       3.1  Selection of detection wavelengthThe UV spectrum showed that bilirubin  had obvious absorbent peak near the wavelength 450 nm after UV-scanning, thus 450 nm was selected as detection wavelength.
       3.2  Selection of mobile phaseThe experiments compared the chromatographic behavior of the mobile phases-methanol and 1% of phosphoric acid solution and tetrahydrofuran (80:8:6, 80:8:12,80:8:18). The result showed that under the conditions of the mobile phases(80:8:12), there was a complete baseline separation among Bilirubin and other components and also a symmetric chromatographic peak. Therefore the mobile phase - methanol and 1% of phosphoric acid solution and tetrahydrofuran(80:8:12) was selected as the best chromatographic condition.
       3.3  ConclusionAccording to the literatures, the quality evaluation standards of Zhenxian Tablets has not set up the method of quality identification, so we added the TLC method for the identification of Rhizoma Acori Tatarinowii, Radix Curcumae and Radix Polygalae. Through the verification of three of samples, this method is recurrent well, and can be used as a reliable tool for quality control. We also choosed the bilirubin as quality control criteria and established the best RP-HPLC method for determining the content of bilirubin in Zhenxian Tablets with the help of the literatures. Through the verification of the experimental methods, this method is accurate, recurrent well and satisfying, therefore it can provide an effective quality evaluation in the production of Zhenxian Tablets.
       【参考文献】
         　　［1］Committee of National Pharmacopoeia,“Pharmacopoeia of People"s Republic of China”［S］.Press of Chemical Industry, Beijing,2005：1.
       
       ［2］Liting XU, Jinghui DING, Taijun SUN. Determination of Bilirubin in Rengongniuhuang and its Preparation ［J］.Herald of Medicine, 2004，2（1）：49.
       
       ［3］Lei ZHANG, Neiqiang LIU, Futang ZHANG. Determination of Bilirubin in Zhenxian Tablets by HPLC ［J］.Chinese Pharmaceutical Affairs, 2003，17(9)：580.