| Honeysuckle is a common Chinese herb. The modern pharmacology research shows that honeysuckle has the functions of anti-bacterial, anti-viral, ect. It has been reported that tens of honeysuckle compound pharmaceutical preparations have been applied to clinic,and the chlorogenic acid is the main active component of honeysuckle.Quality control for drug is an essential attribute and the basis for security and validity of medicine. Quality standard is a main means to monitor drug quality and also a detailed reflection of quality control. Through looking up large number of literatures, it can be found that quality control methods of many honeysuckle compound pharmaceutical preparations have been reported yet. However, the quality control of some honeysuckle compound pharmaceutical preparations has not been reported, or only one or two active components for quality control has been reported. It can not totally evaluate the quality of these drugs. Furthermore, some quality control methods reported before need to be improved too.This research selected three kinds of honeysuckle compound pharmaceutical preparations to study, such as Yinqiao Jiedu tablet, Reduning injection and coryza dripping solution. The main active components were used as the quality control indices. And systematical methodology research of drug quality control based on RP-HPLC method was studied. The author established the assay of RP-HPLC to determine the chlorogenic acid,forsythin and glycyrrhetic acid in Yinqiao Jiedu tablet,the assay of RP-HPLC to determine the chlorogenic acid and gardenin in Reduning injection,and the assay of RP-HPLC to determine the baicalin and ephedrine hydrochloride, the chlorogenic acid and baicalin, and the only chlorogenic acid in coryza dripping solution. The method built in this research has several advantages, such as strong specificity, high sensitivity, good accuracy and stability. Therefore, it can be used for the quality control of the above mentioned honeysuckle compound pharmaceutical preparations.Part OneSimultaneous Determination of Chlorogenic Acid, Forsythin and Glycyrrhetic Acid in Yinqiao Jiedu Tablet by RP-HPLC with Three-Wavelength Detection and Gradient ElutionObjective:To establish a RP-HPLC method for separation and determination of chlorogenic acid, forsythin and glycyr- rhetic acid in Yinqiao Jiedu tablet. A analysis method can be provided for the quality control of Yinqiao Jiedu tablet by using three kinds of active ingredients as indicators. Methods: (1) Optimization of the extraction method: different extraction solvents and extration time were tested for optimization of the extraction efficacy of Yinqiao Jiedu tablet. The most efficient extration method was selected for the extraction of the major chemical components of Yinqiao Jiedu tablet. (2) Optimization of the chromatographic conditions: chromatographic conditions such as stationary phase, mobile phase systems, velocity, gradient programs and detection method were tested for optimization of the chromatographic conditions,so that the chromatographic peaks of chlorogenic acid,forsythin and glycyrrhetic acid were completely separated with other peaks. (3) System suitability test: under the above conditions, the resolution, theoretical plate number and symmetry factor of the three chemical components were studied. (4) Constructing the calibration curve; testing the limits of detection (LOD), precision, stability and reproducibility. (5) Testing the recovery. (6) Determinating the samples.Results: (1) The extraction method: an accurately weighted powder of Yinqiao Jiedu tablet was extracted with 50% methanol by a sonifier at room temperature for 30 min. The extraction method for the major chemical components of Yinqiao Jiedu tablet was efficient. (2) The chromatographic condition: a DiamonsilTM C18 column(250 mm×4.6 mm , 5μm) was used throughout. The mobile phase consisted of acetonitrile (A) and 0.1% phosphoric acid (v/v, B). The gradient program for quantitative analysis was: 0～5 min, 17 % A; 5～10 min, 17 %~30 % A;10～15 min,30 % A;15～16 min,30 %～43.5 % A;16～28 min,43.5 % A;28～29 min,43.5 %～17 % A(A+B=100 %).The flow rate was 1.0 ml/min and the column temperature was 30℃. The detection wavelength was programmed as: chlorogenic acid at 326 nm, forsythin at 228 nm and glycyrrhetic acid at 252 nm. (3) System suitability test:under the above conditions,the peaks of chlorogenic acid,forsythin and glycyrrhetic acid were separated well with the resolution of more than 2, and the theoretical plate number of each was larger than 5000. (4) Specialization: negative control samples were analyzed and the results were compared with that of the sample preparation.The results showed that the chemical components were determined without the interference of other chemical components originated from the coexisting herbs. (5) The calibration curve:the linearities of chlorogenic acid, forsythin and glycyrrhetic acid were respectively good in the range of 0.396~2.77μg(r=0.9995), 0.593~4.15μg(r=0.9999)and 0.558~ 3.91μg (r=0.9999). (6) The limits of detection (LOD): the LOD of chlorogenic acid,forsythin and glycyrrhetic acid were 0.39, 0.65 and 0.61μg/ml. (7) Precision: the RSD% of the peak areas were 0.51%, 0.24%, 0.33%, respectively. (8) Stability:the RSD% of the peak areas were 1.4%, 1.6%, 1.8%, respectively. The sample solution was stable in 24 hours. (9) Reproducibility: the RSD% of the peak areas were 1.8%, 1.2%, 1.5%, respectively. (10) Recovery test: the average recovery rates of chlorogenic acid, forsythin and glycyrrhetic acid were 98.2%, 97.9%, 96.3% and the RSD% were 1.4%, 1.5%, 1.2%, respectively. (11) The established method was applied for determination of the major chemical components in Yinqiao Jiedu tablet of different production batches and different manufactures. The content of each chemical component was calculated according to external standard method.Conclusions: Chlorogenic acid,forsythin and glycyrrhetic acid can be determined simultaneously by a RP-HPLC with three-wavelength detection and gradient elution. This method has several advantages, such as short analysis time, large linear range, high column efficiency, good specialization, good precision and accuracy. Therefore, it can be used for the quality control of Yinqiao Jiedu tablet.Part Two Determination of Chlorogenic Acid and Gardenin in Reduning Injection by RP-HPLCObjective: To establish a RP-HPLC method for deter- mination of chlorogenic acid and gardenin in Reduning injection.Methods: A DiamonsilTM C18 column (250×4.6mm,5μm) was used throughout.The mobile phase consisted of acetonitrile and 0.4% phosphoric acid (10:90); the flow rate was 1.0 ml/min; the column temperature was 30℃; the detection wavelength was 240nm.Results: Under the above conditions, the peaks of chlorogenic acid and gardenin were separated well with the resolution of more than 2. To the chlorogenic acid , theoretical plate number was 9280, and to the gardenin, theoretical plate number was 10455. The linearity of chlorogenic acid was good in the range of 0.194~1.36μg (r=0.9998) with average recovery rate reaching 99.0% (RSD=1.6%). The linearity of gardenin was good in the range of 0.162~1.14μg (r=1.000) with the average recovery rate reaching 101.5 % (RSD=1.5 %).Conclusions: Chlorogenic acid and gardenin can be determined simultaneously by a RP-HPLC method. This method has several advantages, such as short analysis time, large linear range, high column efficiency, good specialization and no interference of the excipient in the Reduning injection. Therefore, it can be used for the quality control of Reduning injection. Part Three(1) Determination of Baicalin and Ephedrine Hydrochloride in Coryza Dripping Solution by RP-HPLC with double-wavelength detection and Gradient elutionObjective: To establish a RP-HPLC method for determi -nation of baicalin and ephedrine hydrochloride in Coryza Dripping solution .Methods: A DiamonsilTM C18 column(250×4.6mm,5μm) was used throughout. The mobile phase consisted of acetonitrile (A) and 1.0% phosphoric acid (contented 0.4% triethylamine ) (B). The gradient program for quantitative analysis was: 0~3 min,12% A;3~12min, 12%~27% A;12~25min,27% A;25~26 min,27%~12% A(A+B=100 %). The time of gradient elution was 35min and the flow rate was 1.0 ml/min. The detection wavelength was programmed as: 0~7min, 210nm; 7~35min, 278nm. The column temperature was 30℃.Results: Under the above conditions, the peaks of baicalin and ephedrine hydrochloride were separated well with the resolution of more than 2.0, and the theoretical plate number of each was larger than 5000. The linearity of baicalin was good in the range of 0.635~3.02μg (r=0.9999) with average recovery rate reaching 101.5%. The linearity of ephedrine hydrochloride was good in the range of 0.203~0.966μg (r=0.9997) with average recovery rate reaching 101.0%. The RSD% of the average recovery rate for baicalin and ephedrine hydrochloride were not more than 1.Conclusions: Baicalin and ephedrine hydrochloride can be determined simultaneously by a RP-HPLC method. The method is special, sensitive and accurate. It can be used for the quality control of Coryza Dripping solution.(2) Determination of Chlorogenic Acid and Baicalin in Coryza Dripping Solution by RP-HPLCObjective: To establish a RP-HPLC method for determination of chlorogenic acid and baicalin in Coryza Dripping solution.Methods: A DiamonsilTM C18 column(250×4.6mm,5μm) was used throughout; the mobile phase consisted of methanol-water -phosphoric acid (52:48:0.1); the flow rate was 1.0 ml/min; the detection wavelength was 320nm; the column temperature was 30℃.Results: The linearity of chlorogenic acid was good in the range of 4.30～34.4μg/ml (r=1.000) with average recovery rate reaching 102.0%. The linearity of baicalin was good in the range of 15.5～108.6μg/ml (r=0.9998) with average recovery rate reaching 100.7%. The RSD% of the average recovery rate for chlorogenic acid and baicalin were not more than 2.Conclusions: Chlorogenic acid and baicalin can be determined simultaneously by a RP-HPLC method. The method is special, sensitive and simple. It is with a certain significance for the quality control of Coryza Dripping solution.(3) Determination of Chlorogenic Acid in Coryza Dripping Solution by RP-HPLCObjective: To establish a RP-HPLC method for determination of chlorogenic acid in Coryza Dripping solution. A rapid analysis method can be provided for the quality control of Coryza Dripping solution.Methods: A DiamonsilTM C18 column(250×4.6mm,5μm) was used throughout; the mobile phase consisted of methanol–water -acetic acid (25:75:1); the flow rate was 1.0 ml/min; the detection wavelength was 328nm.Results: Under the above conditions, the peak of chlorogenic acid was separated well with the resolution of more than 1.7,and the theoretical plate number was larger than 5000.The linearity of chlorogenic acid was good in the range of 4.34～30.4μg/ml (r=0.9995), and the average recovery rate was 100.5% (RSD=0.86 % ). Conclusions: The symmetrical peak shape, high column efficiency and short analysis time can be maked by a RP-HPLC method for the determination of chlorogenic acid in coryza dripping solution. The method is sensitive, simple and accurate. It can be used for the rapid determination of chlorogenic acid. |
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