Studies On Fingerprints Of Gan Jing Injection

Gan Jing Rejection is made from Herba Artemisiae Scopariae,Gardenia jasminoides Ellis,Radix et Rhizoma Rhei .Variety and complexity of chemistry components of TCM are the physical foundation to produce curative action. However, one or several components are always only used in quality evaluation of TCM as index, which always can't reflect its internal quality comprehensively. The modern analytical technique is applied to study fingerprints by using figures to describe the internal chemical information of TCM. In this case, fingerprints just are a new method to reflect the differences of description and quantity of chemical information. It can be used to control the quality of TCM and drugs preparation through integrative information. It's a better method ratified in the international to control the quality of TCM. So it is necessary to develop the fingerprints of Gan Jing Injection to control quality of it thoroughly.Objective:To establish HPLC fingerprints of Gan Jing Injection ; To get the control chromatogram and the fingerprints of Gan Jing Injection collected from different batches were compared so as to establish a sensitive and specific method for controlling the quality of Gan Jing Injection.Methods:(1) Extraction: An optimal extracting condition was chosen by comparing different experimental results. (2)Chromatographic condition: Choose appropriate column and adjust different formulation and proportion of mobile phase and column temperature in order to establish a better fingerprint chromatogram. (3) System suitability test: In this chromatographic condition, calculate the resolution and theoretical plate of baicalin peak. (4) Specificity test: 10μl of blank solution, test solutions and control solutions were transferred precisely and injected into HPLC system, and then every peak within 130 minutes was recorded. (5) Precision test: the same test solutions were taken and injected to the instrument for five times and the relative retention time and peak area were determined,respectively.Meanwhile,similarity of the fingerprints was compared. (6) Reproducibility test: six shares of the same concentration test solution were prepared. In addition, the relative retention time and peak area was determined, respectively. Meanwhile, similarity of the fingerprints was compared. (7) Stability test: take the sample solution to inject to the instrument. Determine the relative retention time and peak area at the 0, 2.5, 5, 7.5, 12 and 24 hour, respectively. Meanwhile, similarity of these fingerprints was compared. (8) Development of fingerprints: the test solution was prepared from 5 batches of Gan Jing Injection, and then their relevant fingerprint chromatograms were obtained.Results:(1) Extraction: one injection was filtrated through membrane (e0.5μm). (2) The HPLC separation was performed on a C18 analytical column gradient eluted with a mixture consisting of acetonitrile and water at the flow rate of 1ml/min with UV detector. The temperature of column was set at 25±0.15℃. Injection volume was 10μl. (3) System suitability test: Under the above condition, the peak of baicalin was separated well with the resolution of more than 1.5 and about 150000 of theoretical plate. (4) Specificity test: It showed that there weren't any disturbance in blank solvent and the peak corresponding to baicalin appeared at the same time that named 26 in the test solution. (5) Precision test: The similarity of all of the fingerprints is more than 0.98, coinciding with the demand of fingerprints. (6) The reproducibility of sample: The similarity of all of the fingerprints is more than 0.96, coinciding with the requirements of fingerprints. The reproducibility is acceptable. (7) The test solution was stable in 24 hours. The similarity of all of the fingerprints is more than 0.98, coinciding with the requirements of fingerprints. (8) Development of relevant fingerprint chromatograms: Fingerprint chromatograms from 5 batches of Gan Jing Injection were got. (9) Data analysis: Hierarchical clustering analysis was performed and the result can significently reflect the quality of Gan Jing Injection from different batches.Conclusion:It is the first time to establish the HPLC-UV fingerprint chromatogram of Gan Jing Injection,compare the fingerprints from different batches,and study their difference with similarity and Hierarchical clustering analysis, then generally evaluate the quality of Gan Jing Injection. More reliable data were provided for the large scale of Gan Jing Injection production. This method is sensitive,quick, and useful to actualize standardization planting. It can be used for the quality control for Gan Jing Injection. Objective: To study the TLC Identification Method and establish a specific method to control the quality of Gan Jing Injection.Methods:(1) Extraction: An optimal extracting condition was chosen by comparing different experimental results. (2) Chromatographic condition: To meet the requirement of TLC study, chose the proper thin-layer vitreous board, detecting methods, and the ratio of the developer. (3) Specificity test: Drop the sample liquor negtive liquor and the contrast liquor with the same consistence on the one thin-layer vitreous board and use thin-layer chromatography to observe the place of the every main fleck, size and color.Results: (1)Extraction: The optimum extracting condition obtained:Add HCl 2 ml and chloroform 10 ml into 15 ml injection, reflux 1 hour, set cold.After being deproteinizated with chloroform,the extractions were evaporated to dry. The residue was dissolved in 1 ml methanol. (2)Chromatographic condition: Drop the sample liquor and the contrast liquor with 5μl on the one siliceous gel thin-layer vitreous board and use petroleum ether(30~60℃)-ethyl formate-formic acid(15:5: 1,upper layer) as the mobile phase,detecting at 365nm. And use thin-layer chromatography to observe the place of the every main fleck, size and color. (3)Specificity test: TLC chromatograms spots were clear not disturbed identification .Conclusion: There is a simple method which can be used to control the qulity of Gan Jing Injection. Objective: To study the TLC Identification Method and establish a specific method to control the quality of Gan Jing Injection.Methods:(1) Extraction: An optimal extracting condition was chosen by comparing different experimental results. (2) Chromatographic condition: To meet the requirement of TLC study, chose the proper thin-layer vitreous board, detecting methods, and the ratio of the developer. (3) Specificity test: Drop the sample liquor negtive liquor and the contrast liquor with the same consistence on the one thin-layer vitreous board and use thin-layer chromatography to observe the place of the every main fleck, size and color.Results:(1)Extraction:Added 2 ml injection into forty percent of sodium hydroxide,2 ml.Place the solution in dry oven and the temperature was kept at 120℃for 2 hours , transfer pH value to 2-3,centrifuge the supernatant were extracted three times with ethyl acetate,withaxtract volume 20 ml everytime.The final solution was evaporated to dryness.The residue was dissolved in 2 ml ethanol. (2)Chromatographic condition: Drop the sample liquor and the contrast liquor with 5μl on the one siliceous gel thin-layer vitreous board and use isooctane-ether-acetic acid -butanol water(10:5:3:1,upper layer) as the mobile phase,detecting at Visible Light. And use thin-layer chromatography to observe the place of the every main fleck, size and color. (3)Specificity test: TLC chromatograms spots were clear not disturbed identification.Conclusion: There is a simple method which can be used to control the qulity of Gan Jing Injection. Objective:To determine the content of chlorogenic acid in GanJing Injection by HPLCMethods : (1)Chromatographic condition: Choose appropriate column and wavelength, different formulation and proportion of mobile phase and adjust optimal flow rate and column temperature in order to establish a better chromatogram. (2) System suitability test: In this chromatographic condition, calculate the resolution and theoretical plate of chlorogenic acid. (3) Specificity test: 10μl of blank solution, test solutions and control solutions were transferred precisely and injected into HPLC system. (4)Linear test: take the same test solution,inject into the HPLC system with 1μl,2μl,5μl,10μl,25μl respectively. Record the retention time and area. Then the regression equation was obtained with the content of chlorogenic acid as abscissa and the relevant peak area as ordinate. (5) Reproducibility test: prepare the test solutions from Gan Jing Injection with the same amount for 5 times in the same way and record the retention time and area. (6) Stability test: inject the sample at 0,2,4,12,24hour, respectively. Record the retention time and area. (7)Rang: prepare 3 samples at the maximum and the minimum concentration respectively, inject the samples. Record the retention time and area. (8) The recovery rate test of added sample.Results:(1) A Shimadzu (Shim-pack VP-ODS) column (4.6 mm×150 mm,5μm) was used as stationary phase and the Acetonitrile:0.1% Phosphoric acid=10:90was used as mobile phase with 327nm for detection wavelength. The flow rate was 1.0 ml·min-1 and the column temperature was 25℃. Injection volume was 10μl. (2) System suitability test: Under the above condition, the peak of chlorogenic acid of the test solution was separated well with the resolution of 2.65 more than 1.5 and about 6700 of theoretical plate. (3) Specificity test: It showed that there weren't any disturbance in blank solvent and the peaks correspoding to chlorogenic acid appeared at the same time in the test solution. (4) Linear test: Chlorogenic acid showed a good linear relationship in the range of 0.089 9~2.2475μg, the regression equation was Y=3485730.91X +7622.40 (r=0.9999);The linear range of chlorogenic was 0.089 9~2.2475μg (r=0.999 9). (5) Reproducibility test: the average content is 0.2708 mg·ml-1,the RSD values is 1.87%(n=6). (6) Stability test: The RSD values is 0.28%(n=5),the experimental results show that the chlorogenic acid are stable within 24 hours. (7)Rang: the average content is 0.2689mg·ml-1,the RSD values is 1.07%(n=6).(8) The average recovery was 99.24% (RSD=1.87%, n=6).Conclusion:The method is simple, accurate, stable and reproducible, and can be applied to quality control of chlorogenic acid. Objective:To determine the content of baicalin in GanJing Injection by HPLCMethods : (1)Chromatographic condition: Choose appropriate column and wavelength, different formulation and proportion of mobile phase and adjust optimal flow rate and column temperature in order to establish a better chromatogram. (2) System suitability test: In this chromatographic condition, calculate the resolution and theoretical plate of chlorogenic acid. (3) Specificity test: 10μl of blank solution, test solutions and control solutions were transferred precisely and injected into HPLC system. (4) Linear test: take the same test solution,inject into the HPLC system with 1μl,2μl,5μl,10μl,25μl respectively. Record the retention time and area. Then the regression equation was obtained with the content of baicalin as abscissa and the relevant peak area as ordinate. (5) Reproducibility test: prepare the test solutions from Gan Jing Injection with the same amount for 5 times in the same way and record the retention time and area. (6) Stability test: inject the sample at 0,2,4,12,24hour, respectively. Record the retention time and area. (7)Rang: prepare 3 samples at the maximum and the minimum concentration respectively, inject the samples. Record the retention time and area. (8) The recovery rate test of added sample. Results:(1) A Shimadzu (Shim-pack VP-ODS) column (4.6 mm×150 mm,5μm) was used as stationary phase and the Acetonitrile:0.1% Phosphoric acid=45:55 was used as mobile phase with 276 nm for detection wavelength. The flow rate was 1.0 ml·min-1 and the column temperature was 25℃. Injection volume was 10μl. (2) System suitability test: Under the above condition, the peak of baicalin of the test solution was separated well with the resolution of 8.14 more than 1.5 and about 4600 of theoretical plate. (3) Specificity test: It showed that there weren't any disturbance in blank solvent and the peaks correspoding to baicalin appeared at the same time in the test solution. (4)Linear test: The linear range of chlorogenic was 0.104 3～2.607 5μg (r=0.999 9). (5) Reproducibility test: the average content is 1.883 mg·ml-1,the RSD values is 0.99%(n=6). (6) Stability test: The RSD values is 0.78%(n=5),the experimental results show that the chlorogenic acid are stable within 24 hours.(7) Rang: the average content is 1.771mg·ml-1,the RSD values is 1.5%(n=6).(8) The average recovery was 98.72% (RSD=1.05%, n=6).Conclusion:The method is simple, accurate, stable and reproducible, and can be applied to quality control of baicalin.