Scutellaria Barbata Medicine And Injection Of Ginkgo Biloba Extract Quality

Part one Studies on the quality of Scutellaria barbata1 Studies on HPLC fingerprints of Scutellaria barbataObjective: To establish HPLC fingerprints of Scutellaria barbata so as to provide a sensitive and specific method for controlling the quality of Scutellaria barbata.Methods: (1) In order to achieve an optimal extracting condition, variables involved in the procedure, such as extraction method, solvent and time were investigated. (2) Appropriate column was chosen, and different formulation, proportion and flow rate of mobile phase were adjusted in order to establish a better fingerprint chromatogram. (3) In this chromatographic condition, the resolution and theoretical plate of scutellarin peak were calculated. (4) The precision was verified by analyzing the same sample solution six times to determine the relative retention and peak area, respectively. Meanwhile, similarity of the fingerprints was compared. (5) The reproducibility test was carried out by analyzing six replicates of the same concentration test solution, and then the relative retention times and peak areas were determined, respectively. Meanwhile, similarity of the fingerprints was compared. (6) The stability test was verified by analyzing the same sample solution to determine the relative retention times and peak areas at 0, 2, 4, 6, 8, 24, 36 and 48 h, respectively. Meanwhile, similarity of these fingerprints was compared. (7) The test solutions were prepared from 23 batches of Scutellaria barbata of different regions, a batch of reference sample and 6 batches of adulterants of different regions and then their relevant fingerprint chromatograms were obtained.Results: (1) The method of supersonic wave-extraction with 60% ethanol as the best solvent for 60 minutes was simple, quick and stable. (2) The HPLC separation was performed on a C18 analytical column gradient eluted with a mixture consisting of acetonitrile, methanol and 0.05% phosphoric acid at the flow rate of 1 mL·min-1 with UV detector. The temperature of column was set at 35℃. Injection volume was 20μL. (3) Under the above conditions, the peak of scutellarin was separated well. The resolution and theoretical plate of scutellarin were more than 1.5 and 4500, respectively. (4) The precision of the method was good. With the peak of scutellarin as reference, the RSD values of relative retention time and peak area were between 0.13%⁰.66% and between 1.1%².8%, respectively. The similarity of all fingerprints was more than 0.97, corresponding to the demand of fingerprints. (5) The reproducibility of the method was good. With the peak of scutellarin as reference, the RSD values of relative retention time and peak area were between 0.05%⁰.33% and between 1.1%².0%, respectively. The similarity of all fingerprints was more than 0.98, corresponding to the requirements of fingerprints. (6) The test solution was stable in 48 hours. With the peak of Scutellarin as reference peak, the RSD values of relative retention time and peak area were between 0.11%⁰.51% and between 1.0%².6%, respectively. The similarity of all fingerprints was more than 0.97, corresponding to the requirements of fingerprints. (7) Fingerprint chromatograms from 30 batches of Scutellaria barbatata and its adulterants from different regions were obtained. (8) Similarity analysis was performed and the results could significantly reflect the quality of Scutellaria barbata from different regions.Conclusion: The HPLC-UV fingerprints of Scutellaria barbta from different regions were established and compared. And then their differences were studied with similarity. This method is sensitive, quick, and useful to actualize standardization planting. It can be used for the quality control for Scutellaria barbata.2 Simultaneous HPLC determination of flavonoids in scutellaria barbataObjective: To establish a reversed phase high performance liquid chromatography method for the simultaneous determination of six major constituents and total flavonoids in Scutellaria barbatata in order to provide one guide line for their quality control.Methods: 1 Simultaneous determination of six flavonoids: (1) In order to achieve quantitative extraction, variables involved in the procedure, such as extraction method, solvent and times were investigated. (2) Appropriate column was chosen, and different formulation, proportion and flow rate of mobile phase were adjusted in order to separate all analytes well. (3) A series of the reference solutions were prepared. A calibration curve was constructed by plotting the peak area of each compound against the concentration of each compound. (4) For each target constituent, the LOD was determined by serial dilution of standard solution. The LOD was typically three times the noise level. (5) The measurement of intra-day and inter-day variability was utilized to assess the precision of the developed method. The intra-day precision was verified by analyzing six replicates of samples under optimal conditions and determining the relative standard deviation (RSD). The inter-day precision was also evaluated with the sample, six folds a day on different three days, and RSD was calculated. (6) The stability was carried out by determining the analytes in same treated sample solutions after storing at room temperature at different time (0, 2, 4, 6, 8, 24 h). (7) The accuracy test was carried out by adding standard solution at three different levels to the powdered samples, three replicates a level. Then the samples were treated according to the sample preparation procedure described above. Recovery was obtained by calculating the percent ratio of determined amount to added amount. And RSD was also calculated. (8) Under above-mentioned conditions, the content of six compounds was determined in Scutellaria barbatata from different regions. 2 Determination of total flavonoids: (1) A series of the reference solutions were prepared. A calibration curve was constructed by plotting the absorbance of scutellarin against the concentrations of scutellarin. (2) Under above conditions, the content of total flavonoids was determined in Scutellaria barbatata from different regions.Results: 1 Simultaneous determination of six flavonoids: (1) The method of refluxing extraction was proved to be the suitable one with 75% methanol as the best solvent, which assured the complete extraction of all the major constituents. (2) Compounds were separated on a C18 column (250 mm×4.6 mm, 5μm). Gradient elution of the analytes was performed using acetonitrile-methanol-0.05% phosphoric acid as mobile phase. The total run time was 50 min. The column temperature was maintained at 35℃and the flow rate was 1.0 mL·min-1. The detector wavelength was set at 335, 350, 288, 335, 275, 275 nm, respectively. Under the above conditions, the peaks of the test solution were separated well with the resolution of more than 1.5. Theoretical plate of scutellarin was more than 6000. (3) All calibration curves showed good linear regression (r>0.999) within test ranges. (4) The limits of detection were 0.056, 0.015, 0.033, 0.037, 0.015, 0.008μg?mL-1, respectively. (5) In this assay, both of the intra- day and inter-day precisions were below 3% (RSD), indicative of the good precision of the method. (6) The analytes were found to be stable within 24 hours and the RSD were 0.68%, 1.2%, 0.95%, 1.1%, 1.9%, 1.1%, respectively. (7) The results of recovery for all target constituents were good. It showed that the recoveries were more than 95%, which indicated that the proposed method were sufficient for determination of the analytes in scutellaria barbata. (8) The results of all target constituents were gained. 2 Determination of total flavonoids: (1) The regression equation was Y=53.15X-2.3×10^-3, r=0.9992 (n=5), the linear range of scutellarin was from 3.667μg·mL^-1 to 18.34μg·mL^-1. (2) The content of total flavonoids of all samples from different regions was determined.Conclusion: It is the first time to establish the RP-HPLC method to determine six flavonoids simultaneously in Scutellaria barbatata. This method is sensitive, quick and can be used for the quality control for Scutellaria barbatata.3 Determination of protocatechuic acid in Scutellaria barbatataObjective: To establish a method for the assay of protocatechuic acid in Scutellaria barbatata in order to provide one guide line for their quality control.Methods: (1) In order to achieve quantitative extraction, variables involved in the procedure, such as extraction method, solvent and times were investigated. (2) Appropriate column was chosen, and different formulation, proportion and flow rate of mobile phase were adjusted in order to separate protocatechuic acid well. (3) A series of the reference solutions were prepared. The calibration curve was constructed by plotting the peak area of protocatechuic acid against the concentration of protocatechuic acid. (4) The LOD was determined by serial dilution of standard solution. The LOD was typically three times the noise level. (5) The stability was carried out by determining the analytes in same treated sample solutions after storing at room temperature for different time (0, 2, 4, 6, 8, 12, 24 h). (6) The precision was verified by analyzing the same sample six times under optimal conditions and determining the relative standard deviation (RSD). (7) The reproducibility was verified by analyzing six replicates of samples under optimal conditions and determining the relative standard deviation (RSD). (8) The accuracy test was carried out by adding standard solution to the powdered samples. Then the samples were treated according to the sample preparation procedure described above. Recovery was obtained by calculating the percent ratio of determined amount to added amount. And RSD was also calculated. (9) Under above-mentioned conditions, the content of protocatechuic acid was determined in Scutellaria barbatata from different regions.Results: (1) The method of refluxing extraction was proved to be the suitable one with 60% ethanol as the best solvent. (2) The chromatographic procedure was carried out using C18 as an analytical column and a mixture of 25 volume of methanol, 75 volume of 0.05% of phosphoric acid as a mobile phase at a flow rate of 1.0 mL·min^-1. The detection wavelength was set at 258 nm. Under the above conditions, the peak of phosphoric acid in the test solution was separated well with the resolution of more than 1.5. Theoretical plate was more than 6000. (3) The regression equation was Y=4.095X-1.401×10^-3, r=0.9998 (n=6), the linear range of protocatechuic acid was from 0.018μg to 0.184μg. (4) The limit of detection was 3 ng. (5) The sample was stable in 24 hours and the RSD was 1.0%. (6) The precision of the method was good and the RSD was 0.41%. (7) The average content of protocatechuic acid was 59.610μg·g^-1 and the RSD was 0.86%, indicative of good reproducibility of the method. (8) The average recovery was 98.2% and the RSD was 0.95%. (9) The data of the content of protocatechuic acid in Scutellaria barbatata from different regions was obtained.Conclusion: It is the first time to establish the RP-HPLC method to determine protocatechuic acid concentration in Scutellaria barbatata. This method is sensitive, quick and can be used for the quality control for Scutellaria barbatata. Part two Studies on the quality of GbE for injection1 Studies on fingerprints of GbE for injectionObjective: To establish HPLC fingerprints of Ginkgo biloba extract (GbE) for injection, so as to provide a sensitive and specific method for controlling the quality of this preparation.Methods: (1) Appropriate column was chosen, and different formulation, proportion and flow rate of mobile phase were adjusted in order to establish a better fingerprint chromatogram. (2) In this chromatographic condition, the resolution and theoretical plate of rutin peak were calculated. (3) In the specificity test, the chromatograms of sample, control and blank sample were compared. And then every peak within 75 minutes was recorded. (4) The precision was verified by analyzing the same sample solution six times to determine the relative retention and peak area, respectively. Meanwhile, similarity of the fingerprints was compared. (5) The reproducibility test was carried out by analyzing six replicates of the same concentration test solution, and then the relative retention times and peak areas were determined, respectively. Meanwhile, similarity of the fingerprints was compared. (6) The stability test was verified by analyzing the same sample solution to determine the relative retention times and peak areas at 0, 2, 4, 6, 8 and 24 h, respectively. Meanwhile, similarity of these fingerprints was compared. (7) The test solutions were prepared from 10 batches of GbE, intermediate product and injection and then their relevant fingerprint chromatograms were obtained. (8) Similarity analysis was performed.Results: (1) The HPLC separation was performed on a C18 analytical column gradient eluted with a mixture consisting of acetonitrile and 0.05% phosphoric acid at the flow rate of 1 mL·min-1 with UV detector. The temperature of column was set at 35℃. Injection volume was 20μL. (2) Under the above conditions, the peak of rutin was separated well. The resolution and theoretical plate of rutin were more than 1.5 and 3500, respectively. (3) The specificity test showed that no interference peaks were found at the retention time of each peak. (4) The precision of the method was good. With the peak of rutin as reference, the RSD values of relative retention time and peak area were between 0.04%⁰.36% and between 0.10%⁰.52%, respectively. The similarity of all fingerprints was more than 0.98, corresponding to the demand of fingerprints. (5) The reproducibility of the method was good. With the peak of rutin as reference, the RSD values of relative retention time and peak area were between 0.03%⁰.31% and between 0.25%⁰.73%, respectively. The similarity of all fingerprints was more than 0.97, corresponding to the requirements of fingerprints. (6) The test solution was stable in 24 hours. With the peak of rutin as reference peak, the RSD values of relative retention time and peak area were between 0.03%⁰.34% and between 0.59%¹.6%, respectively. The similarity of all fingerprints was more than 0.96, corresponding to the requirements of fingerprints. (7) Fingerprint chromatograms from 10 batches of GbE for injection were obtained. (8) Similarity analysis was performed and the results could significantly reflect the quality of GbE for injection of different batches.Conclusion: The HPLC-UV fingerprint chromatograms of GbE for injection were established and compared. And then their difference was studied with similarity. This method is sensitive, quick and useful to control the quality of GbE for injection.2 Determination of flavonoids in GbE for injectionObjective: To establish a method for the determination of flavonoids in GbE for injection in order to provide one guide line for their quality control.Methods: (1) Appropriate column was chosen, and different formulation, proportion and flow rate of mobile phase were adjusted in order to separate all analytes well. (2) In this chromatographic condition, the resolution and theoretical plate of rutin peak were calculated. (3) In the specificity test, the chromatograms of sample, control and blank sample were compared. And then every peak within 45 minutes was recorded. (4) A series of the reference solutions were prepared. A calibration curve was constructed by plotting the peak area of each compound against the concentration of each compound. (5) For each target constituent, the LOD was determined by serial dilution of standard solution. The LOD was typically three times the noise level. (6) The precision was verified by analyzing six replicates of samples under optimal conditions and determining the relative standard deviation (RSD). (7) The reproducibility was verified by analyzing six folds of samples under optimal conditions and determining the relative standard deviation (RSD). (8) The stability was carried out by determining the analytes in same treated sample solutions after storing at room temperature at different time (0, 2, 4, 6, 8, 24 h). (9) The accuracy test was carried out by adding standard solution to six folds of samples. Then the samples were treated according to the sample preparation procedure. Recovery was obtained by calculating the percent ratio of determined amount to added amount. And RSD was also calculated. (10) Under above conditions, the contents of three compounds were determined in GbE, intermediate product and injection of different batches.Results: (1) Compounds were separated on a C18 column (250 mm×4.6 mm, 5μm). The chromatographic procedure was carried out using C18 as an analytical column and a mixture of 50 volume of methanol, 50 volume of 0.4% of phosphoric acid as a mobile phase at a flow rate of 1.0 mL·min^-1. The detection wavelength was set at 360 nm. (2) Under the above conditions, the peaks of the test solution were separated well with the resolution of more than 1.5. Theoretical plate of quercetin was more than 6500. (3) The specificity test showed that no interference peaks were found at the retention times of every peak. (4) All calibration curves showed good linear regression (r>0.999) within test ranges. The regression equations were as follows: Y=9.050×10⁴X-1.827×10⁴, r=0.9993(n=5), Y=8.143×10⁴X-0.7202×10⁴, r=0.9998(n=5) and Y=1.052×10⁵X- 0.4654×10⁴, r=0.9993(n=5).The linear ranges were 5.10⁴⁵1.04μg·mL^-1, 5.0⁵0μg·mL^-1 and 1.38¹3.8μg·mL^-1, respectively. (5) The limits of detection were 5, 8 and 12ng, respectively. (6) In this assay, precision was below 3% (RSD), indicative of the good precision of the method. (7) In this assay, reproducibility was below 3% (RSD), indicative of the good reproducibility of the method. (8) The analytes were found to be stable within 24 hours and the RSD below 3%. (9) The results of recovery for all target constituents were obtained. It showed that the recoveries were more than 98%, which indicated that the proposed method were sufficient for determination of the analytes in GbE for injection. (10) The results of all target constituents were obtained.Conclusion: A RP-HPLC method was established to determine flavonoids simultaneously in GbE for injection. This method is sensitive, quick and can be used for the quality control for GbE for injection.3 Determination of terpenlactones in GbE for injectionObjective: To establish a method for the determination of terpenlactones in GbE for injection in order to provide one guide line for their quality control.Methods: (1) Appropriate column was chosen, and different formulation, proportion and flow rate of mobile phase were adjusted in order to separate all analytes well. (2) Under this chromatographic condition, the resolution and theoretical plate of rutin peak were calculated. (3) In the specificity test, the chromatograms of sample, control and blank sample were compared. And then every peak within 25 minutes was recorded. (4) A series of the reference solutions were prepared. A calibration curve was constructed by plotting the peak area of each compound against the concentration of each compound. (5) For each target constituent, the LOD was determined by serial dilution of standard solution. The LOD was typically three times the noise level. (6) The precision was verified by analyzing six replicates of samples under optimal conditions and determining the relative standard deviation (RSD). (7) The reproducibility was verified by analyzing six folds of samples under optimal conditions and determining the relative standard deviation (RSD). (8) The stability was carried out by determining the analytes in same treated sample solutions after storing at room temperature at different time (0, 2, 4, 6, 8, 24 h). (9) The accuracy test was carried out by adding standard solution to six folds of samples. Then the samples were treated according to the sample preparation procedure. Recovery was obtained by calculating the percent ratio of determined amount to added amount. And RSD was also calculated. (10) Under above conditions, the contents of four compounds were determined in GbE, intermediate product and injection of different batches.Results: (1) The HPLC separation was performed on a C18 analytical column gradient eluted with a mixture consisting of methanol and water at the flow rate of 1 mL·min-1 with ELSD. The temperature of column was set at 35℃. Injection volume was 20μL. (2) Under the above conditions, the peaks of the test solution were separated well with the resolution of more than 1.5. Theoretical plate of bilobalide was more than 5000. (3) The specificity test showed that no interference peaks were found at the retention times of every peak. (4) All calibration curves showed good linear regression (r>0.998) within test ranges. The linear ranges were 0.0412⁰.3708 mg·mL-1, 0.0255⁰.2295 mg·mL-1, 0.0293⁰.2637 mg·mL-1 and 0.0665⁰.5985 mg·mL-1, respectively. The regression equation were as follows: lgY=1.33lgX+7.83 (r=0.9989, n=5), lgY=1.32lgX+7.79 (r=0.9993, n=5), lgY=1.40lgX+8.03 (r=0.9990, n=5) and lgY=1.24lgX+4.81 (r=0.9994,n=5). (5) The limits of detection were 8, 15, 13 and 11 ng, respectively. (6) In this assay, precision was below 3% (RSD), indicative of the good precision of the method. (7) In this assay, reproducibility was below 3% (RSD), indicative of the good reproducibility of the method. (8) The analytes were found to be stable within 24 h and the RSD below 3%. (9) The results of recovery for all target constituents were obtained. They showed that the recoveries were more than 97%, which indicated that the proposed method were sufficient for determination of the analytes in GbE for injection. (10) The results of all target constituents were obtained.Conclusion: A RP-HPLC method was established to determine terpenlactones simultaneously in GbE for injection. This method is sensitive, quick and can be used for the quality control for GbE for injection.