Hebei The Geoherbs Chromatographic Fingerprints Study

Part one Studies on fingerprints of Radix Asteris from Hebei ProvinceRadix Asteris is the dry root or rhizome of Aster tataricus L. f., which belongs to the family of Compositae. It has many effects, such as moistening lung to lower qi, dispersing phlegm and relieving cough, and so on. Triterpenes (shionone, friedelin, epifriedelinol, et al) are the bioactive components. Shionone is one of the bioactive components. Radix Asteris mainly is produced in Hebei province, where the quality of it is best. Radix Asteris of Anguo is always called as"Radix Asteris of Qi". Because of difference of geography and growth environment, the quality is different from different places.Objective: To establish HPLC-UV fingerprints of Radix Asteris from Hebei province and get reference fingerprint, to compare the fingerprints of Radix Asteris collected from different producing areas. To determine the contents of shionone in 17 batches of Radix Asteris, combining with the study of fingerprints so as to establish a specific method for controlling the quality of Radix Asteris.Methods: 1 Establishment of fingerprint (1) Extraction: An optimal extracting condition is chosen by comparing the experimental results. (2) Chromatographic conditions: Choose appropriate column and adjust different formulation, proportion of mobile phase and column temperature in order to obtain a better fingerprint. (3) System suitability test: Under above conditions, calculate the resolution and theoretical plate of shionone peak. (4) In the experiment of precision, prepare sample solution, inject to the apparatus for six times, and determine the relative retention time and peak areas, respectively. (5) In the experiment of reproducibility, prepare six sample solutions of Radix Asteris parallelly, inject each solution for one time in the same way, and determine the relative retention time and peak areas, respectively. (6) In the experiment of stability, prepare sample solution of Radix Asteris and determine the relative retention time and peak areas at 0, 2, 4, 6, 8, 12, 24 h, respectively. (7) Establishment of fingerprint: Prepare each sample solution of Radix Asteris, inject to HPLC, and get their relevant fingerprints. 2 Assay (1) Extraction: The same as"establishment of fingerprint". (2) Chromatographic conditions: Basing on"establishment of fingerprint", adjust different formulation and proportion of mobile phase in order to obtain a better method for assay. (3) System suitability test: Under above conditions, calculate the resolution and theoretical plate of shionone peak. (4) Preparation of standard curve: Prepare a series of the reference solutions of shionone and determine peak areas. Then the regression equation was obtained with the concentrations of shionone as ordinate and the relevant peak areas as abscissa. (5) In the experiment of precision, prepare sample solution of Radix Asteris, inject to the apparatus for six times, determine the peak areas of shionone, and then calculate the contents and RSD value. (6) In the experiment of reproducibility, prepare six sample solutions of Radix Asteris parallelly, inject each solution for one time in the same way, determine the peak areas, and then calculate the contents and RSD value. (7) In the experiment of stability, prepare sample solution of Radix Asteris to determine the peak areas of shionone at 0, 2, 4, 6, 8, 12 and 24 h, and then calculate the contents and RSD value. (8) In the experiment of recovery, transfer 6 shares of Radix Asteris, add the reference solution of shionone, and then calculate the contents of it. (9) Determination of the detection limit: Dilute the reference solution of shionone until the value of S/N is more than or equal to three. The relevant sample size is the detection limit. (10) Assay: Under above-mentioned conditions, determine the contents of shionone in 17 batches of Radix Asteris.Results: 1 Establishment of fingerprint (1) Extraction: The method of supersonic wave-extraction with methanol for 20 min was simple, quick and stable. (2) Chromatographic conditions: The chromatographic procedure for fingerprint was carried out with DiamonsilTM C18 (250 mm×4.6 mm, 5μm) as an analytic column and a mixture consisting of acetonitrile and 0.05% H3PO4 in gradient as mobile phase. The detection wavelength was set at 200 nm. The temperature of column was 30?C. The injection volume was 20μL. (3) System suitability test: Under above conditions, the peak of shionone was separated well with the resolution of more than 1.5 and about 3.0×103 of theoretical plate. (4) The precision of sample was good. Designating shionone peak for reference peak, the RSD values of relative retention time and peak areas were between 0.11%～1.3% and between 0.18%～1.5%, respectively. The semblances were above 0.9. (5) The reproducibility of sample was good and the RSD values of relative retention time and peak areas were between 0.12%～1.1% and between 0.31%～2.9%, respectively. The semblances were above 0.9. (6) The test solution was stable in 24 h. The RSD values of relative retention time and peak areas were between 0.19%～1.7% and between 0.29%～2.1%, respectively. The semblances were above 0.9. (7) Establishment of fingerprint: We have got 20 common peaks from reference fingerprint and the fingerprints of different batches of Radix Asteris. (8) Data analysis: Used similarity to analyze data, the results showed that the semblances of 10 batches of samples from Hebei province were above 0.95. There was no significant difference among samples from Hebei province and obvious difference among samples from different sources. 2 Assay (1) Extraction: The same as"establishment of fingerprint". (2) Chromatographic conditions: Acetonitrile as mobile phase at a flow rate of 2.0 mL/min, and the others were the same as"establishment of fingerprint". (3) System suitability test: Under above conditions, the peak of shionone was separated well with the resolution of more than 1.5 and about 9.8×103 of theoretical plate. (4) The regression equation was: Y=2.500×10-3+9.800×10-8X, r=0.9996(n=5). The linear range was between 0.099～0.895 mg/mL. (5) The precision of sample was good and the RSD value of shionone was 1.4%. (6) In the experiment of reproducibility, the RSD value of shionone was 3.0%. (7) The sample solution was stable in 24 h and the RSD value of shionone was 0.66%. (8) The average recovery of was 99.27%, and the RSD value was 1.1%. (9) The detection limit of shionone was 1 ng. (10) The results showed the contents of shionone in Radix Asteris of different sources were between 0%～0.3880%.Conclusion: We have established the HPLC-UV fingerprints of 10 batches of Radix Asteris from Hebei province, got reference fingerprint, compared the fingerprints of 7 batches of samples from different sources, and studied their difference with semblances. At the same time, we have determined the contents of 17 batches of Radix Asteris from different sources. The reproducibility and precision of the method were good. The method was useful to actualize standardization planting and control the quality of Radix Asteris. Part two Studies on fingerprints of Radix Polygalae from Hebei ProvinceRadix Polygalae is the dry root of Polygala tenuifolia Willd. or Polygala sibirica L., which belongs to the family of Polygalaceae. It has many effects, such as growing in intelligence, eliminating phlegm, detumescence and so on. The onjisaponins are the main components and are mainly analyzed with HPLC-ELSD. Besides, xanthenone, glycolipid are also the main components of Radix Polygalae. In order to show the chemical compositions as many as possible, we analyze the samples with HPLC-UV. Based on the fact, correlating spectrum analysis is performed for Radix Polygalae from famous-region drugs so as to provide objective basis for the quality evaluation of Radix Polygalae from different producing areas.Objective: To establish HPLC-ELSD and HPLC-UV fingerprints of Radix Polygalae from Hebei province and get two reference fingerprints, to compare the fingerprints of Radix Polygalae collected from different producing areas so as to establish a specific method for controlling the quality of Radix Polygalae. Methods: 1 HPLC-ELSD fingerprint (1) Extraction: An optimal extracting condition is chosen according to onjisaponins by comparing the experimental results. (2) Chromatographic conditions: Choose appropriate column and detector; adjust different formulation, proportion of mobile phase and column temperature in order to obtain a better fingerprint. (3) System suitability test: Under above conditions, calculate the resolution and theoretical plate of each peak. (4) In the experiment of precision, prepare sample solution of Radix Polygalae, inject it to the apparatus for six times, and determine the relative retention time and peak areas, respectively. (5) In the experiment of reproducibility, prepare six sample solutions of Radix Polygalae parallelly, inject each solution for one time in the same way, and determine the relative retention time and peak areas, respectively. (6) In the experiment of stability, prepare sample solution of Radix Polygalae and determine the relative retention time and peaks area at 0, 2, 4, 6, 8, 12, 24 h, respectively. (7) Establishment of fingerprints:Prepare each sample solution of Radix Polygalae, inject to apparatus, and get their relevant fingerprints. 2 HPLC-UV fingerprint (1) Extraction: An optimal extracting condition is chosen according to the most chemical compositions by comparing the experimental results. (2) Chromatographic conditions: Choose appropriate column and detector; adjust different formulation, proportion of mobile phase and column temperature in order to obtain a better fingerprint. (3) System suitability test: Under above conditions, calculate the resolution and theoretical plate of each peak. (4) In the experiment of precision, prepare sample solution of Radix Polygalae, inject to the apparatus for six times, and determine the relative retention time and peak areas, respectively. (5) In the experiment of reproducibility, prepare six sample solutions of Radix Polygalae parallelly, inject each solution for one time in the same way, and determine the relative retention time and peak areas, respectively. (6) In the experiment of stability, prepare sample of Radix Polygalae and determine the relative retention time and peak areas at 0, 2, 4, 6, 8, 12, 24 h, respectively. (7) Establishment of fingerprints: Prepare each sample solution of Radix Polygalae, inject to apparatus, and get their relevant fingerprints.Results: 1 HPLC-ELSD fingerprint (1) Extraction: The method of supersonic wave-extraction with 75% methanol for 20 min was simple, quick and stable. (2) Chromatographic conditions: The fingerprints of 19 batches of Radix Polygalae were determined by using of Waters 1525 pump and Waters ELSD 2420 detector. The chromatographic procedure was carried out with DiamonsilTM C18 (250 mm×4.6 mm, 5μm) as an analytical column and a mixture consisting of acetonitrile and 0.2% formic acid in gradient as mobile phase. The flow rate was 1.0 mL/min. The temperature of column was 35?C. The flow rate of N2 was 25 psi. The temperature of tube was 65?C. The temperature of nebulizer was 36?C. The gain was 350. The injection volume was 20μL. (3) System suitability test: Under above conditions, each peak was separated well with the resolution of more than 1.2 and about 6.0×103 of theoretical plate. (4) The precision of sample was good. Designating reference peak, the RSD values of relative retention time and peak areas were between 0.09%～2.0% and between 0.15%～2.5%, respectively. The semblances were above 0.9. (5) The reproducibility of sample was good and the RSD values of relative retention time and peak areas were between 0.16%～2.7% and between 0.31%～2.9%, respectively. The semblances were above 0.9. (6) The test solution was stable in 24 h. The RSD values of relative retention time and peak areas were between 0.10%～1.3% and between 0.29%～2.3%, respectively. The semblances were above 0.9. (7) Establishment of fingerprint: We have got 16 common peaks from reference fingerprint and the fingerprints of different batches of Radix. (8) Data analysis: Used similarity to analyze data, the results showed that the semblances of 10 batches of samples from Hebei province were above 0.85. There was no significant difference among samples from Hebei, Shanxi and Shanxi provinces, and obvious difference among samples from other sources because of the lack of chemical compositions. 2 HPLC-ELSD fingerprint (1) Extraction: The same as"HPLC-UV fingerprints". (2) Chromatographic conditions: The fingerprints of 19 batches of Radix Polygalae were determined by using of Waters 1525 pump and Waters UV 2487 detector. The chromatographic procedure was carried out with DiamonsilTM C18 (250 mm×4.6 mm, 5μm) as an analytical column and a mixture consisting of acetonitrile and 0.2% formic acid in gradient as mobile phase. The flow rate was 1.0 mL/min. The temperature of column was 35?C. The injection volume was 20μL. (3) System suitability test: Under above conditions, each peak was separated well with the resolution of more than 1.0 and about 2.8×103 of theoretical plate. (4) The precision of sample was good. Designating reference peak, the RSD values of relative retention time and peak areas were between 0.07%～1.7% and between 0.13%～1.6%, respectively. The semblances were above 0.9. (5) The reproducibility of sample was good and the RSD values of relative retention time and peak areas were between 0.15%～2.5% and between 0.29%～2.7%, respectively. The semblances were above 0.9. (6) The test solution was stable in 24 h. The RSD values of relative retention time and peak areas were between 0.08%～1.5% and between 0.21%～2.0%, respectively. The semblances were above 0.9. (7) Establishment of fingerprint: We have got 24 common peaks from reference fingerprint and the fingerprints of different batches of Radix. (8) Data analysis: Used similarity to analyze data, the results showed that the semblances of 10 batches of samples from Hebei province were above 0.85. There was no significant difference among samples from Hebei, Shanxi and Shanxi provinces and obvious difference among samples from other sources because of the lack of chemical compositions.Conclusion: We have established the HPLC-ELSD and HPLC-UV fingerprints of 10 batches of Radix Polygalae from Hebei province, got two reference fingerprints, and respectively compared with the fingerprints of 9 batches of samples from different sources, and used similarity to analyze data so as to study their difference with semblances. The reproducibility and precision of the method were good. The method was useful to actualize standardization planting and control the quality of Radix Polygalae. Part three Studies on fingerprints of Radix isatid from Hebei ProvinceRadix isatidis is the dry root of Isatis indigotica Fort., which belongs to the family of crucifer. It has many effects, such as heat-clearing and detoxicating, cooling blood, relieving sore-throat, and so on. Organic acid, amino acids, alkaloids are the main components. We divide the components into water-soluble chemical components and the fat-soluble chemical constituents according to their heteropolarity. In this paper, we establish the fingerprints of water-soluble and the fat-soluble chemical constituents of 20 batches of samples, and evaluate the result in order to control the quality of Radix isatidis. At the same time, we determine the contents of indigo and indirubin in 20 batches of Radix isatidis, combining with the study of fingerprints so as to actualize standardization planting and control the quality of Radix isatidis.Objective: To establish HPLC-UV fingerprints of the water-soluble and the fat-soluble chemical constituents of Radix isatidis from Hebei province and get two reference fingerprints, to compare the fingerprints of Radix isatidis collected from different producing areas, respectively. To determine the contents of indigo and indirubin in 20 batches of Radix isatidis, combining with the study of fingerprints so as to establish a specific method for controlling the quality of Radix isatidis.Methods: 1 Fingerprint of the water-soluble chemical constituents (1) Extraction: An optimal extracting condition is chosen by comparing the experimental results. (2) Chromatographic conditions: Choose appropriate column and adjust different formulation, proportion of mobile phase and column temperature in order to obtain a better fingerprint. (3) System suitability test: Under above conditions, calculate the resolution and theoretical plate of each peak. (4) In the experiment of precision, prepare sample solution, inject to the apparatus for six times, and determine the relative retention time and peak areas, respectively. (5) In the experiment of reproducibility, prepare six sample solutions of Radix isatidis parallelly, inject each solution for one time in the same way, and determine the relative retention time and peak areas, respectively. (6) In the experiment of stability, prepare sample solution of Radix isatidis and determine the relative retention time and peak areas at 0, 2, 4, 6, 8, 12, 24 h, respectively. (7) Establishment of fingerprints: Prepare each sample solution of Radix isatidis, inject to apparatus, and get their relevant fingerprints. 2 Fingerprints of the fat-soluble chemical constituents (1) Extraction: An optimal extracting condition is chosen by comparing the experimental results. (2) Chromatographic conditions: Choose appropriate column and adjust different formulation, proportion of mobile phase and column temperature in order to obtain a better fingerprint. (3) System suitability test: Under above conditions, calculate the resolution and theoretical plate of indigo and indirubin peaks. (4) In the experiment of precision, prepare sample solution, inject to the apparatus for six times, and determine the relative retention time and peak areas, respectively. (5) In the experiment of reproducibility, prepare six sample solutions of Radix isatidis parallelly, inject each solution for one time in the same way, and determine the relative retention time and peak areas, respectively. (6) In the experiment of stability, prepare sample solution of Radix isatidis and determine the relative retention time and peak areas at 0, 2, 4, 6, 8, 12, 24 h, respectively. (7) Establishment of fingerprints: Prepare each sample solution of Radix isatidis, inject to apparatus, and get their relevant fingerprints. 3 Assay (1) Extraction: The same as"fingerprints of the fat-soluble chemical constituents". (2) Chromatographic conditions: Basing on"fingerprints of the fat-soluble chemical constituents", adjust different formulation and proportion of mobile phase in order to obtain a better method for assay. (3) System suitability test: Under above conditions, calculate the resolution and theoretical plate of indigo and indirubin peaks. (4) Preparation of standard curve: Prepare a series of the reference solutions of indigo and indirubin, determine peak areas. Then the regression equation is obtained with the concentrations of indigo and indirubin as abscissa and the relevant peak area as ordinate, respectively. (5) In the experiment of precision, prepare sample solution of Radix isatidis, inject to the apparatus for six times, and determine the relevant peak areas of indigo and indirubin, respectively. Then calculate the contents and RSD values. (6) In the experiment of reproducibility, prepare six sample solutions of Radix isatidis parallelly, inject each concentration solution for one time in the same way, and determine the relevant peak area, respectively. Then calculate the contents and RSD values. (7) In the experiment of stability, prepare sample solution of Radix isatidis to determine the peak areas at 0, 2, 4, 6, 8, 12 and 24 h, respectively. Then calculate the contents and RSD values. (8) In the experiment of recovery, transfer 6 shares of Radix isatidis and add the reference solutions of indigo and indirubin, respectively. Then determine the contents of indigo and indirubin. (9) Determination of the detection limit: Dilute the reference solutions of indigo and indirubin respectively until the values of S/N are more than or equal to three. The relevant sample sizes are the detection limits of indigo and indirubin. (10) Assay: Under above-mentioned conditions, determine the contents of indigo and indirubin in different batches of Radix isatidis, respectively.Results: 1 Fingerprints of the water-soluble chemical constituents (1) Extraction: The method of supersonic wave-extraction with water for 20 min was simple, quick and stable. (2) Chromatographic conditions: The chromatographic procedure for fingerprints was carried out with DiamonsilTM C18 (250 mm×4.6 mm, 5μm). The gradient elution was a mixture consisting of methanol and water as mobile phase. The temperature of column was 30?C. The flow rate was 1.0 mL/min. The detection wavelength was set at 260 nm. The injection volume was 20μL. (3) System suitability test: Under above conditions, each peak was separated well with the resolution of more than 1.2 and about 2.7×103 of theoretical plate. (4) The precision of sample was good. Designating reference peak, the RSD values of relative retention time and peak areas were between 0.09%～1.8% and between 0.15%～2.5%, respectively. The semblances were above 0.9. (5) The reproducibility of sample was good and the RSD values of relative retention time and peak areas were between 0.17%～2.1% and between 0.29%～2.9%, respectively. The semblances were above 0.9. (6) The test solution was stable in 24 h. The RSD values of relative retention time and peak areas were between 0.11%～1.5% and between 0.17%～2.3%, respectively. The semblances were above 0.9. (7) Establishment of fingerprint: We have got 10 common peaks from reference fingerprint and the fingerprints of different batches of Radix isatidis. (8) Data analysis: Used similarity to analyze data, the results showed that the semblances of 20 batches of samples were above 0.850. There was no significant difference among samples from Hebei province and different sources. 2 Fingerprints of the fat-soluble chemical constituents (1) Extraction: The method of refluxing with chloroform for 5 h was simple and stable. (2) Chromatographic conditions: The chromatographic procedure for fingerprints was carried out with DiamonsilTM C18 (250 mm×4.6 mm, 5μm). The gradient elution was a mixture consisting of acetonitrile and 0.05% H3PO4 as mobile phase. The temperature of column was 30?C. The flow rate was 1.0 mL/min. The detection wavelength was set at 300 nm. The injection volume was 20μL. (3) System suitability test: Under above conditions, indigo and indirubin peaks were separated well with the resolution more than 1.5, the theoretical plate of them were 8.3×103 and 1.2×104. (4) The precision of sample was good. Designating indirubin peak for reference peak, the RSD values of relative retention time and peak areas were between 0.25%～2.0% and between 0.58%～2.9%, respectively. The semblances were above 0.9. (5) The reproducibility of sample was good and the RSD values of relative retention time and peak areas were between 0.17%～2.1% and between 0.22%～2.1%, respectively. The semblances were above 0.9. (6) The test solution was stable in 24 h. The RSD values of relative retention time and peak areas were between 0.19%～2.0% and between 0.31%～2.4%, respectively. The semblances were above 0.9. (7) Establishment of fingerprint: We have got 14 common peaks from reference fingerprint and the fingerprints of different batches of Radix isatidis. (8) Data analysis: Used similarity to analyze data, the results showed that the semblances of 10 batches of samples were above 0.850. There was obvious difference among samples from other sources because of the lack of chemical compositions. 3 Assay (1) Extraction: The same as"fingerprints of the chemical constituents". (2) Chromatographic conditions: The chromatographic procedure for fingerprints was carried out with DiamonsilTM C18 (250 mm×4.6 mm, 5μm). The gradient elution was a mixture consisting of acetonitrile and 0.05% H3PO4 as mobile phase. The temperature of column was 30?C. The flow rate was 1.0 mL/min. The detection wavelength was set at 300 nm. The injection volume was 20μL. (3) System suitability test: Under above conditions, the peaks of indigo and indirubin were separated well. The resolutions were 6.15 and 2.78, the theoretical plates were 3.3×103 and 3.8×103. (4) The regression equations of indigo and indirubin were: Y靛蓝=8.646×104X+ 1.219×104 (r=0.9999, n=7);Y靛玉红=8.568×104X+3.146×104 (r=0.9995, n=7), respectively. Their linear ranges were between 0.1488～9.520μg/mL and between 0.2539～16.25μg/mL, respectively. (5) The precision of sample was good and the RSD values of indigo and indirubin were 1.0% and 0.79%, respectively. (6) In the experiment of reproducibility and the RSD values of indigo and indirubin were 1.6% and 1.6%, respectively. (7) The test solution was stable in 24 h and the RSD values of indigo and indirubin were 1.7% and 1.3%, respectively. (8) The average recoveries of indigo and indirubin were 97.8% and 98.5%, and their RSD values were 1.7% and 1.6%, respectively. (9) The detection limits of indigo and indirubin were 2ng and 3 ng, respectively. (10) The results showed the contents of indigo and indirubin in Radix isatidis of different sources were between 0.0459～4.095μg/g and between 6.250～0.0522μg/g, respectively.Conclusion: We have established the HPLC-UV fingerprints of the water-soluble and the fat-soluble chemical constituents of 10 batches of Radix isatidis from Hebei province, got reference fingerprints, compared with the fingerprints of 10 batches of samples from different sources, and studied their difference with semblances. We also have determined the contents of indigo and indirubin in 20 batches of Radix isatidis, combining with the study of fingerprints so as to generally evaluate the quality of Radix isatidis. The reproducibility and precision of the method were good. The method was useful to actualize standardization planting and control the quality of Radix isatidis.