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A quantitative analysis of multi-components by single marker method (QAMS) was established for simultaneous determination of gallic acid, protocatechuic acid, catechin, epicatechin, p-coumaric acid, ferulic acid and phloridzin in Cynomorium songaricum Rupr. The analysis was performed on a ChromCore Polae C18 column (250 mm×4.6 mm, 5 μm) , with a mobile phase consisting of acetonitrile-0.3% phosphoric acid aqueous solution for gradient elution. The volume flow rate, column temperature and sample injection volume were set at 1.0 mL·min-1, 25 ℃, and 40 µL, respectively. The relative correction factors of gallic acid and protocatechuic acid, catechin, epicatechin, p-coumaric acid, ferulic acid and phloridzin were calculated and the durability was also investigated. The contents of these seven compounds in fourteen batches of Cynomorium songaricum Rupr. from different producing areas or batches were determined by external standard method (ESM) and quantitative analysis of multi-components with a single-marker method (QAMS), respectively. SPSS and Origin Pro software were employed for principal components assay, similarity evaluation and cluster analysis. The specificity, precision, repeatability, stability and linear range (R2 > 0.999 0) of the seven components were all good. The average recovery was 96.89%-103.16% and RSD was 0.55%-2.76%. Then gallic acid was chosen as internal reference for calculation the correction factors for the other six components, the average relative correction factors of protocatechuic acid, catechin, epicatechin, p-coumaric acid, ferulic acid and phloridzin were 1.141 5, 0.200 5, 0.208 0, 2.361 9, 1.867 7, 0.204 6, respectively. Student's test results showed that there was no significant difference between the data analyzed by ESM and the data obtained from QAMS method. Through data visualization analysis, the contents of gallic acid, protocatechuic acid, catechin and epicatechin in different samples were significantly different, indicating that these four components might be the main quality markers of Cynomorium songaricum Rupr. for gaving more contributes to the principal components. The cluster analysis showed that samples from Xinjiang and samples from Inner Mongolia were clustered in significantly different categories, meaning that the quality of Cynomorium songaricum Rupr. had great relation with producing areas. The method of QAMS established in this study is a simple, economical and practical method with scientific and applicable charactistics for evaluating the quality of Cynomorium songaricum Rupr. efficiently and scientifically.
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A method for determination of 9 isoflavones in Puerariae Lobatae Radix was established and the accuracy and feasibility of the method were verified. The relative correction factors of eight isoflavonoids,3'-hydroxy puerarin,puerarinapioside,3'-methoxy puerarin,puerarin 6″-O-xyloside,daidzin,genistin,formononetin and daidzein were determined by HPLC method with puerarin as the internal standard. The contents of 9 isoflavonoids in 11 batches of samples were determined by external standard method and QAMS.The accuracy and feasibility of the methods were evaluated by comparison of the quantitative results between external standard method and QAMS. The reproducibility of the relative correction factors was good under different experimental conditions,and there was no significant difference between the external standard method of the 9 compounds and the content of QAMS method. The results showed that using puerarin as an internal standard to simultaneously determine the 8 isoflavonoids mentioned above is accurate and feasible. Thus,it can be used as quality control of Puerariae Lobatae Radix.
Subject(s)
Chromatography, High Pressure Liquid , Drugs, Chinese Herbal , Isoflavones , Plant Roots , Pueraria , Reproducibility of ResultsABSTRACT
OBJECTIVE: To establish a method of quantitative analysis of multi-components by single marker (QAMS) for determining four essential oils (cinnamaldehyde, cinnamyl alcohol, cinnamic acid, 2-methoxy cinnamaldehyde) in Cinnamomum cassia, and provide the experimental base for establishing the quality standard of Cinnamomum cassia. METHODS: Cinnamaldehyde was used as the internal reference standard, and the relative correction factors (RCF) of cinnamyl alcohol, cinnamic acid, and 2-methoxy cinnamaldehyde in Cinnamomum cassia were calculated. The contents of the four components were determined by both external standard method and QAMS. The validity of the QAMS method was evaluated by comparison of the quantitative results of both methods. RESULTS: The RCFs had good reproducibility, relative correction factor 0.673, 0.605 and 1.943, with RSDs of 0.529%, 0.373%, and 0.759%, respectively. No significant differences were found in the quantitative analysis results of cinnamyl alcohol, cinnamic acid, 2-methoxy cinnamaldehyde by using RCF and ESM. CONCLUSION: In the absence of reference substance, the content determination of the four essential oils in Cinnamomum cassia can be realized by QAMS, and this method can be used in the multi-index evaluation of Cinnamomum cassia essential oil constituents. It is suggested that the standard for cinnamaldehyde content be increased to 2.5%, and the contents of total cinnamyl alcohol, cinnamic acid and 2-methoxy cinnamaldehyde be not less than 0.2%.
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Objective To compare the differences between external standard method and relative correction factor method for determination of the flavonoids from Sorbus tianschanica Rupr.Methods Using HPLC external standard method for determination of hyperoside,rutin,isoquercitrin,quercetin-3-O-(6″-O-malonyl)-β-D-glucoside,astragalin and Kaempferol-3-O-(6″-O-malonyl)-β-D-glucopyranoside in Sorbus tianschanica Rupr.,HPLC relative correction factor method was adopted to establish relative correction factor of the other five flavonoids above with hyperoside as reference.The difference was evaluated by comparing the external standard method with the relative correction factor method.Results There was no significant difference between the T test external standard method and relative correction factor method(P>0.05).Conclusion External standard method and relative correction factor method can be used for determination of the flavonoids from Sorbus tianschanica Rupr.,but in the case of lack of reference substance or mass detection,using the relative correction factor method for determination of rutin,hyperoside isoquercitrin,quercetin-3-O-(6″-O-malonyl)-β-D-glucoside,astragalin and kaempferol-3-O-(6″-O-malonyl)-β-D-glucopyranoside in Sorbus tianschanica Rupr.It was more feasible and it can be used as a new quality evaluation method in determination of flavonoid components from Sorbus tianschanica Rupr.
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Objective:To develop a method of quantitative analysis of multi-components by single marker( QAMS) for nine kinds of alkaloids in Xiaohuoluo pills. Methods: An HPLC-QTOF-MS method with an Agilent ZORBAX Extend-C18 RRHT(2. 1 mm × 50 mm,1. 8 μm) column was applied. The flow rate was 0. 21 ml·min-1 . The column temperature was 30 ℃. The mobile phase was methanol (A)-water (B;containing 0. 1％ formic acid and 2. 5 mmol·L-1 ammonium acetate) with gradient elution. The aconitine was used as the internal standard, and the relative correction factor ( RCFs) of hypaconitine, mesaconitine, benzoylaconine, benzoyl-hypaconine, benzoylmesaconine, aconine, hypaconine and mesaconine was respectively established, and the reproducibility inspection on the RCF was performed. The contents of the other 8 kinds of aconitum alkaloids were calculated according to the RCF. At the same time, an external standard method ( ESM) was performed for the content determination of the nine alkaloids. The results of the two methods were compared. The feasibility and accuracy of the QAMS method were verified. Results:Within a certain range,the RCF of hypacontine,mesacontine, benzoylaconine, benzoylhypaconine, benzoyl mesaconine, aconine, hypaconine and mesaconine to aconitine was 1. 736,1. 979,1. 0471,0. 9242,1. 2901,1. 3078,1. 2859,and 1. 0948,respectively. The QAMS method was established for determi-ning alkaloids. There were no significant differences between the results of the QAMS method and those of the external standard method ( ESM) . Conclusion:With the validation of methodology, the method established in our study can be used for the content determina-tion of aconitine, hypaconitine, mesaconitine, benzoylaconine, benzoylhypaconine, benzoylmesaconine, aconine, hypaconine and mesaconine in xiaohuoluo pills.
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To develop the HPLC method for simultaneous determination of febrifugine and isofebrifugine in Dichroa febrifuga root, and on the basis of this, the feasibility of quantitative analysis of multi-component by a single-marker (QAMS) model for the determination of the two alkaloids was investigated. The chromatographic separation was performed on an octadecyl bonded silica gel column with mixed solvent consisting of acetonitrile-water-glacial acetic acid-triethylamine (9∶91∶0.36∶0.745) as mobile phase at a flow rate of 1.0 mL•min⁻¹. The detection wavelength was set at 225 nm, and the column temperature was set at 30 ℃. The linear range of febrifugine and isofebrifugine were 10.7-426 ng and 10.6-424 ng, respectively. Their average recovery were 98.33% (RSD 2.7%) and 100.4% (RSD 1.8%), respectively. On the basis of this established method, febrifugine was used as the internal reference substance to calculate the relative correction factors (RCF) and the relative retention values (RRV) of isofebrifugine to febrifugine. Through a series of methodology evaluations, the two alkaloids were simultaneously assayed only by quantitative determination of febrifugine. This result played the part of demonstration role for the application of QAMS model in the determination of isomers.
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OBJECTIVE:To establish the method for simultaneous determination of 5 saponins in Lonicerae Flos. METHODS:Using macranthoidin B as a reference,HPLC method was adopted to calculate the relative correction factor(RCF)of it to macran-thoidin A,dipsacoside B,macranthoside A and macranthoside B. The contents of above 4 saponins were calculated through RCF. Using the contents of saponins determined by external standard method as measured value,the calculated value was compared with measured value. RESULTS:The linear ranges of macranthoidin A,macranthoidin B,dipsacoside B,macranthoside A and macran-thoside B were 0.316-6.32 μg(r=0.9973),0.453-9.06 μg(r=0.9982),0.231-4.62 μg(r=0.9996),0.342-6.84 μg(r=0.9984) and 0.147-2.94 μg(r=0.9961),respectively. RSDs of precision,stability and reproducibility tests were all lower than 2.0%. The recoveries were 97.74%-104.51%(RSD=2.37%,n=6)、96.70%-103.20%(RSD=2.37%,n=6)、96.12%-103.61%(RSD=2.45%, n=6)、98.80%-104.70%(RSD=2.32%,n=6)、99.21%-102.92%(RSD=1.39%,n=6),respectively. There was no statistical sig-nificance between calculated value and measured value(P>0.05). CONCLUSIONS:The method is simple,precise,stable and re-producible. It can be used for the determination of saponins in Lonicerae Flos.
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Objective: To establish a method of quantitative analysis of multi-components by single marker (QAMS) for determining the flavonoids in Epimedii Herba and improve the level of quality control of Epimedii Herba by applying multi-index components control. Methods: To detect a relative correction factor (RCF) of epimendin A, epimendin B, epimendin C, and icariin at detection wavelength of 270 nm by HPLC in the range of a linear, to investigate the reproducibility of RCF in different chromatographic columns and different instruments as well, to consider icariin as an internal standard, to calculate the contents of epimendin A, epimendin B, and epimendin C in Epimedii Herba by using RCF; Meanwhile, to analyze the content of the four components in 100 Epimedii Herba by external standard method (ESM) and verify the accuracy and scientificalness of QAMS. Results: The RCF had a good reproducibility, which were 1.352, 1.384, and 1.340. The values of RSD were less than 5%; No significant differences were found in the quantitative results of epimendin A, epimendin B, and epimendin C determined by using RCF and ESM. Conclusion: This method could be accurate and reliable, simple and feasible, which could save the reference and costs of testing. It further validates that this approach can be used as a quality control method for the determination of multi-component index in Epimedii Herba.
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Objective: To establish a quantitative analysis method for determining the contents of artemisinin, quercetin, isorhamnetin, Ginkgo esters, and lactones a, b, c in Shuxuening Injection by using multi-components with a single marker (QAMS), and set up methodological evaluation model of the above experiment. Methods: With artemisinin and Ginkgo esters as indexes, the relative correction factor (fk/s) between artemisinin and the other two total flavonoids and the fk/s between Ginkgo esters and lactones a, b, c were established. The contents of artemisinin and Ginkgo esters in Shuxuening Injection were determined by external standard method, the contents of other five components in Shuxuening Injection were calculated by the relative correction factor. The results of the content of five batches of Shuxuening Injection determined by QAMS and tested by external standard method were carried out by t test. Results: The fk/s of quercetin and isorhamnetin with reference to artemisinin were 1.873 and 0.324, the fk/s of ginkgolide A, B, C with reference to Ginkgo esters were 2.280, 1.659, and 1.429. And the repeatability was good under different experimental conditions. There were no significant differences between the calculated value and estimated value on QAMS and external standard method of five batches of Shuxuening Injection, and the results showed that fk/s was authentic. Conclusion: The established correction factor has good repeatability, and could be used for quantitative analysis and quality evaluation of multiple components in Shuxuening Injection.
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Objective To establish QAMS method to determine the contents of three alkaloids in bile processed Coptidis Rhizoma; To compare the results of QAMS with those from external standard method; To prove the feasibility of QAMS.Methods An HPLC method was developed. Berberine hydrochloride was selected as the internal reference substance. 2 relative correction factors (RCF) of berberine hydrochloride to palmatine hydrochloride and to jatrorrhizine hydrochloride were established. Obtained RCFs were used to conduct content calculation (calculated value) to complete QAMS method. At the same time, the contents (measured value) of the three components were also determined by external standard method. Calculated value and measured value were compared.Results The analysis results showed that there was no significant difference between the calculated values and the measured values of the three alkaloids in 10 batches of bile processed Coptidis Rhizoma.Conclusion The QAMS method can be applied in the determination of alkaloids in bile processed Coptidis Rhizoma.
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Objective:To establish a quantitative analysis of multi-components by single marker ( QAMS) method for the determi-nation of six components ( phillyrin, schizandrol, schisandra ester, deoxyschizandrin, schisandrin B and schisandrin C ) in Liuwei Wuling tablets,and verify the accuracy and feasibility of the method applied in the preparation. Methods:The relative correction factor ( f) of schisandrin B, schisandrin C, schisandra ester, schizandrol, phillyrin and deoxyschizandrin was detected by HPLC at the detec-tion wavelength of 230nm, and the contents of the components in Liuwei Wuling tablets were calculated to achieve the multi evaluation. Meanwhile, the contents of the six components were analyzed by an external standard method and the relative error between the calcula-tion of QAMS method ( Wf ) and the result of the external standard method ( Ws ) was calculated to verify the accuracy of QAMS. The reproducibility of the method was also investigated. Results:The QAMS method was established to determine the contents of 6 compo-nents in Liuwei Wuling tablets, and the components in 10 batches of Liuwei Wuling tablets were determined. The differences between the calculated values and the measured values were small ( RSD < 5%) . Conclusion:The QAMS method is simple, effective and ac-curate in determining the contents of 6 effective components in Liuwei Wuling tablets. The quality evaluation model of QAMS for Liuwei Wuling tablets is validated, which can be used for the quality control of Liuwei Wuling tablets and provide reference for the further study.
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Objective To establish a kind of simple ,rapid ,accurate and reliable method to determine the phenobarbital in the biomaterial .Methods We pre‐treated biomaterial by the method that the reagent of acetone∶water (v/v 8∶2) was firstly used to soak the biomaterial ,and then we took use of ethyl acetate as reagent to extract the phenobarbital of the biomaterial in the present of pH=3-4 .We finally employed GC‐MS to determine these samples .On the one hand ,we not only took advantage of the reten‐tion time of the phenobarbital in total ion current (TIC) but also took advantage of the characteristic fragment ions of phenobarbital in mass spectrogram as qualitative basis .On the other hand ,we took advantage of the external standard method as quantitative ba‐sis .Results The method had the characteristics of the simple and easy operation .There was hardly background interference and there was good separation effect in the method .The method also had the characteristics of fast analytical speed such as the retention time of the phenobarbital was 8 .385 min .The characteristic fragment ions of phenobarbital was m/z 204 and m/z 232 .The charac‐teristic fragment ions of m/z 204 was served as quantitative ion fragments and we employed the external standard method to quanti‐fy in the method .In short ,the average recovery rate of the method was 87 .35% .Relative standard deviation (RSD) was 5 .43% in the method .The lowest limit of detection (LLOD) was 0 .005 mg/mL .Conclusion The method showes satisfactory result that it could be applied to determine the phenobarbital of the biomaterial of forensic toxicological analysis .
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Objective:To develop a method of quantitative analysis of multi-components by single marker ( QAMS) for 4 kinds of saponins in spina date seed.Methods:Using jujuboside A as a reference, an HPLC method with a Kromasil C18 column (250 mm × 4.6 mm, 5 μm) was applied, the mobile phase was acetonitrile-0.1% phosphoric acid (30 ∶70), the flow rate was 1.0 ml · min-1 , the detection wavelength was 280 nm, the column temperature was 32℃and the injection volume was 20μl.The relative cor-rection factors among the four kinds of saponins were detected by QAMS .An external standard method was also used to determine the contents of the four saponins ( Wf ) , and the results were compared with those of QAMS ( Ws ) , and then the relative errors were calcu-lated between W f and Ws to evaluate the accuracy of QAMS , and the reproducibility was investigated as well .Results:The established QAMS was used to determine the four kinds of saponins in spina date seed , and totally 14 batches of spina date seed were determined . No significant differences were found out between the calculated values and the determined ones ( RSD<5%) .Conclusion:The vali-dated HPLC method shows the advantages of precision ,reproducibility and reliability .The established QAMS method is feasible , and suitable for the quality control of saponins in spina date seed .
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OBJECTIVE: To develop an HPLC method for simultaneous determination of multiple-components in Hedyotis diffusa Willd. METHODS: The HPLC analysis was carried out on a C18 column (4.6 mm×250 mm, 5 μm) by gradient elution with acetoni-trile-water[both containing 0.1‰ (V/V) acetic acid] as mobile phase at a flow rate of 0.8 mL·min-1, the column temperature at 35°C, and the detection wavelength was set at 238 nm. External standard method and quantitative analysis of multi-components by single marker (QAMS) method were adopted for simultaneous determination of six components in Hedyotis diffusa Willd, respectively. RESULTS: The linear ranges for asperulosidic acid, quercetin-3-O-[2-O-(6-O-E-feruloyl)-β -D-glucopyranosyl]-β-D-glucopyrano-side, kaempferol-3-O-[2-O-(6-O-E-feruloyl)-β-Z) -gfucopyranosyl]-β-D-galactopyranoside, (E)-6-O-p-coumaroyl scandoside methyl ester, (E)-6-O-feruloyl scandoside methyl ester, (Z)-6-O-p-coumaroyl scandoside methyl ester were 2.34-93.50, 2.61-104.33, 0.67-26.69, 3.42-136.84, 0.65-26.07, and 1.10-44.17 μg·mL-1 (r<0.9993), respectively. The RSD values of precision, reproducibility, and sample stability were not more than 2.2%. The average recoveries of the six components were 99.8%-101.1% with RSDs not more than 1.2%. The P values of external standard method and QAMS by paired t-test were greater than 0.05. CONCLUSION: There is no significant difference in the content analysis results of the two methods, which can both used for simultaneous determination of the four iridoids and two flavonoids in Hedyotis diffusa Willd.
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OBJECTIVE: To study the feasibility of quantitative analysis of multi-components by single-marker (QAMS) in determination of flavones in Shuanghuanglian preparations by comparing the results assayed by QAMS and external standard method (ESM). METHODS: The analysis was performed on a Xtimate™ C18 column (4.6 mm × 250 mm, 5 μm)with methanol and water containing 0.2% phosphoric acid as the mobile phase in gradient elution. The flow rate was set at 1.0 mL · min-1. The UV detector wavelength was 274 nm, and the column temperature was 30°C. RESULTS: Within the linear ranges, the analysis results showed no significant difference between QAMS and ESM. CONCLUSION: The QAMS method is credible and will be a new quality evaluation pattern for the determination of multiple flavones in Shuanghuanglian preparations.
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OBJECTIVE:To improve the HPLC method for the content determination of Methotrexate(MTX)for injection.METHODS:The determination was performed on Inertsil C 8 column,mobile phase consisted of acetonitrile-water-acetic acid glacial-triethylamine(14:86:0.5:0.3)at flow rate of 0.8 mL?min-1.UV detection wavelength was set at 306 nm.Metronidazolum was selected as internal standard for internal standard method to replace external standard method stated in Chinese Pharmacopeia.Result of internal standard method was compared with that of external standard method.RESULTS:The linear range of MTX was 0.52～3.64?g?mL-1(r=0.999 7)with an average recovery of 99.77%(RSD=0.42%).Two kinds of methods obtained similar results.CONCLUSION:Improved method is convenient,accurate and reproducible for quality control of MTX.