Quality evaluation methods of Poria based on yield and content of alkali extracted polysaccharide / 中国中药杂志

To improve the quality control methods of Poria and develop and utilize its resources fully, alkaline extraction was used in this study to determine the yield and content of alkali-soluble polysaccharides of Poria. The alkali-soluble extracts of Poria were obtained according to the optimum extraction conditions on the basis of single-factor test, and 30 batches of samples were determined. The structure and chemical composition of the alkali-soluble extracts was characterized by high-performance gel permeation chromatography(HPGPC), Fourier transform infrared spectrometry(FT-IR), nuclear magnetic resonance(NMR) spectroscopy and high-performance liquid chromatography(HPLC) with 1-phenyl-3-methyl-5-pyrazolone(PMP-HPLC). The results showed that the content of the alkali-soluble extracts was in the range of 46.98%-73.86%. The main component was β-(1→3)-glucan, and its molecular mass was about 1.093×10~5. Further, the content of alkali-soluble polysaccharides of Poria was measured by UV-Vis spectrophotometry and HPLC coupled with the evaporative light scattering detector(HPLC-ELSD), and 30 batches of samples were measured. The results indicated that the content of alkali-soluble polysaccharides determined by UV-Vis spectrophotometry was in the range of 73.70%-92.57%, and the content of samples from Hubei province was slightly higher than that from Yunnan province, Anhui province and Hunan province. The content of alkali-soluble polysaccharides determined by HPLC-ELSD was in the range of 51.42%-76.69%, and the samples from Hunan province had slightly higher content than that from the other three provinces. The content determined by UV-Vis spectrophotometry was higher than that by HPLC-ELSD. However, the content determined by HPLC-ELSD was close to that of alkali-soluble extract, which could accurately characterize the content of alkali-soluble polysaccharides in Poria, and the method was simple and repeatable. Therefore, it is recommended that the quantitative analysis method for alkali-soluble extract and alkali-soluble polysaccharides by HPLC-ELSD be used in the quality standards of Poria in Chinese Pharmacopeia.

Determination of eight active components in Buyang Huanwu Decoction by HPLC-DAD-ELSD / 国际中医中药杂志

Objective:To establish an HPLC-DAD-ELSD method for the simultaneous determination of eight main active components in Buyang Huanwu Decoction, including hydroxysafflor yellow A, paeoniflorin, calycosin glycoside, ferulic acid, ononin, calycosin, fermononetin and astragaloside.Methods:Agilent Eclipse XDB-C18 column (250 mm×4.6 mm, 5 μm) was used with acetonitrile-0.1% formic acid as the mobile phase. The flow rate was 1.0 ml/min; the column temperature was 30 ℃; the detection wavelengths were 230 nm (paeoniflorin), 254 nm (calycosin glycoside, ononin, calycosin, fermononetin), 322 nm (ferulic acid) and 403 nm (hydroxysafflor yellow A); the drift tube temperature of the evaporative light scattering detector was 60 ℃; the carrier gas flow rate was 1.6 L/min.Results:Under these conditions, the separation of hydroxysafflor yellow A, paeoniflorin, calycosin glycoside, ferulic acid, ononin, calycosin, fermononetin and astragaloside was good, and the linear relationship was in line with the requirements ( r=0.994 0-0.999 9). The average recovery was 97.8% - 101.4% ( RSD was 1.28% - 3.70%). Conclusion:The method is simple, stable and reproducible, and can be used for the quality control of Buyang Huanwu Decoction.

Simultaneous determination of five components in Anemarrhenae Rhizoma by HPLC-ELSD / 药学实践杂志

Objective To establish a method for the simultaneous determination of new mangiferin, mangiferin, artemisinin BⅡ, icariin and artemisinin A in Anemarrhenae Rhizoma by high performance liquid chromatography-evaporation light scattering detector (HPLC-ELSD). Methods The column was Agilent Poroshell 120 EC-C18. The mobile phase used acetonitrile-0.2% acetic acid water system with gradient elution. Column temperature was 30 ℃. Flow rate was 0.7 ml/min. Evaporative light scattering detector used nitrogen as atomizing gas. The atomizing gas temperature was 40 ℃ and the drift tube temperature was 90 ℃. The nitrogen volume flow rate was 2.00 L/min and the sample volume was 20 μl. Results The five components were able to achieve baseline separation. Neomangiferin, Mangiferin, Anemaponin BⅡ, Baohuoside I , Anemarrhena saponin AⅢwere determined as 24.1-386 μg/ml (r=0.999 3), 23.2-371 μg/ml (r=0.998 6), 54.2-867.2 μg/ml(r=0.995 6), 5.3-84.8 μg/ml (r=0.996 8), 10-160 μg/ml (r=0.998 9) respectively, which showed a good linear relationship within the concentration range. The average recovery rate of the five components was between 101.8% and 105.0%, and the repeatability RSD was less than 2.4%. The content of the above five components in Zhimu medicinal materials were 1.62%, 0.82%, 7.36%, 0.07%, 0.34%, respectively. Conclusion The method is simple, accurate, and highly sensitive, which could be used as the quantitative determination of multiple index components of Anemarrhenae Rhizoma.

Effect of Flower Removal on Content of Three Alkaloids in Different Parts of Fritillaria thunbergii from Different Regions and at Different Growth Stages / 中国实验方剂学杂志

ObjectiveTo study the effect of flower removal on the content of three alkaloids in different parts of Fritillaria thunbergii from different regions and at different growth stages. MethodThe content of peiminine， peimine， and peimisine in the bulb， root， stem， and leaf of F. thunbergii after flower removal and with flower un-removed at different growth stages and in different regions were determined simultaneously by ultra-performance liquid chromatography-evaporative light scattering detection （UPLC-ELSD） method. The UPLC was conducted on ACQUITY UPLC BEH C18 column （2.1 mm × 150 mm， 1.7 μm） with the mobile phase of 0.02% triethylamine aqueous solution （A） and methanol （B）elution gradient（0-2 min， 45%A； 2-5 min， 45%-25%A； 5-7 min， 25%A； 7-17 min， 25%-10%A； 17-20 min， 10%A）， flow velocity of 0.20 mL·min-1， column temperature 35 °C， sample room temperature of 20 °C， and injection volume of 3 µL. The ELSD was carried out at drift tube temperature 45 °C and with the sprayer parameter of 40%. ResultThe flower removal significantly increased the yield of F. thunbergii. At the budding stage， the alkaloid content in the bulb of F. thunbergii from Ningbo in Zhejiang， Pan'an in Zhejiang， and Nantong in Jiangsu after flower removal were significantly higher than that of flowering un-removal treatment， while it showed no significant difference between the flower removal and un-removal treatments for the samples from Fengjie in Chongqing. At the flowering stage， the alkaloid content in the bulb of F. thunbergii from Nantong in Jiangsu after flower removal was significantly higher than that of flower un-removal treatment， while it showed an opposite trend for the samples from Pan'an in Zhejiang and Fengjie in Chongqing and had no significant difference between the two treatments for the samples from Ningbo in Zhejiang. At the bulb expansion stage， the alkaloid content in the bulb of F. thunbergii from Ningbo in Zhejiang and Pan’an in Zhejiang after flower removal were significantly higher than that of flower un-removal treatment， which was opposite for the samples from Nantong in Jiangsu and had no significant difference between the treatments for the samples from Fengjie in Chongqing. At the harvest stage， except for the samples from Pan'an in Zhejiang， the samples from the rest 3 regions showed decreased alkaloid content in the bulb after flower removal compared with that of flower un-removal treatment. The alkaloid content in the leaf was higher than that in the bulb of F. thunbergii at all growth stages and from different origins. ConclusionFlower removal can increase the yield of F. thunbergii. The alkaloid content in the bulb of F. thunbergii with flower removed was higher than that with flower un-removed at the budding stage， while this trend was reversed at the harvest stage. Both the yield and the alkaloid content of F. thunbergii from Pan'an in Zhejiang were increased by flower removal. The above-ground part of F. thunbergii has a potential development value.

Development and validation method for simultaneous quantification of neomycin and polymyxin B by HPLC-ELSD and comparison with microbiological method

The aim of this study is to develop the first simultaneous method for quantification of neomycin and polymyxin B inthe presence of dexamethasone using High Performance Liquid Chromatography (HPLC) with an Evaporative LightScattering Detector (ELSD). The analysis was performed using a phenyl Waters X Bridge column, an evaporationtemperature of 50oC, and a nitrogen pressure of 320 kPa. The mobile phase consists of a combination of methanoland trichloroacetate acid (40 mM, pH 1.70–1.80) in gradient mode, flow rate at 1.0 ml/minute, detector gain of 6,and analysis time of 35 minutes. The linearity was achieved with a concentration of 100–500 µg/ml (r = 0.99955)for neomycin and concentration of 30–100 µg/ml (r = 0.99703) for polymyxin B. Recovery results were obtainedbetween 99.150% and 104.773% for neomycin and 96.538% and 105.139% for polymyxin B. The analysis samplefrom the market was found to be 102.27% for neomycin and 100.79% for polymyxin B. The result was compared tothe standard microbiological method. Based on the T-test results of two samples with a 95% confidence level (α =0.05), it was concluded that there was no significant difference between HPLC-ELSD and microbiological methodsfor determining neomycin and polymyxin B. The HPLC-ELSD method has a potential for routine analysis due toadvantages in terms of increasing precision, accuracy, and shorter testing time.

Analysis on Quality Evaluation Methods of Asparagi Radix Decoction Pieces and Its Standard Decoction / 中国实验方剂学杂志

Objective:To establish the quality evaluation methods of Asparagi Radix decoction pieces and its standard decoction. Method:Ten batches of Asparagi Radix standard decoction were prepared. High performance liquid chromatography-evaporative light scattering detection method (HPLC-ELSD) was established for the determination of protodioscin and protoneodioscin in Asparagi Radix decoction pieces and its standard decoction, and the fingerprint detection of Asparagi Radix decoction pieces with acetonitrile-water as mobile phase for gradient elution. UHPLC-LTQ-Orbitrap-MS/MS was used to identify ten main common peaks in the fingerprint with acetonitrile-0.1% formic acid solution as mobile phase for gradient elution, electrospray ionization (ESI) and positive and negative ion mode scanning were employed, the detection range was m/z 100-1 400. Result:The total content of protodioscin and protoneodioscin in Asparagi Radix decoction pieces was 0.41%-0.72%, and their total content in Asparagi Radix standard decoction was 0.33%-0.59%, the transfer rate of these two components was 73.6%-98.3%. The dry extract yield of the standard decoction was 59.0%-73.0%, and its pH was 4.9-5.6. There were 10 common peaks in the fingerprint, and all of them were saponins, including protoneodioscin, protodioscin, aspacochioside A and its isomer, methyl protodioscin, asparagoside F, (25R)-26-O-β-D-glucopyranosyl-furostan-5, 20-diene-3β, 26-diol-3-O-[α-L-rhamnopyranosyl (1→2)]-[β-D-glucopyranosyl (1→4)-α-L-rhamnopyranosyl (1→4)]-β-D-glucopyranoside, 26-O-β-D-glucopyranosyl-furostan-20 (22)-ene-3β, 26-diol-3-O-[α-L-rhamnopyranosyl (1→2)]-[α-L-rhamnopyranosyl (1→4)]-β-D-glucopyranoside, pseudodiosgenin, aspacochioside C. Conclusion:In this paper, the quality evaluation methods of Asparagi Radix decoction pieces and its standard decoction are established, and these methods are stable and feasible, which can provide reference for the quality control of pharmaceutical preparations containing Asparagi Radix.

Comparison between charged aerosol detector and evaporative light scattering detector for analysis of sugar in Zhusheyong Yiqi Fumai and study on accuracy of methods / 中国中药杂志

Evaporative light scattering detector(ELSD) and charged aerosol detector(CAD) methods were established in this study for the content determination of four kinds of sugars in Zhusheyong Yiqi Fumai(YQFM), and the factors affecting the accuracy of CAD methods were discussed. HPLC-ELSD chromatographic separation was performed on a Shodex Asahipak NH2 P-50 column with acetonitrile-water(75∶25)as the mobile phase, with a flow rate of 0.8 mL·min~(-1), drift tube temperature of 80 ℃. The analysis by HPLC-CAD was performed on the same column with acetonitrile-water as mobile phase for gradient elution, with a flow rate of 0.8 mL·min~(-1), a neb temperature of 45 ℃, and power function(PF) of 1.3. The samples of YQFM were detected by ELSD and CAD respectively. It was found that YQFM was composed of fructose, glucose, sucrose and maltose. The linear relationship of the two methods was good, and the recoveries, reproducibility and stability of these four kinds of sugars measured by the two methods satisfied the requirements of methodology. Both CAD and ELSD detectors were accurate and reliable in detecting saccharides components in YQFM. In addition, it was revealed in this study for the first time that the PF parameter of CAD had an important influence on the accuracy of sugar determination and acted as the key parameter of CAD method. It was also found that for CAD, a non-linear detector, there was no significant difference between the results of linear regression and logarithmic regression.

Assay of Four Saponins in Astragali Radix by HPLC-ELSD / 医药导报

Objective To study the feasibility of high performance liquid chromatography-evaporative light scattering detector ( HPLC-ELSD) in quantitative analysis of multi-components ( QAMS) by single marker. Methods Four saponins in Astragali Radix were simultaneously determined by HPLC-ELSD using external standard method, and malonylastragaloside I served as internal standard. The relative correction factors between internal standard and astragaloside Ⅰ, astragaloside Ⅱ and astragaloside Ⅳ were calculated, and the stability was investigated. Results Astragaloside Ⅳ in Astragali Radix was little, while malonylastragalosideⅠand AstragalosideⅠwere abundant.The relative correction factors lacked stability, so ELSD could not be used in QAMS. Conclusion HPLC-ELSD can precisely determine contents of four saponins in Astragali Radix. The detector needs to be further studied when the components have poor ultraviolet absorption such as saponins by QAMS.

A preliminary study on quality standard and stability forQizhu-Fuzheng Yin / 国际中医中药杂志

Objective To establish the quality standard ofQizhu-Fuzheng Yin, and to conduct a preliminary study on its stability.Methods Corydalis tuber and licorice were identified by TLC. The content of astragaloside was determined by UPLC-ELSD. The initial stability was studied by accelerated test method. Results The spots on TLC plates were clear without interference in the blank reference. The response of astragaloside was linear in the ranges of 36.5-365.0 μg/ml (r2=0.999 2), and the average recovery was 102.8 %, and theRSD was 2.4%. After 1, 2, 3, 6 months tests, the average contents of 3 batches astragaloside were 61.6, 60.4, 60.6μg/ml.ConclusionQizhu-Fuzheng Yin was simple preparation, quality control and stability.

Determination of 20(R)-25-OH-PPT in different parts of species in Panax Linn. by HPLC-ELSD methods / 中草药

Objective: For making full use of our country's rich ginseng resources to look for 25-OH-PPT\ and provide the scientific basis for the innovation and development of ginsenoside hypoglycemic activity. Methods: Using chromatographic column Agilent C18 (250 mm × 4.6 mm, 5 μm); mobile phase was CH3OH-H2O (70:30); Column temperature was 35 ℃; Flow rate was 1.0 mL/min; the boiling chamber temperature of ELSD was 87 ℃; Flow rate of carrier gas was 2.20 L/min; injection volume was 10 μL. Using acetone as the solvent and ultrasonic extraction to extract the medicinal materials, then using the HPLC-ELSD method to determine the contents of 20(R)-25-OH-PPT in different parts of plants in Panax ginseng. Results: 20(R)-25-OH-PPT had a good linear relationship when the concentration was 0.001-0.05 mg/mL. The 20(R)-25-OH-PPT contents in gingseng cauline leaf, gingseng root, gingseng fruit, gingseng flower, cauline leaf of American ginseng, root of American ginseng, and the basal part of stem of P. notoginseng were 3.74%, 2.59%, 0.40%, 2.37%, 0.68%, 0.22%, and 0.39%. The recovery rates were 98.54%, 97.54%, 100.35%, 101.46%, 97.46%, 99.14%, and 99.38%. Conclusion :25(R)-OH-PPT exists in different parts of P. ginseng, and it can be an important basis for the use and development of ginsenoside with hypoglycemic activity.

Application of evaporative light scattering detector in analysis of natural drugs / 药学实践杂志

Objective To summarize the advantage and application of evaporative light scattering detector(ELSD) for the analysis of natural drugs .Methods The application of ELSD in natural medicine analysis was reviewed in the article .Results ELSD in analysis of natural drugs ,natural drugs quality standards research ,fingerprint ,multi‐spectrum combination technology and high‐throughput screening had been widely applied .Conclusion Evaporative light scattering detector had a broad applica‐tion prospect .

Effect of Different Sample Solvents on Determination of Astragaloside Ⅳ by High Performance Liquid Chromatograph_Evaporative Light Scattering Detector / 医药导报

Objective To exPlore the effect of samPle_solVent comPositions on the determination of AstragaliⅣby high Performance liquid chromatograPh_ eVaPoratiVe light scattering detector ( HPLC_ELSD ) . Methods Radix astragali and ganduqing granules serVed as samPles. Methanol,90%methanol,80%methanol,70%methanol,32%acetonitrile,15%acetonitrile, and water were samPle solVents. HPLC_ELSD was used to determine content of astragalosideⅣ. Results The results showed that the 90%methanol solution was an aPProPriate samPle solVent with good system suitability,Precision,accuracy,and,linearity etc. . Conclusion This method benefits the quality control of astragalosideⅣin Radix astragali and agents containing Radix astragali.

Analysis of the chemical composition of polysorbate 80 / 中国药学杂志

OBJECTIVE: To establish a method for the separation and identification of the components in polysorbate 80 and to lay the foundation for identifing the sensitinogen of polysorbate 80. METHODS: Polysorbate 80 samples from both domestic and foreign manufacturers were collected. The separation condition was first established using HPLC-ELSD, and then the chemical composition of each chromatographic peak was identified by HPLC-MS. RESULTS: The chemical components with different types of structures were efficiently separated, and each chromatographic peak was properly assigned. Polysorbate 80 samples of different batches and manufacturers showed different chemical compositions and relative contents. There was illegal blending during the production of polysorbate 80. CONCLUSION: The proposed method is rapid and simple, which is not only suitable for the process control of polysorbate 80 but also lays foundation for further investigation of the physical and chemical properties of the components thus to find the sensitinogen. Copyright 2012 by the Chinese Pharmaceutical Association.

Quantification of six bioactive compounds in Zhenqi Fuzheng preparation by high-performance liquid chromatography coupled with diode array detector and evaporative light scattering detector / 药物分析学报

A simple and accurate high-performance liquid chromatography（HPLC）coupled with diode array detector（DAD）and evaporative light scattering detector（ELSD）was established for the determination of six bioactive compounds in Zhenqi Fuzheng preparation（ZFP）.The monitoring wavelengths were 254,275 and 328 nm.Under the optimum conditions,good separation was achieved,and the assay was fully validated in respect of precision,repeatability and accuracy.The proposed method was successfully applied to quantify the six ingredients in 31 batches of ZFP samples and evaluate the variation by hierarchical cluster analysis（HCA）,which demonstrated significant variations on the content of these compounds in the samples from different manufacturers with different preparation procedures.The developed HPLC method can be used as a valid analytical method to evaluate the intrinsic quality of this preparation.

HPLC-ELSD in determination of celosin A and celosin B contents in Semen celosiae / 第二军医大学学报

Objective: To determine the contents of celosin A and celosin B in Semen celosiae. Methods: HPLC was carried out with an Agilent Alliance, Model 1100, equipped with Elite ODS C18 column (4.6mm X 250mm, 5 μm) and evaporated light scattering detector (ELSD). The mobile phase (acetonitrile-0.1% glacial acetic acid) was eluted in gradient mode. Results: Celosin A and the peak area showed a good linearship within the range of 2. 5-30.0 μg, with the recovery rate being 99.89% (n=6) and RSD being 1.85%. Celonsin B and the peak area showed a good linearship within the range of 1.25-15.0 μg, with the recovery rate being 98.98% (n=6) and the RSD being 1.78%. Contents of celosin A and celosin B in 8 Semen celosiae samples on medical markets were obviously different from each other, ranging from (0.051 2±0.001 6)%, to (0.114 3 ± 0.000 8)% and (0.010 9±0.001 9)% to (0.081 5±0.000 9)%. Conclusion: We have established an HPLC-ELSD method for determining the effective contents of Semen celosiae; the method is accurate and simple.

Application of Normal Phase Liquid Chromatography-Evaporative Light-Scattering Detection for Determination of Paraffin Wax in Food / 分析化学

A method was proposed for the separation and determination of paraffin waxes in food by HPLC-evaporative light scattering detection (ELSD). A normal-phase column was used to separate nonparaffinic and paraffinic materials without resolving the latter into individual components. The t-test method was adopted for the evaluation of mean difference between response factors of n-alkanes in paraffin waxes on ELSD detector. No mean difference was obtained between response factors, which can be used for quantitative determination of paraffin waxes in food. The determination results obtained by HPLC-ELSD were compared with those by GC-MS. The linear range for the determination of paraffin waxes was in the range from 10 to 500 mg/L with a correlation coefficient of 0.9988, and the limit of detection was 1.0 mg/L. With the spiking level of 10, 50 and 100 mg/kg, the recovery ranged from 84.6% to 105.4% and the relative standard deviation ranging from 5.4% to 7.2%. The proposed method is simple, fast and sensitive.

Analysis of Hupehenine in the Total Alkaloids from Fritillaria hupehensis by HPLC-ELSD / 华中科技大学学报（医学）（英德文版）

A reverse-phase high pressure liquid chromatography (HPLC) method with evaporative light scattering detection (ELSD) has been developed for the quantitative analysis of hupehenine in the total alkaloids from Fritillaria hupehensis. Samples were analyzed on a reverse-phase column (Hypersil C-18) with a mobile phase of methanol:water:chloroform: triethylamine (85:15:1:0.6). The ELSD was set at the drift tube temperature of 68.3℃ and gas flow rate of 1.8 L/min. Hupehenine's retention time was 13.7 min with an asymmetry factor of 1.2. The validity of the method has been verified with linearity, limit of detection, accuracy and precision. The logarithmic linear curve was obtained from 8.936 to 134.04 μg/mL (r=0.9993). The detection limit (S/N＞3) of hupehenine was 1.79 μg/mL on the column. Intra-day RSD was 1.42% and inter-day RSD was 2.26% (3 days within a week). The average recovery of hupehenine was 101.50%, and RSD was 1.62%.

Development of Chromatographic Fingerprint of Rhizoma Alismatis Orientalitis by HPLC-ELSD / 中药新药与临床药理

Objective A fingerprint of Rhizoma Alismatis Orientalitis' decoction pieces was established by HPLC-ELSD. Methods The gradient elution mode was applied in chromatographic separation by mobile phase of methanol and water at flow rate of 0.5 mL? min-1 in a Diamonsil TM C18 chromatographic column (250 mm ? 4.6 mm, 5 ? m), and recording time was 70 minute. The parameters of evaporative light scattering detector were set as follows: the temperature of the drift tube was 82.5 ℃ and gas flow rate was 1.9 L ? min-1.The data was analyzed with cluster analysis and similarity calculation software. Results The fingerprint showing 15 common peaks was obtained. The results of cluster analysis and similarity calculation showed there existed differences in fingerprints of 16 batchers of medicinal slices of Rhizoma Alismatis Orientalitis. Conclusion The method is effective and reproducible for the quality control of medicinal slices of Rhizoma Alismatis Orientalitis.