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ObjectiveTo clarify the scientific validity of in vivo pharmacokinetic determination of the whole drug composition in Shenbai nanosuspension in rats， and to provide methodological guidance and theoretical basis for the in vivo study of multi-component complex system of traditional Chinese medicine（TCM） preparations. MethodThe concentration of the overall components， mainly total saponins and total polysaccharides in Shenbai decoction and Shenbai nanosuspension， was determined in rat plasma at different times by area under the absorbance-wavelength curve method（AUAWC）， and the concentration of individual ginsenoside Rg1 was determined by high performance liquid chromatography（HPLC）， and the methodology was verified. The pharmacokinetic parameters of the whole component were compared with those of ginsenoside Rg1 to evaluate the in vivo operational characteristics of the two preparations. ResultThe methodological investigations of AUAWC and HPLC were in accordance with the requirements. AUAWC analysis showed that the overall components in both the decoction group and the nanosuspension group showed a one-compartment model， with half-life（t1/2） of 2.43 h and 2.04 h， respectively. The relative bioavailability of Shenbai nanosuspension was 138.99%. HPLC assay showed that ginsenoside Rg1 in the decoction group and the nanosuspension group showed a two-compartment model， with distribution half-life（t1/2α） of 0.13 h and 2.55 h， and elimination half-life（t1/2β） were 14.28 h and 3.85 h， respectively. The relative bioavailability of Shenbai nanosuspension was 127.49%. Compared with Shenbai decoction， the time to peak（tmax）， peak concentration（Cmax） and area under the drug-time curve（AUC） of the overall components and ginsenoside Rg1 in Shenbai nanosuspension were increased. ConclusionThe established AUAWC can be used for the pharmacokinetic study of the overall components of TCM preparations， which is complementary to the results of individual components measured by HPLC， and can provide useful reference for the in vivo study of new dosage forms of TCM.
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@#Objective To develop a high performance liquid chromatography(HPLC)method for determination of aluminium adjuvant content in vaccine,and verify and preliminarily apply the method.Methods The 8-hydroxyquinoline derivatization method was used for determination. The chromatographic column was phenyl-hexyl column[Luna 5u PhenylHexyl(250 mm × 4. 6 mm)],and the mobile phase was composed of ammonium acetate solution-acetonitrile(with 8-hydroxyquinoline)(60 ∶ 40)containing 20 mg/L ascorbic acid,while eluted at a flow rate of 1. 0 mL/min with the isocratic eluent. The excitation wavelength and the emission wavelength of the fluorescence detector were 380 nm and 520 nm respectively. The column temperature was 40 ℃,and the sample injection was 50 μL. The developed method was verified for the specificity,linear range,accuracy,repeatability,stability and durability,and used to determine the aluminum content in 12 batches of vaccines. The results were compared with those determined by titration in general principle 3106of Chinese Pharmacopoeia(VolumeⅢ,2020 edition).Results No interference peaks appeared in the sample chromatogram,and the non-aluminum adjuvant vaccine components and phosphate buffer had no interference with the determination. The linearity of aluminum standard was good in the concentration range of 6. 25 ～ 100 μg/mL,r = 0. 999 6. The average results of spike recoveries of aluminum content in inactivated hepatitis A vaccine,recombinant hepatitis B vaccine,adsorbed acellular DTP vaccine and inactivated enterovirus 71 vaccine were 98. 32%,100. 85%,101. 09% and 99. 31%,respectively in the verification for accuracy. The relative standard deviations(RSDs) of the determination results of aluminum content in the solution of six samples of the four vaccines in the same batch were 1. 09%,1. 42%,0. 97% and1. 30%,respectively. The RSDs of aluminum content of four vaccine samples stored at room tempe-rature for 0,2,4,6 and8 h were 0. 82%,0. 73%,0. 40% and 0. 48%,respectively. When the ratio of ammonium acetate solution to 8-hydroxyquinoline acetonitrile solution in mobile phase changed within 5%,the fluctuation range of aluminum content of four vaccines was less than 2%. There was no significant difference between the developed HPLC method and the titration method of Chinese Pharmacopoeia(VolumeⅢ,2020 edition)for determination of aluminum content in the 12 batches of vaccine samples.Conclusion A HPLC method for determination of aluminum adjuvant content in vaccines has been successfully established with good specificity,linearity,accuracy,repeatability,stability and durability,simple operation,high degree of automation and less interference of manual factors. It can realize the determination of aluminium content in single dose,which provides an effective means for the rapid and large-scale determination of aluminum content in vaccine products and monitoring the dispensing of semi-final products in the production process.
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ObjectiveTaking Achyranthis Bidentatae Radix（ABR） from different origins as samples， to quantitatively analyze the chemical composition and chromaticity of ABR with different processing degrees， and clarify the correlation and change law between color and composition in the processing process of ABR， so as to provide reference for the quality evaluation of processed products of ABR. MethodThe colorimeter is used to measure the chromaticity values of three kinds of processing degrees of ABR in different origins to show the color value change trend during the processing process， and the color parameters of wine-processed and salt-processed products of ABR with different processing degrees were analyzed by principal component analysis（PCA）， orthogonal partial least squares-discriminant analysis（OPLS-DA） and other analysis methods. The contents of eight representative components of ABR were measured by high performance liquid chromatography（HPLC）， the correlation between chromaticity and each representative component was analyzed by Pearson correlation analysis， and the applicability of the selected eight representative components was further verified by Fisher linear discriminant analysis， and the wine-processed and salt-processed products of ABR with different processing degrees were grouped according to the degree of processing， and 48 samples of wine-processed and salt-processed products with different processing degrees were used as training samples. Taking the contents of 5-hydroxymethylfurfural， polypodine B， β-ecdysterone， 25R-inokosterone， 25S-inokosterone， ginsenoside Ro， chikusetsusaponin Ⅳa and polysaccharides as variables， the discriminant function was established respectively， and 12 samples of wine-processed and salt-processed products of ABR with different processing degrees were back-tested to verify the discriminant function and test the reliability of the function. ResultPCA and OPLS-DA results showed that ABR samples with different processing degrees were classified into clusters， and the results could significantly distinguish different processed products. During the process of wine and salt processing， the contents of 5-hydroxymethylfurfural， ginsenoside Ro， and chikusetsusaponin Ⅳa gradually increased with the deepening of the processing degree， while the contents of polypodine B， β-ecdysterone， 25R-inokosterone， 25S-inokosterone and polysaccharides showed a gradual decreasing trend， indicating these 8 components increased and decreased to different degrees in the process of wine and salt processing. The results of Pearson correlation analysis showed that the 5-hydroxymethylfurfural content of the samples with different processing degrees of wine-processed and salt-processed products were negatively correlated with the brightness value（L*） and the total color difference value（E*ab）（P<0.01）， and positively correlated with the red-green value（a*） and the yellow-blue value（b*）（P<0.01）， and that the content of polypodine B and polysaccharides were positively correlated with L* and E*ab（P<0.01）. The discriminant functions of wine-processed and salt-processed products of ABR were established by Fisher linear discriminant analysis， and their accuracy rates in the training samples were 93.75% and 95.83%， respectively. Twelve test samples of wine-processed and salt-processed products with different processing degree were back substitution， and the correct rate was 100%. ConclusionThe trend of composition and color changes of ABR with different processing degrees in different production areas is relatively consistent， and the color value can better distinguish ABR with different processing degrees， and the color of ABR is related to some representative components in the processing process， indicating that the color can provide reference for the identification of the processing degree of ABR and the prediction of component content.
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Objective To establish a method for simultaneous determination of HPLC fingerprint and multi-target ingredients in Atractylodis Macrocephalae Rhizoma(AMR),in order to provide reference for its quality control.Methods HPLC-DAD multi-wavelength switching method was used to establish fingerprint of AMR,similarity evaluation combined with hierarchical clustering analysis(HCA),principal components analysis(PCA)and discriminant analysis of partial least squares(PLS-DA)were used to carry out chemometric study.The contents of differential component such as atractylenolide Ⅰ,Ⅱ,Ⅲ and atractylon were determined simultaneously.Results The HPLC fingerprint of 37 batches of AMR was established.Nine common peaks were marked,and 4 of them were identified as atractylon,atractylenolide Ⅰ,Ⅱ,Ⅲ.The similarity degrees were between 0.539 and 0.996,the quality of AMR from different origin and different batches varies greatly.Atractylon,atractylenolide Ⅰ,Ⅱ,Ⅲ and one unknown component(peak 9)are the important factors affecting the quality of AMR.Conclusion The combination methods of HPLC fingerprint and simultaneous determinations of multiple components are simple,stable,accurate and reliable,which can provide reference for the quality evaluation of AMR and the improvement of quality standard,as well as lay a foundation for the basic research of its pharmacodynamic substances and related compound.
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ObjectiveTo improve the quality standard of Yuanhu Zhitong oral liquid in order to strengthen the quality control of this oral liquid. MethodThin layer chromatography（TLC） was used for the qualitative identification of Corydalis Rhizoma and Angelicae Dahuricae Radix in Yuanhu Zhitong oral liquid by taking tetrahydropalmatine， corydaline reference substances and Corydalis Rhizoma reference medicinal materials as reference， and cyclohexane-trichloromethane-methanol（5∶3∶0.5） as developing solvent， Corydalis Rhizoma was identified using GF254 glass thin layer plate under ultraviolet light（365 nm）. And taking petroleum ether（60-90 ℃） -ether-formic acid（10∶10∶1） as developing solvent， Angelicae Dahuricae Radix was identified using a silica gel G TLC plate under ultraviolet light（305 nm）. High performance liquid chromatography（HPLC） was performed on a Waters XSelect HSS T3 column（4.6 mm×250 mm， 5 μm） with acetonitrile（A）-0.1% glacial acetic acid solution（adjusted pH to 6.1 by triethylamine）（B） as the mobile phase for gradient elution（0-10 min， 20%-30%A； 10-25 min， 30%-40%A； 25-40 min， 40%-50%A； 40-60 min， 50%-60%A）， the detection wavelength was set at 280 nm， then the fingerprint of Yuanhu Zhitong oral liquid was established， and the contents of tetrahydropalmatine and corydaline were determined. ResultIn the thin layer chromatograms， the corresponding spots of Yuanhu Zhitong oral liquid， the reference substances and reference medicinal materials were clear， with good separation and strong specificity. A total of 12 common peaks were identified in 10 batches of Yuanhu Zhitong oral liquid samples， and the peaks of berberine hydrochloride， dehydrocorydaline， glaucine， tetrahydropalmatine and corydaline. The similarities between the 10 batches of samples and the control fingerprint were all >0.90. The results of determination showed that the concentrations of corydaline and tetrahydropalmatine had good linearity with paek area in the range of 0.038 6-0.193 0， 0.034 0-0.170 0 g·L-1， respectively. The methodological investigation was qualified， and the contents of corydaline and tetrahydropalmatine in 10 batches of Yuanhu Zhitong oral liquid samples were 0.077 5-0.142 9、0.126 1-0.178 2 g·L-1， respectively. ConclusionThe established TLC， fingerprint and determination are simple， specific and reproducible， which can be used to improve the quality control standard of Yuanhu Zhitong oral liquid.
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ObjectiveBased on response surface methodology combined with principal component analysis（PCA）， the optimal decocting process of Moringa oleifera leaf standard decoction was optimized， and its multi-index quality evaluation system was established， in order to provide scientific basis for the quality control of this standard decoction. MethodResponse surface methodology and PCA were used to optimize the decoction process by taking the relative peak areas of 8 characteristic peaks and dry extract yield as indexes. Based on this， the quality of 15 batches of the standard decoction was evaluated by high performance liquid chromatography（HPLC） characteristic chromatogram， determination of major components（neochlorogenic acid， L-tryptophan， cryptochlorogenic acid， vicenin-2， isoquercetin， astragalin）， determination of active parts（total flavonoids， total organic acids， total polysaccharides， total α-amino acids， total sinapine）， dry extract yield， specific gravity and pH. ResultThe optimal decocting process was to soak M. oleifera leaves（100.00 g） for 30 min and decoct twice with the first decoction of 12 times the amount of water for 30 min and the second decoction of 10 times the amount of water for 20 min. Standard decoction containing 0.2 g·mL-1 of crude drug was defined by x¯±30%， the specific gravity was 0.722-1.340， pH was 3.86-7.16， dry extract yield was 23.1%-42.9%， and the alcohol-soluble extract content was 8.26%-15.34%. Calculated according to the dried products of the standard decoction， the contents of neochlorogenic acid， L-tryptophan， cryptochlorogenic acid， vicenin-2， isoquercetin and astragalin were 1.99-3.69， 1.20-2.22， 1.44-2.67， 0.53-0.99， 2.45-4.55， 1.22-2.26 mg·g-1， the relative transfer rates relative to the herbs were 34.37%-63.83%， 62.43%-115.94%， 64.65%-120.06%， 56.98%-105.82%， 37.46%-69.57%， 41.81%-77.64%， respectively. The contents of total flavonoids， total organic acids， total polysaccharides， total α-amino acids， total sinapine were 10.19-18.92， 11.82-21.96， 94.07-174.71， 42.69-79.27， 9.55-17.73 mg·g-1， the relative transfer rates for herbs were 25.72%-47.77%， 41.78%-77.59%， 64.90%-120.54%， 42.30%-78.57%， 34.99%-64.99%， respectively. ConclusionThe optimized decocting technology of M. oleifera leaf standard decoction is stable and feasible， and the established multi-indicator quality evaluation system can lay the foundation for the quality control of this standard decoction.
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Objective To establish an HPLC method for the simultaneous determination of 10 components in the active parts of Uygur medicine Dracocephalum Moldavica L.Methods The determination was performed on a Shim-pack ODS(4.6 mm×250 mm,5 um)column with the mobile phase consisting of acetonitrile(A)-0.5%formic acid(B)in aqueous solution in a gradient elution mode(0-30 min,17%A;30-60 min,17%→ 28%A;60-78 min,28%A)at a flow rate of 1.0 mL·min-1.The temperature of the chromatographic column was 35℃and the detection was monitored by a UV detector at 330 nm.Results Cof-feic acid,p-coumalic acid,cynaroside,luteolin-7-O-β-D-glucuronide,apigenin 7-O-glucuronide,rosmarinic acid,diosmetin7-O-β-D-glucuronide,salvianolic acid A,tilianin,apigenin were well separated under this chromatographic condition,and the linear relation-ship were good in the concentration range examined(r＞0.999 2).The overall recoveries ranged from 91.83%to 106.43%with the RSD ranging from 0.38%to 2.22%.Conclusion The established content determination method is highly accurate and reproduci-ble,and suitable for the analysis and quality control of the active parts of Dracocephalum Moldavica L.
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ObjectiveTo analyze the correlation between 11 small molecule active components and 1 protein component of characteristic processed products with porcine cardiac blood and other products of Salviae Miltiorrhizae Radix et Rhizoma（SMRR） from Menghe medical school and anti-cerebral ischemic oxidative damage， and to identify its key component markers of characteristic processed products with porcine cardiac blood for anti-cerebral ischemic oxidative damage. MethodHigh performance liquid chromatography（HPLC） was established to simultaneously determine the contents of 11 active ingredients in SMRR and its processed products［processed with porcine cardiac blood， porcine blood， wine and transferrin（Tf） in porcine cardiac blood］， and the content of Tf in different processed products of SMRR was detected by enzyme-linked immunosorbent assay（ELISA）. Furthermore， A zebrafish ischemic stroke model was constructed to evaluate the effects of different processed products of SMRR on the behavioral trajectory of cerebral ischemic zebrafish， the neuronal damage of transgenic zebrafish Tg（elavl3：eGFP） brain， as well as the levels of malondialdehyde（MDA） and superoxide dismutase（SOD） in the brain tissues. The hippocampal neurons oxygen-glucose deprivation/reoxygenation（OGD/R）-induced ischemia-hypoxia model was constructed to evaluate the effects of different processed products of SMRR on oxidative damage of neuronal cells by taking lactate dehydrogenase（LDH）， reactive oxygen species（ROS）， MDA and SOD as indexes. Finally， principal component analysis（PCA）， partial least squares-discriminant analysis（PLS-DA） and Spearman correlation analysis were used to analyze the 11 small molecule active components and 1 protein component with efficacy indicators， in order to screen the key components of the characteristic processed products with porcine cardiac blood for cerebral ischemic oxidative damage. ResultCompared with the raw products， the contents of water-soluble and fat-soluble components in processed products of SMRR increased to different degrees， while the content of salvianolic acid A decreased. Compared with the wine-processed products， the contents of salvianolic acid B， danshensu， rosmarinic acid and other components in the porcine cardiac blood-processed products， porcine blood-processed products， Tf-processed products were increased， while the content of salvianolic acid A was decreased. ELISA results showed that there was no significant difference in Tf content between the porcine cardiac blood-processed products， porcine blood-processed products， Tf-processed products. Pharmacological results showed that different processed products of SMRR could improve the behavioral deficits， brain neuronal injury and oxidative stress after ischemic stroke in zebrafish， and the effect of the porcine cardiac blood-processed products was most pronounced. PCA results showed that salvianolic acid B， salvianolic acid A， rosmarinic acid， lithospermic acid， danshensu， tanshinone ⅡA， caffeic acid， cryptotanshinone and tanshinone Ⅰ were the main contributing components of SMRR and its processed products. And the results of correlation analysis showed that the contents of cryptotanshinone， rosmarinic acid， caffeic acid， dihydrotanshinone Ⅰ， salvianolic acid B， tanshinone ⅡA and tanshinone Ⅰ were negatively correlated with MDA level in zebrafish brain tissue， while the contents of lithospermic acid， protocatechuic aldehyde， rosmarinic acid， dihydrotanshinone Ⅰ， salvianolic acid B and Tf were positively correlated with SOD level， and the contents of rosmarinic acid， caffeic acid， dihydrotanshinone Ⅰ， salvianolic acid B， tanshinone ⅡA， tanshinone Ⅰ， danshensu， Tf were positively correlated with neuronal fluorescence intensity in the zebrafish brain. And the contents of lithospermic acid， protocatechuic aldehyde， rosmarinic acid， dihydrotanshinone Ⅰ， salvianolic acid B， tanshinone ⅡA and Tf were negatively correlated with LDH， ROS and MDA levels and positively correlated with SOD level. ConclusionThere are differences in the anti-ischemic oxidative damage effects of SMRR and its different processed products， among which the porcine cardiac blood-processed products has the strongest effect on improving oxidative damage， which may be related to the content changes of salvianolic acid B， danshensu， rosmarinic acid and other components. This study can provide a basis for clarifying the quality markers of SMRR processed with porcine cardiac blood for cerebral ischemia and elucidating its processing mechanism.
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In this study, we evaluated several agronomic characteristics of S. divaricata when grown in various regions in Vietnam, including Northeast, Northwest, and Central Highlands. Among the three regions, S. divaricata grown in Northwestern Vietnam has the highest yield reaching 6.21 tons/ha, with a total active ingredient content of 0.655% (Prim-O-glucosylcimifugin 0.383% and 5-O-methylvisamminoside 0.272%). This is followed by the Central Highlands of which S. divaricata yielded 4.12 tons/ha, with a total active ingredient content is 0.543% (Prim-O-glucosylcimifugin 0.292% and 5-O-methylvisamminoside 0.251%). The lowest yield of S. divaricata was recorded in Northeast with 3.13 tons/ha, of which a total active ingredient content was 0.394% with 0.253% for Prim-O-glucosylcimifugin and 0.141% for 5-O- methylvisamminoside. With the applied analytical conditions, HPLC - DAD chromatograms are obtained with sharp peaks of prim-O-glucosylcimifugin (tR = 7.51 min) and 5-O-methylvisamminoside (tR = 20.23 min) , balanced, clear on the background of the medicinal plants Saposhnikovia divaricata (Turcz.) Schischk. In particular, the applicable analytical conditions allow for simultaneous qualitative and quantitative analysis of both prim-O-glucosylcimifugin and 5- O-methylvisaminoside. The results of building the standard curve prim-O-glucosylcimifugin Y = (16146)X – 4020.3, R2 = 0.9992, standard curve 5-O- methylvisamminoside Y = (20490)X – 6921.8, R2 = 0.9999. The quantitative results of prim-O-glucosylcimifugin and 5-O- methylvisamminoside in all regions were slightly higher than that of the pharmacopoeias of Vietnam, China and Hong Kong with the total active ingredient content not less than 0.24%. Thus, The study concluded that Vietnam is a country that can develop medicinal plants Saposhnikovia divaricata (Turcz.) Schischk
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ObjectiveTo establish the specific chromatogram and thin layer chromatography（TLC） of Qingxin Lianziyin（QXLZY） benchmark samples， in order to clarify the key quality attributes and provide a reference for the quality evaluation of QXLZY. MethodHigh performance liquid chromatography（HPLC） specific chromatogram of QXLZY benchmark samples was developed by using a YMC Hydrosphere C18 column（4.6 mm×250 mm， 5 μm） with the mobile phase of acetonitrile（A）-0.2% formic acid aqueous solution（B） for gradient elution（0-10 min， 5%-20%A； 10-20 min， 20%A； 20-25 min， 20%-24%A； 25-40 min， 24%-30%A； 40-55 min， 30%-50%A； 55-65 min， 50%-100%A； 65-75 min， 100%A； 75-75.1 min， 100%-5%A； 75.1-90 min， 5%A）， and the detection wavelength was 360 nm. Ultra-high performance liquid chromatography-linear ion trap/orbitrap mass spectrometry（UHPLC-LTQ-Orbitrap MS） with electrospray ionization（ESI） was used to identify the components of QXLZY benchmark samples by accurate relative molecular weight and multilevel MS fragment ion information， the detection conditions were positive and negative ion modes and data dependency scanning mode. TLC identification methods for Ophiopogonis Radix， Lycii Cortex， Nelumbinis Semen， Poria， Astragali Radix and Ginseng Radix et Rhizoma in QXLZY were established. ResultA total of 15 characteristic peaks were identified from Glycyrrhizae Radix et Rhizoma， Plantaginis Semen and Scutellariae Radix， and the relative standard deviations of the retention times of 15 characteristic peaks in 15 batches of QXLZY benchmark samples were≤3% with peak 8（baicalin） as the reference peak. A total of 100 compounds， including flavonoids， organic acids， saponins， amino acids and others， were identified in the benchmark samples by UHPLC-LTQ-Orbitrap MS. The established TLC had good separation and was suitable for the identification of Ophiopogonis Radix， Lycii Cortex， Nelumbinis Semen， Poria， Astragali Radix and Ginseng Radix et Rhizoma in QXLZY. ConclusionThe material basis of QXLZY benchmark samples is basically determined by MS designation and source attribution. The established specific chromatogram and TLC of QXLZY are simple， stable and reproducible， which can provide a reference for the development and quality control of QXLZY.
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ObjectiveTo explore the pretreatment methods to promote the enzymatic digestion and extraction of active ingredients from Magnoliae Officinalis Cortex dregs（MOCD）， and to provide a reference basis for the utilization of resource components in MOCD. MethodLiquid chromatography-mass spectrometry（LC-MS） was used for qualitative analysis of resource components in MOCD with an Agilent C18 reversed-phase column（3.0 mm×100 mm， 2.7 µm） at the flow rate of 0.4 mL·min-1， the mobile phase was water（A）-acetonitrile（B） for gradient elution（0-3 min， 25%-48%B； 3-6 min， 48%-59%B； 6-10 min， 59%-80%B； 10-20 min， 80%-90%B； 20-25 min， 90%B）， electrospray ionization（ESI） was employed with negative ion mode scanning and scanning range of m/z 50-1 200. A high performance liquid chromatography（HPLC）， which refered to the determination in the 2020 edition of Chinese Pharmacopoeia， was used for quantitative analysis of resource components in MOCD. Four kinds of pretreatment agents were used to separate the resource components from MOCD， and the mechanism of different pretreatment agents was investigated by field emission scanning electron microscopy（FESEM）， X-ray powder diffraction（XRD） and Fourier transform infrared spectroscopy（FT-IR）. ResultMagnolol， honokiol and lignocellulose were identified as the main resource components of MOCD by qualitative and quantitative analysis. Under the conditions of 1% NaOH， reaction temperature at 80 ℃ and reaction time of 60 min， the concentration of reducing sugar produced by the enzymatic hydrolysis was 32.18 g·L-1， which was 79.8% higher than that of the untreated MOCD. After adding tween-80， the enzymatic hydrolysis time was reduced to 1/3 of the original time， the concentration of reducing sugar was increased by 102.0%. And the total recovery of magnolol and honokiol in the pretreatment solution was 69.23%. ConclusionMagnolol， honokiol and lignocellulosic components in MOCD are valuable for development and utilization， the combination of alkaline pretreatment and tween-80 can realize the recovery and utilization of these three resource components， which can provide a new idea for comprehensive utilization of resource components in MOCD.
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ObjectiveTo establish the specific chromatogram and thin layer chromatography（TLC） identification method of Kaixinsan（KXS） samples， in order to clarify the key quality attributes and provide reference for the quality evaluation of KXS. MethodHigh performance liquid chromatography（HPLC） specific chromatogram of KXS was developed with YMC Hydrosphere C18 column（4.6 mm×250 mm， 5 μm）， the mobile phase was acetonitrile（A）-0.2% formic acid aqueous solution（B） for gradient elution（0-15 min， 2%-20%A； 15-25 min， 20%-25%A； 25-30 min， 25%-30%A； 30-45 min， 30%-31%A； 45-50 min， 31%-44%A； 50-65 min， 44%-45%A； 65-73 min， 45%-75%A； 73-95 min， 75%-100%A； 95-105 min， 100%A； 105-105.1 min， 100%-2%A； 105.1-120 min， 2%A）， the detection wavelength was 320 nm. Ultra high performance liquid chromatography-linear ion trap-electrostatic field orbitrap mass spectrometry（UHPLC-LTQ-Orbitrap MS） was used to identify the chemical components of KXS with electrospray ionization（ESI）， negative ion mode and scanning range of m/z 50-2 000. TLC identification methods for Poria and Ginseng Radix et Rhizoma in KXS were established. ResultThere were 11 common peaks in the specific chromatogram of KXS， attributed to Polygalae Radix， Poria and Acori Tatarinowii Rhizoma. Taking peak 9（α-asarone） as the reference peak， the relative standard deviations of the retention times of 15 batches of KXS samples were<0.2%. A total of 34 compounds were identified by UHPLC-LTQ-Orbitrap MS， including terpenoids， phenylpropanoids， oligosaccharides and ketones. The established TLC had good separation and was rapid， reliable， simple， feasible， suitable for the identification of Poria and Ginseng Radix et Rhizoma in KXS. ConclusionThe specific chromatogram and TLC of KXS are stable and reproducible. The material basis of KXS is basically clarified by MS， which can provide a reference for the development and quality control of KXS.
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ObjectiveTo establish the quality standard for Fraxini Cortex（Fraxinus chinensis） dispensing granules based on standard decoction， and to provide a basis for the quality control of this dispensing granules. MethodHigh performance liquid chromatography（HPLC） specific chromatograms of 15 batches of Fraxini Cortex（F. chinensis） standard decoctions and 3 batches of Fraxini Cortex（F. chinensis） dispensing granules were established with the mobile phase of 0.1% phosphoric acid aqueous solution（A）-acetonitrile（B） for gradient elution（0-10 min， 12%-15%B； 10-30 min， 15%-32%B） and the detection wavelength of 220 nm. And similarity evaluation， cluster analysis and principal component analysis（PCA） were also carried out. HPLC quantitative analysis of multi-components by single marker（QAMS） was established to determine the contents of the main components in the standard decoctions and dispensing granules. The contents of the corresponding components in Fraxini Cortex（F. chinensis） decoction pieces were also detected， and the transfer rates from decoction pieces to standard decoctions and dispensing granules were calculated. ResultThe similarities between specific chromatograms of 15 batches of Fraxini Cortex（F. chinensis） standard decoctions and 3 batches of Fraxini Cortex（F. chinensis） dispensing granules were all>0.9， and 7 common peaks were identified. The results of cluster analysis and PCA showed that there was some differences in the composition of different batches of standard decoctions， but did not show aggregation of origin. As the standard decoctions， the extract rate was 6.18%-11.62%， the contents of esculin， syringin， fraxin， esculetin， fraxetin， calceolarioside B were 44.92-103.51， 1.36-11.87， 33.26-90.73， 4.63-29.75， 2.40-16.86， 2.49-17.35 mg·g-1， and the transfer rates from decoction pieces to standard decoction were 25.21%-42.54%， 52.57%-88.84%， 43.43%-79.45%， 49.15%-88.27%， 49.22%-72.69%， 27.66%-47.67%， respectively. The extract rates of Fraxini Cortex（F. chinensis） dispensing granules were 10.4%-10.7%， the transfer rates of the above six components from decoction pieces to dispensing granules were 42.76%-43.17%， 80.01%-80.90%， 59.59%-59.88%， 51.35%-52.67%， 60.50%-60.93%， 37.98%-38.37%， respectively， which were generally consistent with the transfer rates from decoction pieces to standard decoctions. ConclusionThe established quality control standard of Fraxini Cortex（F. chinensis） dispensing granules based on standard decoctions is reasonable and reliable， which can provide reference for the quality control and process research of this dispensing granules.
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ObjectiveTo evaluate the quality of Mori Cortex from different producing areas by the entropy weight-technique for order preference by similarity to an ideal solution（TOPSIS）， and to provide a new evaluation method for the quality control of Mori Cortex. MethodAccording to the five key indexes of color， thickness， texture， powdery and cortex remain， a subjective scoring table was designed to evaluate the appearance of Mori Cortex. High performance liquid chromatography（HPLC） was used to determine the fingerprint and the contents of multiple components（mulberroside A， chlorogenic acid， oxyresveratrol， mulberroside C， sanggenone D， sanggenone C， morusin）， and chemometrics was used to explore the differential components of Mori Cortex from different habitats. On this basis， TOPSIS was used to comprehensively evaluate the quality of Mori Cortex from different habitats， and SPSS 22.0 software was used to carry out bivariate correlation analysis between thickness and appearance color with contents of seven components of Mori Cortex. ResultThose with lighter color， thicker root bark， tougher texture， sufficient powder and less cortex remain scored higher， and the top five were all from Anhui. The established fingerprint and determination methods were stable and reliable. Partial least squares-discriminant analysis（PLS-DA） screened three components with the variable importance in the projection（VIP） value>1（mulberroside A， sanggenone D， sanggenone C）， which made an important contribution to the difference in the origin of Mori Cortex. Correlation analysis showed that there was a significantly positive correlation between mulberroside C with lightness value（L*） and total chromaticity value（E*ab） and mulberroside A with yellow-blue value（b*）（P<0.05， P<0.01）， a significantly negative correlation between sanggenone C with b* and between morusin with L*（P<0.05， P<0.01）. And there was a significantly negative correlation between mulberroside A， chlorogenic acid， and morusin with thickness（P<0.01）， a clearly negative correlation between sanggenone D with thickness（P<0.05）， a significantly positive correlation between sanggenone C with thickness（P<0.01）. TOPSIS comprehensive scores showed that the samples from Anhui had a good score and ranked high. ConclusionThere are great differences in the quality of Mori Cortex from different habitats， and those with the close habitats show similar characteristics in appearance and component content， and lighter color and less cortex were positively correlated with the quality. Among them， the quality of Mori Cortex from Anhui is relatively good.
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@#Objective To develop and verify a high performance liquid chromatography(HPLC) for determination of residual 4-dimethylamino-pyridine(DMAP)content in pneumococcal polysaccharide-protein conjugate vaccine.Methods A HPLC method for determination of residual DMAP content in pneumococcal polysaccharide-CRM197 protein conjugate vaccine was developed by optimization of type of chromatographic column and composition of mobile phase,verified for specificity,accuracy,repeatability and reproducibility,and determined for limit of detection(LOD),limit of quantitation(LOQ)and linear range.The residual DMAP contents in pneumococcal polysaccharide-CRM197 protein conjugate vaccine of 13 serotypes and the intermediates of 1-cyano-4-dimethylamino pyridinium tetrafluoroborate(CDAP)-activated pneumococcal polysaccharide were determined by the developed method.Results The condition for HPLC was optimized as follows:Sepax HP-C18 chromatographic column(5 μm,4.6 mm × 250 mm)was adopted,using the mixture of 5 mmol/L sodium heptanesulfonate containing 20 mmol/L potassium dihydrogen phosphate(the pH value was adjusted to 3.0 with phosphoric acid)and acetonitrile at a ratio of 87∶13(v/v)as mobile phase at a detection wavelength of 280 nm,a flow rate of 1 mL/min,a sample load of 10 μL and a column temperature of 35 ℃.The mechanism of samples and solvent for pre-treatment showed no interference to the determination result,indicating good specificity of the developed method.The LOD and LOQ were 3.6 and 14.4 ng/mL respectively,while the linear range was 0.05 ～ 10 μg/mL with a R2value of0.999 9.The spike recovery rate was 83% ～ 105%,while the RSDs in repeatability and reproducibility tests were 0.28% ～0.72% and 7.38% respectively.The residual DMAP contents in pneumococcal polysaccharide-CRM197 protein conjugate vaccine of 13 serotypes were 0.135 ～ 1.635 μg/mL.DMAP was detected in the first step of activation of pneumococcal polysaccharide with CDAP,while no CDAP was detected.Conclusion The developed HPLC method is simple,specific,accurate,repeatable and reproducible,which is effective for quality control of pneumococcal polysac-charide-protein conjugate vaccine.
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ObjectiveTo establish the characteristic sugar spectrum of polysaccharides， oligosaccharides and monosaccharides of wild-simulated and transplanted Astragali Radix， and find out the difference of the sugar spectrum between the two， so as to provide a basis for quality evaluation of Astragali Radix. MethodThe relative molecular weight distribution of polysaccharides from 18 batches of wild-simulated Astragali Radix and 12 batches of transplanted Astragali Radix were characterized by high performance liquid chromatography-evaporative light scattering detection（HPLC-ELSD） to establish the characteristic chromatograms of two kinds of polysaccharides. The difference in the peak area ratio of APS-Ⅱ， a polysaccharide component with a relative molecular weight of 10 kDa， in two kinds of Astragali Radix was analyzed， and the critical value of peak area ratio of APS-Ⅱ was determined by receiver operating characteristic（ROC） curve. At the same time， APS-Ⅱ was partially acid-hydrolyzed by trifluoroacetic acid（TFA） to establish characteristic spectra of two kinds of oligosaccharides from Astragali Radix based on HPLC-ELSD， and the characteristics of differential oligosaccharides were found by principal component analysis（PCA） and orthogonal partial least squares-discriminant analysis（OPLS-DA）. Two kinds of APS-Ⅱ were completely acid-hydrolyzed by TFA and derivatized to establish characteristic spectra of two kinds of monosaccharides from Astragali Radix based on HPLC， PCA and OPLS-DA were performed on the peak area ratio of two kinds of monosaccharides to explore the differences in the composition of two kinds of APS-Ⅱ monosaccharides. ResultThe characteristic sugar spectrum of polysaccharides from Astragali Radix showed that the peak area ratio of APS-Ⅱ was the main difference， and the peak area of APS-Ⅱ of wild-simulated and transplanted Astragali Radix were 89.17%-97.17% and 80.14%-91.96%， respectively. The ROC curve determined the critical value of 92.28% for the difference of APS-Ⅱ peak area ratio of the two kinds of Astragali Radix. The multivariate analysis of APS-Ⅱ oligosaccharides revealed that the peak area ratio of oligosaccharides with polymerization degree≥10 was the main difference， which ranged from 11.835%-19.092% for wild-simulated products and 2.778%-7.017% for transplanted products. The results of monosaccharide characteristic sugar spectrum analysis showed that both Astragali Radix species consisted of six monosaccharides， and glucose and arabinose were the differential monosaccharide fractions. The peak area ratios of glucose and arabinose in wild-simulated products were 85%-93.9% and 2.7%-5.8%， respectively， while those of transplanted products were 74.3%-87.3% and 5.3%-10.7%， suggesting that the structures of the two polysaccharide fractions APS-Ⅱ of Astragali Radix may be different. ConclusionThe difference of sugar spectrum between two kinds of Astragali Radix may be related to the content and structure of APS-Ⅱ， and this study may provide a reference for the study of carbohydrates in Astragali Radix and the quality evaluation of medicinal materials.
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ObjectiveTo establish a high performance liquid chromatography（HPLC） fingerprint of Yanghetang benchmark sample， and evaluate its quality with chemometric methods， so as to provide a reference for the quality control of this benchmark sample. MethodHPLC was used to establish the fingerprint of Yanghetang benchmark sample with ZORBAX SB-C18 column（4.6 mm×250 mm， 5 μm）， the mobile phase was consisted of acetonitrile（A） -0.05% phosphoric acid aqueous solution （containing 0.05% triethylamine solution）（B） for gradient elution（0-5 min， 2%-3%A； 5-15 min， 3%-5%A； 15-65 min， 5%-30%A； 65-90 min， 30%-70%A）， the flow rate was 1.0 mL·min-1， the column temperature was 35 ℃， and the detection wavelength was 210， 260 nm. Traditional Chinese Medicine（TCM） Chromatographic Fingerprint Similarity Evaluation System （2012 edition） combined with cluster analysis， principal component analysis（PCA） and partial least squares-discriminant analysis（PLS-DA） were used to evaluate the quality differences between different batches of Yanghetang benchmark samples， and to find the main chemical components responsible for the quality differences. ResultHPLC fingerprint of Yanghetang benchmark sample was established， 13 common peaks were identified and attributed to each common peak， including peaks 2 and 8 from Rehmanniae Radix Praeparata， peaks 10 and 11 from Cinnamomi Cortex， peaks 1， 3-6 from fried Sinapis Semen， peak 13 from Ephedrae Herba， and peaks 7， 9， 12 from Glycyrrhizae Radix et Rhizoma. Eight of them were identified by comparing with control substance， which were 5-hydroxymethylfurfural（peak 2）， sinapine thiocyanate（peak 4）， glycyrrhizin（peak 7）， verbascoside（peak 8）， cinnamic acid（peak 10）， cinnamaldehyde（peak 11）， glycyrrhizic acid（peak 12） and ephedrine hydrochloride（peak 13）. The similarities of the HPLC fingerprints of 15 batches of Yanghetang benchmark samples with the control fingerprint were all greater than 0.80. The three chemometric methods could classify the samples into two categories. Eight differential components were screened out， among which 5-hydroxymethylfurfural， sinapine thiocyanate， verbascoside and ephedrine hydrochloride were identified. ConclusionThe established fingerprint analysis method is accurate， stable and reproducible， which basically reflects the overall chemical composition of Yanghetang benchmark sample， and can provide a basis for establishment of quality standards for compound preparations of this famous classical formula.
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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.
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Poria/chemistry , Spectroscopy, Fourier Transform Infrared , China , Polysaccharides/chemistry , Reference Standards , Chromatography, High Pressure Liquid/methodsABSTRACT
In order to comprehensively evaluate the quality of Viticis Fructus, this study established HPLC fingerprints and evaluated the quality of 24 batches of Viticis Fructus samples from different species by similarity evaluation and multivariate statistical analysis(PCA, HCA, PLS-DA). On this basis, an HPLC method was established to compare the content differences of the main components, including casticin, agnuside, homoorientin, and p-hydroxybenzoic acid. The analysis was performed on the chromatographic column(Waters Symmetry C_(18)) with a gradient mobile phase of acetonitrile(A)-0.05% phosphoric acid solution(B) at the flow rate of 1 mL·min~(-1) and detection wavelength of 258 nm. The column temperature was 30 ℃ and the injection volume was 10 μL. The HPLC fingerprint of 24 batches of Viticis Fructus samples was established with 21 common peaks, and nine peaks were identified. Similarity analysis was carried out based on chromatographic data of 24 batches of chromatographic data of Viticis Fructus, and the results showed that except for DYMJ-16, the similarity of Vitex trifolia var. simplicifolia was ≥0.900, while that of V. trifolia was ≤0.864. In addition, the similarity analysis of two different species showed that the similarity of 16 batches of V. trifolia var. simplicifolia was 0.894-0.997 and that of the eight batches of V. trifolia was between 0.990 and 0.997. The results showed that the similarity of fingerprints of these two species was different, but the similarity between the same species was good. The results of the three multivariate statistical analyses were consistent, which could distinguish the two different species. The VIP analysis results of PLS-DA showed that casticin and agnuside contributed the most to the distinction. The content determination results showed that there was no significant difference in the content of homoorientin and p-hydroxybenzoic acid in Viticis Fructus from different species, but the content of casticin and agnuside was significantly different in different species(P<0.01). The content of casticin was higher in V. trifolia var. simplicifolia, while agnuside was higher in V. trifolia. The findings of this study show that there are differences in fingerprint similarity and component content of Viticis Fructus from different species, which can provide references for the in-depth study of the quality and clinical application of Viticis Fructus.
Subject(s)
Drugs, Chinese Herbal/chemistry , Chromatography, High Pressure Liquid/methods , Fruit/chemistry , Vitex/chemistryABSTRACT
ObjectiveTo develop a quality control method for the simultaneous determination of multiple active components in Nymphaeae Flos aiming at the problems of the single index for quality control and the relatively low overall quality control level. MethodUltra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS/MS）was used to identify and select the index components for quality control with the mobile phase of 0.1% formic acid aqueous solution（A）-acetonitrile（B）for gradient elution （0-2 min， 3%-8%B； 2-4 min， 8%-10%B； 4-13 min， 10%-15%B； 13-19 min， 15%-20%B； 19-26 min， 20%-45%B） at a flow rate of 0.4 mL·min-1， detection wavelength of 350 nm， electrospray ionization（ESI）， negative ion scanning mode， ion source temperature of 120 ℃， scanning range of m/z 100-1 200， transmit collision energy of 6 eV for low-energy scanning and 25-50 eV for high-energy scanning. High performance liquid chromatography（HPLC）was used to establish the quality control method for the simultaneous determination of multi-index components with the mobile phase of 0.2% phosphoric acid aqueous solution（A）-acetonitrile（B） for gradient elution（0-30 min， 12%-15%B； 30-60 min， 15%-22%B； 60-90 min， 22%-40%B）and detection wavelength of 350 nm. The preparation method of the test solution for content determination was refluxing extraction for 60 min with 80 times the amount of 70% methanol. ResultBy comparing the retention time， ultraviolet absorption characteristics， MS and MS/MS spectrometric signals in the samples with the reference substances， 8 active components with high contents， including brevifolincarboxylic acid， ellagic acid， rutin， nicotiflorin， astragalin， quercetin， quercetin-3-methylether and kaempferol， were identified qualitatively from Nymphaeae Flos， which were selected as the index components for quality control. Under the established HPLC conditions， the above 8 components could be well separated（resolution>1.5）， and showed good linearity（r=0.999 9）between the concentration ranges of 1.99-99.6， 1.76-176， 1.52-75.8， 3.60-180， 0.964-96.4， 1.18-118， 1.94-96.8， 1.04-104 mg·L-1 and the peak areas， respectively. The detection limits of them were 10-49 μg·L-1， and the limits of quantitation were 34-164 μg·L-1. The average recoveries were 97.12%-103.1% with the relative standard deviations （RSDs） were 1.1%-2.2%. ConclusionA quality control method for simultaneous determination of the multiple active components in Nymphaeae Flos have been developed， which is simple， accurate and reproducible， and it can provide a scientific basis for the formulation of quality standard of this herb and lay a research foundation for the transformation of Uygur hospital preparations containing Nymphaeae Flos into new drugs.