Comparison of Quality Change During Processing Process of Achyranthis Bidentatae Radix from Different Origins Based on Color-component Correlation Analysis / 中国实验方剂学杂志

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.

Development and verification of a high performance liquid chromatography method for determination of aluminium adjuvant content in vaccine / 中国生物制品学杂志

@#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.

In vivo Pharmacokinetic Analysis of Shenbai Nanosuspension in Rats Based on Overall Composition of Chinese Materia Medica / 中国实验方剂学杂志

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.

STUDY ACCLIMATIZED SEED SAMPLE SAPOSHNIKOVIA DIVARICATA (TURCZ) SCHISCHK IN VIETNAM

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

Analysis on Sugar Spectrum Difference Between Wild-simulated and Transplanted Astragali Radix / 中国实验方剂学杂志

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.

Establishment of A Quality Control Method for Simultaneous Determination of Multiple Active Components in Uygur Medicine of Nymphaeae Flos / 中国实验方剂学杂志

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.

Comparison of HPLC fingerprints and determination of main components of Viticis Fructus from different species / 中国中药杂志

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.

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.

Applicability of an HPLC method for analysis of alcoholic and glycolic Brazilian green-propolis extracts

Abstract Brazilian green propolis has been widely used in food and pharmaceutical products due to its valuable source of phenolic compounds and versatile biological activities. The development and validation of analytical methods are extremely useful for the characterization and quality control of products containing propolis. Therefore, the aim of this study was to optimize, validate and investigate the applicability of a reversed-phase HPLC method for analysis of different types of Brazilian green propolis extracts (glycolic and ethanolic). The method showed to be selective for the propolis phenolic markers. The analysis of variance and residues demonstrated that the method had significant linear regression, without lack of fit. It was also a precise, accurate and robust method, which was of utmost importance to analyze both glycolic and ethanolic extracts and at different concentrations. Moreover, as these products can display most complex matrices to analyze, a valid HPLC method can also prove to be specific and versatile.

Development and verification of high performance liquid chromatography for determination of residual 4-dimethylaminopyridine content in pneumococcal polysaccharide-protein conjugate vaccine / 中国生物制品学杂志

@#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.

Quality Evaluation of Mori Cortex from Different Producing Areas Based on Entropy Weight-TOPSIS Method / 中国实验方剂学杂志

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.

Analysis on Quality Standard of Fraxini Cortex（Fraxinus chinensis） Dispensing Granules Based on Standard Decoction / 中国实验方剂学杂志

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.

Establishment of Specific Chromatogram and Thin-layer Identification Method for Kaixinsan / 中国实验方剂学杂志

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.

Investigation of Pretreatment Methods for Comprehensive Resource Utilization of Magnoliae Officinalis Cortex Dregs / 中国实验方剂学杂志

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.

Quality Evaluation of Aurantii Fructus from Sichuan by Specific Chromatogram Combined with Chemometrics and Grey Correlation Analysis / 中国实验方剂学杂志

ObjectiveHigh performance liquid chromatography （HPLC） was used to establish the specific chromatograms of Aurantii Fructus from different origins， and the quality variability of Aurantii Fructus from Sichuan was analyzed and evaluated by combining entropy weighting method and grey correlation method. MethodHPLC was performed on an Agilent Eclipse Plus C18 column （4.6 mm×250 mm， 5 μm） with a gradient elution of methanol （A）-0.1% phosphoric acid aqueous solution as the mobile phase （0-12 min， 25%-33%A； 12-21 min， 33%-41%A； 21-30 min， 41%-42%A； 30-40 min， 42%-59%A； 40-53 min， 59%-72%A； 53-60 min， 72%A； 60-65 min， 72%-100%A； 65-70 min， 100%A； 70~71 min， 100%-25%A； 71-80 min， 25% A） at a flow rate of 1.0 mL·min-1， the injection volume was 10 μL and the detection wavelength was 330 nm. Fifty batches of Aurantii Fructus samples from different origins （Sichuan， Chongqing， Jiangxi and Hunan） were tested， and the similarity evaluation software is used to generate characteristic profiles and compare them with control profile for peak identification， and then to evaluate the similarity of the samples. IBM SPSS 19.0 and SIMCA 14.1 were used to perform multivariate statistical analysis on the results of the samples， and then the entropy weighting method and grey correlation were used to calculate the overall quality score of samples from Sichuan. ResultHPLC specific chromatogram of Aurantii Fructus was established， and 14 common peaks were identified as eriocitrin， neoeriocitrin， narirutin， naringin， hesperidin， neohesperidin， meranzin hydrate， poncirin， meranzin， marmin， nobiletin， 3，3′，4′，5，6，7，8-heptamethoxyflavone， tangeretin and auraptene. And the similarities between the samples from Sichuan and the control chromatogram were all above 0.980. The samples could be classified into four categories according to their main origins by chemical pattern recognition， and the results of cluster analysis， principal component analysis and orthogonal partial least squares-discriminant analysis were all able to discriminate the samples of different main origins effectively. The comprehensive evaluation results of entropy weighting method combined with grey correlation showed that the quality of Aurantii Fructus from Sichuan varied greatly among different origins， and the quality of Aurantii Fructus from Sichuan was ranked as Bazhong>Luzhou>Chongqing>Neijiang. ConclusionIn this study， the characteristic mapping of Aurantii Fructus from Sichuan is established， and combined with the analytical methods of chemometrics and grey correlation， the quality of samples from different origins can be effectively differentiated， which can provide a reference for the comprehensive evaluation and control of the quality of Aurantii Fructus from Sichuan.

Development and verification of high performance liquidchromatography for determination of ethylenediaminetetraacetic acid disodium salt residue in NMM tumor DNA vaccine / 中国生物制品学杂志

@#Objective To develop and verify a high performance liquid chromatography(HPLC) method for the determination of ethylenediaminetetraacetic acid disodium salt(EDTA-2Na) residues in the bulk of NMM tumor DNA vaccine for the quality control of DNA vaccine.Methods After NMM tumor DNA vaccine bulk was complexed with copper sulfate,a HPLC method for the determination of EDTA-2Na residues was developed with Agilent ZORBA XSB-C18(150 mm × 4.6 mm,5 μm) as the chromatographic column,water,tetrabutylammonium hydroxide 10% and acetonitrile solution(74.5:0.5:25)as the mobile phase.The detection method was as follows:the detection wavelength was 254 nm,the flow rate was 0.8 mL/min,the column temperature was 20 ℃ and the injection volume was 20 μL.The method was verified for the specificity,linear range,limit of detection(LOD),limit of quantification(LOQ),solution stability,durability,accuracy and precision,and used to detect the EDTA-2Na residues in several batches of DNA vaccine bulk.Results When EDTA-2Na control and DNA vaccine bulk with EDTA-2Na reacted with copper sulfate,the absorption peak appeared at around 5.3 min,while no absorption peak was observed when DNA vaccine bulk reacted with copper sulfate;In the range of 4～400 μg/mL,the control solution concentration showed a good linear relationship with the peak area,R~2=0.999 9;The LOD of the method was 10 ng/mL,and the LOQ was 40 ng/mL;The solution of control and sample was stable after placed for 12 h;When the detection conditions changed slightly(different mobile phase ratio,flow rate and column temperature),the influence on the detection results was within acceptable range;The average recovery rate of EDTA-2Na in low,medium and high concentration standard added samples was 101.38% with the RSD of 0.39%;0.1 mg/mL control solution was injected continuously for 6 times,and the peak area RSD was 0.04%.EDTA-2Na was not detected in 6 sample solution,and the peak area RSD of DNA vaccine bulk with EDTA-2Na solution was 0.02%,indicating a good intermediate precision.EDTA-2Na residue was not detected in these batches of DNA vaccine bulk.Conclusion The developed method is simple,accurate,reliable with good specificity,which can be used for the determi-nation of EDTA-2Na residues in DNA vaccine bulk.

HPLC Fingerprint Analysis of Benchmark Sample of Yanghetang / 中国实验方剂学杂志

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.

Establishment of Specific Chromatogram and TLC Identification for Qingxin Lianziyin / 中国实验方剂学杂志

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.

Application and Analysis of High-quality Evaluation for Jianwei Xiaoshi Tablets / 中国实验方剂学杂志

Jianwei Xiaoshi tablets， as a common variety of Chinese patent medicine with "one product with many manufacturers"， have many manufacturers and huge market sales. However， the phenomenon about uneven quality and discrepant price is prominent. Based on this， this study was carried out for the quality evaluation of Jianwei Xiaoshi tablets by applying the high-quality evaluation criteria with the quality as core for Chinese patent medicine， which was based on the full production cycle， from the multi-dimension including raw material selection， production process， quality control， post-marketing research and so on. The evaluation results showed that the quality evaluation scores of Jianwei Xiaoshi tablets from different manufacturers varied greatly （ranging from 35 to 66）， indicating that the quality was significantly different. In the actual production， generally inadequate attention was paid to the quality of raw materials， and the quality of raw materials was insufficient with the score ratio of 43%， especially the poor consistency control of them. The role of good manufacturing practice was obvious， and the scores of production process were generally high with the average score ratio of 62%， and the maximum up to 80%. The technological advancement of the manufacturer was outstanding. The score ratio of quality control was only 31% that the internal quality standard of each manufacture almost stayed at the qualified line， which was equal to the national standard， and the consistency of products was insufficient. The post-marketing research was lacking with the score ratio of 37%. Manufacturers with high brand awareness and market share were upper scores， while the others lagged far behind. The results of this evaluation are in line with the overall prediction， which can provide a reference for the high-quality evaluation of Chinese patent medicine， and supply the scientific data for high-quality and high-price application.

Identification and Quality Evaluation of Dendrobium flexicaule and Its Related Species / 中国实验方剂学杂志

ObjectiveTo identify Dendrobium flexicaule and its related species， and analyze the differences in polysaccharide composition and D-mannose content， so as to provide theoretical basis for the accurate identification and quality control of Dendrobium medicinal materials. MethodNine samples of Dendrobium （S1-S9） were identified by DNA barcoding and infrared spectroscopy， and the contents of polysaccharides and D-mannose were determined by ultraviolet spectrophotometry （UV） and high performance liquid chromatography （HPLC）， respectively. UV detection condition was 488 nm， HPLC detection conditions were the mobile phase of 20 mmol·L-1 ammonium acetate solution-acetonitrile （81.5∶18.5） and the detection wavelength at 250 nm. ResultDNA barcoding results showed that samples S1-S3 were D. nobile， samples S4-S5 were D. officinale， sample S6 was D. huoshanense， and S7-S9 were D. flexicaule. One-dimensional infrared spectroscopy showed that only D. nobile had stable characteristics at the wavenumber of 1 570-1 467 cm-1， showing a "W" shape， while no absorption peak was found at the wavenumber of 842-740 cm-1， but the other Dendrobium samples had stable absorption peaks at the wavenumber of 842-740 cm-1. In the first derivative spectrum， at the wavenumber of 785 cm-1， D. huoshanense presented a "V" shape， while the rest of Dendrobium presented a "W" shape. At the wavenumber of 1 110 cm-1， D. flexicaule had a stable characteristic peak. In the second derivative spectrum， at the wavenumber of 1 125 cm-1， D. officinale presented an "M" shape， and the rest of Dendrobium was approximately "W" shape. The results of determination showed that the contents of polysaccharides in samples S1-S9 were 9.35%， 9.12%， 32.78%， 49.38%， 48.97%， 32.48%， 32.95%， 39.41% and 25.32%， and their contents of D-mannose were 1.39%， 0.47%， 13.57%， 3.04%， 33.85%， 23.57%， 16.64%， 17.47% and 19.49%， respectively. Among them， D. flexicaule had high polysaccharide and D-mannose contents. ConclusionBoth DNA barcoding and infrared spectroscopy can be used to identify D. flexicaule and its related species， and infrared spectroscopy is cost-effective and easy to operate. At the same time， D. flexicaule has high contents of polysaccharides and D-mannose， which can provide a scientific basis for rapid identification of D. flexicaule and its relatives， and provides a reference for its quality control， and resource development and utilization.