Development and verification of cation exchange high performance liquid chromatography for detection of charge variants of pembrolizumab / 中国生物制品学杂志

@#Objective To develop and verify a cation exchange high performance liquid chromatography(CEX-HPLC)method for the detection of charge variants of pembrolizumab.Methods Pembrolizumab was bound to the exchange column matrix by using MabPac SCX-10 column,and the variants with different charges were eluted by gradually increasing the salt concentration of the mobile phase.The specificity,precision,linear range,accuracy and durability of the method were verified,and the charge variants of three batches of pembrolizumab finished products were detected by using the developed method.Results The resolution of the last acidic isomer peak and the first basic isomer peak of pembrolizumab from the main peak were 1.28 and 1.42,respectively.The mobile phase A and preparation buffer had no obvious interference peaks at the peak of the sample;The RSD values of the precision verification were all less than 2.0%;The total peak area,main peak area,acidic isomer peak area and basic isomer peak area of the standard all exhibited good linear relationship with the theoretical dilution concentration with each R~2 of 1.00;The recovery rates of the total peak area and main peak area of the standard at three concentrations were between 96.81% and 106.07%;When pH value of the mobile phase was within the range of 6.30±0.10,the RSD values of the total peak area and main peak area percentage of the standard were1.5% and 1.9%,and when the column temperature was within the range of(35±4) ℃,the RSD values of the total peak area and main peak area percentage of the standard were 0.4% and 0.3%,respectively.The RSD value of the main peak retention time of the three batches of finished products was 0.Conclusion The developed CEX-HPLC method can effectively separate the acidic isomers,main peaks and basic isomers of pembrolizumab with good specificity,precision and accuracy,which can be used for the follow-up research and development of pembrolizumab,the process verification of expanding production and the stability research.

Determination of tetrodotoxin in salted pufferfish by liquid chromatography-tandem mass spectrometry / 预防医学

Objective@#To develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with cation exchange-based solid phase extraction (SPE) for determination of tetrodotoxin (TTX) in salted pufferfish. @*Methods@#Evenly crushed salted pufferfish samples were subjected to ultrasound-assisted extraction with 0.5% acetic acid/50% methanol/water. The extract was cleaned with cation exchange-based SPE cartridge and eluted with 0.3% hydrochloric acid and 50% acetonitrile/water. The eluent was neutralized with ammonia and separated with a Waters XBridgeTM BEH Amide column (150 mm×3.0 mm, 1.7 μm), and determined using LC-MS/MS in a multiple reaction monitoring mode.@*Results@#The matrix effects of TTX were 85.7%-92.4%, and the matrix suppression effect was under effective control following clean-up procedures using the optimized SPE method. The TTX showed a good linear relationship at the range of 2.0 to 4 000 μg/kg, with a correlation coefficient (r2) of 0.999 2. The limits of detection and quantitation for TTX in sample matrix were 1.0 μg/kg and 2.0 μg/kg, respectively. The mean spiked-recovery rates were 81.2% to 96.5% at spiked amounts of 2.0, 200 μg/kg and 2 200 μg/kg, with relative standard deviations (RSDs) of 4.3% to 7.5%. The intraday accuracy and precision of TTX were 84.4% to 95.6% and 4.9% to 5.8% in quality control samples, and the interday accuracy and precision of TTX were 86.1% to 94.9% and 5.5% to 8.5% in quality control samples. The detection of TTX was 60.5% in 38 market-sold salted pufferfish products using the established LC-MS/MS method.@*Conclusion@#The established LC-MS/MS method is effective for accurate quantitative determination of TTX in salted pufferfish.

Evaluation of soil contamination by heavy metals at public cemeteries in the municipality of Lages, southern Brazil / Avaliação da contaminação de solos por metais tóxicos em cemitérios públicos do município de Lages, Sul do Brasil

ABSTRACT The burial of bodies is a potentially polluting activity. Taking this into consideration, the aim of the present study was to verify the compliance of two cemeteries with environmental legislation and to quantify the concentrations of heavy metals in soils affected by burial activities. Physicochemical characterization of the soil was performed by analyzing control samples from areas near the cemeteries. Concentrations of cadmium, lead, chromium, nickel, zinc and copper were determined using high-resolution continuum source atomic absorption spectrometry. The two cemeteries had unsatisfactory properties for the retention of metal cations, with clay percentages ranging from 15.40 to 41.40% and sand percentages ranging from 28.75 to 66.85%. The control samples presented low cation exchange capacity (12.27 to 22.73 cmolc/dm³) and high aluminum (Al3+) saturation (66.74 to 90.16%). Although neither of the two cemeteries had concentrations above the limits established for the metals analyzed by Resolution No. 420/2009 of the National Environment Council, the contaminants may be leaching to groundwater due to inadequate soil characteristics.

RESUMO O sepultamento de corpos é uma atividade potencialmente poluidora. Este trabalho teve como objetivo verificar a adequação das áreas de dois cemitérios públicos à legislação ambiental e à atividade cemiterial e quantificar a concentração de metais pesados nos solos que estão sob influência desses empreendimentos. Realizou-se a caracterização físico-química do solo, com a análise de amostras testemunha de solo de cada cemitério. Também foram determinadas as concentrações dos metais pesados: cádmio, chumbo, cromo, níquel, zinco e cobre, por meio de espectrometria de absorção atômica de alta resolução com fonte contínua. As áreas dos cemitérios apresentam condições insatisfatórias para a retenção de íons catiônicos metálicos, com percentuais de argila variando entre 15,40 e 41,40% e de areia entre 28,75 e 66,85%. Os solos testemunha apresentaram reduzida capacidade de troca de cátions entre 12,27 e 22,73 cmolc/dm³) e elevada saturação por alumínio entre 66,74 e 90,16%. Apesar de nenhum dos cemitérios apresentar concentrações dos metais analisados acima dos limites de prevenção estabelecidos pela Resolução nº 420/2009 do Conselho Nacional do Meio Ambiente, em função das características dos solos, os contaminantes podem estar sendo lixiviados para os recursos hídricos subjacentes.

Determination of five carbohydrate impurities in amino acid bulk drug by HPLC-ELSD / 中国药科大学学报

@#An analytical method was developed for the determination of five carbohydrate impurities in amino acid drug substances by high performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD). Sugar impurities in the amino acid sample were separated and enriched by cation exchange resin. A Lichropher NH2 column (4.6 mm × 250 mm, 5 μm) was used for chromatographic separation, and a gradient elution was performed using acetonitrile-water as mobile phase. The drift tube temperature was 40 oC, the gain value was 8, and nitrogen (350 kPa) was auxiliary gas. Method validation results showed that the limits of detection for fructose, glucose, sucrose, maltose and lactose were in the range of 20.8-75.0 mg/kg and that the limits of quantitation were in the range of 96.2-238.8 mg/kg. Good linear relationship (r ≥ 0.999) were in the linear range for the five sugars, and the recoveries ranged from 84.9%-107.8%. With easy operation, high sensitivity, good precision and reliable accuracy, the method can be used for analysis of residual sugar impurities in amino acid drug bulk drug.

Content Determination of 17 Kinds of Amino Acids in Sargassum and Its Adulterants / 中国药房

OBJECTIVE：To establish the method for content determination of 17 kinds of amino acids in Sargassum and its adulterants，and to carry out cluster analysis ，so as to provide reference for quality control of Sargassum. METHODS ：Totally of 18 batches of sample （S1-S6 as certified product ，S7-S18 as adulterants ）were collected. After acid hydrolysis ，amino acids contents were detected by using automatic amino acid analyzer. The separation was performed on LCAK 06/Na sulfonic acid cation exchange resin column with mobile phase consisted of buffer-regeneration system （gradient elution ）at the flow rate of 0.45 mL/min （elution pump ）and 0.25 mL/min（derivative pump ）. The detection wavelengths were set at 440 nm（proline）and 570 nm（other amino acids ），and the sample size volume was 50 μL. PASW Statistics 18.0 software was used ，and cluster analysis was conducted by using group connection method of cluster analysis with “square Euclidean distance ”as the measurement standard. RESULTS ：17 kinds of amino acids were well separated without interference from blank sample. The linear relationship between mass concentration and peak area was good （all r were over 0.998），and the upper and lower limits of the linear range were 48.06 μg/L （cystine）and 1.501 μg/L（glycine），respectively；RSDs of precision ，reproducibility and stability tests were lower than 2％. The average recoveries were between 90.60％-101.56％（RSDs were 0.88％-2.15％，n＝6）. 17 kinds of amino acids were detected in Sargassum and its adulterants ，among which the contents of glutamic acid ，aspartic acid ，leucine，alanine，glycine and valine were relatively high . Results of cluster analysis showed that 18 batches of sample were clustered into 4 categories，i.e. S 1-S6 into one category；S7-S9 into one category ；S10-S12，S16-S18 into one category ；S13-S15 into one category ；which was consistent with the identification result of Sargassum and its adulterants . CONCLUSIONS ：The method is simple ， rapid， accurate and reproducible，and can be used for the quantitative analysis and identification of amino acids in Sargassum and adulterants.

Chemical attributes of a remineralized Oxisol / Atributos químicos de um Latossolo remineralizado

ABSTRACT: Remineralizers are comminuted rocks that are applied to soil, and their use as an agricultural amendment was regulated in Brazil in 2013. However, mechanisms of action of these materials must be better known to enable them to be best used in agricultural fields. Soil chemical attributes of an Oxisol were monitored after the application of a diabase remineralizer. The increase in exchangeable Na observed was associated with the dissolution of the border of the plagioclase crystals where this element is highly concentrated (albite). Therefore, it was inferred that the time since the application of the remineralizer (1 to 2 years depending on the treatment) was not sufficient to exhaust this crystal volume. Unfortunately, the presence of several sources of Ca-containing minerals in the remineralizer did not allow to infer if the calcic nuclei was dissolving. An increase in effective cation exchange capacity was observed without the concurrent increase in the pH of the soil. The two non-exclusive hypotheses proposed to explain this result were that an extra surface charge has originated on the surface of the newly precipitated oxidic phases and/or from the dissolution of the remineralizer grains. Rapid precipitation of amorphous solids (as measured by the increase in Alo and Feo) would also explain the lack of increase in exchangeable Fe and Al despite the large amount of Al2O3 (11.90%) and Fe2O3 (14.45%) in the remineralizer.

RESUMO: O uso de remineralizadores como insumo agrícola foi regularizado em 2013, mas seus mecanismos de ação precisam ser melhor conhecidos para viabilizar o manejo nos campos agrícolas. Atributos químicos de um Latossolo foram monitorados após remineralização com diabásio. O aumento de Na trocável foi atribuído à dissolução das bordas dos plagioclásios (albita) onde a concentração deste elemento é maior. Infelizmente, não é possível especular se o tempo decorrido desde a aplicação (um a dois anos, dependendo do tratamento) foi suficiente para solubilizar o núcleo cálcico (anortita) destes cristais, já que o remineralizador possui outros minerais fonte de Ca. Houve aumento da capacidade de troca catiônica efetiva sem aumento do pH. As hipóteses propostas para explicar este fenômeno são a precipitação de fases oxídicas amorfas e o aparecimento de cargas elétricas na superfície dos grãos do remineralizador durante sua dissolução. Apesar da concentração de Al2O3 do remineralizador (11,90%) e Fe2O3 (14,45%), não houve aumento destes elementos no complexo de troca, possivelmente por sua rápida precipitação em formas amorfas (Alo e Feo no solo).

Effects of different processing methods on content of crude protein and hydrolytic amino acids in Cervi Cornu Pantotrichum / 中草药

Objective: To compare the contents of protein and amino acids in different parts of Cervi Cornu Pantotrichum (CCP) with different processing methods, in order to provide a guidance for the processing and comprehensive utilization of CCP. Methods: The techniques of Dumas combustion and cation-exchange chromatography were respectively adopted to determinate the contents of crude protein and 17 amino acids in different parts of CCP with different processing methods, and the difference was compared. Results: The content of crude protein in powder slices of CCP with blood was higher than that of CCP with blood (P 0.05). The crude protein content in wax slices of CCP with freeze-drying processing was less than that with boiling processing (P 0.05). The content of crude protein and TAA in wax slices of CCP is both significantly higher than that in powder slices, bee slices (P 0.05). Conclusio:n The difference is existed in content of crude protein and amino acids in CCP with different processing methods. The wax silices of CCP are significantly higher than that of powder slices and bee slices. And the difference in powder slices and bee slices is not significantly. The distribution of crude protein and TAA in different parts of CCP with freeze-drying processing is more uniform than CCP with boiling processing.

Content Determination of Urea in Urea [13C] Capsules by High Performance Cation-exchange Chromatogra-phy / 中国药房

OBJECTIVE:To determine the content of urea in Urea [13C] capsules by high performance cation-exchange chroma-tography (HPCEC). METHODS:The determination was performed on Zorbax 300 SCX column with mobile phases consisting of acetonitrile-0.1% phosphoric acid (20:80,V/V) at the flow rate of 1.0 mL/min. The detection wavelength was set at 200 nm and column temperature was 35 ℃. The sample size was 20 μL. RESULTS:The linear range of urea was 0.0039-1.0030 mg/ml(r=0.9997). The limit of quantitation was 3.918 μg/mL and the limit of detection was 0.975 μg/mL. RSDs of precision,stability and repetitive test were all lower than 2.0%. The recovery ranged 99.3%-101.0%(RSD=0.67%,n=9). CONCLUSIONS:The meth-od is simple,rapid,sensitive and suitable for the content determination of urea in Urea [13C] capsules.

Simultaneous Determination of Contents of 17 Amino Acids in Fritillariae Pallidiflorae Bulbus Produced in Xinjiang by Post-column Derivatization Cation-exchange Chromatography / 中国药房

OBJECTIVE:To establish a method of contents determination of 17 amino acids in Fritillariae Pallidiflorae Bulbus produced in Xinjiang. METHODS:Cation-exchange column was used to separate 17 kinds of amino acids;post-column derivatiza-tion liquid chromatography was used to determine the contents of amino acids:the column was strong sulfonic acid cation exchange resin LCAK06/Na with mobile phase A(weighing trisodium citrate 11.9 g,citric acid 6 g,phenol 1 g,dissolving by water,then adding 65 mL of methanol and 6 mL of concentrated hydrochloric acid,bringing the volume with tap water,adjusting pH to 3.4), mobile phase B(weighing trisodium citrate 11.9 g,NaOH 2.8 g,phenol 1 g,boric acid 5.0 g,adding water to dissolve,adjustingpH to 10.8),gradient elution,flow rate was 0.45 mL/min for A pump and 0.25 mL/min for M pump,the detection wavelength was 440 nm for proline and 570 nm for the remaining amino acids,the injection volume was 50 μL. RESULTS:The linear range were 20~400 nmol/mL of aspartic acid,threonine,serine,glutamic acid,glycine,alanine,valine,methionine,isoleucine,leucine,ty-rosine,phenylalanine,histidine,lysine,arginine,proline,10-200 nmol/mL of cystine(r were higher than 0.9890);RSDs of preci-sion,stability and reproducibility tests were lower than 2.0%;limits of detection were 0.16 nmol/mL except for cystine(0.08 nmol/mL);recovery was 98.5%-99.5%(RSD was 0.06%-0.21%,n=6). CONCLUSIONS:The method is simple with good precision, stability and reproducibility,and can be used for the simultaneous determination of amino acids in Fritillariae Pallidiflorae Bulbus produced in Xinjiang.

Preparation and Quality Evaluation of Carbinoxamine Maleate Sustained-release Suspension / 中国药房

OBJECTIVE:To prepare Carbinoxamine maleate sustained-release suspension,and evaluate its quality. METH-ODS:Using carbinoxamine maleate as raw material,drug-loaded resin was prepared by cation exchange resin;surface coating method was used to finally prepare sustained-release suspension,using Eudragit RS100 as sustained-release coating material to pre-pare sustained-release microparticles. HPLC was conducted to determine the content of carbinoxamine maleate,release degree of original preparations and self-made suspensions was compared,drug-loading capacity was calculated. RESULTS:The drug amount in preparing drug-loaded resin was 2%,reaction temperature was 25 ℃,and reaction time was 4 h;the drug-loading capacity in surface coating was 35%,amount of coating material was 10%,and reaction temperature was 40 ℃. The drug-loading capacities of sustained particles before and after coating were 35.23%,32.72%,respectively;the yield was 96.82%. The carbinoxamine ma-leate in prepared sustained-release suspension accounted for 98.76% of the labeled amount;release degree in 10 h reached about 80%,f2 was 65.73. CONCLUSIONS:Carbinoxamine maleate sustained-release suspension is prepared successfully,and its release is similar to the original preparation.

Enzyme-assisted Extraction and Enrichment of Galanthamine from Lycoris aurea / 中草药·英文版

Objective: To explore the optimum condition for complex enzyme-assisted extraction of galanthamine from Lycoris aurea by L9 (34) orthogonal array design and separation effect of cation exchange resin on galanthamine. Methods: Cellulase and pectinase solution was used as the extraction solvent. The effects of pH value of enzyme, amount of complex enzyme, dissociation time, and enzymatic hydrolysis temperature on the extraction results were investigated. Results: The optimal conditions were obtained as follows: ratio of solid to liquid (g: mL) 1:10, pH value 4.5, amount of complex enzymes 4%, enzymatic hydrolysis temperature 50 °C, and reaction time 2.0 h. Under these conditions, the extraction yield of galanthamine was 0.0294%. In addition, D-001 cation exchange resin was selected for separation of galanthamine. The separation conditions were that adsorption flow rate was 3 BV/h with reagent of pH 2 and the desorption flow rate was 3 BV/h with 70% ethanol solution containing 1.5 mol/L ammonia. After separation, the content of galanthamine was increased to 12.31%. Conclusion: The results provide a reference for industrial production of galanthamine.

Elaboración de bio-resina intercambiadora de cationes a partir de cáscara de plátano o guineo para eliminar metales pesados en agua contaminada / Manufacture of cation exchange bio-resin from banana peel to remove heavy metals in contaminated water

Esta investigación tuvo por objetivo obtener una bio-resina intercambiadora de cationes utilizando cáscaras de guineo o plátano, la cual reduzca la concentración de metales pesados en agua contaminada. A esta bio-resina se le realizaron pruebas fisicoquímicas: densidad seca aparente, pH y solubilidad en agua y solventes orgánicos. Se evaluó su efectividad filtrando agua contaminada con metales pesados, tales como hierro, cromo y níquel (Fe3+, Cr6+ y Ni2+), variando las condiciones de tiempo de contacto, temperatura y el tipo de cáscara. La cuantificación de la concentración de los metales en el agua filtrada se llevó a cabo por espectrofotometría visible. Se llegó a la conclusión que la bio-resina obtenida es efectiva para disminuir la concentración de metales pesados en agua, teniendo especial afinidad química por el cromo hexavalente; metal pesado que logró remover arriba del 90%. Las condiciones óptimas de operación de la bio-resina son a 30°C y 90 minutos de tiempo de contacto con la muestra. Además, las pruebas fisicoquímicas, permitieron tipificarla preliminarmente como una resina de intercambio catiónico débil con un grado de entrecruzamiento bajo.

The objective of this research was to obtain a cation exchange bio-resin using banana peels, which reduces the concentration of heavy metals in contaminated water. Physicochemical tests were carried out on this bio-resin: apparent dry density, pH and solubility in water and organic solvents. Its effectiveness was evaluated by filtering water contaminated with heavy metals, such as iron, chromium and nickel (Fe3 +, Cr6 + and Ni2 +), varying the conditions of contact time, temperature and the type of shell. The quantification of the metal concentration in the filtered water was carried out by visible spectrophotometry. It was concluded that the bio-resin obtained is effective in reducing the concentration of heavy metals in water, having a special chemical affinity for hexavalent chromium; heavy metal that was able to remove over 90%. The optimal operating conditions for the bio-resin are at 30 ° C and 90 minutes of contact time with the sample. Furthermore, physicochemical tests allowed it to be preliminarily typified as a weak cation exchange resin with a low degree of crosslinking.

Determination of Cu, Ni and Zn in Aluminum Food Packaging Materials by Strong Acid Cation Exchange Fiber Solid Phase Extraton-ICP-AES / 中国药师

Objective:To establish the analysis method for the determination of Ni, Cu and Zn in aluminum food packaging mate-rials by inductively coupled plasma atomic emission spectrometry ( ICP-AES) . Methods: A small amount of EDTA was added to the test solution till the concentration at 0. 010 0 mol·L-1 and the pH value was adjusted to 5. 0. Strong acid cation exchange fibers were used for the assisted extraction of Al3+ with ultrasonic. Al3+ could not quickly form a complex anion with EDTA, so it was adsorbed by the strong acid cation exchange fibers. Cu2+, Ni2+ and Zn2+ could form complex anions with EDTA rapidly, so they could not be ad-sorbed by the strong acid cation exchange fibers and were left in the solution to be determined. Results:After the separation, the con-tents of Cu, Ni and Zn were not lost, and the content of residual aluminum was about 170 times of that of Cu, Ni and Zn, which had not interferece with the determination of Cu, Ni and Zn. The sample standard addition recovery was within the range of 98. 3%-104%with RSD of 0. 2%-2. 5%(n=5). The relative errors between the measured values and the certified values of standard substances were less than 5. 0%. Conclusion:The proposed method can be successfully applied in the separation and determination of Cu, Ni and Zn in aluminum cans, barbecue aluminum foil and standard substances.

Content Comparison of 18 Amino Acids in Plancenta Histolysate Determined by Post-column Derivatization Cation-exchange Chromatography and Pre-column Derivatization HPLC / 中国药师

Objective:To compare the content differences of 18 amino acids in plancenta histolysate determined by pre-column de-rivatization HPLC and post-column derivatization cation-exchange chromatography ( AARO) . Methods: The HPLC method was per-formed on a C18 column and 2, 4-dinitro chlorobenzene ( DNCB) was used for pre-column derivatization, and then the determination was carried out after adding 0. 1 mol·L-1 borax buffer (pH=9. 1), and the AARO method was used for the direct determination with a strong acid cation-exchange chromatographic column and post-column derivatization. Results: The RSD for reproducibility of the AARO method was 1. 84%-0. 91%, while that of the HPLC method was 1. 87%-1. 04%. Conclusion:Both AARO method and HPLC method can be used for the quantitative determination of 18 amino acids in plancenta histolysate with the similar results. However, pre-column derivatization HPLC method may cause incomplete derivatization and instable derivatives.

Study on Purification Technology of Total Alkaloid from the Flos of Aconitum kusnezoffii / 中国药房

OBJECTIVE:To optimize the purification technology of total alkaloid from the flos of Aconitum kusnezoffii. METH-ODS:The content of total alkaloid from the flos of A. kusnezoffii was determined by acid-base titration. The purification technology of total alkaloid from the flos of A. kusnezoffii was optimized by ion resin with resin type,mass concentration of loading liquid and exchange speed as factors,maximum adsorption quantity,desorption rate and mass fraction of total alkaloid as index,and verifica-tion test was conducted. RESULTS:The optimal purification technology was as follows as type 732 cation exchange resin,mass concentration of loading liquid 0.32 g/L,exchange speed of 7 column volume(BV)/h. In validation test,the content of total alka-loid was 86.88%(RSD=0.52%,n=3),and desorption rate was 92.81%(RSD=0.40%,n=3)averagely. The extraction trans-port rate of total alkaloid from 3 batches of the flos of A. kusnezoffii was 81.76% and purification transport rate was 89.47% in av-erage. CONCLUSIONS:The established method is stable and feasible,and shows high transport rate.

Separation and purification of total alkaloids from Evodia rutaecarpa using 732 cation exchange resin and investigations on its anti-breast cancer bioactivity / 中国生化药物杂志

Objective To optimize conditions for extraction of alkaloids from Evodia rutaecarpa by means of 732 cation exchange resin and performe its anti-breast cancer bioactivity.Methods The optimum processing route on extraction of alkaloids from Evodia rutaecarpa was investigated by means of 732 cation exchange resin.The performance of 732 cation exchange resin was compared with tranditonal process ( aqueous extraction-ethanol precipitation in extraction-purification process).The alkaloid reagent-potassium mercuric iodide test and MTT test were performed on the crudes.Results Compared with traditional purification process, it was much better to use 732 cation exchange resin approach for extraction of alkaloids from Evodia rutaecarp with high yield(16.81%) The best eluent should be saturated brine with excellent purification.No obvious correlations were found between the toxin of breast cancer cell and concentration of total alkaloids.When the concentration of total alkaloids was 50μmol/mL, cellular survival rate was 68%afterward 24 h.When the concentration of total alkaloids comes to 100 μmol/mL and 150 μmol/mL, cellular survival rates were slightly decreased by 66% and 60%.Conclusion 732 cation exchange resin performes much higher than traditional process in purification of total alkaloids extracted from Evodia rutaecarpa.Simultaneously, the extracted total alkaloids showe remarkable inhibition in breast cancer cell.

Expression and purification of nucleocapsid protein of MERS coronavirus in E.coli / 军事医学

Objective To construct a prokaryotic expression vector pET-22b+with Middle East respiratory syndrome ( MERS) coronavirus nuclocapsid protein( NP) gene and to express and purify N protein.Methods N gene amplified by PCR was inserted into the prokaryotic expression vector pET-22b+.Recombinant plasmid was confirmed using DNA elec-trophoresis and sequencing.NP was expressed in E.coli BL21(DE3) by IPTG induced and purified by cation exchange chromatography using the AKTA purification system.Results The NP gene sequence was proved to be correct by sequen-cing and the protein was expressed in both soluble and insoluble forms in E.coli BL21 ( DE3 ) after IPTG induction.The purity and concentration of recombinant protein was improved obviously by cation exchange chromatography and enrich-ment.Conclusion Recombiant NP of high purity and concentration is purified and will facilitate NP functional research.

Signal Amplification Effect of Non-fluorescent Zinc Sulfide Nanoparticles Cluster for Detection of Trace Proteinsbiological Molecules / 分析化学

Cation exchange (CX) reaction for the non-fluorescent ZnS nanocrystal clusters (NCCs) can be used to detect trace biomolecules . Nano clusters synthetized by hydrothermal synthesis are porous. So they can quickly release large amounts of Zn2+ from through cation exchange ( CX) reaction and nano cluster, generate fluorescent signal under the action of zinc reagent to detect fluorescence. The relationship between the release efficiency, target binding force of Zn2+ and its average diamete was investigated when the average diameter was 44 nm, 86 nm and 144 nm in this experiment. Results showed that the smallest nano cluster exhibited the highest cation exchange efficiency, and 71 percent of Zn2+ closed could be released by microwave radiation within 2 min. When the sandwich method of NCCs of 44-nm was used to detect immunoglobulin E (IgE) in a sandwich assay, the limit of detection (LOD) was 5 ng / L, which was 1000 times lower than that of ELISA. It turns out that CX for the ZnS NCCs is superior to the conventional signaling strategies in its high amplification efficiency, robustness, and biocompatibility.

Determination of 14 Sulfonamides Residue in Milk by On-line Solid Phase Extraction in Cation Exchange Mode/Liquid Chromatography-Tandem Mass Spectrometry / 分析化学

To determine the residue of 14 sulfonamides in milk, a high performance liquid chromatography-tandem mass spectrometry ( HPLC-MS/MS ) method with on-line soild phase extraction ( SPE ) in cation exchange mode was established. 5 g of milk was extracted with 15 mL acetonitrile. Then the extraction was evaporated by 50 ℃ nitrogen and dissolved by 1. 00 mL 0. 2% formic acid. The dissolution was enriched and purified by MS/MS cation exchange on-line SPE column on a double ternary liquid chromatography, and eluted by the mixed solution of 2% ammonia methanol and 0. 2% formic acid (50:50, V/V). The compounds were separated by an octadecyl silica bonded column and determined by the tandem mass spectrometry. The results showed that the linearity of 14 sulfonamides was good in the range of 0 . 1–10 μg/kg ( r≥0 . 9995 ) . The LOD of the method was 0. 05 μg/kg, while the LOQ was 0. 1 μg/kg. The recoveries of the 14 sulfonamides were in the range of 60 %-90 %, while the inter-batch and intra-batch RSDs were all lower than 10%. The method was proved to be more convenient, economical and stable than the traditional SPE column method.

Analysis of alkaloids in Nelumbo nucifera leaves by solid phase extraction-rapid resolution liquid chromatography-quadrupole-time of flight mass spectrometry / 中草药

Objective: A novel method for the analysis of alkaloids in Nelumbo nucifera leaves was established by SPE-RRLC-Q-TOF mass spectrometry. Methods: The crude sample was extracted by 1% HCl with ultrasound-assisted extraction, then purified by SPE column, and eluted with ammonia-methanol (5:95). After concentration, the residue was dissolved by methanol solution. The real sample was analyzed by RRLC-Q-TOF. A Welch Materials C18 column was applied in the RRLC separation using acetonitrile and water as mobile phase. The elutes were detected by Q-TOF to obtain the MS spectra with extract molecular weights under positive ion mode. Results: Nine alkaloids were identified. Conclusion: This method can be used to rapidly determine the alkaloids of N. nucifera leaves.