Optimization and verification of size exclusion chromatography-high performance liquid chromatography for determination of recombinant human growth hormone-Fc immunofusion protein polymer / 中国生物制品学杂志

@#Objective To optimize and verify the size exclusion chromatography-high performance liquid chromatography(SEC-HPLC) method for the determination of recombinant human growth hormone(rhGH)-Fc immunofusion protein polymer.Methods The multimer content of rhGH-Fc immunofusion protein was detected by SEC-HPLC.The detection conditions(salt concentration,mobile phase pH,flow rate,column temperature and column model) were optimized to observe the separation effect of the target proteins and polymers.The system suitability,specificity,linearity and range,precision,accuracy and limit of quantification of the method were verified.Results The optimized method was to use TSK-gel G2000SW_(x1)column(5 μm,7.8 mm × 300 mm),mobile phase of 50 mmol/L phosphate buffer(pH 6.80),detection wavelength of280 nm,injection volume of 100 μL,flow rate of 0.6 mL/min and column temperature of 45 ℃.The resolution of rhGHFc immunofusion protein and polymer,the theoretical plate number and the tailing factor all met the requirements;the peak time of rhGH-Fc immunofusion protein was the same as that of the control,while the peak time of GH national standard was different from that of the control,and the protein buffer showed no peak;the concentration of rhGH-Fc immunofusion protein was in the range of 0.307～1.842 mg/mL with good linear correlation between the peak area integral value and the injection volume(R~2=0.999 4);the RSD of peak area and purity in repeatability verification were 0.7% and 0.1%,respectively;the RSD of intermediate precision verification was 0.8%;the average recovery rate of accuracy verification was 99.1% with the RSD of 1.9%;the limit of quantification was 6 μg/mL.Conclusion The optimized SEC-HPLC method was used to detect the content of polymer in rhGH-Fc immunofusion protein with improved accuracy,and the column efficiency and separation were in accordance with the relevant requirements of Chinese Pharmacopoeia(Volume Ⅳ,2020edition),which could be used for the detection of polymer content in samples.

Quantification of complete viral particles in inactivated avian influenza virus antigen by high performance size exclusion chromatography coupled with multi-angle laser light scattering / 生物工程学报

We developed a method for accurate quantification of the intact virus particles in inactivated avian influenza virus feedstocks. To address the problem of impurities interference in the detection of inactivated avian influenza virus feedstocks by direct high performance size exclusion chromatography (HPSEC), we firstly investigated polyethylene glycol (PEG) precipitation and ion exchange chromatography (IEC) for H5N8 antigen purification. Under the optimized conditions, the removal rate of impurity was 86.87% in IEC using DEAE FF, and the viral hemagglutination recovery was 100%. HPSEC was used to analyze the pretreated samples. The peak of 8.5-10.0 min, which was the characteristic adsorption of intact virus, was analyzed by SDS-PAGE and dynamic light scattering. It was almost free of impurities and the particle size was uniform with an average particle size of 127.7 nm. After adding antibody to the IEC pretreated samples for HPSEC detection, the characteristic peak disappeared, indicating that IEC pretreatment effectively removed the impurities. By coupling HPSEC with multi-angle laser scattering technique (MALLS), the amount of intact virus particles in the sample could be accurately quantified with a good linear relationship between the number of virus particles and the chromatographic peak area (R2=0.997). The established IEC pretreatment-HPSEC-MALLS assay was applied to accurate detection of the number of intact virus particles in viral feedstocks of different subtypes (H7N9), different batches and different concentrations, all with good applicability and reproducibility, Relative standard deviation < 5%, n=3.

Mechanism of Astragali Radix Vesicle-like Nanoparticles for Reducing Blood Glucose in db/db Diabetic Mice by Regulating Gut Microbiota / 中国实验方剂学杂志

Objective:To study the extraction method and characteristics of vesicle-like nanoparticles （VLNs） in Astragali Radix decoction， and to explore the mechanism of the VLNs in reducing blood glucose by regulating the gut microbiota of db/db diabetic mice. Method:Ultracentrifugation and size exclusion chromatography were used to enrich VLNs from Astragali Radix decoction， and the morphology， particle size and concentration of the VLNs were analyzed by transmission electron microscope and nanoparticle tracking analyzer. The db/db diabetic mice were randomly divided into model group， Astragali Radix VLNs high-， medium- and low-dose （21.1， 10.6， 5.3 g·kg^-1） groups and metformin group （0.25 g·kg^-1） according to their blood glucose levels. There were 7 mice in each group， and another 7 C57BL/6 mice were set as the normal group. The mice were given intragastrically for 3 weeks （once a day）， and the changes of fasting blood glucose were observed every week. Hematoxylin-eosin （HE） staining was used to observe the pathological morphology of liver and pancreas of diabetic mice. The feces of mice were collected for 16S rRNA diversity detection of intestinal microbes. Result:The size of the nanoparticles obtained by the two methods was about 200 nm. Astragali Radix VLNs extracted by ultracentrifugation had a typical saucer-like shape with the concentration of 3.0×10¹¹ particles·mL^-1. The morphology of Astragali Radix VLNs obtained by size exclusion chromatography was relatively poor with the concentration of 2.2×10¹¹ particles·mL^-1. After 3 weeks of administration， compared with the model group， Astragali Radix VLNs high-， medium- and low-dose groups could significantly reduce the fasting blood glucose of diabetic mice （<italic>P</italic><0.05， <italic>P</italic><0.01）. The VLNs could improve the gut microbiota dysbiosis， significantly decrease the ratio of Firmicutes/Bacteroidota， and increase the relative abundance of beneficial bacteria and reduce the relative abundance of harmful bacteria. Conclusion:Astragali Radix VLNs may reduce the blood glucose of db/db diabetic mice by adjusting the ratio of Firmicutes/Bacteroidota in the intestinal flora.

Quality Evaluation of Tremella fuciformis Polysaccharides from Different Sources / 中国实验方剂学杂志

Objective:To establish a method for the determination of polysaccharide and monosaccharide composition of Tremella fuciformis, and to analyze the difference of polysaccharide content in T. fuciformis from different sources and cultivation methods, so as to provide reference for the quality determination.Method:High performance size exclusion chromatography coupled with multi-angle laser light scattering and refractive index detection (HPSEC-MALLS-RID) was employed to determine the content and relative molecular weight distribution of T. fuciformis polysaccharides. The monosaccharide types and proportions of T. fuciformis polysaccharides were analyzed by 1-phenyl-3-methyl-5-pyrazolone (PMP) precolumn derivative high performance liquid chromatography (HPLC).Result:The weight-average relative molecular weight (Mw) and the content of polysaccharides in T. fuciformis cultivated by cut-log from different sources were distributed in 2.618×106-3.503×106 Da and 307.12-609.06 g·kg-1, respectively. These two parameters of polysaccharides in T. fuciformis with substitute cultivation from different sources were 2.723×106-3.886×106 Da and 366.38-647.37 g·kg-1, respectively. The T. fuciformis polysaccharides mainly consisted of mannose, glucuronic acid, glucose, galactose, xylose and fucose, their ratios in samples with cut-log and substitute cultivation were 4.4∶0.7∶1.0∶0.2∶1.4∶1.6 and 4.4∶0.8∶1.0∶0.1∶1.5∶1.5, respectively. The contents of the above six monosaccharides in 39 batches of T. fuciformis from different sources were mannose of 36.71-191.31 g·kg-1, glucose of 10.46-76.10 g·kg-1, galactose of 1.00-6.72 g·kg-1, xylose of 16.73-70.54 g·kg-1, glucuronic acid of 9.74-32.12 g·kg-1, fucose of 17.16-68.20 g·kg-1.Conclusion:The content of polysaccharides in T. fuciformis from different sources has a certain difference, the developed method can be used as a routine method for the quality evaluation of polysaccharides in T. fuciformis.

Analysis of polymer impurities in ceftazidime raw materials and preparations / 药学学报

To establish a method for the determination of polymer impurities in ceftazidime raw materials and preparations, a ceftazidime degradation solution containing polymer impurities was prepared by forced polymerization. Polymer impurities in the degradation solution were separated and identified by high performance gel chromatography and the column switching-LC-MSn method. A new RP-HPLC method for ceftazidime polymer was established and validated with a Phenomenex Gemini-C18 column using a mobile phase gradient elution of 0.02 mol·L-1 phosphate buffer, methanol and acetonitrile. The results showed that when using this high performance gel chromatography method some small molecular weight impurities were co-eluted with the polymers, resulting in a poor specificity and poor quantitative accuracy. But when using the RP-HPLC method, four polymer impurities were detected in the 25-45 min time range with good specificity, sensitivity and robustness, including two ceftazidime dimers, trimers, and derivatives. Therefore, the described RP-HPLC method is suitable for the quality control of polymer impurities in ceftazidime, and ceftazidime degradation solution can be used as suitable solution for analysis of ceftazidime polymers.

Analysis of polymer impurities in cefixime raw materials and preparations / 药学学报

To establish a method for the determination of polymer impurities in cefixime raw materials and preparations, a cefixime degradation solution containing polymer impurities was prepared by forced polymerization. Polymer impurities in the degradation solution were separated and identified by high performance gel chromatography and the column switching-LC-MSn method. A new RP-HPLC method for cefixime polymer was established and validated with a Phenomenex Gemini-C18 column using a mobile phase gradient elution of 0.5% formic acid-water solution and 0.5% formic acid-acetonitrile solution. The results showed that when using this high performance gel chromatography method some small molecular weight impurities were co-eluted with the polymers, resulting in a poor specificity and poor quantitative accuracy. But when using the RP-HPLC method, three polymer impurities were detected with good specificity, sensitivity and robustness, including two cefixime dimers, and dehydrate dimer. Therefore, the described RP-HPLC method is suitable for the quality control of polymer impurities in cefixime, and cefixime degradation solution can be used as suitable solution for analysis of cefixime polymers.

Promotion of self-nucleic acid fragments on the assembly of foot-and-mouth disease virus-like particles / 生物工程学报

The special nucleic acid fragments, 5' untranslated region (5' UTR) and internal ribosome entry site (IRES) of foot-and-mouth disease virus (FMDV), which interact with the capsid proteins, were selected as scaffolds to investigate the assembly efficiency of foot-and-mouth disease (FMD) virus-like particles (VLPs). The assembled product was characterized by evaluation of particle size, surface potential, gel retardation assay, nuclease digestion experiments, size-exclusion chromatography, transmission electron microscopy and circular dichroism analysis. The results confirmed that the 5' UTR and IRES of FMDV co-assembled with the FMD VLPs and facilitated the assembly efficiency of FMD-VLPs. It demonstrates that the assembly efficiency of 75S particles of VLPs-5'UTR was significantly higher than those of the VLPs (P<0.001) and VLPs-IRES group (P<0.01). Comparatively the assembly efficiency of 12S particles of VLPs-IRES was significantly higher than those of the VLPs (P<0.000 1) and VLPs-5'UTR (P<0.000 1). It showed that the 5' UTR represented more effective in facilitating the assembly of VLPs. This study proposes an optimized strategy for improving the assembly efficiency of VLPs for the development of VLPs vaccine.

Inter-laboratory Comparison of UPLC Determination of Polymers in Human Albumin and Evaluation of the Method Applicability / 中国药学杂志

OBJECTIVE: To conduct an inter-laboratory comparison of UPLC method for polymer determination in human serum albumin and verify the method applicability. METHODS: National reference of human serum albumin and 20 samples of human serum albumin from domestic and foreign manufactures were distributed to six laboratories in order to carry out inter-laboratory comparison and demonstration of applicability. UPLC was used to determine the content of polymer and HPLC method was used for parallel comparison. RESULTS: There was no significant difference in the determination results between the UPLC method and current HPLC method in four laboratories (P>0.05) equipped with both HPLC and UPLC. The mean values of 21 samples measured with UPLC method by six laboratories were matched with those measured with HPLC method by four laboratories (t test P>0.05). The mean values of standard deviation (SD) and relative standard deviation (RSD) for 21 samples by UPLC method were only 0.06 and 1.02% respectively. The mean values of standard deviation (SD) and relative standard deviation (RSD) were 0.14 and 2.33% for parallel 21 samples determination by HPLC method, suggesting that the difference of UPLC test results between laboratories was smaller. CONCLUSION: The results of UPLC method are in good agreement with those of HPLC method. UPLC method is more effective and efficient, with smaller inter-laboratory difference, thus is significantly better than the HPLC method.

Vaccine pretreatment for quantification of 146S antigen in foot-and-mouth disease vaccines by high performance size exclusion chromatography / 生物工程学报

We developed a pre-treatment method to remove interfering substances during quantification of 146S antigens in foot-and-mouth disease (FMD) vaccines by high performance size exclusion chromatography (HPSEC). Three methods, including ultracentrifugation, PEG precipitation and nuclease digestion, were optimized and compared for removal efficiency of the interfering impurities in FMD vaccines. Under optimized conditions, the 146S contents in two batches of FMD vaccines were determined to be 7.1 and 7.6 μg/mL by ultracentrifugation, 9.7 and 10.4 μg/mL by PEG precipitation, and 10.5 and 10.4 μg/mL by nuclease digestion. The optimal condition for nuclease digestion using Benzonase determined by response surface method was as follows: appending Benzonase into 200 μL of antigen phase to a final concentration of 421 U/mL and incubating at 25.1 °C for 1.29 h. This method has advantages including efficient removal of the interfering impurities, fast processing speed, and mild operating conditions. Then 12 bathes of FMD vaccines with different serotypes produced by 4 manufacturers were tested to verify the established treatment method. Results showed the method was applicable to various FMD vaccines with good reproducibility (RSD<5.3%, n=3). The developed method removed interference from impurities during quantification of 146S, and therefore would broaden the application of HPSEC in vaccine quality control and ensure the accuracy and reliability.

Establishment of a Method for Determination of Molecular-Size Distribution of Human Serum Albumin by Ultra Performance Liquid Chromatography / 中国药学杂志

OBJECTIVE: To establish a determination method of molecular-size distribution of human serum albumin (HSA) by ultra performance liquid chromatography (UPLC). METHODS: An UPLC method was developed to specifically determine the polymers and other components in HSA on UPLC PROTEIN BEH SEC analysis column with Waters Alliance UPLC system and Waters UPLC TUV detector. The separation was performed using a mobile phase consisting of PBS at a flow rate of 0.6 mL•min-1 and the UV detection wavelength was set at 280 nm. HSA samples were diluted to different concentrations (0.5-20 mg•mL-1) to confirm the optimal concentration range of the injection. The change of component percentage and the linear relationship between HSA concentration and chromatographic peak height were confirmed and the molecular-size distribution was calculated by area normalization method. Within the optimum injection concentration range, the national control sample for HSA was diluted to 12 mg•mL-1 and tested by UPLC method and the methodology was confirmed. Twenty batches of HSA samples were determined by both UPLC and existing HPLC methods, and the samples were determined in parallel. The consistency of the methods was compared and the method was reconfirmed. RESULTS: The UPLC retention time of HSA polymer was 1.469 min, of dimer was 1.972 min, and of monomer was 2.267 min, respectively. The resolution of dimer and monomer was 2.20 and the USP tailing of monomer peak was 1.18 respectively. In the range of the injection concentrations, 0.5-20 mg•mL-1, there was linear relationship between the concentrations of the components of 11 HAS samples, including polymer, dimer and monomer peak, and the peak area%, peak height, peak area, and the squares of linear correlation coefficient were all greater than 0.997 0. The components peak area percentage of HSA samples remained relatively stable within the concentration range of 10-16 mg•mL-1 (total injection amount of 100-160 μg). The RSDs of the percentage of polymers were 0.00% (n=3, 10 mg•mL-1), 0.10% (n=3, 12 mg•mL-1), and 0.10% (n=3, 16 mg•mL-1), respectively. The UPLC method was used to determine the national control sample for human albumin of 12 mg•mL-1, and the mean value of peak area% was 5.62% (n=10). The results were consistent with those of the parallel determination by HPLC (5.58%), both of which were in accordance with the quality control range of the national standard for human albumin. The RSD of the percentage of the peak area of the polymers in national standard for human albumin by UPLC was 0.40% (n=10). The HPLC and UPLC methods were used to determine the polymer peak area percentage of 20 batches of HSA samples from 7 domestic and foreign enterprises at the concentration of 12 mg•mL-1. The correlation coefficient of the two methods was 0.996 0 (P> 0.05) and there was no significant difference between the two methods (P>0.05). CONCLUSION: Compared with the traditional HPLC method, the detection time of HSA SEC by the proposed UPLC method is shortened by at least 10 times, and the accuracy and repeatability of the determination are satisfactory. UPLC method can save much more analysis time, is simple and much faster, and can be used for high-throughput determination of molecular-size distribution of human serum albumin.

Determination of Polymerized in Cefotaxime Sodium and Cefotaxime Sodium for Injection by HPSEC / 中国药学杂志

OBJECTIVE: To establish a high performance size exclusion chromatography(HPSEC) method for the separation and analysis of polymers in cefotaxime sodium and cefotaxime sodium for injection, and determine the structures of the impurities by LCMS. METHODS: HPSEC was performed by using Sepax SRT SEC-150(7.8 mm × 300 mm, 5 μm) column. The mobile phase was 0.1 mol•L-1 disodium hydrogen phosphate and 0.1 mol•L-1 phosphate buffer solution. The flow rate was 0.8 mL•min-1, the detection wavelength was set at 235 nm, the injection volume was 10 μL, and the column temperature was maintained at 35 ℃. The concentration of polymers was quantified by external standard method. The LC-MS/MSn system conditions were as following: the mobile phase was 20 mmol•L-1 amonium acetate, the flow rate was 0.8 mL•min-1, ESI source with positive and negative ion scan was utilized, the scanning range was m/z 200 - 1 600, and the post-column diversion ratio was 1:4. RESULTS: Eight impurity peaks were obtained in total; the resolutions were all greater than 1. 5. The linear range of cefotaxime was 1 - 100 μg•mL-1 (r = 1.000 0). The RSD repeatability was 1.2%(n = 6). The limit of detection was 0.2 μg and the limit of quantitation was 0.4 μg. Three polymers were identified by LC-MS. CONCLUSION: The HPSEC method can be used for the quantitative and qualitative analyses of individual polymer impurities. It is also sensitive for the control of polymers in cefotaxime.

Using size-exclusion chromatography to quantify the 146S antigen in inactivated foot-and-mouth disease vaccine / 生物工程学报

The aim of this study is to quantify the 146S antigen in foot-and-mouth disease virus (FMDV) inactivated vaccine by size-exclusion chromatography (SEC). The analysis was performed on a TSKgel G4000SWXL column (7.8 mm×30 cm), with a pH 7.2 buffer salt system as the mobile phase. The flow rate was 0.6 mL/min, the injection volume was 100 μL and the detection wavelength was 259 nm. The calibration curve was established by using purified inactivated FMDV (serotype O) 146S antigen; 3 batches of vaccine formulated by inactivated antigen solution were tested to verify the accuracy, reproducibility, specificity and tolerability of the method. At last 16 batches of vaccine were determined by the SEC method. Results showed a good linearity between peak area and concentration of 146S antigen in the range between 0.56 and 67.42 μg/mL (R2=0.996, n=10), and the average recovery rate of 146S antigen in the 3 batches of vaccine formulated in lab were 93.6% (RSD=2.7%, n=3), 102.3% (RSD=2.6%, n=3), and 95.5% (RSD=5.1%, n=3). The method was proved accurate and reliable with good reproducibility (RSD=0.5%, n=6), and applied to determine 16 batches of the commercial FMDV vaccine. According to the above results, the SEC method is high effective for 146S antigen quantify in the inactivated FMDV vaccine and would provide strong support for the vaccine quality control.

Proteolytic activity of recombinant DegP from Chromohalobacter salexigens BKL5

Background: DegP is a serine protease that specifically cleaves and refolds unfolding proteins in the periplasmic space of the cells. To date, there is no information regarding DegP from halophilic bacteria. Chromohalobacter salexigens BKL5 is a moderately halophilic bacterium that has the ability to grow in a media containing more than 15% salt. Therefore, the objectives of this work were to clone and overexpress DegP-encoding gene from C. salexigens BKL5 and characterize its biochemical properties. Results: DegP-encoding gene was overexpressed in Escherichia coli BL21(DE3) CodonPlus in an active form. SDS-PAGE analysis showed that the molecular weight of the recombinant DegP was 45 kDa. Size-exclusion chromatography analysis suggested that recombinant DegP was present in two multimeric states, hexameric and dodecameric, with molecular weights of 297.9 and 579.12 kDa, respectively. Both conformations were enzymatically active when casein was used as substrate for enzymatic assay. Circular dichroism analysis showed that recombinant DegP was composed of 0.21­0.29 helical content, which was comparable to the helical content in the crystal structure of E. coli DegP. The basic/acidic residue ratio of recombinant DegP was 0.56, which was slightly higher than that of DegP from extreme halophiles (average, 0.45) but significantly lower than that of DegP from nonhalophiles (average, 0.94). Conclusions: Recombinant DegP from C. salexigens BKL5 showed proteolytic activity when ß-casein was used as a substrate. In silico analysis indicated that recombinant DegP had characteristics similar to those of halophilic proteins depending on its amino acid composition.

Measurement of total phenolic content and antioxidant activity of aerial parts of medicinal plant Coronopus didymus

Objective To evaluate the total phenolic content and compare the antioxidant activity of various solvent extracts and fractions from the aerial parts of Coronopus didymus through various assays. Methods Total phenolic content was determined using the Folin-Ciocalteu assay and the in vitro antioxidant activity of a number of different extracts was investigated in a dose-dependent manner with three different methods: the 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and ferric reducing antioxidant power (FRAP) assays. A flavone was isolated from the most active ethanolic extract with high antioxidant activity using size exclusion chromatography. IC

Measurement of total phenolic content and antioxidant activity of aerial parts of medicinal plant Coronopus didymus

OBJECTIVE@#To evaluate the total phenolic content and compare the antioxidant activity of various solvent extracts and fractions from the aerial parts of Coronopus didymus through various assays.@*METHODS@#Total phenolic content was determined using the Folin-Ciocalteu assay and the in vitro antioxidant activity of a number of different extracts was investigated in a dose-dependent manner with three different methods: the 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and ferric reducing antioxidant power (FRAP) assays. A flavone was isolated from the most active ethanolic extract with high antioxidant activity using size exclusion chromatography. IC values were calculated for the DPPH and ABTS methods. The FRAP activity was assessed in terms of μM Fe (II) equivalent.@*RESULTS@#The phenolic content was found to be highest in the ethanol extract (CDA Et; 47.8 mM GAE) and the lowest in the dichloromethane extract (CDA DCM; 3.13 mM GAE). The ethanol extract showed high radical scavenging activity towards DPPH and ABTS radicals with IC values of (7.80 × 10) and (4.32 × 10) μg/mL, respectively. The most active ethanol extract had a FRAP value of 1921.7 μM Fe (II) equivalent. The isolated flavone F10C (5,7,4'-trihydroxy-3'-methoxy flavone) was far more effective for scavenging free radicals in the DPPH and ABTS assays with IC of 43.8 and 0.08 μg/mL, than the standard trolox, with IC values of 97.5 and 21.1 μg/mL, respectively. In addition, the flavone F10C and the standard ascorbic acid had FRAP values of 1621.7 and 16 038.0 μM Fe (II) equivalents, respectively.@*CONCLUSIONS@#The total phenolic content of extracts in decreasing order is ethanol extract (CDA Et) > acetone extract (CDA ACE) > phenolic extract (CDA MW) > n-hexane extract (CDA nHX)> chloroform extract (CDA CHL) > dichloromethane extract (CDA DCM). The ordering of extracts in terms of antioxidant activity from highest to lowest is CDA Et > CDA MW > CDA DCM > CDA CHL > CDA ACE > CDA nHX in DPPH, ABTS and FRAP assays. A significant relationship is found between antioxidant potential and total phenolic content, suggesting that phenolic compounds are the major contributors to the antioxidant activity of C. didymus.

Content Determination of Polymer in Acitve Pharmacentical lngredient Cefatrizine Propylene Glycol by High Performance Size Exclusion Chromatography / 中国药房

OBJECTIVE:To establish a method for the content determination of polymer in cefatrizine propylene glycol. METHODS:High performance sephadex gel chromatography was performed on the column of Sephadex G-10 with mobile phase A of 0.01 mol/L phosphate buffer [0.01 mol/L Disodium hydrogen phosphate solution-0.01 mol/L Sodium dihydrogen phosphate solution (61∶39,V/V)](pH7.0)and mobile phase B of water at a flow rate of 1.0 ml/min,the detection wavelength was 254 nm,column temperature was 30℃,and volume injection was 200μl. RESULTS:The linear range of polymer was 2.07-103.30 mg/ml(r=0.999 4);the limit of quantitation of 10.4 ng,limit of detection was 4.1 ng;RSDs of precision and reprodicibility tests were lower than 3%. CONCLUSIONS:The method is specific with high sensitivity and good reproducibility,and can be used for the content determination of polymer in active pharmacentical ingredient cefatrizine propylene glycol.

Preparation of anticoagulant peptide from casein and study on thrombolysis and anticoagulation in vitro / 中草药

Objective: To study the preparation and separation method for anticoagulant peptide and the effect of anticoagulation and thrombolysis in vitro. Methods: The casein was hydrolyzed to prepare anticoagulant peptide using the mixed four enzymes such as papain, pineapple proteinase, neutral protease, and alkali protease. The anticoagulant peptide was extracted using immobilized thrombin. The effect of haemolysis and anticoagulation in vitro was investigated through the New Zealand rabbits experiments. Results: The conditions of preparation anticoagulant peptide were as follows: quality of casein was 15%, papain proteinase, pineapple proteinase, neutral protease, and alcalase dosage were 1500, 2400, 1000, and 1250 U/(g casein), respectively, temperature was 50℃, pH value was 7.0, and hydrolysis time was 4 h. The conditions for the extraction of anticoagulant peptide were as follows: the initial concentration was 6 ATU (Anti Thrombin Unit)/mL, temperature was 30℃, pH value was 5.0, and time was 30 min. Anti-extraction temperature was 30℃, pH value was 7.6, and time was 40 min. The purified anticoagulant peptide was analyzed via high performance size exclusion chromatography. The molecular weight of purified anticoagulant peptide was equal to N-Hippuryl-His-Leu hydrate and the main components were three peptides. The time of anticoagulation was more than 72 h and the time of hemolysis was 24 h in vitro. Conclusion: The main components of anticoagulant peptides are three peptides. The effect of hemolysis and anticoagulation in vitro is good.

Determination of high molecular weight substance in ginkgo diterpene lactone extraction by HPSEC-RI / 中草药

Objective: To establish a high performance size exclusion chromatography (HPSEC) method for the determination of high molecular weight substances in ginkgo diterpene lactone extraction. Methods: The chromatographic column Phenomenex Biosep-SEC-S2000 (300 mm × 7.8 mm, 5 μm) was used with 0.05 mol/L sodium sulfate as the mobile phase at a flow rate of 0.7 mL/min, with refractive index detector (RID), and the temperature of the column was 30℃. High molecular weight substances in samples were calculated with area normalization method. Results: In the range of 2 500-84 400 of substance molecular weights, a linearity was achieved (r = 0.998 3). The average recovery rate was 96.8%, and RSD was 1.5%. The high molecular weight substances in five batches of ginkgo diterpene lactone extraction were not detected. Conclusion: This method is simple, quick, accurate, and suitable for the quality control of high molecular weight substances in ginkgo diterpene lactone extraction.

Research on the determination of molecular weight distribution and the peptide content of Ganlong capsule / 中国生化药物杂志

Objective To establish the molecular weight distribution of Ganlong capsule by HPSEC the content of the peptide determined by Lowry and Methods The superdex peptide 10/300 GL (10 mm ×300 mm) column was used.The pH=6.0 and phosphate buffer of 0.05 mol/L was used as the mobile phase, containing 0.1 mol/L NaCl.The flow rate was set at 0.7 mL/min;The column temperature was 25℃;The detection wavelength was 214 nm.Results The content of the peptide ranged from 0.08 mg to 0.4 mg ( r =0.9996 ) .The RSDs of measurement precision of molecular weight and content were 0.08% and 0%(n=6), respectively.The RSDs of the repeatability were 1.3% and 1.1%(n=6);The regression equation of standard material was logMr =5.1455 -0.0871tR, r =0.9983,the relative molecular weight ranged from 2.68 ×102 Da ~5.73 ×103 Da(r =0.9983). Conclusion on The method is simple and rapid for determining the peptide content and the molecular weight distribution of Ganlong capsule.It can be used quality control method for Ganlong capsule.

Determination of Impurities Including Polymers in Latamoxef Sodium by HPSEC / 中国药师

Objective:To establish a high performance size-exclusion chromatography ( HPSEC) method for the determination of impurities including polymers in latamoxef sodium. Methods:The analysis was performed on a Zenix SEC-150 column(7. 8 mm × 300 mm, 3 μm)with the mobile phase of 0. 005 mol·L-1 phosphate buffer solution [0. 005 mol·L-1 disodium hydrogen phosphate-0. 005 mol·L-1 sodium dihydrogen phosphate (61∶39), pH 7. 0] at a flow rate of 0. 8 ml·min-1. The detection wavelength was set at 254 nm. The column tempretrue was 25℃ and the injection volume was 10μl. Results:The impurities including polymers in latamoxef so-dium were completely separated from latamoxef. The linear range of latamoxef was 0. 98-97. 73 μg·ml-1(r=0. 999 9). The limit of quantitation of latamoxef was 2. 9 ng, and the detection limit was 1. 0 ng. The linear range of the total impurities was 0. 45-2. 8 mg· ml-1(r=0. 999 5). Conclusion: The established method is accurate, rapid and reproducible, and suitable for the determination of impurities including polymers in latamoxef sodium.