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Objective:To establish a method for isolation and purification of neuraminidase from influenza vaccine and to prepare reference substance for quantitative detection of neuraminidase.Methods:Functional magnetic particles with specific affinity for neuraminidase were prepared. The method for separation and purification of neuraminidase was established based on the magnetic particles. The separation and purification conditions were optimized. The purity of neuraminidase was analyzed and the specificity was verified. The enzyme activity was determined and the protein was quantified.Results:The functional magnetic particles modified with 4-aminophenanthroline were successfully prepared and the method for isolation and purification of neuraminidase based on the magnetic particles was established. The purity of neuraminidase was 98.7%. The concentrations of neuraminidase isolated and purified from the monovalent stock solution of H1N1, H3N2, B/Victoria and B/Yamagate vaccines were 71.50, 100.58, 64.11 and 37.68 μg/ml, respectively, and the enzyme activity remained.Conclusions:The method for isolation and purification of influenza virus neuraminidase was established and the corresponding reference substance was prepared.
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This study attempts to develop a reference substance for the live bacteria count of Streptococcicosis live vaccines in order to evaluate the validity of live bacterial count in inspection and testing. We prepared a batch of live Streptococcus suis reference substance for live bacterial count, tested their physical property, purity, vacuum degree, remaining moisture, and determined their homogeneity, thermal stability and transportation stability. Moreover, we organized collaborative calibration to assign count values to the reference substance and determine the shelf life of the reference substance in 12 months. The results showed that the physical property, the purity, the remaining moisture and the vacuum degree of the reference substance were all in compliance with the requirements of the Chinese Veterinary Pharmacopoeia. The homogeneity test showed that the coefficient of variation of the count of the reference substance was less than 10%, indicating a good homogeneity. Transportation stability test showed that the reference substance remained active after 72 h transportation in summer and winter with the package of styrofoam boxes and ice packs. Thermal stability test showed that the reference substance could be stored for up to 3 months at -20 °C, or up to 21 days at 4 °C. According to the collaborative calibration, the reference vaccine was assigned a count value range of (8.5-12.1)×107 CFU/ampoule. The shelf life test showed that the reference substance was stable for 12 months when stored at -70 °C. The reference substance could provide a reference for the live bacterial count of Streptococcicosis live vaccines. Moreover, it could also be used as a reference to evaluate the quality of corresponding agar media.
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Bacterial Load , Reference Standards , Vaccines, AttenuatedABSTRACT
With the in-depth study of related substances and the development of consistency evaluation of generic drugs, relative correction factors are gaining increasing attention. By analyzing the domestic and foreign literature on correction factors in recent years, this paper describes the correction factor component, the current measurement method and its application. The rules and key points of use of an impurity correction factor and its determination and application are described, and some problems in its determination and application are discussed, providing a reference and basis for the standardization of research on impurity correction factors in the future.
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OBJECTIVE: To carry out HPTLC and HPLC fingerprint analysis of 18 batches of Ganoderma samples using two kinds of reference substance of Ganoderma extract, G. lucidum Extract Reference Substance(CZERS) and G. sinense Extract Reference Substance(ZZERS). METHODS: HPTLC Fingerprint was used to analyze triterpene acids and sterols in Ganoderma with chloroform-acetonitrile-methanol-formic acid (13∶2∶0.5∶0.5, develop 3 times) and cyclohexane-ethyl acetate-methanol-formic acid (15∶5∶0.5∶0.5, develop 2 times) respectively. HPLC Fingerprint analysis was conducted using Kromasil 100-5 C18 column (4.6 mm×250 mm, 5 μm) kept at 25 ℃. Mobile phase A was acetonitrile and B was 0.02% phosphoric acid; gradient elution procedure was as follows: 0-40 min, 29%→33% A; 40-70 min, 33%→65%A; 70-105 min, 65%→100%A; 105-120 min, 100% A; flow rate was 1.0 mL•min-1. DAD detector was adopted with detection wavelength set at 244 nm. The injection volume was 10 μL. RESULTS: By using ERS and fingerprint analysis, G. lucidum, G. sessile and G. lucidum could be distinguished. The components of G. lucidum in different species and growth patterns were different. CONCLUSION: There are many varieties of G. lucidum, which can be divided into wild and artificial cultures, and the culture media of artificial culture are different, which leads to the difference of individual components of different G. lucidum. Fingerprint analysis based on ERS of specific varieties are more suitable for the overall quality control of G. lucidum.
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OBJECTIVE: To investigate the hygroscopicity of vitamin B12 and the related problems of its chemical reference substance (CRS) that should be paid attention to. METHODS: UV absorption coefficient was determined at 361 nm, using water as solvent. Dynamic vapor absorption analysis (DVS) was applied to evaluate the moisture sorption trend and capacities of vitamin B12 under different humilities. RESULTS: Owing to the strong hygroscopicity of vitamin B12, deviation in the process of weighing might result in smaller UV absorption coefficient compared with the actual value, especially for dried and low moisture content raw materials in high-humidity environment. CONCLUSION: In the study of drug quality control for vitamin B12, the strong hygroscopicity of the raw material should be paid attention to, particularly in the research and application of vitamin B12 CRS.
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OBJECTIVE： To establish a UHPLC method for determination of the contents of 11-keto-β-boswellic acid(KAB) and 11-keto-β-acetyl-boswellic acid(AKBA)in Frankincense and explore the suitability and accuracy of substitute reference substance method with DRS origin software for qualitative and quantitative determination of chromatographic peaks. METHODS ：The samples were separated by UHPLC for determination of AKBA and KBA. AKBA was used as a reference to investigate the accuracy of KBA identification using DRS origin software on 19 different C18 columns. The RSDs of relative correction factors were calculated for different detection wavelengths and instruments.The relative correction factor method and the external standard method were selected for quantification and the differences were compared. RESULTS: The established method met the requirements of methodology and the average recovery was 100.21%(n=6) with RSD of 2.47%. The DRS origin software can be used to accurately determine the chromatographic peaks. The correct factor of AKBA vs. KBA was 0.936 and it was consistent under different conditions. There were no significant differences between the content calculated by the relative correction factor method and by the external standard method. CONCLUSION： This method is intelligent, feasible, reliable and economical, and can be used for the determination of frankincense content.
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OBJECTIVE: To propose a holistic strategy for quality control of Chinese patent medicines, and establish an HPLC analytical method for Tongzhi Surunjiang preparation according to the strategy. METHODS: The strategy contained three steps.The first step was multi-wavelength chromatographic detection.The second step was multivariate analysis for identification and assay. The third step was to establish substitute reference substance method.The preparations were extracted by ultrasound with methanol, chromatography was performed on ODS column with gradient elution with acetonitrile and 0.1%phosphoric acid aqueous solution.The detection wavelengths were set at 270, 350, 410 and 440 nm.The radar chart, HCA heatmap, principal component analysis and cosine similarity were used for data analysis.At last, linear calibration using two reference substances, relative retention time method and PDA spectrum method were used for peak identification, and relative correction factor method was used for quantitative analysis. RESULTS: The multi-components determination method and fingerprint analysis met the method validation requirements. Data analysis showed that there were some differences among the samples of different manufacturers. Strong characteristic peaks for classification were gallic acid, chebulinic acid, chebulea fructus, sennae folium, sennae folium and crocin.CONCLUSION: The method is specific, with low cost, and could be used to accurately control the quality of Tongzhi Surunjiang preparation.
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Objective A variety of techniques were adopted to establish a standardization method of Astragalus polysaccharides (APs) reference substance. This reference substance was used for quality control of APs for injection. Methods The total sugar content was quantified by UV spectrophotometry ration. The content of monosaccharide was determined by HPLC at detective wavelength of 250 nm after derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP). The high performance size exclusion chromatography method was used for the determination of molecular weight and distribution. TLC was applied to check the free monosaccharide. Then the glycosidic linkage position and configuration were detected by using gas chromatography tandem mass spectrometry and nuclear magnetic resonance spectroscopy (1H-NMR). A number of experimental parameters were applied to control the quality of APs reference substance. Results The APs was composed of rhamnose (Rha), galactose acid (GalA), glucose (Glc), galactose (Gal), and arabinose (Arb), and Glc was the main component and free monosaccharide were not detected in APs. The result of molecular weight test showed that there were four chromatographic peaks in APs, of which the main peak (peak 3) MW was 12 531 and the peak area ratio was 83%. The amount of sugar was 94% by UV test in which the glc was calculated as reference substance. There were eight main types of glycosidic bonds detected by GC-MS. Among them, the 1,4-glucose linkage was the main one. The glycosidic bond confirmed by NMR was α configuration. Conclusion The standardization method of APs reference substance has been established in this study. The identification method and determination of sugar amount are accurate and reliable. In the further research, APs reference substance will be used in quality control of APs products.
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Traditional Chinese medicine (TCM) reference drug is a new form of TCM standard reference substance. The purpose of this guideline is to guide the establishment of the reference drug and standardize its investigation and application in national drug standards. Definition of TCM reference drug was specified and relating guideline and technical requirement were introduced in this paper. Its application in quality control of TCM was analyzed and the developing train was proposed. There is a wide prospect for the application of reference drug in quality control of TCM. Thus it has practical significance to explore and conduct the quality evaluation system by using TCM reference drug as the reference substance.
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Using reversed-phase high performance liquid chromatography, nine ginsenosides were simultaneously separated on an UltimateC₁₈ column with high-resolution and high purity of each chromatographic peak. Adopting the QAMS quality evaluation model for traditional Chinese medicines, ginsenoside Rb₁ was used as the internal reference substance, and the relative correction factors (RCFs) and the relative retention values (RTRs) of ginsenosides Rg₁, Re, Rf, Rb₁, Rc, Rb₂, Rb₃, Rd and 20 (S)-ginsenoside Rg₃ to ginsenoside Rb₁ were calculated individually. Through a series of methodology evaluations, and positioned by the red ginseng reference chromatograph and RTVs, nine ginsenosides in red ginseng were simultaneously assayed only by quantitative determined ginsenoside Rb₁.
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This study aims to establish quality standards of Aleuritopteris Herba (AH), which could supply scientific evidence for the quality control of AH. The morphological and microscopic identification characters were reformulated. The tests of water content, total ash, acid-insoluble ash and ethanol-soluble extractives of AH were carried out according to the methods recorded in appendix of Chinese Pharmacopeia (2010 edition, volume 1). The TLC method was established by using aleuritopesis A [2,19-diol（2β,4α）-16-enekaureniod] and reference herb as references. With preparation of aleuritopesis A[2,19-diol（2β,4α）-16-enekaureniod] reference substance, the content of aleuritopesis A in AH was determined by HPLC. As a result, the macroscopic identification, microscopic features and TLC methods were specific and simple. The water content, total ash, acid-insoluble ash and ethanol-soluble extractive and the content of aleuritopesis A of all samples varied in the ranges of 8.8%-10.9%, 7.6%-11.4%, 2.5%-4.2%, 9.3%-10.2% and 0.56%-0.71%, respectively. The improved quality standard can be used to evaluate and guarantee the quality of AH comprehensively.
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Substitute reference substance method is an effective approach for quality control of multiple components in accordance with the characteristics of traditional Chinese medicines. The purpose of the guideline is to guide the establishment of substitute reference substance method, prove the conformance of the method to the requirements for testing, and standardize the study method and its application in national drug standards. The topics of the guideline include the definition and classification of substitute reference substance method, the principles and approaches of quantitative analysis, the identification and confirmation of chromatographic peaks, and technical requirements. When substitute reference substance method is used for fingerprint identification or multiple components assay in traditional Chinese medicines, the analytical method can be validated following the guideline.
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@#(±)-Praeruptorin A(PA)was used as the standard reference substance to establish a new method of substitute for reference substance. The contents of both(±)-praeruptorin B(PB)and(+)-praeruptorin E(PE)were calculated by the method. The major content of coumarins in different processing materials could be gained by determining the content of(±)-praeruptorin A, which could provide scientific evidence for the research on pharmacology discrepancy and quality standards. The method was carried out on a Welch Materials Ultimate XB-C18 column(4. 6 mm×250 mm, 5 μm), with a gradient mobile phase of methanol and water at the flow rate of 1. 0 mL/min. The column temperature was 30 °C and detection wavelength was set at 322 nm. According to this method, the relative correlation factors(f)of(±)-praeruptorin B and(+)-praeruptorin E were determined as 0. 787 2 and 0. 969 0, respectively. After determine the contents of 15 batches of materials from different sources, no significant differences between substitute method and external standard method were observed. It was a feasible and credible method to determine the content of major coumarins in Peucedani Radix with different processing materials. The results showed lower contents of coumarins in processing materials than in raw materials.
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OBJECTIVE:To establish a preparative separation method for euphol reference substance in Euphorbia pekinensis. METHODS:Parts of petroleum ether extraction from E. pekinensis ethanol extract were separated by silica gel column chromatogra-phy with petroleum ether-ethyl acetate(95∶5-70∶30,V/V)by gradient elution. The enriched fractions of euphol were collected. With the methanol repeated recrystallization,nuclear magnetic resonance spectroscopy(NMR)method,mass spectrometry(MS)meth-od and other spectroscopic methods were used to identify the chemical structures,and thin layer chromatography (TLC),UV, HPLC-UV,HPLC-MS were combined to detect the mass fraction. RESULTS:The mass fraction of euphol reference substance sepa-rated from E. pekinensis was>99%. CONCLUSIONS:The reference substance of euphol acquired by this method meet the relative requirements of the chemical reference substance in the content dertermination of TCM new drug quality standard. It provides chemi-cal reference substance for the quality control of E. pekinensis and prescription preparations containing E. pekinensis and the basic research of effective substances.
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OBJECTIVE: An international collaborative study involving fourteen laboratories has taken place, organized by the European Directorate on Quality of Medicines (EDQM) and National Institute for Biological Standards and Control(NIBSC) to provide supporting data for the establishment of replacement batches of calibration chemical reference substance (CRS) for low molecular weight heparin (LMWH). METHODS: The study was organized in two phases: A prequalification (phase 1, performed in three laboratories in 2005) followed by an international collaboration study (phase 2). Our institute (National Institutes for Food and Drug Control, NIFDC) took part in the phase 2 study started in March 2006. The molecular mass parameters were determined for seven different LMWH samples using the current CRS (CRS1) and two batches of candidate replacement material (cCRS2 and cCRS3) with a defined number average molecular mass (Mn) of 3700 determined in phase 1. RESULTS: The calculated values of cCRS2 and cCRS3 were systematically different from the values calculated using CRS1 with its assigned Mn of 3700. Using the raw data supplied by other participants, the molecular mass parameters were recalculated using cCRS2 and cCRS3 with values for Mn of 3800 and 3900. The calculated values using these Mn values agreed more closely with those calculated using CRS1, supporting the fact that the candidates, though similar in view of the production processes, differed slightly from CRS1 in terms of molecular mass distribution. CONCLUSION: The establishment of cCRS2 and cCRS3 could be recommended with an assigned Mn value of 3 800 that is consistent with both the phase 1 results and the determination result of current CRS1.
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Objective: To establish a separation method for naringin and neohesperidin reference substances from Citrus aurantium. Methods: Naringin and neohesperidin monomers in C. aurantium were isolated and purified by macroporous resin and medium-low- pressure preparative chromatography. Results: The contents of the prepared naringin and neohesperidin reached to 98.76% and 99.50%, respectively. Conclusion: This method is effective for the preparation of naringin and neohesperidin with high purity. It could be used to prepare the reference substances for the qualitative and quantitative analyses of Chinese materia medica.
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OBJECTIVE: to establish a national reference substance for chondroitin sulfate sodium. METHODS: Identification of chondroitin sulfate sodium was carried out by infrared(IR) and high performance liquid chromatography(HPLC). A high performance size exclusion chromatography in conjunction with refractive index detector and multi-angle laser light scatter(MALLS) was used to determine the molecular weight and molecular weight distribution of chondroitin sulfate sodium. The content of chondroitin sulfate sodium was calculated by mass balance method. RESULTS: The content of chondroitin sulfate sodium reference substance was 99.1%. CONCLUSION: The establishment of national reference substance of chondroitin sulfate sodium can effectively control the quality of the product. Copyright 2012 by the Chinese Pharmaceutical Association.
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Objective: To establish a separation method of tricin reference substance from Bambusa textilis. Methods: After extracted with ethyl acetate, the extract of B. textilis was isolated and purified by silica gel column chromatography and preparative HPLC. Tricins were identified by melting point, UV, and IR spectroscopy. Results: The content of tricin was over 98% by normalization method of HPLC. Conclusion: The developed method is simple with lower cost, by which tricin can be used as reference substances for the qualitative and quantitative analyses of Chinese herbal medicine.
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AIM:To prepare a compound as the chemical reference substance of Guangjinqiancao Zonghuangtong Capsule.METHODS:To apply general column chromatography combined with preparative HPLC to isolate the target compound,to use analytic HPLC to determine the purity,stability and its content in the capsule,and to employ spectroscopic analysis (UV,IR,ESI-MS,1H-NMR,13C-NMR,DEPT,1H-13CCOSY,1H-1HCOSY,1DHOHAHA.1D.NOE,HMBC) to elucidate the structure of the isolated compound.RESULTS:The obtained compound was identified as isoschaftoside with the purity of over 99%, which was stable within 3 months at ambient temperature.As for isosehaftoside solution.it was stable within 8 h at ambient temperature.Its content in the capsule was above 3.0%.CONCLUSION:Isoschaftoside is a qualified reference substance for analytic assay ofGuangjinqiancao Zonghuangtong Capsule,and can be isolated from Desmodium styracifolium(Osb.)Merr.
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Object To establish an isolation method of theanine reference substance by preparative HPLC. Methods After raw material extracted by water, the water phase was extracted by chloroform saturated with water, then concentrated on a thermostat. After centrifuged, supernatant was isolated and purified by preparative HPLC. Fraction was frozen and dried by Flexi Drier. Crude product was rinsed by methanol. The purity of product was determined by analytical HPLC. Results The purity of product is higher than 98% and theanine yield from raw material by this method exceeds 60%. Conclusion The developed method is simple, rapid, and at low production cost. The product owns the quality of reference substance.