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To establish and optimize a method for the detection of recombinant human midkine (rhMK) activity and verify its methodology, cell counting kit-8 (cck-8) method was used to measure the proliferation activity of rat knee chondrocytes. The specificity, accuracy, precision, linearity and robustness of the method were also verified in this study. The established method was proven to have good specificity because the buffer of rhMK and recombinant human interleukin-1 receptor antagonist have no obvious active effect; the recoveries of the samples with relative activities of 50%, 75%, 100%, 125%, 150% were in the range of 80.0% to 124.0% by statistical analysis, the relative standard deviations (RSD) of relative potency were all within 20%, the linear correlation coefficient, R2 ≥ 0.98, suggesting that the accuracy, precision and linearity of the method were good; the robustness correlation coefficient, R2 ≥ 0.92 and the ratio of maximum to minimum of sigmoidal dose-response were no less than 1.5, indicating that robustness of the methods was good. In conclusion, a bioactivity measurement method for rhMK was established and fully validated in this study and it provides a reliable method for the bioactivity analysis of rhMK routine samples during the development. This study was approved by the Animal Care and Use Committee of Shanghai Model Organisms Center, Inc. (approval number: 2019-0008-06).
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Objective To experimentally verify the precision and accuracy of determining tritiated water in ambient air using the desiccant adsorption sampling–high temperature negative pressure desorption of liquid water (containing HTO)–liquid Scintillation counter method, and to provide technical support for developing standard methods for monitoring tritiated water in ambient air. Methods The relative standard deviation and recovery of multi-group samples were verified by collecting, testing, and analyzing environmental samples with different activity concentrations. The uncertainty of the method was evaluated, the main uncertainty components were identified, and the reliability of measurement results was analyzed. Through experimental comparison of different methods, the differences in the test results of different methods were examined. Results The relative standard deviation of multiple samples ranged from 6.7% to 7.9%, the recovery ranged from 95.7% to 97.3%, and the uncertainty was greatly affected by the sample counting rate, with no significant difference as compared to condensation sampling method. Conclusion The precision and accuracy of this method meet the requirements of environmental authorities for monitoring tritiated water in ambient air, and it can be widely used in the monitoring of tritiated water in ambient air.
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This study constructed a LHCGR-CRE-luc-HEK293 transgenic cell line according to the activation of the cAMP signaling pathway after recombinant human chorionic gonadotropin binding to the receptor. The biological activity of recombinant human chorionic gonadotropin was assayed using a luciferase assay system. The relative potency of the samples was calculated using four-parameter model. And the method conditions were optimized to validate the specificity, relative accuracy, precision and linearity of the method. The results showed that there was a quantitative potency relationship of human chorinonic gonadotropin (hCG) in the method and it was in accordance with the four-parameter curve. After optimization, the conditions were determined as hCG dilution concentration of 2.5 μg·mL-1, dilution ratio of 1∶4, cell number of 10 000-15 000 cells/well, and induction time of 6 h. The method had good specificity, relative accuracy with relative bias ranging from -8.9% to 3.4%, linear regression equation correlation coefficient of 0.996, intermediate precision geometric coefficient of variation ranging from 3.3% to 15.0%, and linearity range of 50% to 200%. This study successfully established and validated a reporter gene method to detect hCG biological activity, which can be used for hCG biological activity assay and quality control.
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Objectives@#The aim of this study is to establish a Reversed Phase – High Performance Liquid Chromatographic (RP-HPLC) method for the quantification of Rhein from Cassia fistula L. leaves.@*Methods@#A Shimadzu system equipped with a C18 Column (150 x 4.6 mm, 5 μm) with an isocratic elution of Acetonitrile (solvent A) and 0.1% trifluoroacetic acid aqueous solution (solvent B) (Merck, 1.08178.0050) with a 55:45 ratio, respectively and a flow rate of 1.0 mL/min and sample injection of 10 μL detection was done at 230 nm. Standard solution of Rhein (Chengdu Biopurify) was prepared for method development. This study was validated using the guidelines set under “ICH Topic Q2 R2 or the Validation of Analytical Procedures”. Procedures for linearity, precision, accuracy, limit of detection, and limit of quantitation were performed.@*Results@#The retention time of Rhein standard was determined at 5.10 minutes. LOD and LOQ were determined to be 1.278 mcg/mL and 3.872 mcg/mL, respectively with good linearity (R2 ≥0.996) with a linear range of 2.5-20 ug/mL of the Rhein standard. The accuracy of the method was determined based on % recovery method and ranged from 94.75%-100.32% (intraday, n=3) with %RSD of 0.71. The intraday precision %RSD was 2.92 (n=6) while interday precision %RSD was 3.75 (n=3). The method was able to check the Rhein quantity among 10 samples of Cassia fistula L. leaves from different locations in the Philippines.@*Conclusion@#The method was found to be sensitive and accurate for the quantification of Rhein. The method was found to be useful for the quantification of the amount of Rhein and can be used as a Quality Control tool for the assessment of Cassia fistula.
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Cassia , Cromatografía Líquida de Alta PresiónRESUMEN
Abstract Introduction: Mitotane (o,p'-DD) is the drug of choice for Adrenocortical Carcinomas (ACC) and its measurement in plasma is essential to control drug administration. Objective: To develop and validate a simple, reliable and straightforward method for mitotane determination in plasma samples. Method: Drug-free plasma samples were collected in potassium-ethylenediamine tetraacetate (K-EDTA) tubes and spiked with 1.0, 2.5, 10.0, 25.0 and 50.0 µg/mL of mitotane (DDD). The p,p'-DDD was used as an Internal Standard (IS) and was added at 25.0 µg/mL concentration to all samples, standards and controls. Samples were submitted to protein precipitation with acetonitrile and then centrifuged. 50 uL of the supernatant was injected into an HPLC system coupled to a Diode Array Detector (DAD). DDD and IS were detected at 230 nm in a 12 min isocratic mode with a solvent mixture of 60 % acetonitrile and 40 % formic acid in water with 0.1 % pump mixed, at 0.6 mL/min flow rate, in a reversed-phase (C18) chromatographic column kept at 28°C. The sensitivity, selectivity, precision, presence of carry-over, recovery and matrix-effect, linearity, and method accuracy were evaluated. Results: The present study's method resulted in a symmetrical peak shape and good baseline resolution for DDD (mitotane) and 4,4'-DDD (internal standard) with retention times of 6.0 min, 6.4 mim, respectively, with resolutions higher than 1.0. Endogenous plasma compounds did not interfere with the evaluated peaks when blank plasma and spiked plasma with standards were compared. Linearity was assessed over the range of 1.00 -50.00 µg/mL for mitotane (R2 > 0.9987 and a 97.80 %-105.50 % of extraction efficiency). Analytical sensitivity was 0.98 µg/mL. Functional sensitivity (LOQ) was 1.00 µg/L, intra-assay and inter-assay coefficient of variations were less than 9.98 %, and carry-over was not observed for this method. Recovery ranged from 98.00 % to 117.00 %, linearity ranged from 95.00 % to 119.00 %, and high accuracy of 89.40 % to 105.90 % with no matrix effects or interference was observed for mitotane measurements. Patients' sample results were compared with previous measurements by the GC-MS method with a high correlation (r = 0.88 and bias = −10.20 %). Conclusion: DDD determination in plasma samples by the developed and validated method is simple, robust, efficient, and sensitive for therapeutic drug monitoring and dose management to achieve a therapeutic index of mitotane in patients with adrenocortical cancer.
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A sensitive and selective liquid chromatography-mass spectrometry (LC-MS/MS) method using multiple reaction monitoring (MRM) mode was developed and validated for trace analysis of 4-(chloromethyl)-1, 2-dimethoxybenzene (Veratryl chloride) a potential genotoxic impurity in Ivabradine hydrochloride (IVB). Chromatographic separation was performed on a Poroshell 120EC C18 (50 × 3.0 mm, 2.7 ?m) column using a mixture of 10 mM ammonium formate and acetonitrile in isocratic elution mode at a 0.25 mL/min flow rate. A simple pre-column derivatization with di-ethylamine was employed for the derivatization of the veratryl chloride. The developed LC-MS/MS method was linear and accurate in the 1.5–10.0 ppm concentration range with r2 ? 0.999 and percent recoveries greater than 90%. The developed method was precise with RSD (%) of not more than 4.5%. In-silico genotoxicity and carcinogenicity potential of veratryl chloride was assessed using ICH M7 principles found to be positive. The developed method can identify and quantify veratryl chloride in IVB, hence can be applied by quality control labs of pharmaceutical industries for trace quantification.
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The goal of this work was to explore the prospect of standardized application of an in-vitro bioactivity assay for recombinant human follicle-stimulating hormone based on a reporter gene. The relative accuracy, intermediate precision, linearity and applicable range of the method were validated according to the General Rules of Chinese Pharmacopoeia 2020 edition Volume IV (9401). Three laboratories used this method to determine the in-vitro biological activities of six batches of drug product and three batches of drug substance manufactured by two different companies. The consistency of the potency determined by three laboratories, the intra-laboratory precision and inter-laboratory precision were analyzed. The method was optimized during the collaborative validation. The results of method validation meet the requirements of the General Rules of Chinese Pharmacopoeia 2020 edition Volume IV (9401). Aiming to resolve the problems found in the collaborative validation, the medium for cell seeding, the pre-diluted buffer solution of standard and sample, and the means of removing and discarding supernatant after stimulation were optimized. After optimization, there was no significant difference in the bioactivity among the different laboratories (P > 0.05), indicating statistical equivalency. Intra-laboratory and inter-laboratory precision were good and the geometric coefficient of variation (GCV%) were both less than 15%. In conclusion, the reporter gene assay has good intra-laboratory repeatability and inter-laboratory reproducibility and is suitable for analyzing recombinant human follicle-stimulating hormone drug product and drug substance by different manufacturers. It is expected to be used as a standardized method for the determination of the in-vitro bioactivity of such products.
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In this study, we established a novel bioassay to determine the activity of polyethylene glycolated recombinant human growth hormone (PEG-rhGH) using Nb2-11 cells. We performed experimental condition optimization and methodological verification, and then detected the relative potency of PEG-rhGH products using this method. We demonstrated that the bioactivity of PEG-rhGH in promoting Nb2-11 cell proliferation displays a dose-response relationship, which conformed to the four-parameter model. Using PEG-rhGH reference as a control, we analyzed the relative potency of six batches of PEG-rhGH products, as well as linearity, regression and parallelism of the obtained curves. The relative potency of six batches of PEG-rhGH products was 95% to 105%. These results implied that the new bioassay established may be employed in quality control of PEG-rhGH products.
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Given that impurities may affect the quality and safety of drug products,impurity identification and profiling is an integral part of drug quality control and is particularly important for newly developed medications such as solriamfetol,which is used to treat excessive daytime sleepiness.Although the high-performance liquid chromatography analysis of commercial solriamfetol has revealed the presence of several impurities,their synthesis,structure elucidation,and chromatographic determination have not been reported yet.To bridge this gap,we herein identified,synthesized,and isolated eight process-related solriamfetol impurities,characterized them using spectroscopic and chromatographic tech-niques,and proposed plausible mechanisms of their formation.Moreover,we developed and validated a prompt impurity analysis method based on ultrahigh-performance liquid chromatography with UV detection,revealing that its selectivity,linearity,accuracy,precision,and quantitation limit meet the acceptance criteria of method validation stipulated by the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use.Thus,the developed method was concluded to be suitable for the routine analysis of solriamfetol substances.
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Objective To establish a high-performance liquid chromatography-tandem mass spectrometry(LC-MS/MS)method for the simultaneous determination of seven vitamin Bs(VBs)in rat plasma.Methods The plasma samples were pretreated using the protein precipitation method.The chromatographic separation was achieved using an Agilent ZORBAX SB-Aq column(2.1 mm×150 mm,3.5 μm),with a mobile phase composed of 0.01%formic acid(A)and methanol(B)in a gradient elution mode.The flow rate was set at 0.3 mL·min-1 and the column temperature was maintained at 30℃.Mass spectrometric detection was performed using positive ion mode in multiple reaction monitoring.2,4,5,6-Deuteronicotinamide was used as the internal standard.Results Methodological validation results showed that the lowest limits of quantitation(LOQs)for the seven VBs ranged from 7.5 to 300 ng/mL,while the highest LOQs ranged from 1 000 to 20 000 ng·mL-1.Within a certain concentration range,all compounds showed a good linearity(r2>0.992 2).The accuracies ranged from 86.0%to 117.6%,and both intra-and inter-batch precision were less than 20%.Additionally,all analytes demonstrated extraction recoveries greater than 86.1%.No significant matrix effects were observed,and the stability was good.The established validation method was successfully applied to the pharmacokinetics study of single intragastric administration of VB solution in SD rats.Conclusion The analytical method established in this study is simple,rapid,sensitive,and exclusive for the simultaneous determination of VB in real plasma samples.This LC-MS/MS method provides a new analytical tool for further exploring the physiological and pathological effects of VB.
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@#Objective To develop and validate the real-time fluorescent quantitative PCR(Q-PCR)method for the detection of 8 murine viruses. Methods The specificity,sensitivity and precision of the Q-PCR method were verified by four laboratories,and the virus simulated contamination test and blind sample detection were carried out simultaneously,of which the detection results were compared. The Q-PCR method was used to detect 26 batches of monoclonal antibody cell lines for SARS-CoV-2 vaccine production and 15 batches of other products of murine origin. Results The Q-PCR method used for detecting 8 kinds of murine viruses had no cross reaction with the same family and genus or other murine viruses. Except the sensitivity of laboratory 2 to ectromelia virus(EctV/Mouse Pox,MPV)was 2 × 10~2copies/μL,the sensitivity of laboratory 2 to other 7 viruses and 3 other laboratories to 8 murine viruses was 2 × 10~1copies/μL. Except the inter-assay CV of the copy number of mouse adenovirus(MAdV)detected by laboratory 3 was 37. 58%,the intra-assay and inter-assay CVs of the Ct and copy number of other 7 viruses detected by laboratory 3 and those of 8 viruses detected by other 3 laboratories were all less than 25%.The sensitivity of virus simulated contamination test met the parameter requirements. The coincidence rate of blind sample detection results by 4 laboratories was 100%. All the 26 batches of monoclonal antibody cell lines for SARS-CoV-2 vaccine production and 15 batches of other murine derived products were negative for 8 murine viruses. Conclusion Q-PCR method for murine virus has good specificity,sensitivity and precision,and can be used for the detection of murine derived biological products.
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【Objective】 To develop and verify high performance liquid chromatography (HPLC) detection for tromethamine (Tris) residues in human coagulation factor Ⅷ. 【Methods】 Alanine was used as internal standard, and AQC for pre-column derivation. Inertsil® ODS-SP was adopted, and acetic acid-sodium acetate buffer and acetonitrile were used for gradient elution at the flow rate of 1 mL/min and column temperature of 37℃. The 25 μL sample was loaded and determined by UV-detector with detection wavelength at 248 nm. This method was then verified. 【Results】 Glycine, sodium citrate and calcium chloride showed no interference with the detection of tromethamine (Tris) residues. The recovery rate of spike samples was within 90.0%~100.8%. The RSD in repeatability test were 2.5%, 0.7% and 1.2%, respectively, and in intermediate precision test 0.8%. The tromethamine (Tris) at concentrates of (0.5~4.0)μg/mL showed good linear relationship to the peak area to internal standard, of which the regression equation was Y=0.012 9X-0.000 782, R=0.999 97.The quantitative detection limit was 0.04μg/mL. 【Conclusion】 The HPLC for determination of tromethamine (Tris) residues in human coagulation factor Ⅷ was successfully developed, which showed good linearity as well as high specificity, precision and accuracy.
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In this study, butaselen-2,6-dimethyl-β-cyclodextrin inclusion complexes were prepared by saturated aqueous solution method to improve the solubility of butaselen, so as to obtain its injection solutions. The content of butaselen in the inclusions was determined by high performance liquid chromatography (HPLC), and then the preparation process was optimized by orthogonal design using the inclusion ratio as an indicator. X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR) and scanning electron microscope (SEM) were used to verify the structure of the inclusions. The effects of the inclusions on the solubility and stability of butaselen were also investigated. The results showed that the optimized preparation process with a mass ratio of 1∶340, an encapsulation time of 3 h and an encapsulation temperature of 70 ℃ resulted in an encapsulation ratio of (91.24 ± 0.42) %, and the results of XRD, FTIR and SEM demonstrated the formation of inclusion complexes. The developed HPLC method is rapid, simple, accurate, applicable, specific and reproducible for the determination of butaselen content in butaselen cyclodextrin inclusion complexes, which can lay the foundation for the development of new butaselen dosage forms and clinical applications and provide technical support.
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Aim To study the application of HL-60 monocyte activation test in the pyrogen detection of freeze-dried rabies vaccine for human use.Methods The established HL-60 mononuclear cell activation test (MAT) was transferred between laboratories and the method was verified; referring to the interference test in the photometric method of the bacterial endotoxin test method of the Chinese Pharmacopoeia, HL-60- IL- 6 MAT was used to detect the recovery and pyrogen content of 13 batches of freeze-dried rabies vaccine for human use.Results The linearity of the amount of 1L-6 secreted by HL-60 cells, which stimulated by dif¬ferent concentrations of endotoxin standards was above 0.95; the calculated minimum detection limit was not more than 0.125 EU • mL"1 ; the recovery experiment with a solution containing 0.5 and 1.0 EU • mL"1 of endotoxin was performed to cheek the accuracy of the method.HL-60-IL-6 was used to detect 13 hatches of Freeze-dried rahies vaccine for human use, and the re¬covery of endotoxin was between 50 % to 200%.It was consistent that HL-60-IL-6 with pyrogens and en¬dotoxin test for 4 batches of freeze-dried rabies vaccine for human use which pyrogens and endotoxin test failed and the 3 batches of water for injection.Conclusion The HL-60 MAT using IL-6 as a detection indicator is suitable for the detection of pyrogenic substances in freeze-dried rabies vaccine for human use.
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A contaminação microbiana pode comprometer a eficácia e a segurança dos produtos farmacêuticos. Os testes de contagem microbiana são utilizados para avaliar a qualidade microbiológica de produtos farmacêuticos não estéreis, exigidos pela maioria dos compêndios farmacopeicos. Apesar disso, raramente é considerada a avaliação da incerteza de medição para testes de contagem microbiana, o que pode levar a falsas decisões quanto à conformidade/nãoconformidade. Neste trabalho avaliamos os efeitos de matriz nos testes de contagem microbiana e sua avaliação de incerteza top-down, e avaliamos a incerteza da medição utilizando a abordagem bottom-up, além de que estimamos os riscos do consumidor ou do produtor devido à incerteza da medição. As incertezas combinada e expandida são calculadas empregando-se a abordagem topdown consideraram a exatidão (recuperação) e a precisão como os principais componentes de incerteza. O componente de incerteza da exatidão foi o mais relevante em 59% das amostras estudadas, enquanto a precisão foi a principal fonte de incerteza em apenas 41% das amostras, sendo observado que quanto maior a interferência da matriz, maior o fator de incerteza e, consequentemente, maior a assimetria para o intervalo em torno da medida. A partir da abordagem bottom-up, foram identificadas e quantificadas três principais fontes de incerteza: fator de diluição, volume plaqueado e contagem das placas. A contribuição dessas fontes de incerteza depende do valor medido da carga microbiana em produtos farmacêuticos, a contribuição do fator de diluição e das incertezas do volume plaqueado aumentam com o aumento do valor medido, enquanto a contribuição da contagem das placas diminui com o aumento do valor medido. Foi possível avaliar o risco de decisões falsas devido à incerteza de medição, por meio das estimativas dos riscos do consumidor ou do produtor. Os riscos foram avaliados utilizando-se o método Monte Carlo. Portanto, foi demonstrado a relevância da avaliação da incerteza de medição para garantir a confiabilidade dos resultados dos testes de contagem microbiana e a apoiar a tomada de decisões quando a avaliação da conformidade/não-conformidade dos produtos farmacêuticos não estéreis
Microbial contamination can compromise the efficacy and safety of pharmaceutical products. Microbial counting tests are used to assess the microbiological quality of non-sterile pharmaceutical products required by most pharmacopoeia compendiums. Despite this, measurement uncertainty assessment for microbial count tests is rarely considered, which can lead to false compliance/non-compliance decisions. In this work we evaluated the matrix effects on microbial counting tests and their top-down uncertainty assessment, and evaluated measurement uncertainty using the bottom-up approach, inaddition to estimating the consumer's or producer's risks due to measurement uncertainty. The combined and expanded uncertainties calculated using the top-down approach considered accuracy (recovery) and accuracy as the main components of uncertainty. The uncertainty component of accuracy was the most relevant in 59% of the samples studied, while accuracy was the main source of uncertainty in only 41% of the samples, being observed that the greater the interference of the matrix, the greater the uncertainty factor and, consequently, the greater the asymmetry for the interval around the measurement. From the bottom-up approach, three main sources of uncertainty were identified and quantified: dilution factor, platelet volume and plaque count. The contribution of these sources of uncertainty depends on the measured value of microbial load in pharmaceutical products, the contribution of the dilution factor and uncertainties of the plated volume increase with the increase in the measured value, while the contribution of plate counting decreases with the increase of the measured value. It was possible to assess the risk of false decisions due to measurement uncertainty by estimating consumer or producer risks. The risks were evaluated using the Monte Carlo method. Therefore, the relevance of measuring uncertainty assessment has been demonstrated to ensure the reliability of microbial count test results and to support decision-making when assessing non-sterile pharmaceutical conformity/non-compliance
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Preparaciones Farmacéuticas/análisis , Eficacia , Incertidumbre , Reproducibilidad de los Resultados , Gestión de la Calidad Total/métodos , AdaptabilidadRESUMEN
In this study,we developed a simple screening procedure for the determination of 18 anthelmintics(including benzimidazoles,macrocyclic lactones,salicylanilides,substituted phenols,tetrahydropyr-imidines,and imidazothiazoles)in five animal-derived food matrices(chicken muscle,pork,beef,milk,and egg)using liquid chromatography-tandem mass spectrometry.Analytes were extracted using acetonitrile/1％acetic acid(milk and egg)and acetonitrile/1％acetic acid with 0.5 mL of distilled water(chicken muscle,pork,and beef),and purified using saturated n-hexane/acetonitrile.A reversed-phase analytical column and a mobile phase consisting of(A)10 mM ammonium formate in distilled water and(B)methanol were used to achieve optimal chromatographic separation.Matrix-matched standard calibration curves(R2≥0.9752)were obtained for concentration equivalent to ×1/2,×1,×2,×3,×4,and ×5 fold the maximum residue limit(MRL)stipulated by the Korean Ministry of Food and Drug Safety.Recoveries of 61.2-118.4％,with relative standard deviations(RSDs)of ≤19.9％(intraday and interday),were obtained for each sample at three spiking concentrations(×1/2,×1,and ×2 the MRL values).Limits of detection,limits of quantification,and matrix effects were 0.02-5.5 μg/kg,0.06-10 μg/kg,and-98.8 to 13.9％(at 20 μg/kg),respectively.In five samples of each food matrix(chicken muscle,pork,beef,milk,and egg)purchased from large retailers in Seoul that were tested,none of the target analytes were detected.It has therefore been shown that this protocol is adaptable,accurate,and precise for the quantification of anthelmintic residues in foods of animal origin.
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The high performance liquid chromatography-fluorescence micelle assay (HPLC-FMA) method for the content determination of polysorbate 80 in monoclonal antibody drugs was validated to study its applicability and transferability between various laboratories, and the feasibility to be included in the Chinese Pharmacopoeia. Both J.T. Baker and Nanjing Well-sourced polysorbate 80 was used in the collaborative validation of polysorbate 80 content analysis in seven different laboratories. The results show that when the protein concentration was no more than 20 mg·mL-1 and the concentration of polysorbate 80 ranged from 0.05 to 0.5 mg·mL-1, the method had good specificity. The recovery rates of the spiked samples ranged from 92.20% to 117.70% for J.T.Baker and from 93.90% to 117.20% for Nanjing Well. The intra-laboratory precision (%RSD) was less than 4.30% for J.T. Baker, and less than 2.60% for Nanjing Well, while the overall precision was less than 5.45% for J.T. Baker, and less than 6.70% for Nanjing Well. The linear correlation coefficient was more than 0.98 for J.T. Baker and more than 0.99 for Nanjing Well. The results of the collaborative validation prove that the HPLC-FMA method has good accuracy, precision, linearity, and specificity, and could be used for release control analysis of polysorbate 80 content in monoclonal antibodies across different laboratories.
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Introduction@#One of the main ingredients of Anar-5 tablets is Piper longium L. Piperine alkaloids are the main active ingredients of the Piper longum and have anti-inflammatory, antioxidant and gastric protection properties.In the framework of the standardization study of Anar-5 tablets, a method was developed to determine the content of piperine in highly perpormance liquid chromatography, and then it was sought to include it in the method of analysis of Anar-5 drugs.@*Goal@#Quantitative determination of piperine in Anar-5 tablets and validate the method@*Material and Methods@#The research was conducted in the Chemistry and Chemical Technology Laboratory of the Research Center of the Institute of Traditional Medicine and Technology. And Anar-5 tablets (serial number 04012020) that are produced for experimental were used in the research. The standard substance, piperine alkaloids, was purchased from Green Chemistry.Purification of HPLC (organic solvent methanol, 99.9%, distilled water) was used. The EX 1600 HP/ PUMP high-performance liquid chromatography instrument (column Arcus EP+-C18, 5µm, 4.6x250 mm) and the organic solvent filter 0.45 μm syringe filter were used. The methodology related to this research was discussed and approved at the online meeting of the Ethics Committee of the Academy of Sciences on January 26, 2021. SPSS 16 software was used to statistically program the survey results.@*Results @#According to the above method, the retention time of the standard piperine is 10.38± 0.02 minutes, and the retention time of the piperine in Anar-5 tablets is 10.42±0.033 minutes. Relative velocity deviation RSD 1.077%, accuracy 0.65446±0.0068mg, stability 0.61298±0.013mg, capture time 10.42±0.033 minutes, relative standard deviation RSD≤2%, specificity 10.35 minutes, The equation of a line constructed with a standard curve is y=43360x-33587 and the correlation coefficient R2=0.9989. The piperine content of Anar-5 tablets was determined to be 0.61298±0.013 mg. The LOD and LOQ for piperine were in 2.268 μg/ml and 6.873 μg/ml, respectively.@*Conclusion@#The content of piperine in Anar-5 tablets can be determined by the HPLC method, and the appropriate conditions for this method have been established. The HPLC method is unique, accurate, linear, and stable, and meets ICH Q2 (R1) guideline criteria.
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Introduction@#Calycosin-7-O-β-D-glucoside is a glycosyloxyisoflavone that is calycosin substituted by a beta-D-glucopyranosyl residue at position at 7 via a glycosidic linkage. calycosin-7-O-β-D-glucoside, a calycosin derivative compound derived from Astragali Radix, has protective effect against ischemia/ reperfusion injury as well as bacterial endotoxin-induced vascular cell injury. A joint research team of the “Tsombo Pharm” Co., LTD and the Drug research Institute is conducting an experiment to produce a solution of “Astragalus mongholicus” injection prepared by Astragalus mongholicus bunge.@*Goal @#The aim of this study was to develop the validation method of Calycosin-7-O-β-D-glucoside in “Astragalus mongholicus” injection. @*Material and Methods@#As a test sample “Astragalus mongholicus” injection was produced by “Tsombo pharma” Co., LTD. The starndard Calycosin-7-O-β-D-glucoside was supplied from Xilong Scientific Co., Ltd. The reagent were high-performance liquid chromatography (HPLC) grade acetonitrile, formic acid, methanol and purified water. Shimadzu HPLC (CMB-20 A, UV detector Shimadzu SPD-20A was used as the analytical instrument and the analysis conditions were as follows Table 1.@*Results@#The calibration curves for Calycosin-7-O-β-D-glucoside were made by plotting the peak area versus the concentration for each analyte using regression analysis. Each calibration curve was obtained using six levels of concentrations in the range 12.5-100µg/ml. The linear correlation coefficient (R2) for all calibration curves was higher than 0.9981 for all analytes. The limit of detection and limit of quantitation for Calycosin-7-O-β-D-glucoside were in 10.37 µg/ml and 31.45 µg/ml. Accuracy and precision were assessed by analyzing five sets of samples, independently prepared at low (50%) middle (100%) and high (150%) concentrations. The RSD values of both repeatability and intermediate precision were below 0.68% and 0.618% the accuracy remaining between 95.55 to 101.71%. The resulting accuracy data were satisfactory for the quantitative analysis of Calycosin-7-O-β-D-glucoside in “Astragalus mongholicus” injection.@*Conclusions@#Finally, this method can be employed conveniently, reliably and successfully for the estimation of Calycosin-7-O-β-D-glucoside for routine quality contral and stability studies in “Astragalus mongholicus” injection.
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【Objective】 To develop and verify a kinetic method for the determination of prekallikrein activator (PKA) content. 【Methods】 The optimal reaction conditions were determined by comparing the factors of pH and ionic strength of different sample dilution buffers, incubation time of each procedure, and incubation temperature. The accuracy, specificity, precision, linearity, stability and durability of the method were validated. 【Results】 The sample was diluted with 0.05 mol/L Tris-HCl buffer (pH8.5, containing 0.15 mol/L NaCl) and incubated by prekallikrein (PK) at 37℃ for 20 min. After that, the substrate S-2302 was added. Within 10 min before the measurement, the absorbance change rate reached △A405/min. The validation results indicated that the linear range of the method was (0.5~4.0)IU/mL, while the recovery of calibration standard was 96.9%~103.7% with the R2 value more than 0.99. The specificity test showed that human serum albumin, excipients of intravenous human immunoglobulin (pH4), low pH and protein content had no significant effect on the detection of PKA, The recovery rates of standard sample solution in the specificity experiment were 98.0% (0.9% sodium chloride solution), 95.3% (0.46% sodium caprylate solution), 96.7% (10% maltose solution, pH4.0), 94.0%(20%BSA), and 94.0%(5%BSA, pH4.0), respectively. The accuracy and precision of the method can meet requirements in the range between 0.5 and 4.0 IU/mL. The inter-batch recovery rate of quality control samples were between 96.4%~109.5% with the coefficients of variation(CV) between 0.2%~6.9%, while the intra-batch recovery rate were between 101.5%~102.9% with the CV between 2.6%~5.9%. The linearity, accuracy and precision of the assay can meet the requirements when PK and S-2302 were placed at room temperature for less than 6 hours, with the recovery rate of quality control samples between 94.9%~109.9%. The end-point method and kinetic method were used to determine the PKA in 20 batches of human serum albumin, and the consistency showed that there was no significant difference between the two methods(P>0.05). 【Conclusion】 A kinetic method for determination of PKA content with good linearity, specificity, accuracy, precision, stability and durability has been established. Compared with the method in ChP, the new method is more convenient, accurate and rapid to determine the content of PKA in human albumin and human immunoglobulin (pH4) for intravenous injection.