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Aim To establish a batch of endotoxin standard for baeterial endotoxin detection of insoluble samples.Methods Candidate A and candidate B were prepared by freeze -drying bacterial endotoxin without excipient.The two batches of candidates were calibrated by three methods, including 13 laboratories for gel method, 9 laboratories for kinetic-turbidimetric assay and 5 laboratories for kinetic chromogenic assay.Results After statistical analysis, the geometric mean values of gel method, kinetic-turbidimetric assay and kinetic chromogenic assay calibration of candidate A were 680.1 EU, 827.0 EU and 800.8 EU, with RSD of 22.4%, 16.2% and 16.7%, respectively.The P value of variance analysis of calibration results of the three methods was 0.067, showing no significant difference.The weighted mean of potency was 774.0 EU (95% confidence interval 721.0 - 831.0, FL% 7.10).The geometric mean values of the calibration of candidate B by gel method, kinetic-turbidimetric assay and kinetic chromogenic assay method were 1 640.6 EU, 1 828.6 EU and 3 224.8 EU, with RSD of 33.9% , 47.0% and 54.4% , respectively.The P val¬ue of variance analysis of the calibration results of the three methods was 0.030, showing significant differ¬ence.Chi-square test was used to correct the weight of each method , and weighted average of the results of the three methods was used to obtain a corrected weighted average efficiency value of 1 822.7 EU (95% confi¬dence interval 1 548.7 -2 145.2, FL% 16.4).Can¬didate B was eliminated based on the results.Conclu¬sion Candidate A has become the first batch of na¬tional standard bacterial endotoxin (for insoluble sam¬ples only) approved by National Standard Substance Committee of China, and the potency is 700 EU.
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Objective: In order to evaluate the reliability and feasibility of pueraria reference extractive substance (RES) used in biological sample, the pharmacokinetics of 3′‑hydroxy puerarin (3′-HP), puerarin, 3′‑methoxy puerarin (3′-MP), and daidzein-8-C-apiosyl-(1-6)-glucoside (DAG) in beagle plasma following oral administration of Yufeng Ningxin Tablet were quantitated. Methods: A reliable and sensitive high-performance liquid chromatography-tandem triple quadrupole mass spectrometry (HPLC-QQQ-MS/MS) method developed with chromatographic separation was operated on a Merck C18 column, and acetonitrile-5 mmol/L ammonium was used as mobile phase in gradient elution. The plasma samples were deproteinized by acetone, detected by triple quadrupole mass spectrometry with an electrospray ionization interface, and quantified using selected ion monitoring mode. The pharmacokinetic parameters were calculated by Winnonlin 4.1. Results: The calibration curves of the reference extractive substance and standard substance methods were linear over the ranges 0.0417–11.3309 µg/mL and 0.0394–10.0000 µg/mL. The intra-day and inter-day precision of the two methods at three concentrations were less than 13.63%, and the average recoveries of 3′-HP, puerarin, 3′-MP, and DAG were more than 70.67%. The RSD of the mean plasma concentrations of the analytes calculated by the two methods was less than 5%, and cos (ϑ) = =1.000. Among the analytes, puerarin showed the highest blood concentration [(940 ± 185) ng/mL] and the longest retention time [(5 ± 1) h] in the dog's bodies. Conclusion: Pueraria reference extractive substance can be seen as an alternative to the standard substance to overcome the scarcity of standard substance for the analysis of biological samples.
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Objective To establish a real‐time quantitative PCR method for the detection of cytokine receptor‐like factor 2 (CRLF2) expression .Methods Specific primers amplification target gene CRLF2 and housekeeping genes ABL were designed ,the purified PCR products were performed the TA cloning .After bacterial colony PCR screening and sequencing ,then the recombinant plasmids DNA was extracted and measured by using UV spectrophotometer and converted to copies/mL concentration .Finally it was diluted for preparing the plasmid standard substance ,then the standard curve was drawn for observing the sensitivity and linear rang ,meanwhile the stability of the plasmid DNA was evaluated .This method was initially applied to detect the CRLF2 level of bone marrow mononuclear cells in 10 cases of healthy children and 10 cases of newly diagnosed acute lymphoblastic leukemia (ALL) .Results CRLF2 PCR product had a single specific melting curve;the linear detection range of the standard substance was 103 - 108 copies /ml;the plasmid standard substance by freeze‐thawing for 3 times remained stable;the CRLF2 level of clinical sample was within the linear detection range of standard substance .Conclusion The real‐time quantitative PCR method for CRLF2 established by our laboratory has good specificity ,linearity range and stability ,which can be applied to the quantitative detection of CRLF2 gene in clinical ALL children .
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OBJECTIVE:To establish the quality standard of Belamcandin standard substance.METHODS:Different indices of standard Belamcandin were examined with quantitative and quantitative analyses according to the corresponding measures stipulated in the Appendix of pharmacopoeia of people's republic of China(part Ⅱ)published in 2005.RESULTS:The specific rotatory power of 3 batches of standard Belamcandin was —28?～—30?([?]25 D,c0.11,Pyridine);the E1% 1 cmwas 790～830;the melting point was 252～254 ℃ and the ash content was less than 0.5%.The content of Belamcandin in the products was above 99.5% as revealed by HPLC.CONCLUSION:All of the indices were shown to meet the standards for standard substances and the established standard can be used for the quality control of standard Belamcandin.