FRAX486_1232030-35-1_DataSheet_MedChemExpress
国家药监局关于发布仿制药参比制剂目录（第三十五批）的通告
国家药监局关于发布仿制药参比制剂目录（第三十五
批）的通告
文章属性
•【制定机关】国家药品监督管理局
•【公布日期】2020.12.29
•【文号】国家药品监督管理局公告2020年第92号
•【施行日期】2020.12.29
•【效力等级】部门规范性文件
•【时效性】现行有效
•【主题分类】药政管理
正文
国家药品监督管理局公告
2020年第92号
国家药监局关于发布仿制药参比制剂目录（第三十五批）的
通告
经国家药品监督管理局仿制药质量和疗效一致性评价专家委员会审核确定，现发布仿制药参比制剂目录（第三十五批）。
特此通告。
附件：仿制药参比制剂目录（第三十五批）
国家药监局
2020年12月29日附件
仿制药参比制剂目录（第三十五批）。
CFDA发布的仿制药参比制剂目录汇总
Amoxicillin for Suspension/Clamoxyl Amoxicillin Capsules/Amoxil Amoxicillin Capsules/Amoxil Amoxicillin Chewable Tablets/Amoxicillin Amoxicillin Chewable Tablets/Amoxicillin Amoxicillin Chewable Tablets/Amoxicillin Amoxicillin Chewable Tablets/Amoxicillin Amoxicillin and Clavulanate Potassium for Suspension/Augmentin Amoxicillin and Clavulanate Potassium for Suspension/Augmentin
1-19 1-20 1-21 3-16
8-8 8-219 8-9 7-1 4-33 3-27 3-26 4-31 4-32 7-97 6-35 3-13 3-13 4-20 4-20 4-19 4-19 3-14 3-15 10-1 10-271 3-18 3-19 4-26
8-10 8-11 8-12 8-13 8-14
8-17
Aminobutyric Acid Tablets/GAMMALON Amlodipine Besylate and Atorvastatin Calcium Tablets/-Amlodipine Besylate and Atorvastatin Calcium Tablets/-Amlodipine Besylate and Atorvastatin Calcium Tablets/-Olanzapine Orally Disintegrating Tablets/ Zyprexa Olanzapine Orally Disintegrating Tablets/ Zyprexa Olanzapine Orally Disintegrating Tablets/ Zyprexa Olanzapine Orally Disintegrating Tablets/ Zyprexa Olanzapine Tablets/ Zyprexa Olanzapine Tablets/ Zyprexa Olanzapine Tablets/ Zyprexa Oxcarbazepine Tablets/Trileptal Oxcarbazepine Tablets/Trileptal Oxcarbazepine Tablets/Trileptal Orlistat Capsules/Alli Orlistat Capsules/Xenical
FDA批准的放射性药物都有哪些？
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marked manuscript
Quality evaluation of Flos Lonicerae through a simultaneous determination of seven saponins by HPLC with ELSDXing-Yun Chai1, Song-Lin Li2, Ping Li1*1Key Laboratory of Modern Chinese Medicines and Department of Pharmacognosy, China Pharmaceutical University, Nanjing, 210009, People’s Republic of China2Institute of Nanjing Military Command for Drug Control, Nanjing, 210002, People’s Republic of China*Corresponding author: Ping LiKey Laboratory of Modern Chinese Medicines and Department of Pharmacognosy, China Pharmaceutical University, Nanjing 210009, People’s Republic of China.E-mail address: lipingli@Tel.: +86-25-8324-2299; 8539-1244; 135********Fax: +86-25-8532-2747AbstractA new HPLC coupled with evaporative light scattering detection (ELSD) method has been developed for the simultaneous quantitative determination of seven major saponins, namely macranthoidinB (1), macranthoidin A (2), dipsacoside B (3), hederagenin-28-O-β-D-glucopyranosyl(6→1)-O-β-D- glucopyranosyl ester (4), macranthoside B (5), macranthoside A (6), and hederagenin-3-O-α-L-arabinopyranosyl(2→1)-O-α-L-rhamnopyranoside (7)in Flos Lonicerae, a commonly used traditional Chinese medicine (TCM) herb.Simultaneous separation of these seven saponins was achieved on a C18 analytical column with a mixed mobile phase consisting of acetonitrile(A)-water(B)(29:71 v/v) acidified with 0.5% acetic acid. The elution was operated from keeping 29%A for 10min, then gradually to 54%B from 10 to 25 min on linear gradient, and then keep isocratic elution with 54%B from 25 to 30min.The drift tube temperature of ELSD was set at 106℃, and with the nitrogen flow-rate of 2.6 l/min. All calibration curves showed good linear regression (r2 0.9922) within test ranges. This method showed good reproducibility for the quantification of these seven saponins in Flos Lonicerae with intra- and inter-day variations of less than 3.0% and 6.0% respectively. The validated method was successfully applied to quantify seven saponins in five sources of Flos Lonicerae, which provides a new basis of overall assessment on quality of Flos Lonicerae.Keywords: HPLC-ELSD; Flos Lonicerae; Saponins; Quantification1. IntroductionFlos Lonicerae (Jinyinhua in Chinese), the dried buds of several species of the genus Lonicera (Caprifoliaceae), is a commonly used traditional Chinese medicine (TCM) herb. It has been used for centuries in TCM practice for the treatment of sores, carbuncles, furuncles, swelling and affections caused by exopathogenic wind-heat or epidemic febrile diseases at the early stage [1]. Though four species of Lonicera are documented as the sources of Flos Lonicerae in China Pharmacopeia (2000 edition), i.e. L. japonica, L. hypoglauca,L. daystyla and L. confusa, other species such as L. similes and L. macranthoides have also been used on the same purpose in some local areas in China [2]. So it is an important issue to comprehensively evaluate the different sources of Flos Lonicerae, so as to ensure the clinical efficacy of this Chinese herbal drug.Chemical and pharmacological investigations on Flos Lonicerae resulted in discovering several kinds of bioactive components, i.e. chlorogenic acid and its analogues, flavonoids, iridoid glucosides and triterpenoid saponins [3]. Previously, chlorogenic acid has been used as the chemical marker for the quality evaluation of Flos Lonicerae,owing to its antipyretic and antibiotic property as well as its high content in the herb. But this compound is not a characteristic component of Flos Lonicerae, as it has also been used as the chemical marker for other Chinese herbal drugs such as Flos Chrysanthemi and so on[4-5]. Moreover, chlorogenic acid alone could not be responsible for the overall pharmacological activities of Flos Lonicerae[6].On the other hand, many studies revealed that triterpenoidal saponins of Flos Lonicerae possess protection effects on hepatic injury caused by Acetaminophen, Cd, and CCl4, and conspicuous depressant effects on swelling of ear croton oil [7-11]. Therefore, saponins should also be considered as one of the markers for quality control of Flos Lonicerae. Consequently, determinations of all types of components such as chlorogenic acid, flavonoids, iridoid glucosides and triterpenoidal saponins in Flos Lonicerae could be a better strategy for the comprehensive quality evaluation of Flos Lonicerae.Recently an HPLC-ELSD method has been established in our laboratory for qualitative and quantitative determination of iridoid glucosides in Flos Lonicerae [12]. But no method was reported for the determination of triterpenoidal saponins in Flos Lonicera. As a series studies on the comprehensive evaluation of Flos Lonicera, we report here, for the first time, the development of an HPLC-ELSD method for simultaneous determination of seven triterpenoidal saponins in the Chinese herbal drug Flos Lonicerae, i.e.macranthoidin B (1), macranthoidin A (2), dipsacoside B (3), hederagenin-28-O-β-D-glucopyranosyl(6→1)-O-β-D- glucopyranosyl ester (4), macranthoside B (5), macranthoside A (6), and hederagenin-3-O-α-L-arabinopyranosyl(2→1)-O-α-L-rhamnopyranoside (7) (Fig. 1).2. Experimental2.1. Samples, chemicals and reagentsFive samples of Lonicera species,L. japonica from Mi county, HeNan province (LJ1999-07), L. hypoglauca from Jiujang county, JiangXi province (LH2001-06), L. similes from Fei county, ShanDong province (LS2001-07), L. confuse from Xupu county, HuNan province (LC2001-07), and L. macranthoides from Longhu county, HuNan province (LM2000-06) respectively, were collected in China. All samples were authenticated by Dr. Ping Li, professor of department of Pharmacognosy, China Pharmaceutical University, Nanjing, China. The voucher specimens were deposited in the department of Pharmacognosy, China Pharmaceutical University, Nanjing, China. Seven saponin reference compounds: macranthoidin B (1), macranthoidin A (2), dipsacoside B (3), hederagenin-28-O-β-D-glucopyranosyl(6→1)-O-β-D- glucopyranosyl ester (4), macranthoside B (5), macranthoside A (6), and hederagenin-3-O-α-L-arabinopyranosyl(2→1)-O-α-L-rhamnopyranoside (7) were isolated previously from the dried buds of L. confusa by repeated silica gel, sephadex LH-20 and Rp-18 silica gel column chromatography, their structures were elucidated by comparison of their spectral data (UV, IR, MS, 1H- NMR and 13C-NMR) with references [13-15]. The purity of these saponins were determined to be more than 98% by normalization of the peak areas detected by HPLC with ELSD, and showed very stable in methanol solution.HPLC-grade acetonitrile from Merck (Darmstadt, Germany), the deionized water from Robust (Guangzhou, China), were purchased. The other solvents, purchased from Nanjing Chemical Factory (Nanjing, China) were of analytical grade.2.2. Apparatus and chromatographic conditionsAglient1100 series HPLC apparatus was used. Chromatography was carried out on an Aglient Zorbax SB-C18 column（250 4.6mm, 5.0µm）at a column temperature of 25℃.A Rheodyne 7125i sampling valve (Cotati, USA) equipped with a sample loop of 20µl was used for sample injection. The analog signal from Alltech ELSD 2000 (Alltech, Deerfield, IL, USA)was transmitted to a HP Chemstation for processing through an Agilent 35900E (Agilent Technologies, USA).The optimum resolution was obtained by using a linear gradient elution. The mobile phase was composed of acetonitrile(A) and water(B) which acidified with 0.5% acetic acid. The elution was operated from keeping 29%A for 10min, then gradually to 54%B from 10 to 25 min in linear gradient, and back to the isocratic elution of 54%B from 25 to 30 min.The drift tube temperature for ELSD was set at 106℃and the nitrogen flow-rate was of 2.6 l/min. The chromatographic peaks were identified by comparing their retention time with that of each reference compound tried under the same chromatographic conditions with a series of mobile phases. In addition, spiking samples with the reference compounds further confirmed the identities of the peaks.2.3. Calibration curvesMethanol stock solutions containing seven analytes were prepared and diluted to appropriate concentration for the construction of calibration curves. Six concentrationof the seven analytes’ solution were injected in triplicate, and then the calibration curves were constructed by plotting the peak areas versus the concentration of each analyte. The results were demonstrated in Table1.2.4. Limits of detection and quantificationMethanol stock solution containing seven reference compounds were diluted to a series of appropriate concentrations with methanol, and an aliquot of the diluted solutions were injected into HPLC for analysis.The limits of detection (LOD) and quantification (LOQ) under the present chromatographic conditions were determined at a signal-to-noise ratio (S/N) of 3 and 10, respectively. LOD and LOQ for each compound were shown in Table1.2.5. Precision and accuracyIntra- and inter-day variations were chosen to determine the precision of the developed assay. Approximate 2.0g of the pulverized samples of L. macranthoides were weighted, extracted and analyzed as described in 2.6 Sample preparation section. For intra-day variability test, the samples were analyzed in triplicate for three times within one day, while for inter-day variability test, the samples were examined in triplicate for consecutive three days. Variations were expressed by the relative standard deviations. The results were given in Table 2.Recovery test was used to evaluate the accuracy of this method. Accurate amounts of seven saponins were added to approximate 1.0g of L. macranthoides,and then extracted and analyzed as described in 2.6 Sample preparation section. The average recoveries were counted by the formula: recovery (%) = (amount found –original amount)/ amount spiked ×100%, and RSD (%) = (SD/mean) ×100%. The results were given in Table 3.2.6. Sample preparationSamples of Flos Lonicerae were dried at 50℃until constant weight. Approximate 2.0g of the pulverized samples, accurately weighed, was extracted with 60% ethanol in a flask for 4h. The ethanol was evaporated to dryness with a rotary evaporator. Residue was dissolved in water, followed by defatting with 60ml of petroleum ether for 2 times, and then the water solution was evaporated, residue was dissolved with methanol into a 25ml flask. One ml of the methanol solution was drawn and transferred to a 5ml flask, diluted to the mark with methanol. The resultant solution was at last filtrated through a 0.45µm syringe filter (Type Millex-HA, Millipore, USA) and 20µl of the filtrate was injected to HPLC system. The contents of the analytes were determined from the corresponding calibration curves.3. Results and discussionsThe temperature of drift tube and the gas flow-rate are two most important adjustable parameters for ELSD, they play a prominent role to an analyte response. In ourprevious work [12], the temperature of drift tube was optimized at 90°C for the determination of iridoids. As the polarity of saponins are higher than that of iridoids, more water was used in the mobile phase for the separation of saponins, therefore the temperature for saponins determination was optimized systematically from 95°C to 110°C, the flow-rate from 2.2 to 3.0 l/min. Dipsacoside B was selected as the testing saponin for optimizing ELSD conditions, as it was contained in all samples. Eventually, the drift tube temperature of 106℃and a gas flow of 2.6 l/min were optimized to detect the analytes. And these two exact experimental parameters should be strictly controlled in the analytical procedure [16].All calibration curves showed good linear regression (r2 0.9922) within test ranges. Validation studies of this method proved that this assay has good reproducibility. As shown in Table 2, the overall intra- and inter-day variations are less than 6% for all seven analytes. As demonstrated in Table 3, the developed analytical method has good accuracy with the overall recovery of high than 96% for the analytes concerned. The limit of detection (S/N=3) and the limit of quantification (S/N=10) are less than 0.26μg and 0.88μg respectively (Table1), indicating that this HPLC-ELSD method is precise, accurate and se nsitive enough for the quantitative evaluation of major non- chromaphoric saponins in Flos Lonicerae.It has been reported that there are two major types of saponins in Flos Lonicerae, i.e. saponins with hederagenin as aglycone and saponins with oleanolic acid as the aglycone [17]. But hederagenin type saponins of the herb were reported to have distinct activities of liver protection and anti-inflammatory [7-11]. So we adoptedseven hederagenin type saponins as representative markers to establish a quality control method.The newly established HPLC-ELSD method was applied to analyze seven analytes in five plant sources of Flos Lonicerae, i.e. L. japonica,L. hypoglauca,L. confusa,L. similes and L. macranthoides(Table 4). It was found that there were remarkable differences of seven saponins contents between different plant sources of Flos Lonicerae. All seven saponins analyzed could be detected in L. confusa and L. hypoglauca, while only dipsacoside B was detected in L. japonica. Among all seven saponins interested, only dipsacoside B was found in all five plant species of Flos Lonicerae analyzed, and this compound was determined as the major saponin with content of 53.7 mg/g in L. hypoglauca. On the other hand, macranthoidin B was found to be the major saponin with the content higher than 41.0mg/g in L. macranthoides,L. confusa, and L. similis, while the contents of other analytes were much lower.In our previous study [12], overall HPLC profiles of iridoid glucosides was used to qualitatively and quantitatively distinguish different origins of Flos Lonicerae. As shown in Fig.2, the chromatogram profiles of L. confusa, L. japonica and L. similes seem to be similar, resulting in the difficulty of clarifying the origins of Flos Lonicerae solely by HPLC profiles of saponins, in addition to the clear difference of the HPLC profiles of saponins from L. macranthoides and L. hypoglauca.Therefore, in addition to the conventional morphological and histological identification methods, the contents and the HPLC profiles of saponins and iridoids could also be used as accessory chemical evidence toclarify the botanical origin and comprehensive quality evaluation of Flos Lonicerae.4. ConclusionsThis is the first report on validation of an analytical method for qualification and quantification of saponins in Flos Lonicerae. This newly established HPLC-ELSD method can be used to simultaneously quantify seven saponins, i.e. macranthoidin B, macranthoidin A, dipsacoside B, hederagenin-28-O-β-D-glucopyranosyl(6→1)-O-β-D- glucopyranosyl ester, macranthoside B, macranthoside A, and hederagenin-3-O-α-L-arabinopyranosyl(2→1)-O-α-L-rhamnopyranoside in Flos Lonicerae. Together with the HPLC profiles of iridoids, the HPLC-ELSD profiles of saponins could also be used as an accessory chemical evidence to clarify the botanical origin and comprehensive quality evaluation of Flos Lonicerae.AcknowledgementsThis project is financially supported by Fund for Distinguished Chinese Young Scholars of the National Science Foundation of China (30325046) and the National High Tech Program（2003AA2Z2010）.[1]Ministry of Public Health of the People’s Republic of China, Pharmacopoeia ofthe People’s Republic of China, V ol.1, 2000, p. 177.[2]W. Shi, R.B. Shi, Y.R. Lu, Chin. Pharm. J., 34(1999) 724.[3]J.B. Xing, P. Li, D.L. Wen, Chin. Med. Mater., 26(2001) 457.[4]Y.Q. Zhang, L.C. Xu, L.P. Wang, J. Chin. Med. Mater., 21(1996) 204.[5] D. Zhang, Z.W. Li, Y. Jiang, J. Pharm. Anal., 16(1996) 83.[6]T.Z. Wang, Y.M. Li, Huaxiyaoxue Zazhi, 15(2000) 292.[7]J.ZH. Shi, G.T. Liu. Acta Pharm. Sin., 30(1995) 311.[8]Y. P. Liu, J. Liu, X.SH. Jia, et al. Acta Pharmacol. Sin., 13 (1992) 209.[9]Y. P. Liu, J. Liu, X.SH. Jia, et al. Acta Pharmacol. Sin., 13 (1992) 213.[10]J.ZH. Shi, L. Wan, X.F. Chen.ZhongYao YaoLi Yu LinChuang, 6 (1990) 33.[11]J. Liu, L. Xia, X.F. Chen. Acta Pharmacol. Sin., 9 (1988) 395[12]H.J. Li, P. Li, W.C. Ye, J. Chromatogr. A 1008(2003) 167-72.[13]Q. Mao, D. Cao, X.SH. Jia. Acta Pharm. Sin., 28(1993) 273.[14]H. Kizu, S. Hirabayashi, M. Suzuki, et al. Chem. Pharm. Bull., 33(1985) 3473.[15]S. Saito, S. Sumita, N. Tamura, et al. Chem Pharm Bull., 38(1990) 411.[16]Alltech ELSD 2000 Operating Manual, Alltech, 2001, p. 16. In Chinese.[17]J.B. Xing, P. Li, Chin. Med. Mater., 22(1999) 366.Fig. 1 Chemical structures of seven saponins from Lonicera confusa macranthoidin B (1), macranthoidin A (2), dipsacoside B (3), hederagenin-28-O-β-D-glucopyranosyl(6→1)-O-β-D- glucopyranosyl ester (4), macranthoside B (5), macranthoside A (6), and hederagenin-3-O-α-L-arabinopyranosyl(2→1)-O-α-L-rhamnopyranoside (7)Fig. 2Representative HPLC chromatograms of mixed standards and methanol extracts of Flos Lonicerae.Column: Agilent Zorbax SB-C18 column（250 4.6mm, 5.0µm）, temperature of 25℃; Detector: ELSD, drift tube temperature 106℃, nitrogen flow-rate 2.6 l/min.A: Mixed standards, B: L. confusa, C: L. japonica, D: L. macranthoides, E: L. hypoglauca, F: L. similes.Table 1 Calibration curves for seven saponinsAnalytes Calibration curve ar2Test range(μg)LOD(μg)LOQ(μg)1 y=6711.9x－377.6 0.9940 0.56–22.01 0.26 0.882 y=7812.6x－411.9 0.9922 0.54–21.63 0.26 0.843 y=6798.5x－299.0 0.9958 0.46–18.42 0.22 0.724 y=12805x－487.9 0.9961 0.38–15.66 0.10 0.345 y=4143.8x－88.62 0.9989 0.42–16.82 0.18 0.246 y=3946.8x－94.4 0.9977 0.40–16.02 0.16 0.207 y=4287.8x－95.2 0.9982 0.42–16.46 0.12 0.22a y: Peak area; x: concentration (mg/ml)Table 2 Reproducibility of the assayAnalyteIntra-day variability Inter-day variability Content (mg/g) Mean RSD (%) Content (mg/g) Mean RSD (%)1 46.1646.2846.2246.22 0.1346.2245.3647.4226.33 2.232 5.385.385.165.31 2.405.285.345.045.22 3.043 4.374.304.184.28 2.244.284.464.024.255.204 nd1)-- -- nd -- --5 1.761.801.821.79 1.701.801.681.841.77 4.706 1.281.241.221.252.451.241.341.201.26 5.727 tr2)-- -- tr -- -- 1): not detected; 2): trace. RSD (%) = (SD/Mean) ×100%Table 3 Recovery of the seven analytesAnalyteOriginal(mg) Spiked(mg)Found(mg)Recovery(%)Mean(%)RSD(%)1 23.0823.1423.1119.7122.8628.1042.7346.1351.0199.7100.699.399.8 0.722.692.672.582.082.913.164.735.515.7698.197.6100.698.8 1.632.172.152.091.732.182.623.884.404.6598.8103.297.799.9 2.94nd1)1.011.050.980.981.101.0297.0104.8104.1102.0 4.250.880.900.910.700.871.081.561.752.0197.197.7101.898.9 2.660.640.620.610.450.610.751.081.211.3397.796.796.096.8 0.97tr2)1.021.101.081.031.111.07100.9102.799.1100.9 1.81): not detected; 2): trace.a Recovery (%) = (Amount found –Original amount)/ Amount spiked ×100%, RSD (%) = (SD/Mean) ×100%Table 4 Contents of seven saponins in Lonicera spp.Content (mg/g)1 2 3 4 5 6 7 L. confusa45.65±0.32 5.13±0.08 4.45±0.11tr1) 2.04±0.04tr 1.81±0.03 L. japonica nd2)nd 3.44±0.09nd nd nd nd L. macranthoides46.22±0.06 5.31±0.13 4.28±0.10 tr 1.79±0.03 1.25±0.03 tr L. hypoglauca11.17±0.07 nq3)53.78±1.18nd 1.72±0.02 2.23±0.06 2.52±0.04 L. similes41.22±0.25 4.57±0.07 3.79±0.09nd 1.75±0.02tr nd 1): trace; 2): not detected.. 3) not quantified owing to the suspicious purity of the peak.。
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HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationFLUOXETINE HClC17H18F3NO•HClM.W. = 345.79CAS — 59333-67-4STABILITY INDICATINGA S S A Y V A L I D A T I O NMethod is suitable for:ýIn-process controlþProduct ReleaseþStability indicating analysis (Suitability - US/EU Product) CAUTIONFLUOXETINE HYDROCHLORIDE IS A HAZARDOUS CHEMICAL AND SHOULD BE HANDLED ONLY UNDER CONDITIONS SUITABLE FOR HAZARDOUS WORK.IT IS HIGHLY PRESSURE SENSITIVE AND ADEQUATE PRECAUTIONS SHOULD BE TAKEN TO AVOID ANY MECHANICAL FORCE (SUCH AS GRINDING, CRUSHING, ETC.) ON THE POWDER.ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationTABLE OF CONTENTS INTRODUCTION........................................................................................................................ PRECISION............................................................................................................................... System Repeatability ................................................................................................................ Method Repeatability................................................................................................................. Intermediate Precision .............................................................................................................. LINEARITY................................................................................................................................ RANGE...................................................................................................................................... ACCURACY............................................................................................................................... Accuracy of Standard Injections................................................................................................ Accuracy of the Drug Product.................................................................................................... VALIDATION OF FLUOXETINE HCl AT LOW CONCENTRATION........................................... Linearity at Low Concentrations................................................................................................. Accuracy of Fluoxetine HCl at Low Concentration..................................................................... System Repeatability................................................................................................................. Quantitation Limit....................................................................................................................... Detection Limit........................................................................................................................... VALIDATION FOR META-FLUOXETINE HCl (POSSIBLE IMPURITIES).................................. Meta-Fluoxetine HCl linearity at 0.05% - 1.0%........................................................................... Detection Limit for Fluoxetine HCl.............................................................................................. Quantitation Limit for Meta Fluoxetine HCl................................................................................ Accuracy for Meta-Fluoxetine HCl ............................................................................................ Method Repeatability for Meta-Fluoxetine HCl........................................................................... Intermediate Precision for Meta-Fluoxetine HCl......................................................................... SPECIFICITY - STABILITY INDICATING EVALUATION OF THE METHOD............................. FORCED DEGRADATION OF FINISHED PRODUCT AND STANDARD..................................1. Unstressed analysis...............................................................................................................2. Acid Hydrolysis stressed analysis..........................................................................................3. Base hydrolysis stressed analysis.........................................................................................4. Oxidation stressed analysis...................................................................................................5. Sunlight stressed analysis.....................................................................................................6. Heat of solution stressed analysis.........................................................................................7. Heat of powder stressed analysis.......................................................................................... System Suitability stressed analysis.......................................................................................... Placebo...................................................................................................................................... STABILITY OF STANDARD AND SAMPLE SOLUTIONS......................................................... Standard Solution...................................................................................................................... Sample Solutions....................................................................................................................... ROBUSTNESS.......................................................................................................................... Extraction................................................................................................................................... Factorial Design......................................................................................................................... CONCLUSION...........................................................................................................................ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationBACKGROUNDTherapeutically, Fluoxetine hydrochloride is a classified as a selective serotonin-reuptake inhibitor. Effectively used for the treatment of various depressions. Fluoxetine hydrochloride has been shown to have comparable efficacy to tricyclic antidepressants but with fewer anticholinergic side effects. The patent expiry becomes effective in 2001 (US). INTRODUCTIONFluoxetine capsules were prepared in two dosage strengths: 10mg and 20mg dosage strengths with the same capsule weight. The formulas are essentially similar and geometrically equivalent with the same ingredients and proportions. Minor changes in non-active proportions account for the change in active ingredient amounts from the 10 and 20 mg strength.The following validation, for the method SI-IAG-206-02 , includes assay and determination of Meta-Fluoxetine by HPLC, is based on the analytical method validation SI-IAG-209-06. Currently the method is the in-house method performed for Stability Studies. The Validation was performed on the 20mg dosage samples, IAG-21-001 and IAG-21-002.In the forced degradation studies, the two placebo samples were also used. PRECISIONSYSTEM REPEATABILITYFive replicate injections of the standard solution at the concentration of 0.4242mg/mL as described in method SI-IAG-206-02 were made and the relative standard deviation (RSD) of the peak areas was calculated.SAMPLE PEAK AREA#15390#25406#35405#45405#55406Average5402.7SD 6.1% RSD0.1ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::PRECISION - Method RepeatabilityThe full HPLC method as described in SI-IAG-206-02 was carried-out on the finished product IAG-21-001 for the 20mg dosage form. The method repeated six times and the relative standard deviation (RSD) was calculated.SAMPLENumber%ASSAYof labeled amountI 96.9II 97.8III 98.2IV 97.4V 97.7VI 98.5(%) Average97.7SD 0.6(%) RSD0.6PRECISION - Intermediate PrecisionThe full method as described in SI-IAG-206-02 was carried-out on the finished product IAG-21-001 for the 20mg dosage form. The method was repeated six times by a second analyst on a different day using a different HPLC instrument. The average assay and the relative standard deviation (RSD) were calculated.SAMPLENumber% ASSAYof labeled amountI 98.3II 96.3III 94.6IV 96.3V 97.8VI 93.3Average (%)96.1SD 2.0RSD (%)2.1The difference between the average results of method repeatability and the intermediate precision is 1.7%.HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationLINEARITYStandard solutions were prepared at 50% to 200% of the nominal concentration required by the assay procedure. Linear regression analysis demonstrated acceptability of the method for quantitative analysis over the concentration range required. Y-Intercept was found to be insignificant.RANGEDifferent concentrations of the sample (IAG-21-001) for the 20mg dosage form were prepared, covering between 50% - 200% of the nominal weight of the sample.Conc. (%)Conc. (mg/mL)Peak Area% Assayof labeled amount500.20116235096.7700.27935334099.21000.39734463296.61500.64480757797.52000.79448939497.9(%) Average97.6SD 1.0(%) RSD 1.0ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::RANGE (cont.)The results demonstrate linearity as well over the specified range.Correlation coefficient (RSQ)0.99981 Slope11808.3Y -Interceptresponse at 100%* 100 (%) 0.3%ACCURACYACCURACY OF STANDARD INJECTIONSFive (5) replicate injections of the working standard solution at concentration of 0.4242mg/mL, as described in method SI-IAG-206-02 were made.INJECTIONNO.PEAK AREA%ACCURACYI 539299.7II 540599.9III 540499.9IV 5406100.0V 5407100.0Average 5402.899.9%SD 6.10.1RSD, (%)0.10.1The percent deviation from the true value wasdetermined from the linear regression lineHPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::ACCURACY OF THE DRUG PRODUCTAdmixtures of non-actives (placebo, batch IAG-21-001 ) with Fluoxetine HCl were prepared at the same proportion as in a capsule (70%-180% of the nominal concentration).Three preparations were made for each concentration and the recovery was calculated.Conc.(%)Placebo Wt.(mg)Fluoxetine HCl Wt.(mg)Peak Area%Accuracy Average (%)70%7079.477.843465102.27079.687.873427100.77079.618.013465100.0101.0100%10079.6211.25476397.910080.8011.42491799.610079.6011.42485498.398.6130%13079.7214.90640599.413080.3114.75632899.213081.3314.766402100.399.618079.9920.10863699.318079.3820.45879499.418080.0820.32874899.599.4Placebo, Batch Lot IAG-21-001HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::VALIDATION OF FLUOXETINE HClAT LOW CONCENTRATIONLINEARITY AT LOW CONCENTRATIONSStandard solution of Fluoxetine were prepared at approximately 0.02%-1.0% of the working concentration required by the method SI-IAG-206-02. Linear regression analysis demonstrated acceptability of the method for quantitative analysis over this range.ACCURACY OF FLUOXETINE HCl AT LOW CONCENTRATIONThe peak areas of the standard solution at the working concentration were measured and the percent deviation from the true value, as determined from the linear regression was calculated.SAMPLECONC.µg/100mLAREA FOUND%ACCURACYI 470.56258499.7II 470.56359098.1III 470.561585101.3IV 470.561940100.7V 470.56252599.8VI 470.56271599.5(%) AverageSlope = 132.7395299.9SD Y-Intercept = -65.872371.1(%) RSD1.1HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationSystem RepeatabilitySix replicate injections of standard solution at 0.02% and 0.05% of working concentration as described in method SI-IAG-206-02 were made and the relative standard deviation was calculated.SAMPLE FLUOXETINE HCl AREA0.02%0.05%I10173623II11503731III10103475IV10623390V10393315VI10953235Average10623462RSD, (%) 5.0 5.4Quantitation Limit - QLThe quantitation limit ( QL) was established by determining the minimum level at which the analyte was quantified. The quantitation limit for Fluoxetine HCl is 0.02% of the working standard concentration with resulting RSD (for six injections) of 5.0%. Detection Limit - DLThe detection limit (DL) was established by determining the minimum level at which the analyte was reliably detected. The detection limit of Fluoxetine HCl is about 0.01% of the working standard concentration.ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::VALIDATION FOR META-FLUOXETINE HCl(EVALUATING POSSIBLE IMPURITIES)Meta-Fluoxetine HCl linearity at 0.05% - 1.0%Relative Response Factor (F)Relative response factor for Meta-Fluoxetine HCl was determined as slope of Fluoxetine HCl divided by the slope of Meta-Fluoxetine HCl from the linearity graphs (analysed at the same time).F =132.7395274.859534= 1.8Detection Limit (DL) for Fluoxetine HClThe detection limit (DL) was established by determining the minimum level at which the analyte was reliably detected.Detection limit for Meta Fluoxetine HCl is about 0.02%.Quantitation Limit (QL) for Meta-Fluoxetine HClThe QL is determined by the analysis of samples with known concentration of Meta-Fluoxetine HCl and by establishing the minimum level at which the Meta-Fluoxetine HCl can be quantified with acceptable accuracy and precision.Six individual preparations of standard and placebo spiked with Meta-Fluoxetine HCl solution to give solution with 0.05% of Meta Fluoxetine HCl, were injected into the HPLC and the recovery was calculated.HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::META-FLUOXETINE HCl[RECOVERY IN SPIKED SAMPLES].Approx.Conc.(%)Known Conc.(µg/100ml)Area in SpikedSampleFound Conc.(µg/100mL)Recovery (%)0.0521.783326125.735118.10.0521.783326825.821118.50.0521.783292021.55799.00.0521.783324125.490117.00.0521.783287220.96996.30.0521.783328526.030119.5(%) AVERAGE111.4SD The recovery result of 6 samples is between 80%-120%.10.7(%) RSDQL for Meta Fluoxetine HCl is 0.05%.9.6Accuracy for Meta Fluoxetine HClDetermination of Accuracy for Meta-Fluoxetine HCl impurity was assessed using triplicate samples (of the drug product) spiked with known quantities of Meta Fluoxetine HCl impurity at three concentrations levels (namely 80%, 100% and 120% of the specified limit - 0.05%).The results are within specifications:For 0.4% and 0.5% recovery of 85% -115%For 0.6% recovery of 90%-110%HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::META-FLUOXETINE HCl[RECOVERY IN SPIKED SAMPLES]Approx.Conc.(%)Known Conc.(µg/100mL)Area in spikedSample Found Conc.(µg/100mL)Recovery (%)[0.4%]0.4174.2614283182.66104.820.4174.2614606187.11107.370.4174.2614351183.59105.36[0.5%]0.5217.8317344224.85103.220.5217.8316713216.1599.230.5217.8317341224.81103.20[0.6%]0.6261.3918367238.9591.420.6261.3920606269.81103.220.6261.3920237264.73101.28RECOVERY DATA DETERMINED IN SPIKED SAMPLESHPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::REPEATABILITYMethod Repeatability - Meta Fluoxetine HClThe full method (as described in SI-IAG-206-02) was carried out on the finished drug product representing lot number IAG-21-001-(1). The HPLC method repeated serially, six times and the relative standard deviation (RSD) was calculated.IAG-21-001 20mg CAPSULES - FLUOXETINESample% Meta Fluoxetine % Meta-Fluoxetine 1 in Spiked Solution10.0260.09520.0270.08630.0320.07740.0300.07450.0240.09060.0280.063AVERAGE (%)0.0280.081SD 0.0030.012RSD, (%)10.314.51NOTE :All results are less than QL (0.05%) therefore spiked samples with 0.05% Meta Fluoxetine HCl were injected.HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::Intermediate Precision - Meta-Fluoxetine HClThe full method as described in SI-IAG-206-02 was applied on the finished product IAG-21-001-(1) .It was repeated six times, with a different analyst on a different day using a different HPLC instrument.The difference between the average results obtained by the method repeatability and the intermediate precision was less than 30.0%, (11.4% for Meta-Fluoxetine HCl as is and 28.5% for spiked solution).IAG-21-001 20mg - CAPSULES FLUOXETINESample N o:Percentage Meta-fluoxetine% Meta-fluoxetine 1 in spiked solution10.0260.06920.0270.05730.0120.06140.0210.05850.0360.05560.0270.079(%) AVERAGE0.0250.063SD 0.0080.009(%) RSD31.514.51NOTE:All results obtained were well below the QL (0.05%) thus spiked samples slightly greater than 0.05% Meta-Fluoxetine HCl were injected. The RSD at the QL of the spiked solution was 14.5%HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationSPECIFICITY - STABILITY INDICATING EVALUATIONDemonstration of the Stability Indicating parameters of the HPLC assay method [SI-IAG-206-02] for Fluoxetine 10 & 20mg capsules, a suitable photo-diode array detector was incorporated utilizing a commercial chromatography software managing system2, and applied to analyze a range of stressed samples of the finished drug product.GLOSSARY of PEAK PURITY RESULT NOTATION (as reported2):Purity Angle-is a measure of spectral non-homogeneity across a peak, i.e. the weighed average of all spectral contrast angles calculated by comparing all spectra in the integrated peak against the peak apex spectrum.Purity Threshold-is the sum of noise angle3 and solvent angle4. It is the limit of detection of shape differences between two spectra.Match Angle-is a comparison of the spectrum at the peak apex against a library spectrum.Match Threshold-is the sum of the match noise angle3 and match solvent angle4.3Noise Angle-is a measure of spectral non-homogeneity caused by system noise.4Solvent Angle-is a measure of spectral non-homogeneity caused by solvent composition.OVERVIEWT he assay of the main peak in each stressed solution is calculated according to the assay method SI-IAG-206-02, against the Standard Solution, injected on the same day.I f the Purity Angle is smaller than the Purity Threshold and the Match Angle is smaller than the Match Threshold, no significant differences between spectra can be detected. As a result no spectroscopic evidence for co-elution is evident and the peak is considered to be pure.T he stressed condition study indicated that the Fluoxetine peak is free from any appreciable degradation interference under the stressed conditions tested. Observed degradation products peaks were well separated from the main peak.1® PDA-996 Waters™ ; 2[Millennium 2010]ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationFORCED DEGRADATION OF FINISHED PRODUCT & STANDARD 1.UNSTRESSED SAMPLE1.1.Sample IAG-21-001 (2) (20mg/capsule) was prepared as stated in SI-IAG-206-02 and injected into the HPLC system. The calculated assay is 98.5%.SAMPLE - UNSTRESSEDFluoxetine:Purity Angle:0.075Match Angle:0.407Purity Threshold:0.142Match Threshold:0.4251.2.Standard solution was prepared as stated in method SI-IAG-206-02 and injected into the HPLC system. The calculated assay is 100.0%.Fluoxetine:Purity Angle:0.078Match Angle:0.379Purity Threshold:0.146Match Threshold:0.4272.ACID HYDROLYSIS2.1.Sample solution of IAG-21-001 (2) (20mg/capsule) was prepared as in method SI-IAG-206-02 : An amount equivalent to 20mg Fluoxetine was weighed into a 50mL volumetric flask. 20mL Diluent was added and the solution sonicated for 10 minutes. 1mL of conc. HCl was added to this solution The solution was allowed to stand for 18 hours, then adjusted to about pH = 5.5 with NaOH 10N, made up to volume with Diluent and injected into the HPLC system after filtration.Fluoxetine peak intensity did NOT decrease. Assay result obtained - 98.8%.SAMPLE- ACID HYDROLYSISFluoxetine peak:Purity Angle:0.055Match Angle:0.143Purity Threshold:0.096Match Threshold:0.3712.2.Standard solution was prepared as in method SI-IAG-206-02 : about 22mg Fluoxetine HCl were weighed into a 50mL volumetric flask. 20mL Diluent were added. 2mL of conc. HCl were added to this solution. The solution was allowed to stand for 18 hours, then adjusted to about pH = 5.5 with NaOH 10N, made up to volume with Diluent and injected into the HPLC system.Fluoxetine peak intensity did NOT decrease. Assay result obtained - 97.2%.ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationSTANDARD - ACID HYDROLYSISFluoxetine peak:Purity Angle:0.060Match Angle:0.060Purity Threshold:0.099Match Threshold:0.3713.BASE HYDROLYSIS3.1.Sample solution of IAG-21-001 (2) (20mg/capsule) was prepared as per method SI-IAG-206-02 : An amount equivalent to 20mg Fluoxetine was weight into a 50mL volumetric flask. 20mL Diluent was added and the solution sonicated for 10 minutes. 1mL of 5N NaOH was added to this solution. The solution was allowed to stand for 18 hours, then adjusted to about pH = 5.5 with 5N HCl, made up to volume with Diluent and injected into the HPLC system.Fluoxetine peak intensity did NOT decrease. Assay result obtained - 99.3%.SAMPLE - BASE HYDROLYSISFluoxetine peak:Purity Angle:0.063Match Angle:0.065Purity Threshold:0.099Match Threshold:0.3623.2.Standard stock solution was prepared as per method SI-IAG-206-02 : About 22mg Fluoxetine HCl was weighed into a 50mL volumetric flask. 20mL Diluent was added. 2mL of 5N NaOH was added to this solution. The solution was allowed to stand for 18 hours, then adjusted to about pH=5.5 with 5N HCl, made up to volume with Diluent and injected into the HPLC system.Fluoxetine peak intensity did NOT decrease - 99.5%.STANDARD - BASE HYDROLYSISFluoxetine peak:Purity Angle:0.081Match Angle:0.096Purity Threshold:0.103Match Threshold:0.3634.OXIDATION4.1.Sample solution of IAG-21-001 (2) (20mg/capsule) was prepared as per method SI-IAG-206-02. An equivalent to 20mg Fluoxetine was weighed into a 50mL volumetric flask. 20mL Diluent added and the solution sonicated for 10 minutes.1.0mL of 30% H2O2 was added to the solution and allowed to stand for 5 hours, then made up to volume with Diluent, filtered and injected into HPLC system.Fluoxetine peak intensity decreased to 95.2%.ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationSAMPLE - OXIDATIONFluoxetine peak:Purity Angle:0.090Match Angle:0.400Purity Threshold:0.154Match Threshold:0.4294.2.Standard solution was prepared as in method SI-IAG-206-02 : about 22mg Fluoxetine HCl were weighed into a 50mL volumetric flask and 25mL Diluent were added. 2mL of 30% H2O2 were added to this solution which was standing for 5 hours, made up to volume with Diluent and injected into the HPLC system.Fluoxetine peak intensity decreased to 95.8%.STANDARD - OXIDATIONFluoxetine peak:Purity Angle:0.083Match Angle:0.416Purity Threshold:0.153Match Threshold:0.4295.SUNLIGHT5.1.Sample solution of IAG-21-001 (2) (20mg/capsule) was prepared as in method SI-IAG-206-02 . The solution was exposed to 500w/hr. cell sunlight for 1hour. The BST was set to 35°C and the ACT was 45°C. The vials were placed in a horizontal position (4mm vials, National + Septum were used). A Dark control solution was tested. A 2%w/v quinine solution was used as the reference absorbance solution.Fluoxetine peak decreased to 91.2% and the dark control solution showed assay of 97.0%. The difference in the absorbance in the quinine solution is 0.4227AU.Additional peak was observed at RRT of 1.5 (2.7%).The total percent of Fluoxetine peak with the degradation peak is about 93.9%.SAMPLE - SUNLIGHTFluoxetine peak:Purity Angle:0.093Match Angle:0.583Purity Threshold:0.148Match Threshold:0.825 ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationSUNLIGHT (Cont.)5.2.Working standard solution was prepared as in method SI-IAG-206-02 . The solution was exposed to 500w/hr. cell sunlight for 1.5 hour. The BST was set to 35°C and the ACT was 42°C. The vials were placed in a horizontal position (4mm vials, National + Septum were used). A Dark control solution was tested. A 2%w/v quinine solution was used as the reference absorbance solution.Fluoxetine peak was decreased to 95.2% and the dark control solution showed assay of 99.5%.The difference in the absorbance in the quinine solution is 0.4227AU.Additional peak were observed at RRT of 1.5 (2.3).The total percent of Fluoxetine peak with the degradation peak is about 97.5%. STANDARD - SUNLIGHTFluoxetine peak:Purity Angle:0.067Match Angle:0.389Purity Threshold:0.134Match Threshold:0.8196.HEAT OF SOLUTION6.1.Sample solution of IAG-21-001-(2) (20 mg/capsule) was prepared as in method SI-IAG-206-02 . Equivalent to 20mg Fluoxetine was weighed into a 50mL volumetric flask. 20mL Diluent was added and the solution was sonicated for 10 minutes and made up to volume with Diluent. 4mL solution was transferred into a suitable crucible, heated at 105°C in an oven for 2 hours. The sample was cooled to ambient temperature, filtered and injected into the HPLC system.Fluoxetine peak was decreased to 93.3%.SAMPLE - HEAT OF SOLUTION [105o C]Fluoxetine peak:Purity Angle:0.062Match Angle:0.460Purity Threshold:0.131Match Threshold:0.8186.2.Standard Working Solution (WS) was prepared under method SI-IAG-206-02 . 4mL of the working solution was transferred into a suitable crucible, placed in an oven at 105°C for 2 hours, cooled to ambient temperature and injected into the HPLC system.Fluoxetine peak intensity did not decrease - 100.5%.ED. N0: 04Effective Date:APPROVED::。
QPCR及QRT-PCR系列产品
Invitrogen的ICFC系列产品促销1．QPCR及QRT-PCR系列产品Invitrogen公司专门为中国客户提供的定量PCR试剂盒，结合了 UDG 防止残余污染技术和SYBR® Green I 荧光染料（存在于SYBR® Green I荧光定量PCR试剂盒中），在美国接受了严格的质量监控，可提供极高灵敏度的目的序列定量检测，线性剂量低，反应浓度范围很大。
qPCR Supermix-- 即用型反应剂，专为高特异性、实时定量DNA扩增设计UDG-- 防止携带污染物，减少克隆片段假阳性结果ROX参考染料-- 适用ABI仪器的校正染料产品信息活动时间：即日起至2009年4月30日2．Gibco南美胎牛血清即日起凡优惠价¥1780购买Gibco胎牛血清500ml（目录号:C2027050）即可获赠送价值¥250现金抵用券。
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产品信息活动时间：即日起至2009年4月30日独特的采集方式：GIBCO采用无菌心脏穿刺的方式采血原装直送，避免污染：原产地采集、加工、检测、包装。
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产品信息活动时间：即日起至2009年5月31日附：pCR载体优点及图谱：3’-T突出端可直接连接Taq扩增的PCR产物可选择T7或T7和Sp6启动子进行体外RNA转录和测序侧向EcoRⅠ位点的通用多接头位点方便了插入片段的切离可以选择卡那霉素或氨苄青霉素进行筛选非常简便的蓝/白克隆筛选具有M13正向和反向引物位点，方便测序4．GIBCO液体培养基系列产品创立近50年的历史，品质优秀，产品种类丰富；为了中国用户利益，特建立国内生产线；所有产品，从原材料到生产全部按照GIBCO质量标准进行，每批均送抵美国公司总部质检合格后，才在国内销售。
分子对接参考文献
Synthesis and SAR of 20,30-bis-O -substituted N 6,50-bis-ureidoadenosine derivatives:Implications for prodrug delivery and mechanism of actionJadd R.Shelton a ,Christopher E.Cutler a ,Megan S.Browning a ,Jan Balzarini b ,Matt A.Peterson a ,⇑a Department of Chemistry and Biochemistry,Brigham Young University,Provo,UT 84602-5700,United States bRega Institute for Medical Research,KU Leuven,B-3000Leuven,Belgiuma r t i c l e i n f o Article history:Received 5June 2012Revised 1August 2012Accepted 13August 2012Available online 21August 2012Keywords:Purine nucleosidesBio-active adenosine derivatives Antiproliferative nucleosides BMPR1b inhibitorsa b s t r a c tA series of 20,30-bis-O -silylated or -acylated derivatives of lead compound 3a (20,30-bis-O -tert -butyldi-methylsilyl-50-deoxy-50-(N -methylcarbamoyl)amino-N 6-(N -phenylcarbamoyl)adenosine)were prepared and evaluated for antiproliferative activity against a panel of murine and human cancer cell lines (L1210,FM3A,CEM,and HeLa).20,30-O -Silyl groups investigated included triethylsilyl (10a ),tert -butyldi-phenylsilyl (10b ),and triisopropylsilyl (10c ).20,30-O -Acyl groups investigated included acetyl (13a ),ben-zoyl (13b ),isobutyryl (13c ),butanoyl (13d ),pivaloyl (13e ),hexanoyl (13f ),octanoyl (13g ),decanoyl (13h ),and hexadecanoyl (13i ).IC 50values ranged from 3.0±0.3to >200l g/mL,with no improvement relative to lead compound 3a .Derivative 10a was approximately equipotent to 3a ,while compounds 13e –g were from three to fivefold less potent,and all other compounds were significantly much less active.A desilylated derivative (50-deoxy-50-(N -methylcarbamoyl)amino-N 6-(N -phenylcarbamoyl)adeno-sine;5)and several representative derivatives from each subgroup (10a –10c ,13a –13c )were screened for binding affinity for bone morphogenetic protein receptor 1b (BMPR1b).Only compound 5showed appre-ciable affinity (K d =11.7±0.5l M),consistent with the inference that 3a may act as a prodrug depot form of the biologically active derivative 5.Docking studies (Surflex Dock,Sybyl X 1.3)for compounds 3a and 5support this conclusion.Ó2012Elsevier Ltd.All rights reserved.As part of research directed toward the design,synthesis,and biological evaluation of potential inhibitors of HIV integrase,we discovered potent antiproliferative activities associated with a new class of N 6,50-bis-ureidoadenosine derivatives exemplified by compounds 1–3(Fig.1).1IC 50values for 1–3a (R =Ph)ranged from approximately 1–8l M against a majority of the human cancer cell lines in the NCI-60.IC 50values for 3b –i ranged from 3–182l g/mL against a panel of tumor cell lines consisting of murine leukemia (L1210),murine mammary carcinoma (FM3A),human T-lympho-cyte (CEM),and human cervix carcinoma (HeLa).Preliminary SAR studies revealed that for optimal cytostatic activities (low l M),the N 6-and 50-urea moieties are required,and substitution with at least one 20(30)tert -butyldimethylsilyl (TBS)group is also neces-sary.Interestingly,compounds 5and 6were essentially inactive against the NCI-60screen at 10l M concentrations.Similarly,50-carbamates 4a –i were significantly less active than the analogous 50-ureas (3a –i )against L1210,FM3A,CEM,and HeLa—in spite of the fact that 4a –i possess nearly identical substitutions as the 50-ureas.1aThe above observations support the conclusion that the 20,30-O-TBS groups are necessary,but not sufficient,for biological activity and have prompted us to investigate the role of the 20,30-O -substi-tution in this class of compounds.Herein we report the synthesis and antiproliferative activities for a series of variously substituted 20,30-O -derivatives of the most potent of these compounds (3a ),and draw preliminary conclusions from the mechanistic implica-tions of this SAR study.The synthesis begins with 50-azido-50-deoxyadenosine (7)and gives 20,30-bis-O -silylated or 20,30-bis-O -acylated products in good to excellent yields (Scheme 1).The synthesis is very straightforward and is amenable to scale-up.Silylation of 7with triethylsilylchlo-ride,tert -butyldiphenylsilylchloride,or triisopropylsilylchloride gave compounds 8a –c in 42–60%yield.Acylation of compounds 8a –c with phenylisocyanate gave N 6-phenylurea derivatives 9a –c (54–82%).A one-pot,two-step reaction sequence involving reduc-tion of the 50-azido group of compounds 9a –c followed by acylation with the relatively safe and innocuous methylisocyanate surrogate,N-methyl p -nitrophenylcarbamate,2gave 10a –c in 66–77%yield.20,30-Bis-O -acylated compounds 13a –c and 13d –i were obtained via two different pounds 13a –c were obtained in good yields via a five-step protocol analogous to the one employed in preparing 10a –c .However,the more lipophilic 20,30-bis-O -acylated compounds 13g –i were obtained in very low yields following this procedure.An alternative route involving one step from compound 5was investigated.This route was generally much more efficient,0960-894X/$-see front matter Ó2012Elsevier Ltd.All rights reserved./10.1016/j.bmcl.2012.08.050Corresponding author.E-mail address:matt_peterson@ (M.A.Peterson).and yields for13d–i ranged from46–63%(the highest yield for13e was26%,even with this more efficient method,presumably due to the steric bulk of the pivaloyl esters).As a point of comparison,only trace amounts of13i were obtained when thefive-step sequence—steps e,f,b,c,and d—was attempted.Finally,compounds14a–c were obtained in moderate to good yields(31–66%)by treating 11a–c with the aforementioned one-pot,two-step reduction/acyla-tion(steps c and d).The antiproliferative activities for compounds 3a,4a,10a–c,13a–i,and14a–c are shown in Table1.Interestingly, the IC50values for20,30-bis-O-triethylsilyl derivative10a were very similar to those for the20,30-bis-O-TBS derivative3a.In contrast, IC50values for20,30-bis-O-tert-butyldiphenylsilyl and/or20,30-bis-O-triisopropylsilyl derivatives(10b and10c,respectively),were significantly inferior to3a.Acyl derivatives13a–i were generally much less active than3a,especially the O-benzoyl,O-decanoyl, and O-hexadecanoyl derivatives(13b,13h,and13i,respectively). The O-pivaloyl,O-hexanoyl,and O-octanoyl derivatives(13e,13f, and13g,respectively)exhibited nearly equivalent antiproliferative activities,but IC50values for these compounds were from three to fivefold higher than those for pounds14a–c (each of which lacks the N6-phenylurea)showed generally lower antiproliferative activity than their corresponding N6-substituted analogues(13a–c).Recently,we demonstrated that compound5(Fig.1)binds to the ATP-binding site of bone morphogenetic protein receptor1b (BMPR1b)with low l M affinity(K d=11.7±0.5l M).1a When screened against a panel of441protein kinases,compound5 exhibited its greatest activity against BMPR1b,inhibiting binding of BMPR1b to an ATP-binding site ligand by approx.50%at 10l M pound3a,in contrast,did not bind to BMPR1b at concentrations as high as30l M.1a BMPR1b is a trans-membrane receptor with serine/threonine protein kinase activity. The ATP-binding domain lies within the cytoplasm and phosphor-ylates downstream targets(SMADs1,5,and8),which in turn regulate expression of inhibitor of differentiation gene1(Id1).3 Overexpression of Id1has been reported in a number of cancers, including lung,4breast,5colon,6ovarian,7pancreas,8prostate,9 and renal cancers.10Downregulation,inhibition,and/or inactiva-tion of Id1have been shown to induce apoptosis in several of these cancers.11Inhibition of BMPR1b by the desilylated analogue of3a, compound5,could constitute a plausible mechanism for the broad-spectrum antiproliferative activity exhibited by compound 3a.12In this context,compound3a would most likely serve as a prodrug form of the active species,desilylated derivative com-pound5.A commonly used strategy for enhancing membrane permeabil-ity of nucleosides has been to increase the lipophilicity by protect-ing hydroxyls as acetyl,benzoyl,or isobutyryl esters that are cleaved once the compound has crossed the cell membrane.13 TBS-protection has been shown to enhance the activities of a num-ber of antiproliferative compounds,and activities of several of these compounds have been positively correlated with the increased lipophilicity of the biologically active derivative.14 TBS-protected cytidine has been shown to facilitate transport of guanosine50-monophosphate through a model membrane(in con-junction with a lipophilic phosphonium ion co-carrier),15and sily-lated nucleosides have been shown to penetrate the blood–brain barrier where it is presumed they are desilylated to generate the active species.16The lipophilic20,30-bis-O-TBS groups could en-hance membrane permeability of compound3a and serve as a pro-drug depot form of the active derivative compound5.Docking studies performed using the Surflex docking program (Sybyl X1.3)are supportive of such an interpretation.17As illus-trated in Fig.2,the highest ranked pose for compound5is oriented within the ATP binding cleft of BMPR1b(pdb3mdy)with the50-urea undergoing hydrogen bonding interactions with the highly conserved catalytic triad18(Lys231,Glu244,Asp350;Fig.2). The N6-phenyl urea moiety in this pose is oriented toward the sol-vent accessible surface,which is consistent with the relative lack of sensitivity of the antiproliferative activity of3a–i to the substitu-tion pattern in the N6-urea moiety.1a In contrast,the top ranked pose for compound3a had nearly the opposite orientation to com-pound5,with the N6-phenyl urea moiety undergoing nonpolar binding interactions with the‘gatekeeper’residue(Leu277;blue residue;Fig.2)near the end of the catalytic cleft,in close proximity to the catalytic triad.In this pose,the very hydrophobic20,30-bis-O-TBS groups are exposed to the solvent accessible surface.If such a pose were biologically relevant,substitution at the N6-urea posi-tion would be expected to have a much greater effect on the bio-logical activity than the negligible effect that was observed experimentally.(The nature of the R group in3a–i had very little impact on their antiproliferative activities).1a Furthermore,the hydrophobic effect resulting from protrusion of the very nonpolar TBS groups into the aqueous environment would contribute to an unfavorable entropic term in the overall free energy of binding.Consistent with these modeling results is the aforementioned observation that compound5binds to BMPR1b with K d=11.7±0.5l M),while compound3a did not bind at concentrations as high as30l M(Fig.3A and3B,respectively).1a The negative impact of the 20,30-O-substitution on binding was also illustrated for several rep-resentative members of the presently discussed series of20,30-O-derivatives of3a,none of which showed appreciable binding to BMPR1b in a competitive inhibition of binding experiment19at 10l M concentrations(Fig.3C).The relative reactivity of silyl pro-tecting groups toward hydrolysis(TES>TBS TIPS>TBDPS)20is in harmony with these results,and is consistent with a mechanism involving cleavage of the silyl moiety before the nucleoside deriva-tive can interact with its primary biological receptor.21 In conclusion,we have developed efficient methods for the preparation of a variety of20,30-O-substituted derivatives ofour 6068J.R.Shelton et al./Bioorg.Med.Chem.Lett.22(2012)6067–6071recently discovered antiproliferative N 6,50-bis-ureidoadenosine compounds.Bis-O -protection of 50-azido-50-deoxyadenosine with either silyl or acyl protecting groups,followed by sequential acyl-ation of the N 6and 50-amino groups (with phenylisocyanate or N-methyl p -nitrophenylcarbamate,respectively)gave 20,30-O -substi-tuted derivatives of lead compound 3a (10a –c and 13a –c )in good to excellent yields.An alternative route from the more advanced intermediate compound 5gave 13d –i more efficiently than the route applied for 13a –c .Screening of compounds 10a –c ,13a –i ,and 14a –c against a panel of murine and human cancer cell lines did not reveal any improved activity relative to lead compound 3a .Several representative 20,30-O -substituted derivatives were shown to lack binding affinity for BMPR1b at concentrations near the K d for desilylated analogue 5.Taken together,these results sug-gest that the role of the TBS group in compound 3a may be to facil-itate membrane permeability.Cleavage of the TBS groups within the cytoplasm could give rise to the active derivative (5)which previously published screening data 1a suggest may target BMPR1b as its primary biomolecular target.BMPR1b is part of the BMP-sig-naling pathway that regulates expression of Id1.Overexpression of Id1has been reported in numerous cancers.4–10Inhibition of the BMP-signaling cascade by desilylated derivative 5may account for the broad-spectrum activity of compound 3a.Table 1Inhibitory effects of the test compounds on the proliferation of murine leukemia cells (L1210),murine mammary carcinoma cells (FM3A),human T-lymphocyte cells (CEM)and human cervix carcinoma cells (HeLa)CompoundIC 50a (l g/ml)L1210FM3A CEM HeLa 3a 3.8±0.3 5.9±1.18.3±2.9 3.2±0.24a 160±56>200>200P 20010a 3.8±0.1 3.0±0.3 4.2±0.2 3.7±0.410b >200>200P 200104±7110c >200>200142±81P 20013a 97±17150±39107±8>20013b 154±3061±2>200>20013c 29±444±428±073±1313d 20±218±12958±2513e 9.7±3.515±12017±113f 9.5±0.320±110±215±513g 11±032±112±416±913h >100140±16>100>10013i >100>200>100>10014a 112±31>200>200>20014b 16±136±319±840±714c87±1107±1388±3399±14a50%Inhibitory concentration or compound concentration required to inhibit tumor cell proliferation by 50%.J.R.Shelton et al./Bioorg.Med.Chem.Lett.22(2012)6067–60716069We are currently designing50-analogues that may more fully exploit interactions with the catalytic triad(Lys231,Glu244,Asp350)and gatekeeper residues(Leu277),which may lead to en-hanced binding,as indicated by the docking study,and thus,in-creased antiproliferative activity.AcknowledgmentsGenerous support from the BYU Cancer Research Center and BYU College of Physical and Mathematical Sciences and the KU Leuven(GOA10/14)to J.B.is gratefully acknowledged.10010010010053100100Figure2.Docking results for3a and5docked into the active site of BMPR1b(pdb3mdy).Yellow residues:catalytic triad(K231,E244,D350);blue residue:gate-keeper(L277);magenta tube:G-loop or activation loop(I210,G211,K212,G213,R214,Y215,G216);magenta ribbon:hinge region(I278,T279,D280,Y281,H282,E283,N284,G285,S286).18(A)Space-filling model of highest ranked pose ofcompound5.(B)Tube model of highest ranked pose of compound5(G-Loopomitted for clarity).(C)Space-filling model of highest ranked pose of compound3aChem.Lett.22(2012)6067–6071Supplementary dataSupplementary data(experimental procedures and NMR data for all new for compounds)associated with this article can be found,in the online version,at /10.1016/j.bmcl. 2012.08.050.References and notes1.(a)Shelton,J.R.;Cutler,C.E.;Oliveira,M.;Balzarini,J.;Peterson,M.A.Bioorg.Med.Chem.2012,20,1008;(b)Peterson,M.A.;Oliveira,M.;Christiansen,M.A.;Cutler,C.E.Bioorg.Med.Chem.Lett.2009,19,6775;(c)Peterson,M.A.;Oliveira, M.;Christiansen,M. A.Nucleosides Nucleotides Nucleic2009,28,394;(d) Peterson,M.A.;Ke,P.;Shi,H.;Jones,C.;McDougal,B.R.;Robinson,W.E.Nucleosides Nucleotides Nucleic2007,26,499.2.Peterson,M.A.;Shi,H.;Ke,P.Tetrahedron Lett.2006,47,3405.3.(a)Ruzinova,M.B.;Benezra,R.Trends Cell Biol.2003,13,410;(b)Ying,Q.L.;Nichols,J.;Chambers,I.;Smith,A.Cell2003,115,281;(c)Korchynskyi,O.;ten Dijke,P.J.Biol.Chem.2002,277,4883;(d)López-Rovira,T.;Chalaux, E.;Massagúe,J.;Rosa,J.L.;Ventura,F.J.Biol.Chem.2002,277,3176.4.Cheng,Y.J.;Tsai,J.W.;Hsieh,K.C.;Yang,Y.C.;Chen,Y.J.;Huang,M.S.;Yuan,S.S.Cancer Lett.2011,307,191.5.Schoppmann,S.F.;Schindl,M.;Bayer,G.;Aumayr,K.;Dienes,J.;Horvat,R.;Rudas,M.;Gnant,M.;Jakesz,R.;Birner,P.Int.J.Cancer2003,104,677.6.Zhao,Z.R.;Zhang,Z.Y.;Zhang,H.;Jiang,L.;Wang,M.W.;Sun,X.F.Oncol.Rep.2008,19,419.7.Schindl,M.;Schoppmann,S.F.;Ströbel,T.;Heinzl,H.;Leisser,C.;Horvat,R.;Birner,P.Clin.Cancer Res.2003,9,779.8.Lee,K.T.;Lee,Y.W.;Lee,J.K.;Choi,S.H.;Rhee,J.C.;Paik,S.S.;Kong,G.Br.J.Cancer2004,90,1198.9.Ling,M.T.;Lau,T.C.;Zhou,C.;Chua,C.W.;Kwok,W.K.;Wang,Q.;Wang,X.;Wong,Y.C.Carcinogenesis2005,26,1668.10.Li,X.;Zhang,Z.;Xin,D.;Chua,C.W.;Wong,Y.C.;Leung,S.C.L.;Na,Y.;Wang,X.Histopathology2007,50,484.11.(a)Wong,Y.-C.;Wang,X.;Ling,M.-T.Apoptosis2004,9,279;(b)Ling,M.-T.;Kwok,W.K.;Fung,M.K.;Wang,X.H.;Wong,Y.C.Carcinogenesis2006,27,205;(c)Ling,Y.X.;Tao,J.;Fang,S.F.;Hui,Z.;Fang,Q.R.Eur.J.Cancer Prev.2011,20,9;(d)Mern,D.S.;Hoppe-Seyler,K.;Hoppe-Seyler,F.;Hasskarl,J.;Burwinkel,B.Breast Cancer Res.2010,124,623;(e)Mern,D.S.;Hasskarl,J.;Burwinkel,B.Br.J.Cancer2010,103,1237.12.Shelton,J.R.;Burt,S.R.;Peterson,M.A.Bioorg.Med.Chem.Lett.2011,21,1484.13.(a)Li,F.;Maag,H.;Alfredson,T.J.Pharm.Sci.2008,97,1109;(b)Mackman,R.L.;Cihlar,T.Ann.Rep.Med.Chem.2004,305.14.(a)Pungitore,C.R.;León,L.G.;García,C.;Martín,V.S.;Tonn,C.E.;Padrón,J.M.Bioorg.Med.Chem.Lett.2007,17,1332;(b)Donadel,O.J.;Martín,T.;Martín,V.S.;Villarc,J.;Padrón,J.M.Bioorg.Med.Chem.Lett.2005,15,3536;(c)Szilágyi,A.;Fenyvesi,F.;Majercsik,O.;Pelyvás,I.F.;Bácskay,I.;Fehér,P.;Váradi,J.;Vecsernyés,M.;Herczegh,P.J.Med.Chem.2006,49,5626.15.Lee,S.B.;Choo,H.;Hong,J.–I.J.Chem.Res.1998,304.16.Montana,J.G.;Bains,W.Internatl.Patent App.PCT/GB2003/005056,2003;Internatl.Pub.WO2004/050666A1.17.Surflex has been validated as a robust molecular docking method.In terms ofdocking accuracy,it performs as well as other commonly used methods;and in terms of screening utility,its performance has been shown to be superior to other methods for which comparative data are available(a)Jain, A.N.J.Comput.Aided Mol.Des.2007,21,281;(b)Jain,A.N.J.Med.Chem.2003,46,499.18.BMPR1b is a member of the TGF b super family of protein kinases.BMPR1b(also known as Alk6)has68%sequence homology with Alk5(unpublished results).Assignments for the catalytic triad,gatekeeper,G-loop,and hinge region are consistent with published assignments for Alk5and for known sequences for protein kinases in general(a)Goldberg,F.W.;Ward,R.A.;Powell,S.J.;Debreczeni,J.É.;Norman,R.A.;Roberts,N.J.;Dishington,A.P.;Gingell,H.J.;Wickson,K.F.;Roberts,A.L.J.Med.Chem.2009,52,7901;(b) Ghose,A.K.;Herbertz,T.;Pippin,D.A.;Salvino,J.M.;Mallamo,J.P.J.Med.Chem.2008,51,5149.19.Fabian,M.A.;Biggs,W.H.I.I.I.;Treiber,D.K.;Atteridge,C.E.;Azimioara,M.D.;Benedetti,M.G.;Carter,T.A.;Ciceri,P.;Edeen,P.T.;Floyd,M.;Ford,J.M.;Galvin,M.;Gerlach,J.L.;Grotzfeld,R.M.;Herrgard,S.;Insko,D.E.;Insko,M.A.;Lai,A.G.;Lélias,J.-M.;Mehta,S.A.;Milanov,Z.V.;Velasco,A.M.;Wodicka,L.M.;Patel,H.K.;Zarrinkar,P.P.;Lockhart,D.J.Nature Biotech.2005,23,329.20.Nelson,T.D.;Crouch,R.D.Synthesis1996,1031.21.The possibility exists that BMPR1b may not be the primary biomolecular targetfor this class of compounds.However,from a panel of441protein kinases, compound5bound to BMPR1b with greatest affinity(see Ref.1a).Thus, amongst this class of receptors,BMPR1b certainly shows greatest potential.Optimization of binding to BMPR1b could lead to discovery of more potent derivatives and/or discovery of additional related inhibitors.J.R.Shelton et al./Bioorg.Med.Chem.Lett.22(2012)6067–60716071。
川楝素对TRAIL抗肝癌活性及其机制研究
川楝素对TRAIL抗肝癌活性及其机制研究史杰;王芳【摘要】目的观察川楝素对肿瘤坏死因子相关凋亡诱导配体(TRAIL)抗肝癌活性影响并研究其机制.方法将HepG2人肝癌细胞分为对照组、川楝素组、TRAIL组、川楝素+TRAIL组及川楝素+TRAIL+DR5小干扰RNA (DR5 siRNA)组,CCK-8法检测HepG2细胞活力,流式细胞术检测HepG2细胞的凋亡和线粒体膜电位,Western blot实验和流式细胞术检测HepG2细胞表面DR5表达水平及Caspase-8、Caspase-3活化水平.结果 TRAIL+川楝素组HepG2相对细胞活力(0.42±0.04),显著低于TRAIL单治疗组(0.84±0.07,P＜0.05)和川楝素+TRAIL+DR5 siRNA组(0.76±0.06,P＜0.05).TRAIL+川楝素组HepG2细胞凋亡率(32.7±2.4)％,显著高于TRAIL单治疗组[(9.3±0.9)％,P＜0.05]和川楝素+TRAIL+DR5 siRNA组[(13.8±1.1)％,P＜0.05].川楝素处理对HepG2细胞Bcl-2家族蛋白的表达无明显影响,但能显著提高HepG2细胞表面DR5表达水平.TRAIL+川楝素组HepG2细胞的相对线粒体膜电位(0.26±0.02),显著低于TRAIL单治疗组(0.78±0.06,P＜0.05)和川楝素+TRAIL+DR5 siRNA组(0.68±0.05,P＜0.05).TRAIL+川楝素组HepG2细胞Caspase-8表达水平(0.37±0.04),显著高于TRAIL单治疗组(0.11±0.04,P＜0.05)及川楝素+TRAIL+DR5 siRNA组(0.14±0.02,P＜0.05);TRAIL+川楝素组HepG2细胞Caspase-3表达水平(0.42±0.04),显著高于TRAIL单治疗组(0.13±0.02,P＜0.05)及川楝素+TRAIL+DR5 siRNA组(0.17±0.02,P＜0.05).结论川楝素可上调肝癌细胞死亡受体5(DR5)表达,提高TRAIL抗肿瘤活性.【期刊名称】《浙江中西医结合杂志》【年(卷),期】2017(027)011【总页数】5页(P936-939,946)【关键词】肝细胞肝癌;川楝素;DR5;TRAIL【作者】史杰;王芳【作者单位】浙江省慈溪市人民医院检验科宁波 315300;浙江医院检验科杭州310007【正文语种】中文肝癌是预后很差的恶性肿瘤，致死率非常高[1]。
Phanta超高保真酶
1.引物3’端最后一个碱基选择C或G； 2.引物3’端最后8个碱基应避免出现连续错配； 3.引物3’端尽量避免出现发夹结构； 4.引物Tm值控制在55℃-65℃之间； 5.引物额外附加序列，即与模板非配对序列，不应参与引物Tm值计算； 6.引物GC含量控制在40%-60%之间； 7.正向引物和反向引物Tm值以及GC含量尽可能一致。
应用实例
1.反应体系配制：
所有操作请在冰上进行，各组分解冻后请充分摇匀。为了防止PhantaTM Super-Fidelity DNA Polymerase的校对活性降解引物，请将聚合酶最后加入反应体系中。各组 分使用完毕后及时放回-20℃。5 × SF Buffer请勿长时间敞口放置。
ddH2O 5 × SF Buffer (with 10 mM MgSO4) 25 mM MgSO4a dNTP Mix (10 mM each)b
a. 对于大多数PCR反应，Mg2+最佳终浓度为1.5-2 mM。体系中已含有终浓度为2 mM Mg2+，如有需要，可用25 mM MgSO4，以0.2-0.5 mM为间隔向上摸索Mg2+最佳 使用浓度。
b. 请勿使用dUTP和带有尿嘧啶的引物或模板。 c. 扩增子GC含量>60%时加入终浓度3%的DMSO有可能会有助于扩增。 d. 推荐仅当扩增子GC含量>60%且优化条件也无法正常扩增时使用；可能会降低保真度。 e. 不同模板最佳反应浓度有所不同，下表为50 μl反应体系推荐模板使用量：
DMSOc
5 × PCR Enhancerd
模板DNAe
引物1 (10 μM)
引物2 (10 μM)
PhantaTM Super-Fidelity DNA Polymerase (1 U/μl)f
GC测定盐酸普拉克索中三乙胺残留量
Ia医药生化试剂
浓度
纯度
型号
规格
500ml/瓶 5g/瓶
单位
瓶 瓶 瓶 瓶 瓶 支 支 瓶 瓶 瓶 瓶 瓶 箱 瓶 合 瓶 瓶 瓶 甁 瓶 支 支 瓶 支 瓶 瓶 瓶 支 克拉玛尔 Sigma
品牌/厂家
科伦制药有限公司
解剖
组胚
分析纯 生化试剂 生化试剂 生化试剂 生化试剂 60~80目 分析纯 分析纯 医用 进口分装 进口分装
北京义翘神舟生物
Ia101
北京义翘神舟生物
Ia102
北京义翘神舟生物
Ia103 Ia104 Ia105 Ia106 Ia107 Ia108 Ia109 Ia110 Ia111 Ia112 Ia113 Ia114 Ia115 Ia116 Ia117 Ia118 Ia119 Ia120 Ia121 Ia122
浓度
纯度
型号
规格
100克/瓶
单位
瓶 瓶 瓶 支 瓶 瓶 瓶 瓶
品牌/厂家
whatman分装 北京索莱宝科技有限公司 上海原叶 福州迈新生物技术开发有限公 司 吉诺生物医药 西陇化工股份有限公司 国药集团化学试剂有限公司 赛齐（上海）生物工程有限公 司 GIBCO 汕头市西陇化工厂有限公司 北京艾德莱生物科技有限公司 上海生工 天根生化科技有限公司 厂家：上海生工 天根生化科技有限公司 索莱宝 福州迈新生物技术开发有限公 司 厂家：上海生工 NEB 上海生工 中天美行 天津光复 汕头市西陇化工厂有限公司 福州迈新生物技术开发有限公 司 北京索莱宝科技有限公司 NEB公司
浓度
纯度
分析纯 AR 分析纯 AR 分析纯 分析纯 生化试剂 化学纯
型号
规格
10ml/支 100ml/瓶 250ml/瓶 25毫升/瓶 100ml/瓶 100毫升/瓶 100毫升/瓶
化学仿制药参比制剂目录(第六十七批)
株式会社
未进口原研药品
67-22
普瑞巴林缓释片
Pregabalin Extended release Tablets/Lyrica Cr
165mg
Pf Prism CV/UPJOHN US 2 LLC
未进口原研药品
美国橙皮书
67-23
普瑞巴林缓释片
Pregabalin Extended release Tablets/Lyrica Cr
国际公认的同种药品
美国橙皮书
67-34
头孢地尼干混悬剂
Cefdinir for Oral Suspension
125mg/5mL
Aurobindo Pharma Limited
美国橙皮书
67-35
蔗糖铁注射液
Iron Sucrose Injection/Venofer
5ml:100mg铁和1.6g蔗糖
0.25%
（10ml:25mg）
Hospira Inc
未进口原研药品
美国橙皮书
67-27
左卡尼汀口服溶液
Levocarnitine Oral Solution/CARNITOR SF
10ml：1g
Leadiant Biosciences, Inc.
未进口原研药品
美国橙皮书
67-28
碳酸氢钠血滤置换液（钾4mmol/L）
Hemofiltration Replacement Fluid of Sodium Bicarbonate
（4mmol/L Potassium Calcium free）/PrismaSol
5000ml（250ml/4750ml）
BAXTER HEALTHCARE CORP
Bioanalytical Method ValidationGuidance for Indust
Guidance for Industry Bioanalytical Method ValidationU.S. Department of Health and Human ServicesFood and Drug AdministrationCenter for Drug Evaluation and Research (CDER)Center for Veterinary Medicine (CVM)May 2001BPGuidance for Industry Bioanalytical Method ValidationAdditional copies are available from:Drug Information Branch (HFD-210)Center for Drug Evaluation and Research (CDER)5600 Fishers Lane, Rockville, MD 20857 (Tel) 301-827-4573Internet at /cder/guidance/index.htmorCommunications Staff (HFV-12)Center for Veterinary Medicine (CVM)7500 Standish Place, Rockville, MD 20855 (Tel) 301–594-1755Internet at /cvmU.S. Department of Health and Human ServicesFood and Drug AdministrationCenter for Drug Evaluation and Research (CDER)Center for Veterinary Medicine (CVM)May 2001BPTable of ContentsI.INTRODUCTION (1)II.BACKGROUND (1)A.F ULL V ALIDATION (2)B.P ARTIAL V ALIDATION (2)C.C ROSS-V ALIDATION (3)III.REFERENCE STANDARD (4)IV.METHOD DEVELOPMENT: CHEMICAL ASSAY (4)A.S ELECTIVITY (4)B.A CCURACY, P RECISION, AND R ECOVERY (5)C.C ALIBRATION/S TANDARD C URVE (5)D.S TABILITY (6)E.P RINCIPLES OF B IOANALYTICAL M ETHOD V ALIDATION AND E STABLISHMENT (8)F.S PECIFIC R ECOMMENDATIONS FOR M ETHOD V ALIDATION (10)V.METHOD DEVELOPMENT: MICROBIOLOGICAL AND LIGAND-BINDING ASSAYS (11)A.S ELECTIVITY I SSUES (11)B.Q UANTIFICATION I SSUES (12)VI.APPLICATION OF VALIDATED METHOD TO ROUTINE DRUG ANALYSIS (13)A CCEPTANCE C RITERIA FOR THE R UN (15)VII.DOCUMENTATION (16)A.S UMMARY I NFORMATION (16)B.D OCUMENTATION FOR M ETHOD E STABLISHMENT (17)C.A PPLICATION TO R OUTINE D RUG A NALYSIS (17)D.O THER I NFORMATION (19)GLOSSARY (20)GUIDANCE FOR INDUSTRY1Bioanalytical Method ValidationI.INTRODUCTIONThis guidance provides assistance to sponsors of investigational new drug applications (INDs), new drug applications (NDAs), abbreviated new drug applications (ANDAs), and supplements in developing bioanalytical method validation information used in human clinical pharmacology, bioavailability (BA), and bioequivalence (BE) studies requiring pharmacokinetic (PK) evaluation. This guidance also applies to bioanalytical methods used for non-human pharmacology/toxicology studies and preclinical studies. For studies related to the veterinary drug approval process, this guidance applies only to blood and urine BA, BE, and PK studies.The information in this guidance generally applies to bioanalytical procedures such as gas chromatography (GC), high-pressure liquid chromatography (LC), combined GC and LC mass spectrometric (MS) procedures such as LC-MS, LC-MS-MS, GC-MS, and GC-MS-MS performed for the quantitative determination of drugs and/or metabolites in biological matricessuch as blood, serum, plasma, or urine. This guidance also applies to other bioanalytical methods, such as immunological and microbiological procedures, and to other biological matrices, such as tissue and skin samples.This guidance provides general recommendations for bioanalytical method validation. The recommendations can be adjusted or modified depending on the specific type of analytical method used. II.BACKGROUND1 This guidance has been prepared by the Biopharmaceutics Coordinating Committee in the Center for Drug Evaluation and Research (CDER) in cooperation with the Center for Veterinary Medicine (CVM) at the Food and Drug Administration.This guidance has been developed based on the deliberations of two workshops: (1) Analytical Methods Validation: Bioavailability, Bioequivalence, and Pharmacokinetic Studies (held on December 3B5, 19902 ) and (2) Bioanalytical Methods Validation C A Revisit With a Decade of Progress (held on January 12B14, 20003).Selective and sensitive analytical methods for the quantitative evaluation of drugs and their metabolites (analytes) are critical for the successful conduct of preclinical and/or biopharmaceutics and clinical pharmacology studies. Bioanalytical method validation includes all of the procedures that demonstrate that a particular method used for quantitative measurement of analytes in a given biological matrix, such as blood, plasma, serum, or urine, is reliable and reproducible for the intended use. The fundamental parameters for this validation include (1) accuracy, (2) precision, (3) selectivity, (4) sensitivity, (5) reproducibility, and (6) stability. Validation involves documenting, through the use of specific laboratory investigations, that the performance characteristics of the method are suitable and reliable for the intended analytical applications. The acceptability of analytical data corresponds directly to the criteria used to validate the method.Published methods of analysis are often modified to suit the requirements of the laboratory performing the assay. These modifications should be validated to ensure suitable performance of the analytical method. When changes are made to a previously validated method, the analyst should exercise judgment as to how much additional validation is needed. During the course of a typical drug development program, a defined bioanalytical method undergoes many modifications. The evolutionary changes to support specific studies and different levels of validation demonstrate the validity of an assay’s performance. Different types and levels of validation are defined and characterized as follows:A.Full Validation•Full validation is important when developing and implementing a bioanalytical method for the first time.•Full validation is important for a new drug entity.• A full validation of the revised assay is important if metabolites are added to an existing assay for quantification.B.Partial ValidationPartial validations are modifications of already validated bioanalytical methods. Partial validation can range from as little as one intra-assay accuracy and precision determination to a nearly full2 Workshop Report: Shah, V.P. et al., Pharmaceutical Research: 1992; 9:588-592.3 Workshop Report: Shah, V.P. et al., Pharmaceutical Research: 2000; 17:in press.validation. Typical bioanalytical method changes that fall into this category include, but are not limited to:•Bioanalytical method transfers between laboratories or analysts•Change in analytical methodology (e.g., change in detection systems)•Change in anticoagulant in harvesting biological fluid•Change in matrix within species (e.g., human plasma to human urine)•Change in sample processing procedures•Change in species within matrix (e.g., rat plasma to mouse plasma)•Change in relevant concentration range•Changes in instruments and/or software platforms•Limited sample volume (e.g., pediatric study)•Rare matrices•Selectivity demonstration of an analyte in the presence of concomitant medications•Selectivity demonstration of an analyte in the presence of specific metabolitesC.Cross-ValidationCross-validation is a comparison of validation parameters when two or more bioanalytical methods are used to generate data within the same study or across different studies. An example of cross-validation would be a situation where an original validated bioanalytical method serves as thereference and the revised bioanalytical method is the comparator. The comparisons should be done both ways.When sample analyses within a single study are conducted at more than one site or more than one laboratory, cross-validation with spiked matrix standards and subject samples should be conducted at each site or laboratory to establish interlaboratory reliability. Cross-validation should also be considered when data generated using different analytical techniques (e.g., LC-MS-MS vs.ELISA4) in different studies are included in a regulatory submission.All modifications should be assessed to determine the recommended degree of validation. The analytical laboratory conducting pharmacology/toxicology and other preclinical studies for regulatory submissions should adhere to FDA=s Good Laboratory Practices (GLPs)5 (21 CFR part 58) and to sound principles of quality assurance throughout the testing process. The bioanalytical method for human BA, BE, PK, and drug interaction studies must meet the criteria in 21 CFR 320.29. The analytical laboratory should have a written set of standard operating procedures (SOPs) to ensure a complete system of quality control and assurance. The SOPs should cover all aspects of analysis from the time the sample is collected and reaches the laboratory until the results of the analysis are reported. The SOPs also should include record keeping, security and chain of sample custody4 Enzyme linked immune sorbent assay5 For the Center for Veterinary Medicine, all bioequivalence studies are subject to Good Laboratory Practices.(accountability systems that ensure integrity of test articles), sample preparation, and analytical tools such as methods, reagents, equipment, instrumentation, and procedures for quality control and verification of results.The process by which a specific bioanalytical method is developed, validated, and used in routine sample analysis can be divided into (1) reference standard preparation, (2) bioanalytical method development and establishment of assay procedure, and (3) application of validated bioanalytical method to routine drug analysis and acceptance criteria for the analytical run and/or batch. These three processes are described in the following sections of this guidance.III.REFERENCE STANDARDAnalysis of drugs and their metabolites in a biological matrix is carried out using samples spiked with calibration (reference) standards and using quality control (QC) samples. The purity of the reference standard used to prepare spiked samples can affect study data. For this reason, an authenticated analytical reference standard of known identity and purity should be used to prepare solutions of known concentrations. If possible, the reference standard should be identical to the analyte. When this is not possible, an established chemical form (free base or acid, salt or ester) of known purity can be used. Three types of reference standards are usually used: (1) certified reference standards (e.g., USP compendial standards); (2) commercially supplied reference standards obtained from a reputable commercial source; and/or (3) other materials of documented purity custom-synthesized by an analytical laboratory or other noncommercial establishment. The source and lot number, expiration date, certificates of analyses when available, and/or internally or externally generated evidence of identity and purity should be furnished for each reference standard.IV.METHOD DEVELOPMENT: CHEMICAL ASSAYThe method development and establishment phase defines the chemical assay. The fundamental parameters for a bioanalytical method validation are accuracy, precision, selectivity, sensitivity, reproducibility, and stability. Measurements for each analyte in the biological matrix should be validated. In addition, the stability of the analyte in spiked samples should be determined. Typical method development and establishment for a bioanalytical method include determination of (1) selectivity, (2) accuracy, precision, recovery, (3) calibration curve, and (4) stability of analyte in spiked samples.A.SelectivitySelectivity is the ability of an analytical method to differentiate and quantify the analyte in thepresence of other components in the sample. For selectivity, analyses of blank samples of theappropriate biological matrix (plasma, urine, or other matrix) should be obtained from at leastsix sources. Each blank sample should be tested for interference, and selectivity should be ensured at the lower limit of quantification (LLOQ).Potential interfering substances in a biological matrix include endogenous matrix components, metabolites, decomposition products, and in the actual study, concomitant medication and other exogenous xenobiotics. If the method is intended to quantify more than one analyte, each analyte should be tested to ensure that there is no interference.B.Accuracy, Precision, and RecoveryThe accuracy of an analytical method describes the closeness of mean test results obtained by the method to the true value (concentration) of the analyte. Accuracy is determined by replicate analysis of samples containing known amounts of the analyte. Accuracy should be measured using a minimum of five determinations per concentration. A minimum of three concentrations in the range of expected concentrations is recommended. The mean value should be within 15% of the actual value except at LLOQ, where it should not deviate by more than 20%. The deviation of the mean from the true value serves as the measure of accuracy.The precision of an analytical method describes the closeness of individual measures of an analyte when the procedure is applied repeatedly to multiple aliquots of a single homogeneous volume of biological matrix. Precision should be measured using a minimum of five determinations per concentration. A minimum of three concentrations in the range of expected concentrations is recommended. The precision determined at each concentration level should not exceed 15% of the coefficient of variation (CV) except for the LLOQ, where it should not exceed 20% of the CV. Precision is further subdivided into within-run, intra-batch precision or repeatability, which assesses precision during a single analytical run, and between-run, inter-batch precision or repeatability, which measures precision with time, and may involve different analysts, equipment, reagents, and laboratories.The recovery of an analyte in an assay is the detector response obtained from an amount of the analyte added to and extracted from the biological matrix, compared to the detector response obtained for the true concentration of the pure authentic standard. Recovery pertains to the extraction efficiency of an analytical method within the limits of variability. Recovery of the analyte need not be 100%, but the extent of recovery of an analyte and of the internal standard should be consistent, precise, and reproducible. Recovery experiments should be performed by comparing the analytical results for extracted samples at three concentrations (low, medium, and high) with unextracted standards that represent 100% recovery.C.Calibration/Standard CurveA calibration (standard) curve is the relationship between instrument response and known concentrations of the analyte. A calibration curve should be generated for each analyte in thesample. A sufficient number of standards should be used to adequately define the relationship between concentration and response. A calibration curve should be prepared in the same biological matrix as the samples in the intended study by spiking the matrix with known concentrations of the analyte. The number of standards used in constructing a calibration curve will be a function of the anticipated range of analytical values and the nature of theanalyte/response relationship. Concentrations of standards should be chosen on the basis of the concentration range expected in a particular study. A calibration curve should consist of a blank sample (matrix sample processed without internal standard), a zero sample (matrix sample processed with internal standard), and six to eight non-zero samples covering the expected range, including LLOQ.1.Lower Limit of Quantification (LLOQ)The lowest standard on the calibration curve should be accepted as the limit ofquantification if the following conditions are met:C The analyte response at the LLOQ should be at least 5 times the responsecompared to blank response.C Analyte peak (response) should be identifiable, discrete, and reproducible witha precision of 20% and accuracy of 80-120%.2.Calibration Curve/Standard Curve/Concentration-ResponseThe simplest model that adequately describes the concentration-response relationshipshould be used. Selection of weighting and use of a complex regression equation should be justified. The following conditions should be met in developing a calibration curve:C#20% deviation of the LLOQ from nominal concentrationC#15% deviation of standards other than LLOQ from nominal concentrationAt least four out of six non-zero standards should meet the above criteria, including the LLOQ and the calibration standard at the highest concentration. Excluding thestandards should not change the model used.D.StabilityDrug stability in a biological fluid is a function of the storage conditions, the chemical properties of the drug, the matrix, and the container system. The stability of an analyte in a particular matrix and container system is relevant only to that matrix and container system and should not be extrapolated to other matrices and container systems. Stability procedures should evaluate the stability of the analytes during sample collection and handling, after long-term (frozen at theintended storage temperature) and short-term (bench top, room temperature) storage, and after going through freeze and thaw cycles and the analytical process. Conditions used in stability experiments should reflect situations likely to be encountered during actual sample handling and analysis. The procedure should also include an evaluation of analyte stability in stock solution.All stability determinations should use a set of samples prepared from a freshly made stock solution of the analyte in the appropriate analyte-free, interference-free biological matrix. Stock solutions of the analyte for stability evaluation should be prepared in an appropriate solvent at known concentrations.1.Freeze and Thaw StabilityAnalyte stability should be determined after three freeze and thaw cycles. At least three aliquots at each of the low and high concentrations should be stored at the intendedstorage temperature for 24 hours and thawed unassisted at room temperature. Whencompletely thawed, the samples should be refrozen for 12 to 24 hours under the sameconditions. The freeze–thaw cycle should be repeated two more times, then analyzedon the third cycle. If an analyte is unstable at the intended storage temperature, thestability sample should be frozen at -700C during the three freeze and thaw cycles.2.Short-Term Temperature StabilityThree aliquots of each of the low and high concentrations should be thawed at roomtemperature and kept at this temperature from 4 to 24 hours (based on the expectedduration that samples will be maintained at room temperature in the intended study) and analyzed.3.Long-Term StabilityThe storage time in a long-term stability evaluation should exceed the time between the date of first sample collection and the date of last sample analysis. Long-term stabilityshould be determined by storing at least three aliquots of each of the low and highconcentrations under the same conditions as the study samples. The volume of samples should be sufficient for analysis on three separate occasions. The concentrations of allthe stability samples should be compared to the mean of back-calculated values for the standards at the appropriate concentrations from the first day of long-term stabilitytesting.4.Stock Solution StabilityThe stability of stock solutions of drug and the internal standard should be evaluated at room temperature for at least 6 hours. If the stock solutions are refrigerated or frozenfor the relevant period, the stability should be documented. After completion of thedesired storage time, the stability should be tested by comparing the instrumentresponse with that of freshly prepared solutions.5.Post-Preparative StabilityThe stability of processed samples, including the resident time in the autosampler, should be determined. The stability of the drug and the internal standard should be assessedover the anticipated run time for the batch size in validation samples by determiningconcentrations on the basis of original calibration standards.Although the traditional approach of comparing analytical results for stored samples with those for freshly prepared samples has been referred to in this guidance, other statistical approaches based on confidence limits for evaluation of an analyte=s stability in abiological matrix can be used. SOPs should clearly describe the statistical method andrules used. Additional validation may include investigation of samples from dosedsubjects.E.Principles of Bioanalytical Method Validation and Establishment•The fundamental parameters to ensure the acceptability of the performance of a bioanalytical method validation are accuracy, precision, selectivity, sensitivity,reproducibility, and stability.• A specific, detailed description of the bioanalytical method should be written. This can be in the form of a protocol, study plan, report, and/or SOP.•Each step in the method should be investigated to determine the extent to which environmental, matrix, material, or procedural variables can affect the estimation of analyte in the matrix from the time of collection of the material up to and including the time ofanalysis.•It may be important to consider the variability of the matrix due to the physiological nature of the sample. In the case of LC-MS-MS-based procedures, appropriate steps should be taken to ensure the lack of matrix effects throughout the application of the method,especially if the nature of the matrix changes from the matrix used during method validation.• A bioanalytical method should be validated for the intended use or application. All experiments used to make claims or draw conclusions about the validity of the methodshould be presented in a report (method validation report).•Whenever possible, the same biological matrix as the matrix in the intended samples should be used for validation purposes. (For tissues of limited availability, such as bone marrow, physiologically appropriate proxy matrices can be substituted.)•The stability of the analyte (drug and/or metabolite) in the matrix during the collection process and the sample storage period should be assessed, preferably prior to sampleanalysis.•For compounds with potentially labile metabolites, the stability of analyte in matrix from dosed subjects (or species) should be confirmed.•The accuracy, precision, reproducibility, response function, and selectivity of the method for endogenous substances, metabolites, and known degradation products should beestablished for the biological matrix. For selectivity, there should be evidence that thesubstance being quantified is the intended analyte.•The concentration range over which the analyte will be determined should be defined in the bioanalytical method, based on evaluation of actual standard samples over the range,including their statistical variation. This defines the standard curve.• A sufficient number of standards should be used to adequately define the relationship between concentration and response. The relationship between response and concentration should be demonstrated to be continuous and reproducible. The number of standards used should be a function of the dynamic range and nature of the concentration-responserelationship. In many cases, six to eight concentrations (excluding blank values) can define the standard curve. More standard concentrations may be recommended for nonlinear than for linear relationships.•The ability to dilute samples originally above the upper limit of the standard curve should be demonstrated by accuracy and precision parameters in the validation.•In consideration of high throughput analyses, including but not limited to multiplexing, multicolumn, and parallel systems, sufficient QC samples should be used to ensure control of the assay. The number of QC samples to ensure proper control of the assay should be determined based on the run size. The placement of QC samples should be judiciously considered in the run.•For a bioanalytical method to be considered valid, specific acceptance criteria should be set in advance and achieved for accuracy and precision for the validation of QC samples over the range of the standards.F.Specific Recommendations for Method Validation•The matrix-based standard curve should consist of a minimum of six standard points, excluding blanks, using single or replicate samples. The standard curve should cover the entire range of expected concentrations.•Standard curve fitting is determined by applying the simplest model that adequately describes the concentration-response relationship using appropriate weighting and statistical tests for goodness of fit.•LLOQ is the lowest concentration of the standard curve that can be measured with acceptable accuracy and precision. The LLOQ should be established using at least five samples independent of standards and determining the coefficient of variation and/orappropriate confidence interval. The LLOQ should serve as the lowest concentration on the standard curve and should not be confused with the limit of detection and/or the low QC sample. The highest standard will define the upper limit of quantification (ULOQ) of an analytical method.•For validation of the bioanalytical method, accuracy and precision should be determined using a minimum of five determinations per concentration level (excluding blank samples).The mean value should be within ±15% of the theoretical value, except at LLOQ, where it should not deviate by more than ±20%. The precision around the mean value should not exceed 15% of the CV, except for LLOQ, where it should not exceed 20% of the CV.Other methods of assessing accuracy and precision that meet these limits may be equally acceptable.•The accuracy and precision with which known concentrations of analyte in biological matrix can be determined should be demonstrated. This can be accomplished by analysis ofreplicate sets of analyte samples of known concentrations C QC samples C from anequivalent biological matrix. At a minimum, three concentrations representing the entire range of the standard curve should be studied: one within 3x the lower limit of quantification (LLOQ) (low QC sample), one near the center (middle QC), and one near the upperboundary of the standard curve (high QC).•Reported method validation data and the determination of accuracy and precision should include all outliers; however, calculations of accuracy and precision excluding values that are statistically determined as outliers can also be reported.•The stability of the analyte in biological matrix at intended storage temperatures should be established. The influence of freeze-thaw cycles (a minimum of three cycles at twoconcentrations in triplicate) should be studied.•The stability of the analyte in matrix at ambient temperature should be evaluated over a time period equal to the typical sample preparation, sample handling, and analytical run times.•Reinjection reproducibility should be evaluated to determine if an analytical run could be reanalyzed in the case of instrument failure.•The specificity of the assay methodology should be established using a minimum of six independent sources of the same matrix. For hyphenated mass spectrometry-basedmethods, however, testing six independent matrices for interference may not be important.In the case of LC-MS and LC-MS-MS-based procedures, matrix effects should beinvestigated to ensure that precision, selectivity, and sensitivity will not be compromised.Method selectivity should be evaluated during method development and throughout methodvalidation and can continue throughout application of the method to actual study samples.•Acceptance/rejection criteria for spiked, matrix-based calibration standards and validation QC samples should be based on the nominal (theoretical) concentration of analytes.Specific criteria can be set up in advance and achieved for accuracy and precision over therange of the standards, if so desired.V.METHOD DEVELOPMENT: MICROBIOLOGICAL AND LIGAND-BINDING ASSAYSMany of the bioanalytical validation parameters and principles discussed above are also applicable to microbiological and ligand-binding assays. However, these assays possess some unique characteristics that should be considered during method validation.A.Selectivity IssuesAs with chromatographic methods, microbiological and ligand-binding assays should be shown to be selective for the analyte. The following recommendations for dealing with two selectivity issues should be considered:1.Interference From Substances Physiochemically Similar to the Analyte•Cross-reactivity of metabolites, concomitant medications, or endogenouscompounds should be evaluated individually and in combination with the analyteof interest.•When possible, the immunoassay should be compared with a validated reference method (such as LC-MS) using incurred samples and predetermined criteria foragreement of accuracy of immunoassay and reference method.。
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国家药监局关于批准注册121个医疗器械产品的公告(2021年3月)
国家药监局关于批准注册121个医疗器械产品的公告(2021年3月)文章属性•【制定机关】国家药品监督管理局•【公布日期】2021.04.15•【文号】国家药品监督管理局公告2021年第55号•【施行日期】2021.04.15•【效力等级】部门规范性文件•【时效性】现行有效•【主题分类】药政管理正文国家药品监督管理局公告2021年第55号国家药监局关于批准注册121个医疗器械产品的公告(2021年3月)2021年3月，国家药品监督管理局共批准注册医疗器械产品121个。
其中，境内第三类医疗器械产品77个，进口第三类医疗器械产品18个，进口第二类医疗器械产品24个，港澳台医疗器械产品2个（具体产品见附件）。
特此公告。
附件：2021年3月批准注册医疗器械产品目录国家药监局2021年4月15日附件2021年3月批准注册医疗器械产品目录序号产品名称注册人名称注册证编号境内第三类医疗器械1导引系统先健科技（深圳）有限公司国械注准202130301522一次性使用防针刺精密过滤输液器带针上海宝舜医疗器械有限公司国械注准202131401533导引导管南京沃福曼医疗科技有限公司国械注准20213030154 4血液透析滤过器威海威高血液净化制品有限公司国械注准20213100155 5一次性使用压力延长管深圳市保安医疗用品有限公司国械注准20213030156 6硬脑膜修补片北京博辉瑞进生物科技有限公司国械注准20213130157 7一次性使用单向阀多通连接江苏省华星医疗器械实业有限公国械注准器司202131401588椎间融合器深圳市沃尔德外科医疗器械技术有限公司国械注准202131301599一次性使用精密过滤输液器带针淄博侨森医疗用品股份有限公司国械注准2021314016010椎间融合器武汉德骼拜尔外科植入物有限公司国械注准2021313016111不可吸收带线锚钉北京市富乐科技开发有限公司国械注准20213130162 12一次性使用静脉留置针苏州鑫康道医疗科技有限公司国械注准20213140163 13冠脉球囊扩张导管苏州莱诺医疗器械有限公司国械注准20213030164 14一次性使用输液器带针天津市远东医材有限公司国械注准20213140165 15外周球囊扩张导管北京永益润成科技有限公司国械注准20213030166 16一次性使用体外循环管道常州市康心医疗器械有限公司国械注准20213100167 17钛合金手足锁定接骨板系统创美得医疗器械（天津）有限公司国械注准20213130168 18一次性使用血管内成像导管苏州阿格斯医疗技术有限公司国械注准20213060169 19一次性内镜用注射针诸暨市鹏天医疗器械有限公司国械注准20213140170 20分段控弯导引系统先健科技（深圳）有限公司国械注准20213030171 21一次性使用血液透析管路健帆生物科技集团股份有限公司国械注准2021310017222可折叠人工晶状体天津世纪康泰生物医学工程有限公司国械注准2021316017323乙型肝炎病毒核酸测定试剂上海仁度生物科技有限公司国械注准盒（RNA捕获探针法）2021340017424一次性使用电子输尿管肾盂内窥镜导管北京北方腾达科技发展有限公司国械注准2021306017525新型冠状病毒2019-nCoV核酸检测试剂盒（荧光PCR法）杭州迪安生物技术有限公司国械注准2021340017626儿童手部X射线影像骨龄辅助评估软件杭州依图医疗技术有限公司国械注准2021321017727病人监护仪通用电气医疗系统（中国）有限公司国械注准2021307017828三维腹腔内窥镜山东威高手术机器人有限公司国械注准2021306017929单光子发射计算机断层成像系统滨松光子医疗科技（廊坊）有限公司国械注准2021306018030一次性使用热活检钳诸暨市鹏天医疗器械有限公司国械注准2021301018131一次性使用高频十二指肠乳头切开刀杭州莱恩瑟特医疗技术有限公司国械注准2021301018232生物安全柜苏州安泰空气技术有限公司国械注准2021322018333一次性使用Y型连接阀套装厦门鑫康顺医疗科技有限公司国械注准2021303018434预充式导管冲洗器山东赛克赛斯生物科技有限公司国械注准20213140185 35锚钉系统大博医疗科技股份有限公司国械注准20213130186 36泡沫敷料广州润虹医药科技股份有限公司国械注准2021314018737一次性使用精密过滤避光输液器山东新华安得医疗用品有限公司国械注准2021314018838软性亲水接触镜江苏天眼医药科技股份有限公司国械注准2021316018939一次性使用无菌注射器带针江苏采纳医疗科技有限公司国械注准2021314019040注射用交联透明质酸钠凝胶杭州科腾生物制品有限公司国械注准20213130191 41血液透析浓缩液四川威力生医疗科技有限公司国械注准2021310019242一次性使用防针刺静脉输液针山东威高集团医用高分子制品股份有限公司国械注准2021314019343一次性使用避光输液器河南曙光汇知康生物科技股份有限公司国械注准2021314019444一次性使用精密过滤输液器带针南阳市久康医疗器械有限公司国械注准2021314019545一次性使用无菌自毁式注射器聚民生物科技有限公司国械注准2021314019646夹子装置诸暨市鹏天医疗器械有限公司国械注准2021302019747一次性使用无菌注射针江苏采纳医疗科技有限公司国械注准20213140198 48颅内支撑导管北京久事神康医疗科技有限公司国械注准20213030199 49软性亲水接触镜江苏天眼医药科技股份有限公司国械注准2021316020050一次性使用无菌注射器带针成都市新津事丰医疗器械有限公司国械注准2021314020151一次性使用静脉留置针佳康医用器材（青岛）有限公司国械注准20213140202 52颅内球囊扩张导管依奈德医疗技术（上海）有限公司国械注准2021303020353一次性使用压力延长管山东威高集团医用高分子制品股份有限公司国械注准2021314020454颅内球囊扩张导管浙江归创医疗器械有限公司国械注准2021303020555远端通路导引导管珠海通桥医疗科技有限公司国械注准20213030206 56导管鞘沛嘉医疗科技（苏州）有限公司国械注准20213030207 57一次性使用注射笔用针头甘甘医疗科技江苏有限公司国械注准2021314020858麻醉系统通用电气医疗系统（中国）有限公司国械注准2021308020959肺炎CT影像辅助分诊与评估软件北京推想科技有限公司国械注准2021321021060肺炎CT影像辅助分诊与评估软件杭州深睿博联科技有限公司国械注准2021321021161医用血管造影X射线机东软医疗系统股份有限公司国械注准20213060212 62复合陡脉冲治疗设备上海睿刀医疗科技有限公司国械注准2021309021363放射治疗轮廓勾画软件海创时代（深圳）医疗科技有限公司国械注准2021321021464口腔种植手术导航定位设备北京柏惠维康科技有限公司国械注准20213010215 65超导型磁共振成像系统康达洲际医疗器械有限公司国械注准2021306021666人附睾蛋白4测定试剂盒（化学发光法）深圳市亚辉龙生物科技股份有限公司国械注准2021340021767胃泌素释放肽前体测定试剂盒（化学发光法）深圳市亚辉龙生物科技股份有限公司国械注准2021340021868前列腺酸性磷酸酶测定试剂盒（化学发光免疫分析法）苏州长光华医生物医学工程有限公司国械注准2021340021969神经元特异性烯醇化酶测定试剂盒（化学发光免疫分析法）苏州长光华医生物医学工程有限公司国械注准2021340022070乙型肝炎病毒前S1抗原检迈克生物股份有限公司国械注准测试剂盒（直接化学发光法）2021340022171人JAK2-V617F基因突变检测试剂盒（荧光PCR法）迈杰转化医学研究（苏州）有限公司国械注准2021340022272一次性使用血液透析器广州市恩德氏医疗制品实业有限公司国械注准2021310022373流出道单瓣补片北京佰仁医疗科技股份有限公司国械注准20213130224 74一次性使用静脉留置针威海洁瑞医用制品有限公司国械注准20213140225 75软聚硅酮泡沫敷料江苏诺瓦立医疗用品有限公司国械注准2021314022676幽门螺杆菌23S rRNA基因突变检测试剂盒（PCR-荧光探针法）上海芯超生物科技有限公司国械注准2021340022777新型冠状病毒2019-nCoV核酸检测试剂盒（荧光PCR法）重庆中元汇吉生物技术有限公司国械注准20213400228进口第三类医疗器械78血液透析用中心静脉导管及附件Covidien llc国械注进2021310005779钛夹AESCULAP AG国械注进20213020058 80注射用交联透明质酸钠凝胶Q-Med AB国械注进2021313005981颅内取栓支架Micro Therapeutics,Inc.dba ev3Neurovascular国械注进2021303006082游离前列腺特异性抗原校准品Siemens Healthcare DiagnosticsProducts Limited国械注进2021340007383丙型肝炎病毒（HCV）核酸（RNA）检测试剂盒（实时荧光PCR法）Cepheid AB国械注进2021340007484抗CD20（L26）鼠单克隆抗体试剂（免疫组织化学Ventana Medical Systems, Inc.国械注进20213400075法）85牙科种植体Zimmer Dental, Inc.国械注进20213170076 86空心纤维血液透析器Vital Healthcare Sdn. Bhd.国械注进20213100077 87一次性负压装置及护创敷料Smith & Nephew Medical Ltd国械注进20213140090 88双极器械ERBE Elektromedizin GmbH国械注进2021301009189一次性使用高频圈套器オリンパスメディカルシステムズ株式会社国械注进2021301009290麻醉系统Dr?gerwerk AG & Co. KGaA国械注进20213080093 91X射线计算机体层摄影设备Siemens Healthcare GmbH国械注进20213060094 92关节内窥镜（?）国械注进20213060095 93电子下消化道内窥镜富士フイルム株式会社国械注进20213060096 94植入式给药装置专用针Fresenius Kabi AG国械注进20213140097 95微导管Vascular Solutions LLC国械注进20213030098进口第二类医疗器械96胰岛素样生长因子-1检测试剂盒（电化学发光法）Roche Diagnostics GmbH国械注进2021240006197免疫球蛋白E校准品DiaSys Diagnostic Systems GmbH国械注进2021240006298纤维蛋白原质控品（低值）Instrumentation LaboratoryCompany国械注进2021240006399一次性使用直线型切割吻合器及渐进式钉匣Covidien llc国械注进20212020064100血糖仪OSANG Healthcare Co.,Ltd.国械注进20212220065 101血糖仪OSANG Healthcare Co.,Ltd.国械注进20212220066102内窥镜图像处理及照明装置オリンパスメディカルシステムズ株式会社国械注进20212060067103内镜清洗消毒器STEELCO SPA国械注进20212110068 104电动产床タカラメディカル株式会社国械注进20212180069 105角膜内皮显微镜株式会社コーナン?メディカル国械注进20212160070106肺功能测试系统GANSHORN Medizin ElectronicGmbH国械注进20212070071107牙科低压电动马达株式会社ナカニシ国械注进20212170072108抗β2糖蛋白1结构域1IgG抗体检测试剂盒（化学发光免疫分析法）INOVA Diagnostics, Inc.国械注进20212400078109胱抑素C校准液Siemens Healthcare DiagnosticsInc.国械注进20212400079110降钙素原校准品Ortho-Clinical Diagnostics国械注进20212400080111胰岛素样生长因子-I测定试剂盒（化学发光法）Siemens Healthcare DiagnosticsProducts Limited国械注进20212400081112降钙素原质控品Ortho-Clinical Diagnostics国械注进20212400082113喷砂洁牙机SATELEC A Company ofACTEON Group国械注进20212170083114种植体稳固度检测仪Osstell AB国械注进20212170084 115多导睡眠记录系统SOMNOmedics GmbH国械注进20212070085 116一次性使用超声内窥镜水囊HOYA株式会社豪雅株式会社国械注进20212060086 117多导睡眠记录系统SOMNOmedics GmbH国械注进20212070087 118全自动微生物质谱检测系统ASTA Corporation国械注进20212220088119齿科铸造合金Eisenbacher Dentalwaren EDGmbH国械注进20212170089港澳台医疗器械120软性亲水接触镜望隼科技股份有限公司国械注许20213160003 121软性亲水接触镜星歐光學股份有限公司国械注许20213160004。
胸水中LPS、IL-35、RORα在结核性胸腔积液中的表达变化及临床意义
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Product Name:
FRAX486CAS No.:
1232030-35-1Cat. No.:
HY-15542B MWt:
513.39Formula:
C25H23Cl2FN6O Purity :>98%
Solubility:Mechanisms:
Biological Activity:
References:
Caution: Not fully tested. For research purposes only
Medchemexpress LLC
[1]. Dolan BM, et al. Rescue of fragile X syndrome phenotypes in Fmr1 KO mice by the small-molecule PAK
inhibitor FRAX486. Proc Natl Acad Sci U S A. 2013 Apr 2;110(14):5671-6.[2]. Hayashi-Takagi A, et al. PAKs inhibitors ameliorate schizophrenia-associated dendritic spine deterioration
in vitro and in vivo during late adolescence. Proc Natl Acad Sci U S A. 2014 Apr 29;111(17):6461-6.FRAX486 is a selective inhibitor of group I PAKs with IC50s of 8.25/39.5, /55.3 nM for PAK1/PAK2/PAK3
respectivelt; less potent for PAK4(IC50=779 nM).
IC50 value: 8.25/39.5, /55.3 nM(PAK1/PAK2/PAK3) [1]
Target: PAKs inhibitor in vitro: FRAX486 is an inhibitor of PAK, with excellent selectivity for group I over group II PAKs [1]. Different from W-7 and KN-62, FRAX355 and FRAX486 did not completely block the NMDA-R activation-mediated spine enlargement in the presence of control RNAi, suggesting that these FRAX compounds act less
aversely to the synaptic plasticity [2].in vivo: Mice were administered FRAX486 at a concentration of 20 mg/kg, and brain tissue was collected 8 h after drug treatment. Indeed, the phenotype was rescued by treatment with FRAX486 8 h after a single treatment because spine density was decreased to levels similar to WT mice but significantly l...
Pathways:Cell Cycle/DNA Damage; Target:PAKs Product Data Sheet
CHCL3 <8 mg/ml
11D e e r P a r kD r i v e , S u i t e 102D M o n m o u t h J u n c t i o n , N J 08852,U S A
E m a i l : i n f o @m e d c h e m e x p r e s s .c o m W e b : w w w .m e d c h e m e x p r e s s .c o m。